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Evaluation of commercial RNA extraction kits for the isolation of viral MS2 RNA from soil
Journal of Virological Methods 168 (2010) 44–50
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Evaluation of commercial RNA extraction kits for the isolation of viral MS2 RNA from soil Shauna M. Dineen a,b, Roman Aranda IV a,b, Marianne E. Dietz a,b, Douglas L. Anders c, James M. Robertson b,∗ a b c
Visiting Scientist, Federal Bureau of Investigation Laboratory, 2501 Investigation Parkway, Quantico, VA 22135, USA Counterterrorism and Forensic Science Research Unit, Federal Bureau of Investigation Laboratory, 2501 Investigation Parkway, Quantico, VA 22135, USA Hazardous Materials Science Response Unit, Federal Bureau of Investigation Laboratory, 2501 Investigation Parkway, Quantico, VA 22135, USA
a b s t r a c t Article history: Received 23 December 2009 Received in revised form 9 April 2010 Accepted 19 April 2010 Available online 24 April 2010 Keywords: RNA Virus RT-PCR Extraction Detection Soil
Nucleic acid extraction is a critical step in the detection of an unknown biological agent. However, success can vary depending on the isolation and identification methods chosen and the difficulty of extraction from environmental matrices. In this work, bacteriophage MS2 RNA was extracted from three soil matrices, sand, clay, and loam, using five commercially available kits: the PowerSoilTM Total RNA Isolation, E.Z.N.A.® Soil RNA, FastRNA® Pro Soil-Direct, FastRNA® Pro Soil-Indirect, and IT 1-2-3 Platinum PathTM kits. Success of the isolation was determined using an MS2-specific RT-PCR assay. The reproducibility and sensitivity of each method in the hands of both experienced and novice users were assessed and compared. Cost, operator time, and storage conditions were also considered in the evaluation. The RNA isolation method that yielded the best results, as defined by reproducibility and sensitivity, was the E.Z.N.A.® Soil RNA kit for sand, the IT 1-2-3 Platinum PathTM Sample Purification kit for clay, and the FastRNA® Pro Soil-Indirect kit for loam. However, if time and storage conditions are important considerations, the IT 1-2-3 Platinum PathTM kit may be appropriate for use with all soils since the kit has the shortest processing time and fewest temperature requirements. Published by Elsevier B.V.
1. Introduction The detection and identification of an unknown biological agent is a critical component of an environmental analysis or bioterrorism response plan. The accidental or deliberate release of a biological agent into the environment can cause illness or death in humans, animals, or plants. Bacteria such as Bacillus anthracis and ricin toxin from Ricinus communis have been used as biological agents (Rotz et al., 2002). Pathogenic viruses can also serve as agents because they are highly infectious and can be readily transmitted after dispersal into air, water, food, or soil (Brassard et al., 2009; Garnier et al., 2009; Rzezutka and Cook, 2004). With the high morbidity and mortality rates associated with some pathogenic agents, a rapid response to either an outbreak or intentional release is essential (Rotz et al., 2002; Sinclair et al., 2008). For the response plan, methods to isolate and identify biological pathogens should be chosen based on their effectiveness and efficiency at recovering and detecting target organisms from the sample collected. Compared to time-consuming cell culture assays, real-time polymerase chain reaction (PCR) provides a sensitive, specific, and
∗ Corresponding author. Tel.: +1 703 632 4555; fax: +1 703 632 4500. E-mail address: [email protected] (J.M. Robertson). 0166-0934/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.jviromet.2010.04.014
much more rapid means to detect biological agents in environmental samples (Broussard, 2001). Through the use of target-specific primers and a fluorogenic probe, real-time PCR is able to amplify and detect trace amounts of DNA or reverse-transcribed RNA from a specific agent. However, successful detection first requires efficient extraction and purification of the DNA or RNA from the environmental sample (Kuske et al., 1998). Unfortunately, nucleic acids of biological agents can be extremely difficult to detect, particularly in certain environmental matrices. Many environmental matrices contain substances that co-extract with nucleic acids and inhibit the enzymatic processes involved in PCR. Soils are particularly problematic because they are rich in humic substances (Schneegurt et al., 2003). Humic substances, which include humic acids, fulvic acids, and humins, represent a complex mixture of aromatic and aliphatic groups in large molecules that are brown to black in color, absorb light in the 300–400 nm range, and are the major constituents of soil organic matter (Alm et al., 2000; Miller, 2001). With physical–chemical characteristics similar to those of nucleic acids, humic substances are difficult to remove and often co-purify with DNA or RNA from soil (Dong et al., 2006). This contamination affects downstream applications since as little as 0.1 ng l−1 humic acid can inhibit Taq DNA polymerase during PCR (Kermekchiev et al., 2009; Tebbe and Vahjen, 1993; Tsai and Olson, 1991). For the detection and identi-
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fication of a microorganism or virus in an environmental sample, the presence of such PCR inhibitors may potentially lead to falsenegative results (Dreier et al., 2005). Thus, the efficiency of the method used to extract nucleic acid from soil is determined not only by the amount of nucleic acid recovered, but also by the success in removing PCR-inhibitory substances. Numerous procedures exist for the isolation and purification of DNA and RNA from soil. The isolation procedures vary in the timing of cell lysis, occurring either in situ (direct method) or following the separation of the microorganisms from the environmental matrix (indirect method) (Frostegard et al., 1999; Steffan et al., 1988). In addition to the timing and method of cell lysis (chemical, mechanical, or a combination of both), the isolation procedures also vary in how the nucleic acids are purified, often through steps such as phenol/chloroform extraction, cesium chloride density gradient centrifugation, chemical flocculation, and/or gel filtration (Arbeli and Fuentes, 2007; Braid et al., 2003; Miller, 2001; Miller et al., 1999; Persoh et al., 2008). Procedures that include these time-consuming steps are either too cumbersome or too slow for obtaining rapid results in response to an infectious outbreak or suspected bioterrorism attack. Commercial DNA extraction kits have previously been compared for their ability to extract DNA from bacteria in soil samples (Sagova-Mareckova et al., 2008; Whitehouse and Hottel, 2007). Studies suggest that the selection of an appropriate extraction and purification procedure depends on the physical and chemical characteristics of the soil matrix, such as organic matter, clay content, and pH (Burgmann et al., 2001; Sagova-Mareckova et al., 2008). Similar results have been shown with different laboratory procedures used to extract microbial RNA from soil and sediment (Borneman and Triplett, 1997; Mendum et al., 1998; Ogram et al., 1995; Sessitsch et al., 2002; Tsai and Olson, 1991). However, commercial RNA extraction kits have yet to be compared for their relative effectiveness with different soil matrices. In this study, five commercially available kits were evaluated for their ability to extract bacteriophage MS2 RNA from three different soils: sand, clay, and loam, which varied in organic content, pH, and particle size. Each soil was spiked with three different amounts of MS2 phage. MS2 (family Leviviridae, genus Levivirus, species Enterobacteria phage MS2) is a small, male-specific bacteriophage of Escherichia coli often used as a surrogate for pathogenic RNA viruses, particularly to indicate enteric virus contamination of wastewater (Dreier et al., 2005; Olson et al., 2004). Three spiking amounts of MS2 served in the determination of a detection threshold for each kit. The efficiency of extraction was evaluated using an MS2-specific real-time reverse transcription-PCR (RT-PCR) assay (O’Connell et al., 2006). Beyond the comparison of the five kits, two PCR-cleanup methods were evaluated for their ability to remove PCR inhibitors from the kit extracts. The results of this study aid in the selection of an appropriate RNA extraction method and, if necessary, an additional purification step for a given soil sample. The labor, time, and cost required for each method were also compared. 2. Materials and methods 2.1. Soils Three different soils were collected in Quantico, VA in August 2008. Each of the soil samples was sieved using a fiberglass mesh with 1 mm by 1.5 mm openings to remove large, non-soil materials and stored at 4 ◦ C. Soils were not autoclaved in order to represent a real-world scenario more realistically. The soils were analyzed and classified by A&L Eastern Laboratories (Richmond, VA) for particle size, pH, and organic content. Organic content was determined using the Walkley–Black procedure (Walkley, 1935). Characteristics of the three soils are listed in Table 1.
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The soil textures were found to be sand, sandy clay, and sandy loam and are referred to as “sand”, “clay”, and “loam”, respectively. 2.2. MS2 phage and titer determination MS2 bacteriophage strain 15597-B1 and its Escherichia coli (E. coli) host 15597 were purchased from American Type Culture Center (ATCC, Manassas, VA). Media and titer determination procedures were followed according to ATCC instructions. For use in spiking experiments, 100 l aliquots of MS2 bacteriophage were stored in 10% glycerol at −80 ◦ C. 2.3. Soil spiking The three non-autoclaved soils were spiked with four different amounts of MS2 bacteriophage, 0× or “non-spiked”, 0.1×, 1×, and 10×, where 1× corresponds to 3 × 108 plaque-forming units (PFU) per gram of soil. To account for the variation in soil sample size processed by the five kits, the amount of MS2 phage added to each soil was adjusted such that the MS2 PFU per gram soil remained consistent across all kits for a given spiking amount. Spiked soils were mixed thoroughly and then subjected to one of the five extraction kit protocols. 2.4. RNA extraction Five commercially available RNA extraction kits were evaluated in this study. Four kits employ a direct extraction method in which lysis occurs in the presence of the soil (Frostegard et al., 1999; Steffan et al., 1988). The fifth kit uses an indirect method in which the target organisms are separated from the soil prior to lysis. RNA extractions were performed according to the manufacturers’ instructions. All extracted RNA samples were immediately stored at −80 ◦ C until evaluation via RT-PCR analysis. The PowerSoilTM Total RNA Isolation kit (MO BIO, Carlsbad, CA, USA) utilizes bead beating in the extraction procedure. Spiked soils were treated with a series of kit solutions and underwent bead beating on a vortexer to lyse viral particles. Total RNA was isolated using phenol: chloroform and then captured on columns packed with proprietary matrix, washed, and eluted with the kit elution buffer. The E.Z.N.A.® Soil RNA kit (Omega Bio-Tek, Norcross, GA, USA) also combines bead beating by vortexing with phenol: chloroform extraction to isolate RNA from soil. Glass beads and a series of buffers were added to spiked soils and then vortexed. Total RNA was isolated using phenol: chloroform and precipitated using isopropanol. RNA was resuspended in diethylpyrocarbonate (DEPC) water and re-extracted using phenol: chloroform. RNA samples were mixed with ethanol and then transferred to spin columns that bind RNA. RNA was washed with wash buffer solutions and then eluted from columns using DEPC water. The FastRNA® Pro Soil-Direct kit (MP Biomedicals, Solon, OH, USA) uses bead beating as well as a proprietary RNAMATRIX® to release, extract, and purify total RNA from soil. Spiked soils were added to a lysing matrix, treated with lysis buffer, and subjected to bead beating in the FastPrep® instrument. RNA was isolated using phenol: chloroform and precipitated using isopropanol. RNA was washed with ethanol and resuspended in DEPC water. RNA was bound to the RNAMATRIX® , washed, and eluted in DEPC water. The FastRNA® Pro Soil-Indirect kit (MP Biomedicals, Solon, OH, USA) uses an indirect method to extract RNA from soil. Five milliliters of water were added to spiked soils, mixed, and incubated for 10 min. Samples were centrifuged for 5 min at 450 revolutions per minute (rpm) to pellet soil and large particles. Supernatants were filtered through kit-included cheesecloth and then cen-
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Table 1 Characteristics of the soils tested. Soil referred to in text as
pH
Organic matter (%)a
P (ppm)
K (ppm)
Mg (ppm)
Ca (ppm)
Sand Clay Loam
5.8 5.2 4.3
0.1 0.2 8.5
6 3 35
12 133 114
30 195 90
100 100 390
a
Composition (%)
Texture classification
Sand
Silt
Clay
96 52 70
4 10 28
0 38 2
Sand Sandy clay Sandy loam
Percent by mass; determined by the Walkley–Black method (1935).
trifuged at 5000 rpm for 5 min to pellet microorganisms. Pelleted material was resuspended in kit solution and then added to the lysing matrix. The remaining steps were the same as those for the FastRNA® Pro Soil-Direct kit, including bead beating, phenol: chloroform extraction, RNA binding and elution from the RNAMATRIX® . The IT 1-2-3 Platinum PathTM Sample Purification kit (Idaho Technology Inc., Salt Lake City, UT, USA) combines bead beating with magnetic bead technology. Spiked soils were treated with lysis buffer and underwent bead beating on a vortexer. Following centrifugation and protein precipitation, RNA was bound to magnetic beads, washed, and eluted in kit elution buffer. Quadruplicate extractions were performed by experienced users for each soil matrix at the four MS2 spiking amounts (0–10×) using each of the five extraction kits. Duplicate extractions were performed by a novice user (defined as having no prior knowledge or use of the technique) for each soil spiked at the 10× amount of MS2 phage using each of the five extraction kits. 2.5. Additional purification of RNA extracts To assess each kit’s ability to remove humic substances, which absorb light in the 300–400 nm range, the ND-1000 spectrophotometer (NanoDrop, Thermo Scientific, Wilmington, DE, USA) was used to measure the absorbance at 320 nm for each of the extracts from non-spiked soils (0×). In addition, extracts from the nonspiked soils were spiked with commercially-purified MS2 RNA (Roche, Indianapolis, IN, USA) such that the final concentration of MS2 RNA in the extracts was 0.01 ng l−1 (the same concentration as the positive control MS2 RNA). Spiked extracts were evaluated by RT-PCR. The level of PCR inhibition observed in the extracts suggested that an additional cleanup step should be used to remove PCR inhibitors. Two cleanup kits were evaluated: the RNeasy MinElute Cleanup kit (Qiagen, Valencia, CA, USA) and the OneStepTM PCR Inhibitor Removal kit (Zymo Research, Orange, CA, USA). Following the manufacturers’ instructions, 50 l of each sand or clay RNA extract was further purified using the RNeasy MinElute Cleanup kit while 50 l of each loam RNA extract was further purified using the OneStepTM PCR Inhibitor Removal kit. The RNeasy MinElute Cleanup kit combines guanidineisothiocyanate lysis with silica-membrane purification. Lysis buffer containing guanidine-isothiocyanate and -mercaptoethanol was added to 50 l of each sand or clay RNA extract. Samples were mixed with ethanol and transferred to silica-membrane spin columns that bound the RNA. The RNA was washed and then eluted in 14 l of RNase-free water. The OneStepTM PCR Inhibitor Removal kit employs a proprietary column matrix to remove PCR inhibitors from RNA and DNA preparations. Fifty microliters of each loam RNA extract was applied to a prepared column matrix and centrifuged at 8000 × g for 1 min. The filtered RNA was collected. 2.6. RT-PCR detection of MS2 RNA The successful purification of MS2 RNA was detected via realtime fluorogenic reverse transcription-PCR using the TaqMan® EZ
RT-PCR Core Reagents (Applied Biosystems, Foster City, CA, USA). RT-PCR was performed in a total volume of 25 l with the following reagents: 0.02 to 10 l kit-purified MS2 RNA extract, 1× TaqMan® EZ Buffer (Applied Biosystems, Foster City, CA, USA), 3 mM Mn(OAc)2 , 0.3 mM deoxynucleoside triphosphate mixture with 0.6 mM dUTP, 0.2 M primers, 0.1 M probe, 0.25 mg ml−1 bovine serum albumin, 1 U rTth polymerase, 0.25 U uracil-nglycosylase (UNG), and DNase- and RNase-free water (molecular biology grade, MO BIO Laboratories, Carlsbad, CA, USA). The primers were 632F, 5 -GTCGCGGTAATTGGCGC-3 , and 708R, 5 GGCCACGTGTTTTGATCGA-3 , as described for the MS2-specific Assay 1 by O’Connell et al. (2006). The probe was 650P, 5 AGGCGCTCCGCTACCTTGCCCT-3 , labeled on the 5 end with FAM and the 3 end with TAMRA (O’Connell et al., 2006). The RT-PCR was performed on the SmartCycler (Cepheid, Sunnyvale, CA, USA) using the following program: 50 ◦ C for 2 min, 60 ◦ C for 30 min, 95 ◦ C for 5 min, and 40 cycles of 94 ◦ C for 20 s and 62 ◦ C for 1 min (optics on). Reactions were performed in duplicate along with negative and positive controls. The positive control was run using commerciallypurified MS2 RNA that had been purchased (Roche, Indianapolis, IN, USA) and diluted with DNase- and RNase-free water to a concentration of 0.01 ng l−1 and stored in 10 l aliquots at −80 ◦ C. The negative control contained DNase- and RNase-free water. Commercially-purified MS2 RNA was tested to determine the cycle number beyond which non-specific amplification is observed (data not shown). Amplification was determined to be detectable if the cycle threshold (CT ) value was less than 36. 3. Results 3.1. Comparison of RNA extraction kits In order to investigate the ability of each kit to remove PCR inhibitors, the extracts from non-spiked soils were spiked with a known amount of MS2 RNA and evaluated by RT-PCR. Consistent with their high A320 measurements, loam extracts from the PowerSoilTM Total RNA Isolation and FastRNA® Pro Soil-Direct kits showed the highest levels of RT-PCR inhibition. Table 2 shows the amplification results for quadruplicate extracts from soils spiked with MS2 phage at amounts ranging from 107 to 109 PFU g−1 soil. Amplification results were affected by the volume of RNA extract used in the RT-PCR reaction. Some extracts, particularly those from loam, required dilution for amplification to be observed (Fig. 1 and Table 3). Sand and clay extracts were more likely to show amplification when undiluted in the RT-PCR reaction except for the PowerSoilTM Total RNA extracts from sand, which showed amplification when 0.2 l extract was used per reaction (Table 3). Amplification was non-detectable for all samples extracted from non-spiked (0×) soils using each of the five kits. The IT 1-2-3 Platinum PathTM kit yielded detectable RNA from all four replicates of each soil type spiked with the highest amount of MS2 (109 PFU g−1 ) (Table 2). The E.Z.N.A.® Soil RNA kit also showed success with all replicate extracts from sand spiked with the highest amount of MS2 and yielded cycle threshold (CT ) values lower
S.M. Dineen et al. / Journal of Virological Methods 168 (2010) 44–50
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Table 2 Qualitative RT-PCR amplification results for extracts from spiked soils. Soil type
Concentration of MS2 spiked into soil (PFU g−1 )
No. of successful RT-PCR amplifications for replicate samples (n = 4) extracted using the following kitsa , b FastRNA® Pro Soil-Direct kit
FastRNA® Pro Soil-Indirect kit
PowerSoilTM Total RNA Isolation kit
E.Z.N.A.® Soil RNA kit
IT 1-2-3 Platinum PathTM Sample Purification kit
Sand
0 107 108 109
− − − ++++
− + − ++++
− − − +++
− +++ ++++ ++++
− + + ++++
Clay
0 107 108 109
− − − +
− + + +
− − − −
− − − ++
− + + ++++
Loam
0 107 108 109
− − − ++
− ++++ ++++ ++++
− ++++ ++ ++
− ++ ++ ++++
− ++ ++++ ++++
a
Amplification was non-detectable for all samples extracted from non-spiked soils using each of the five kits. All four replicate extracts showed non-detectable amplification (−); one of four replicate extracts showed detectable amplification (+); two of four replicate extracts showed detectable amplification (++); three of four replicate extracts showed detectable amplification (+++); all four replicate extracts showed detectable amplification (++++). b
than those observed for the positive control (Table 3). When sand was spiked with the lower amounts of MS2 (107 and 108 PFU g−1 ), the E.Z.N.A.® Soil RNA kit yielded more replicate extracts showing detectable RT-PCR amplification than any of the other kits (Table 2). From all replicates of loam spiked with any amount of MS2, the FastRNA® Pro Soil-Indirect kit yielded detectable RNA that produced average extract CT values similar to those obtained for the positive control (Table 3). Extraction results were not dependent on the user or the user’s experience. Amplification results from two experienced users and a novice user were similar (data not shown). 3.2. Evaluation of RNA purification methods Each extract was further purified using one of two methods designed to remove PCR inhibitors. Approximately 15% (7/48) of the clay extracts in which RNA was previously non-detectable showed detectable amplification following cleanup with the RNeasy MinElute Cleanup kit. The RNeasy MinElute Cleanup also resulted in detectable amplification for 32% (10/31) of the previously nondetectable sand extracts. Following RNeasy MinElute cleanup, RNA
Fig. 1. Effect of RNA extract dilution on RT-PCR amplification results. For each soil, the proportion of extracts showing detectable RT-PCR amplification is shown for each volume of extract used per 25 l RT-PCR reaction (0.02, 0.2, 2, and 10 l). For a given soil, each proportion represents extracts from all kits and all MS2 spiking concentrations that had shown detectable amplification with at least one of the four extract volumes in RT-PCR.
Table 3 RT-PCR results for extracts from spiked soils. Soil type
Concentration of MS2 spiked into soil (PFU g-1 )
Volume of RNA extract used in 25 l RT-PCR reaction (l)
FastRNA® Pro Soil-Direct kit
E.Z.N.A.® Soil RNA kit
IT 1-2-3 Platinum PathTM Sample Purification kit
n
CT Ratio ± SD
n
CT Ratio ± SD
n
CT Ratio ± SD
n
CT Ratio ± SD
10 108 109
10 10 0.2–10
0 0 4
NDc ND 1.65 ± 0.07
1 0 4
1.95 ND 1.41 ± 0.22
0 0 3
ND ND 1.25 ± 0.02
3 4 4
1.87 ± 0.12 1.54 ± 0.07 0.88 ± 0.01
1 1 4
1.78 1.85 1.74 ± 0.02
Clay
107 108 109
10 10 10
0 0 1
ND ND 1.87
1 1 1
1.94 1.76 1.80
0 0 0
ND ND ND
0 0 2
ND ND 1.80 ± 0.03
1 1 4
1.87 1.76 1.82 ± 0.14
Loam
107 108 109
0.02–2 0.02–2 0.02–2
0 0 2
ND ND 1.10 ± 0.02
4 4 4
1.19 ± 0.01 1.03 ± 0.07 0.91 ± 0.002
4 2 2
1.29 ± 0.01 1.07 ± 0.01 1.05 ± 0.03
2 2 4
1.36 ± 0.02 1.33 ± 0.05 1.11 ± 0.07
2 4 4
1.58 ± 0.02 1.45 ± 0.05 1.36 ± 0.07
a
CT Ratio ± SDb
PowerSoilTM Total RNA Isolation kit
Sand
7
na
FastRNA® Pro Soil-Indirect kit
Number of replicates (out of four) showing detectable amplification. Average extract CT values were only calculated from replicates that produced detectable amplification results. Each ratio was produced by dividing the average extract CT value by the average positive control CT value (obtained in each RT-PCR assay by running a positive control using the same volume per reaction as the extract). A lower ratio suggests either a higher RNA yield or a lower concentration of humic substances in the extract. c ND, not detected. All replicate extracts showed non-detectable amplification. b
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Table 4 Kit parameters and logistics during RNA extraction. Extraction kit
Cost per extraction ($)a
Soil sample size (g)
Approximate processing time (h)
Consumables/ Additional reagents
Refrigerated reagents
Reaction temperatures required
Additional equipment requiredb
FastRNA® Pro Soil-Direct kit FastRNA® Pro Soil-Indirect kit
7.21
0.5–1
2.5
Yes
RT, 4 ◦ C, −20 ◦ C
FastPrep® bead beater
7.21
1
2.5
Yes
RT, 4 ◦ C, − 20◦ C
FastPrep® bead beater
PowerSoilTM Total RNA Isolation kit
8.84
≤2
3
Yes
RT, 4 ◦ C, −20 ◦ C, 45 ◦ C
Vortex adapter
E.Z.N.A.® Soil RNA kit
7.87
2
3.5
Yes
RT, 65 ◦ C, 4 ◦ C, −20 ◦ C, 50 ◦ C
None
IT 1-2-3 Platinum PathTM Sample Purification kit
11.25
0.5
<1
Tubes, ethanol, isopropanol Tubes, ethanol, chloroform, isopropanol Phenol: chloroform: isoamyl alcohol Tubes, ethanol, phenol, chloroform:isoamyl alcohol None
No
RT
PickPen® 1-M
a b
Approximate free market price. In addition to centrifuge, microcentrifuge, water bath and/or vortexer.
was detected in 100% (10/10) of the PowerSoilTM Total RNA Isolation kit sand extracts in which RNA was previously non-detectable. Of the loam extracts showing non-detectable amplification, only 10% (2/20) showed amplification following cleanup with the OneStepTM PCR Inhibitor Removal kit. 3.3. Time and cost analysis The processing time and cost per sample were determined for each RNA extraction method (Table 4). The PowerSoilTM Total RNA Isolation and the FastRNA® Pro Soil-Direct and -Indirect kits require approximately 2.5–3 h to complete an extraction. The E.Z.N.A.® Soil RNA kit requires slightly more time to complete (3.5 h) whereas the IT 1-2-3 Platinum PathTM Sample Purification kit requires the least amount of time to process (<1 h). The Qiagen RNeasy MinElute Cleanup and Zymo Research OneStepTM PCR Inhibitor Removal kits require approximately 15 and 10 min, respectively, to complete purification. The IT 1-2-3 Platinum PathTM Sample Purification kit is the most expensive, costing approximately $11.25 per sample (Table 4). The other four extraction methods are comparable to each other in cost ($7.21–8.84 per sample). For the Qiagen RNeasy MinElute Cleanup and Zymo Research OneStepTM PCR Inhibitor Removal kits, the cost per sample is $5.20 and $1.90, respectively. As shown in Table 4, only the IT 1-2-3 Platinum PathTM Sample Purification kit does not require low or high temperatures and uses the fewest number of accessories and additional consumables, thus making this kit compatible with field analysis. The other four kits are not all-inclusive, requiring additional consumables and reagents from the user as well as multiple reaction temperatures. In terms of additional instruments, the PowerSoilTM Total RNA Isolation kit requires a vortex adapter for 15 ml conical tubes while the two FastRNA® kit protocols recommend the use of a FastPrep® bead beater (Table 4). The PickPen® 1-M used in the IT 1-2-3 Platinum PathTM Sample Purification kit is a hand-held magnetic tool designed for the transfer of magnetic particles. 4. Discussion Soils are a challenging matrix for PCR analysis because humic substances are often co-extracted with nucleic acids (Schneegurt et al., 2003). Humic substances inhibit the polymerase in RT-PCR, preventing an accurate determination of RNA yield and potentially resulting in false-negative amplification results (Kermekchiev et al., 2009; Tebbe and Vahjen, 1993). In this study, kits were com-
pared via MS2-specific RT-PCR for their ability to extract and purify viral MS2 RNA from soil (O’Connell et al., 2006). Since soils vary widely in their physical and chemical properties, three different soils (sand, clay, and loam) ranging in pH (4.3–5.8) and organic matter (0.1–8.5%) were tested (Table 1). Kits varied in their ability to extract purified RNA from the three soil matrices. The IT 1-2-3 Platinum PathTM kit could reproducibly detect MS2 RNA from all soil types spiked with MS2 at 109 PFU g−1 soil (Table 2). At the lower spiking amounts of MS2 (107 and 108 PFU g−1 ), however, the E.Z.N.A.® Soil RNA kit showed greater success than the IT 1-2-3 Platinum PathTM kit in reproducibly extracting purified RNA from sand. For loam, the results from the IT 1-2-3 Platinum PathTM kit extractions were nearly comparable to those from the FastRNA® Pro Soil-Indirect kit. However, in this qualitative assessment, MS2 RNA could only be detected in two of the four IT 1-2-3 Platinum PathTM kit extracts from loam spiked with the lowest amount of MS2 (107 PFU g−1 ) (Table 2). In comparison, MS2 RNA was detected in all four FastRNA® Pro Soil-Indirect kit extracts from loam spiked with the same amount of MS2. In addition, relative to the CT values obtained for the positive control, loam extract CT values were lower for the FastRNA® Pro Soil-Indirect kit than the IT 1-2-3 Platinum PathTM kit (Table 3). These results suggest that the FastRNA® Pro Soil-Indirect kit yielded extracts that either contained more accessible MS2 RNA and/or fewer PCR-inhibitory substances. Although additional studies are required to establish any significant difference between the two kits, these results may suggest that the FastRNA® Pro Soil-Indirect kit provides greater sensitivity with the loam soil. Of the five kits tested, only the FastRNA® Pro Soil-Indirect kit uses an indirect extraction procedure, likely explaining the detection of MS2 RNA in all four extracts from loam spiked with all three amounts of MS2 (107 –109 PFU g−1 ) (Tables 2 and 3). Viral lysis followed by RNA extraction can either be performed in situ (direct method) or following separation from the soil matrix (indirect method) (Frostegard et al., 1999). While the direct method generally results in higher yields of nucleic acids (due to the adherence of microorganisms to soil particles), the indirect method limits the exposure of released RNA to humic substances and thus improves the purity of the extracted RNA (Steffan et al., 1988). The use of the indirect method is particularly appropriate for soils that contain high levels of humic substances. Inhibitory humic substances are formed during the decomposition of organic matter and include humic acids, which are increasingly released with low pH (Alm et al., 2000; Burgmann et al., 2001; Miller, 2001; Sagova-Mareckova
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et al., 2008). With the highest percentage of organic matter (8.5%) and the lowest pH (4.3), the loam soil likely contained the highest level of humic substances (Table 1) and was thus suited for the indirect method (Table 2). Despite the use of the indirect method, FastRNA® Pro SoilIndirect kit extracts from loam still required dilution prior to RT-PCR amplification. In fact, as illustrated in Fig. 1, only 0.02–2 l of the loam extracts from any of the kits could be added to each 25 l RT-PCR reaction for amplification to be detected (Table 3). By alleviating the inhibition of the polymerase, dilution of the extract was often sufficient for amplification to be detected (Tsai and Olson, 1992; Wilson, 1997). In some cases, amplification remained nondetectable even after dilution, suggesting that either the RNA yield was too low or the level of PCR-inhibiting humic substances was still too high to permit detectable amplification. In these cases, a commercially available kit was used to remove humic substances. However, out of the twenty loam extracts for which dilution was not sufficient and amplification still remained non-detectable, only two showed detectable amplification following cleanup with the OneStepTM PCR Inhibitor Removal kit. Compared to loam, sand and clay extracts tended to show detectable amplification when undiluted in RT-PCR (Fig. 1 and Table 3). The only exceptions were the PowerSoilTM Total RNA Isolation kit extracts from sand, which likely retained inhibitory substances during the isolation process since only 0.2 l could be added to the 25 l RT-PCR reaction for detectable amplification to be observed. In the majority of the sand and clay extracts, however, the level of humic substances was likely low and thus the RT-PCR amplification was dependent on the RNA yield. For clay, many of the extracts showed non-detectable amplification, suggesting low RNA yield (Table 3). Clay content has previously been shown to negatively influence the recovery of RNA or DNA since nucleic acids adsorb to clay particles when released from bacteria or viruses (Frostegard et al., 1999; Goring and Bartholomew, 1952; Paget et al., 1992). Since low RNA yield was a larger concern than the inhibitory effect of humic substances with sand and clay extracts, the Qiagen RNeasy MinElute Cleanup kit was used, with limited success, to purify and concentrate RNA present in those extracts. Of note, however, is that all non-detectable PowerSoilTM Total RNA Isolation kit extracts from sand showed detectable amplification following cleanup. These extracts likely contained inhibitory substances, which were diluted out in some samples but required removal by the Qiagen RNeasy MinElute Cleanup kit in others. For all soils, prior evaluation of the crude extracts (both diluted and undiluted) by RT-PCR helped determine if cleanup was necessary. The selection of an extraction kit largely depends on the soil characteristics. Sample size may also be an important consideration, particularly in cases where the concentration of the target microorganism is suspected to be low and it would be advantageous to process a larger soil sample. However, the five kits evaluated in this study process soil samples comparable in size, ranging from 0.5 g to 2 g (Table 4). To process larger samples of soil, the manufacturers’ instructions suggest processing replicate soil samples in parallel and then pooling the purified RNA. Based on the trends observed in this assessment, the RNA isolation method that yielded the best results, as defined by reproducibility and sensitivity, was the E.Z.N.A.® Soil RNA kit for sand, the IT 1-2-3 Platinum PathTM Sample Purification kit for clay, and the FastRNA® Pro Soil-Indirect kit for loam. However, additional studies are required to establish any significant differences in kit sensitivity and reproducibility. In addition to soil type, other factors such as processing time, extra external equipment, and reaction temperatures required should also be considered in the kit selection. The PowerSoilTM Total RNA Isolation, E.Z.N.A.® Soil RNA, FastRNA® Pro Soil-Direct, and FastRNA® Pro Soil-Indirect kits are
49
comparable in cost ($7.21–$8.84 per extraction), processing time (approximately 2.5–3.5 h), reagent refrigeration, and reaction temperatures required (Table 4). The IT 1-2-3 Platinum PathTM kit, on the other hand, requires the least amount of processing time (<1 h) and does not require any additional reagents, refrigeration, or reaction temperatures (other than room temperature). With its potential for portability and high throughput, the IT 1-2-3 Platinum PathTM kit is appropriate for use in the field, particularly when there is a large number of samples or a constraint on sample testing time. However, if reagents, storage conditions, and processing time are less of a concern, the other kits, at least for the sand and loam soils, may provide greater sensitivity with lower detection thresholds (Tables 2 and 3). The isolation and identification of a microorganism is a critical step in the analysis of an environmental sample. Our results suggest that soil characteristics, such as clay and organic matter content, should be assessed and considered prior to the selection of an appropriate nucleic acid extraction and purification method. These methods vary in their ability to remove humic acids and other PCR-inhibitory substances from the final extracts, thereby influencing the success of real-time PCR identification. Kermekchiev et al. (2009) has recently identified novel mutant Taq and Klentaq enzymes that show resistance to PCR inhibitors. Combining an appropriate nucleic acid extraction method with a real-time PCR assay utilizing a resistant enzyme may significantly facilitate the detection and identification of a microorganism in an environmental sample. Acknowledgements The views and conclusions contained in this document are those of the authors and should not be implied as necessarily representing the official policies, either expressed or implied, of the U.S. government. This is publication 09-35 of the Federal Bureau of Investigation (FBI). Names of commercial manufacturers are provided for identification only, and inclusion does not imply endorsement by the FBI. Thanks to Robert D. Koons, Keith Monson, and Christopher Ehrhardt (FBI CFSRU) for useful discussions. Shauna M. Dineen, Roman Aranda IV, and Marianne E. Dietz participated in this research through the Oak Ridge Institute for Science and Education program. References Alm, E.W., Zheng, D., Raskin, L., 2000. The presence of humic substances and DNA in RNA extracts affects hybridization results. Appl. Environ. Microbiol. 66, 4547–4554. Arbeli, Z., Fuentes, C.L., 2007. Improved purification and PCR amplification of DNA from environmental samples. FEMS Microbiol. Lett. 272, 269–275. Borneman, J., Triplett, E.W., 1997. Molecular microbial diversity in soils from eastern Amazonia: evidence for unusual microorganisms and microbial population shifts associated with deforestation. Appl. Environ. Microbiol. 63, 2647–2653. Braid, M.D., Daniels, L.M., Kitts, C.L., 2003. Removal of PCR inhibitors from soil DNA by chemical flocculation. J. Microbiol. Methods 52, 389–393. Brassard, J., Lamoureux, L., Gagne, M., Poitras, E., Trottier, Y., Houde, A., 2009. Comparison of commercial viral genomic extraction kits for the molecular detection of food borne viruses. Can. J. Microbiol. 55, 1016–1019. Broussard, L.A., 2001. Biological agents: weapons of warfare and bioterrorism. Mol. Diagn. 6, 323–333. Burgmann, H., Pesaro, M., Widmer, F., Zeyer, J., 2001. A strategy for optimizing quality and quantity of DNA extracted from soil. J. Microbiol. Methods 45, 7–20. Dong, D., Yan, A., Liu, H., Zhang, X., Xu, Y., 2006. Removal of humic substances from soil DNA using aluminium sulfate. J. Microbiol. Methods 66, 217– 222. Dreier, J., Stormer, M., Kleesiek, K., 2005. Use of bacteriophage MS2 as an internal control in viral reverse transcription-PCR assays. J. Clin. Microbiol. 43, 4551–4557. Frostegard, A., Courtois, S., Ramisse, V., Clerc, S., Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., Simonet, P., 1999. Quantification of bias related to the extraction of DNA directly from soils. Appl. Environ. Microbiol. 65, 5409–5420. Garnier, L., Gaudin, J.C., Bensadoun, P., Rebillat, I., Morel, Y., 2009. Real-time PCR assay for detection of a new simulant for poxvirus biothreat agents. Appl. Environ. Microbiol. 75, 1614–1620.
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Goring, C., Bartholomew, W., 1952. Adsorption of mononucleotides, nucleic acids, and nucleoproteins by clays. Soil Sci. 74, 149–164. Kermekchiev, M.B., Kirilova, L.I., Vail, E.E., Barnes, W.M., 2009. Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples. Nucleic Acids Res. 37, e40. Kuske, C.R., Banton, K.L., Adorada, D.L., Stark, P.C., Hill, K.K., Jackson, P.J., 1998. Smallscale DNA sample preparation method for field PCR detection of microbial cells and spores in soil. Appl. Environ. Microbiol. 64, 2463–2472. Mendum, T., Sockett, R., Hirsch, P., 1998. The detection of Gram-negative bacterial mRNA from soil by RT-PCR. FEMS Microbiol. Lett. 164, 369– 373. Miller, D.N., 2001. Evaluation of gel filtration resins for the removal of PCR-inhibitory substances from soils and sediments. J. Microbiol. Methods 44, 49–58. Miller, D.N., Bryant, J.E., Madsen, E.L., Ghiorse, W.C., 1999. Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Appl. Environ. Microbiol. 65, 4715–4724. O’Connell, K.P., Bucher, J.R., Anderson, P.E., Cao, C.J., Khan, A.S., Gostomski, M.V., Valdes, J.J., 2006. Real-time fluorogenic reverse transcription-PCR assays for detection of bacteriophage MS2. Appl. Environ. Microbiol. 72, 478– 483. Ogram, A., Sun, W., Brockman, F.J., Fredrickson, J.K., 1995. Isolation and characterization of RNA from low-biomass deep-subsurface sediments. Appl. Environ. Microbiol. 61, 763–768. Olson, M.R., Axler, R.P., Hicks, R.E., 2004. Effects of freezing and storage temperature on MS2 viability. J. Virol. Methods 122, 147–152. Paget, E., Monrozier, L., Simonet, P., 1992. Adsorption of DNA on clay minerals: protection against DNase I and influence on gene transfer. FEMS Microbiol. Lett. 97, 31–40. Persoh, D., Theuerl, S., Buscot, F., Rambold, G., 2008. Towards a universally adaptable method for quantitative extraction of high-purity nucleic acids from soil. J. Microbiol. Methods 75, 19–24. Rotz, L.D., Khan, A.S., Lillibridge, S.R., Ostroff, S.M., Hughes, J.M., 2002. Public health assessment of potential biological terrorism agents. Emerg. Infect. Dis. 8, 225–230.
Rzezutka, A., Cook, N., 2004. Survival of human enteric viruses in the environment and food. FEMS Microbiol. Rev. 28, 441–453. Sagova-Mareckova, M., Cermak, L., Novotna, J., Plhackova, K., Forstova, J., Kopecky, J., 2008. Innovative methods for soil DNA purification tested in soils with widely differing characteristics. Appl. Environ. Microbiol. 74, 2902–2907. Schneegurt, M.A., Dore, S.Y., Kulpa Jr., C.F., 2003. Direct extraction of DNA from soils for studies in microbial ecology. Curr. Issues Mol. Biol. 5, 1–8. Sessitsch, A., Gyamfi, S., Stralis-Pavese, N., Weilharter, A., Pfeifer, U., 2002. RNA isolation from soil for bacterial community and functional analysis: evaluation of different extraction and soil conservation protocols. J. Microbiol. Methods 51, 171–179. Sinclair, R., Boone, S.A., Greenberg, D., Keim, P., Gerba, C.P., 2008. Persistence of category A select agents in the environment. Appl. Environ. Microbiol. 74, 555–563. Steffan, R.J., Goksoyr, J., Bej, A.K., Atlas, R.M., 1988. Recovery of DNA from soils and sediments. Appl. Environ. Microbiol. 54, 2908–2915. Tebbe, C.C., Vahjen, W., 1993. Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. Appl. Environ. Microbiol. 59, 2657–2665. Tsai, Y.L., Olson, B.H., 1991. Rapid method for direct extraction of DNA from soil and sediments. Appl. Environ. Microbiol. 57, 1070–1074. Tsai, Y.L., Olson, B.H., 1992. Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction. Appl. Environ. Microbiol. 58, 2292–2295. Walkley, A., 1935. An examination of methods for determining organic carbon and nitrogen in soils. J. Agric. Sci. 25, 598–609. Whitehouse, C.A., Hottel, H.E., 2007. Comparison of five commercial DNA extraction kits for the recovery of Francisella tularensis DNA from spiked soil samples. Mol. Cell. Probes 21, 92–96. Wilson, I.G., 1997. Inhibition and facilitation of nucleic acid amplification. Appl. Environ. Microbiol. 63, 3741–3751.
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Evaluation of commercial RNA extraction kits for the isolation of viral MS2 RNA from soil Shauna M. Dineen a,b, Roman Aranda IV a,b, Marianne E. Dietz a,b, Douglas L. Anders c, James M. Robertson b,∗ a b c
Visiting Scientist, Federal Bureau of Investigation Laboratory, 2501 Investigation Parkway, Quantico, VA 22135, USA Counterterrorism and Forensic Science Research Unit, Federal Bureau of Investigation Laboratory, 2501 Investigation Parkway, Quantico, VA 22135, USA Hazardous Materials Science Response Unit, Federal Bureau of Investigation Laboratory, 2501 Investigation Parkway, Quantico, VA 22135, USA
a b s t r a c t Article history: Received 23 December 2009 Received in revised form 9 April 2010 Accepted 19 April 2010 Available online 24 April 2010 Keywords: RNA Virus RT-PCR Extraction Detection Soil
Nucleic acid extraction is a critical step in the detection of an unknown biological agent. However, success can vary depending on the isolation and identification methods chosen and the difficulty of extraction from environmental matrices. In this work, bacteriophage MS2 RNA was extracted from three soil matrices, sand, clay, and loam, using five commercially available kits: the PowerSoilTM Total RNA Isolation, E.Z.N.A.® Soil RNA, FastRNA® Pro Soil-Direct, FastRNA® Pro Soil-Indirect, and IT 1-2-3 Platinum PathTM kits. Success of the isolation was determined using an MS2-specific RT-PCR assay. The reproducibility and sensitivity of each method in the hands of both experienced and novice users were assessed and compared. Cost, operator time, and storage conditions were also considered in the evaluation. The RNA isolation method that yielded the best results, as defined by reproducibility and sensitivity, was the E.Z.N.A.® Soil RNA kit for sand, the IT 1-2-3 Platinum PathTM Sample Purification kit for clay, and the FastRNA® Pro Soil-Indirect kit for loam. However, if time and storage conditions are important considerations, the IT 1-2-3 Platinum PathTM kit may be appropriate for use with all soils since the kit has the shortest processing time and fewest temperature requirements. Published by Elsevier B.V.
1. Introduction The detection and identification of an unknown biological agent is a critical component of an environmental analysis or bioterrorism response plan. The accidental or deliberate release of a biological agent into the environment can cause illness or death in humans, animals, or plants. Bacteria such as Bacillus anthracis and ricin toxin from Ricinus communis have been used as biological agents (Rotz et al., 2002). Pathogenic viruses can also serve as agents because they are highly infectious and can be readily transmitted after dispersal into air, water, food, or soil (Brassard et al., 2009; Garnier et al., 2009; Rzezutka and Cook, 2004). With the high morbidity and mortality rates associated with some pathogenic agents, a rapid response to either an outbreak or intentional release is essential (Rotz et al., 2002; Sinclair et al., 2008). For the response plan, methods to isolate and identify biological pathogens should be chosen based on their effectiveness and efficiency at recovering and detecting target organisms from the sample collected. Compared to time-consuming cell culture assays, real-time polymerase chain reaction (PCR) provides a sensitive, specific, and
∗ Corresponding author. Tel.: +1 703 632 4555; fax: +1 703 632 4500. E-mail address: [email protected] (J.M. Robertson). 0166-0934/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.jviromet.2010.04.014
much more rapid means to detect biological agents in environmental samples (Broussard, 2001). Through the use of target-specific primers and a fluorogenic probe, real-time PCR is able to amplify and detect trace amounts of DNA or reverse-transcribed RNA from a specific agent. However, successful detection first requires efficient extraction and purification of the DNA or RNA from the environmental sample (Kuske et al., 1998). Unfortunately, nucleic acids of biological agents can be extremely difficult to detect, particularly in certain environmental matrices. Many environmental matrices contain substances that co-extract with nucleic acids and inhibit the enzymatic processes involved in PCR. Soils are particularly problematic because they are rich in humic substances (Schneegurt et al., 2003). Humic substances, which include humic acids, fulvic acids, and humins, represent a complex mixture of aromatic and aliphatic groups in large molecules that are brown to black in color, absorb light in the 300–400 nm range, and are the major constituents of soil organic matter (Alm et al., 2000; Miller, 2001). With physical–chemical characteristics similar to those of nucleic acids, humic substances are difficult to remove and often co-purify with DNA or RNA from soil (Dong et al., 2006). This contamination affects downstream applications since as little as 0.1 ng l−1 humic acid can inhibit Taq DNA polymerase during PCR (Kermekchiev et al., 2009; Tebbe and Vahjen, 1993; Tsai and Olson, 1991). For the detection and identi-
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fication of a microorganism or virus in an environmental sample, the presence of such PCR inhibitors may potentially lead to falsenegative results (Dreier et al., 2005). Thus, the efficiency of the method used to extract nucleic acid from soil is determined not only by the amount of nucleic acid recovered, but also by the success in removing PCR-inhibitory substances. Numerous procedures exist for the isolation and purification of DNA and RNA from soil. The isolation procedures vary in the timing of cell lysis, occurring either in situ (direct method) or following the separation of the microorganisms from the environmental matrix (indirect method) (Frostegard et al., 1999; Steffan et al., 1988). In addition to the timing and method of cell lysis (chemical, mechanical, or a combination of both), the isolation procedures also vary in how the nucleic acids are purified, often through steps such as phenol/chloroform extraction, cesium chloride density gradient centrifugation, chemical flocculation, and/or gel filtration (Arbeli and Fuentes, 2007; Braid et al., 2003; Miller, 2001; Miller et al., 1999; Persoh et al., 2008). Procedures that include these time-consuming steps are either too cumbersome or too slow for obtaining rapid results in response to an infectious outbreak or suspected bioterrorism attack. Commercial DNA extraction kits have previously been compared for their ability to extract DNA from bacteria in soil samples (Sagova-Mareckova et al., 2008; Whitehouse and Hottel, 2007). Studies suggest that the selection of an appropriate extraction and purification procedure depends on the physical and chemical characteristics of the soil matrix, such as organic matter, clay content, and pH (Burgmann et al., 2001; Sagova-Mareckova et al., 2008). Similar results have been shown with different laboratory procedures used to extract microbial RNA from soil and sediment (Borneman and Triplett, 1997; Mendum et al., 1998; Ogram et al., 1995; Sessitsch et al., 2002; Tsai and Olson, 1991). However, commercial RNA extraction kits have yet to be compared for their relative effectiveness with different soil matrices. In this study, five commercially available kits were evaluated for their ability to extract bacteriophage MS2 RNA from three different soils: sand, clay, and loam, which varied in organic content, pH, and particle size. Each soil was spiked with three different amounts of MS2 phage. MS2 (family Leviviridae, genus Levivirus, species Enterobacteria phage MS2) is a small, male-specific bacteriophage of Escherichia coli often used as a surrogate for pathogenic RNA viruses, particularly to indicate enteric virus contamination of wastewater (Dreier et al., 2005; Olson et al., 2004). Three spiking amounts of MS2 served in the determination of a detection threshold for each kit. The efficiency of extraction was evaluated using an MS2-specific real-time reverse transcription-PCR (RT-PCR) assay (O’Connell et al., 2006). Beyond the comparison of the five kits, two PCR-cleanup methods were evaluated for their ability to remove PCR inhibitors from the kit extracts. The results of this study aid in the selection of an appropriate RNA extraction method and, if necessary, an additional purification step for a given soil sample. The labor, time, and cost required for each method were also compared. 2. Materials and methods 2.1. Soils Three different soils were collected in Quantico, VA in August 2008. Each of the soil samples was sieved using a fiberglass mesh with 1 mm by 1.5 mm openings to remove large, non-soil materials and stored at 4 ◦ C. Soils were not autoclaved in order to represent a real-world scenario more realistically. The soils were analyzed and classified by A&L Eastern Laboratories (Richmond, VA) for particle size, pH, and organic content. Organic content was determined using the Walkley–Black procedure (Walkley, 1935). Characteristics of the three soils are listed in Table 1.
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The soil textures were found to be sand, sandy clay, and sandy loam and are referred to as “sand”, “clay”, and “loam”, respectively. 2.2. MS2 phage and titer determination MS2 bacteriophage strain 15597-B1 and its Escherichia coli (E. coli) host 15597 were purchased from American Type Culture Center (ATCC, Manassas, VA). Media and titer determination procedures were followed according to ATCC instructions. For use in spiking experiments, 100 l aliquots of MS2 bacteriophage were stored in 10% glycerol at −80 ◦ C. 2.3. Soil spiking The three non-autoclaved soils were spiked with four different amounts of MS2 bacteriophage, 0× or “non-spiked”, 0.1×, 1×, and 10×, where 1× corresponds to 3 × 108 plaque-forming units (PFU) per gram of soil. To account for the variation in soil sample size processed by the five kits, the amount of MS2 phage added to each soil was adjusted such that the MS2 PFU per gram soil remained consistent across all kits for a given spiking amount. Spiked soils were mixed thoroughly and then subjected to one of the five extraction kit protocols. 2.4. RNA extraction Five commercially available RNA extraction kits were evaluated in this study. Four kits employ a direct extraction method in which lysis occurs in the presence of the soil (Frostegard et al., 1999; Steffan et al., 1988). The fifth kit uses an indirect method in which the target organisms are separated from the soil prior to lysis. RNA extractions were performed according to the manufacturers’ instructions. All extracted RNA samples were immediately stored at −80 ◦ C until evaluation via RT-PCR analysis. The PowerSoilTM Total RNA Isolation kit (MO BIO, Carlsbad, CA, USA) utilizes bead beating in the extraction procedure. Spiked soils were treated with a series of kit solutions and underwent bead beating on a vortexer to lyse viral particles. Total RNA was isolated using phenol: chloroform and then captured on columns packed with proprietary matrix, washed, and eluted with the kit elution buffer. The E.Z.N.A.® Soil RNA kit (Omega Bio-Tek, Norcross, GA, USA) also combines bead beating by vortexing with phenol: chloroform extraction to isolate RNA from soil. Glass beads and a series of buffers were added to spiked soils and then vortexed. Total RNA was isolated using phenol: chloroform and precipitated using isopropanol. RNA was resuspended in diethylpyrocarbonate (DEPC) water and re-extracted using phenol: chloroform. RNA samples were mixed with ethanol and then transferred to spin columns that bind RNA. RNA was washed with wash buffer solutions and then eluted from columns using DEPC water. The FastRNA® Pro Soil-Direct kit (MP Biomedicals, Solon, OH, USA) uses bead beating as well as a proprietary RNAMATRIX® to release, extract, and purify total RNA from soil. Spiked soils were added to a lysing matrix, treated with lysis buffer, and subjected to bead beating in the FastPrep® instrument. RNA was isolated using phenol: chloroform and precipitated using isopropanol. RNA was washed with ethanol and resuspended in DEPC water. RNA was bound to the RNAMATRIX® , washed, and eluted in DEPC water. The FastRNA® Pro Soil-Indirect kit (MP Biomedicals, Solon, OH, USA) uses an indirect method to extract RNA from soil. Five milliliters of water were added to spiked soils, mixed, and incubated for 10 min. Samples were centrifuged for 5 min at 450 revolutions per minute (rpm) to pellet soil and large particles. Supernatants were filtered through kit-included cheesecloth and then cen-
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Table 1 Characteristics of the soils tested. Soil referred to in text as
pH
Organic matter (%)a
P (ppm)
K (ppm)
Mg (ppm)
Ca (ppm)
Sand Clay Loam
5.8 5.2 4.3
0.1 0.2 8.5
6 3 35
12 133 114
30 195 90
100 100 390
a
Composition (%)
Texture classification
Sand
Silt
Clay
96 52 70
4 10 28
0 38 2
Sand Sandy clay Sandy loam
Percent by mass; determined by the Walkley–Black method (1935).
trifuged at 5000 rpm for 5 min to pellet microorganisms. Pelleted material was resuspended in kit solution and then added to the lysing matrix. The remaining steps were the same as those for the FastRNA® Pro Soil-Direct kit, including bead beating, phenol: chloroform extraction, RNA binding and elution from the RNAMATRIX® . The IT 1-2-3 Platinum PathTM Sample Purification kit (Idaho Technology Inc., Salt Lake City, UT, USA) combines bead beating with magnetic bead technology. Spiked soils were treated with lysis buffer and underwent bead beating on a vortexer. Following centrifugation and protein precipitation, RNA was bound to magnetic beads, washed, and eluted in kit elution buffer. Quadruplicate extractions were performed by experienced users for each soil matrix at the four MS2 spiking amounts (0–10×) using each of the five extraction kits. Duplicate extractions were performed by a novice user (defined as having no prior knowledge or use of the technique) for each soil spiked at the 10× amount of MS2 phage using each of the five extraction kits. 2.5. Additional purification of RNA extracts To assess each kit’s ability to remove humic substances, which absorb light in the 300–400 nm range, the ND-1000 spectrophotometer (NanoDrop, Thermo Scientific, Wilmington, DE, USA) was used to measure the absorbance at 320 nm for each of the extracts from non-spiked soils (0×). In addition, extracts from the nonspiked soils were spiked with commercially-purified MS2 RNA (Roche, Indianapolis, IN, USA) such that the final concentration of MS2 RNA in the extracts was 0.01 ng l−1 (the same concentration as the positive control MS2 RNA). Spiked extracts were evaluated by RT-PCR. The level of PCR inhibition observed in the extracts suggested that an additional cleanup step should be used to remove PCR inhibitors. Two cleanup kits were evaluated: the RNeasy MinElute Cleanup kit (Qiagen, Valencia, CA, USA) and the OneStepTM PCR Inhibitor Removal kit (Zymo Research, Orange, CA, USA). Following the manufacturers’ instructions, 50 l of each sand or clay RNA extract was further purified using the RNeasy MinElute Cleanup kit while 50 l of each loam RNA extract was further purified using the OneStepTM PCR Inhibitor Removal kit. The RNeasy MinElute Cleanup kit combines guanidineisothiocyanate lysis with silica-membrane purification. Lysis buffer containing guanidine-isothiocyanate and -mercaptoethanol was added to 50 l of each sand or clay RNA extract. Samples were mixed with ethanol and transferred to silica-membrane spin columns that bound the RNA. The RNA was washed and then eluted in 14 l of RNase-free water. The OneStepTM PCR Inhibitor Removal kit employs a proprietary column matrix to remove PCR inhibitors from RNA and DNA preparations. Fifty microliters of each loam RNA extract was applied to a prepared column matrix and centrifuged at 8000 × g for 1 min. The filtered RNA was collected. 2.6. RT-PCR detection of MS2 RNA The successful purification of MS2 RNA was detected via realtime fluorogenic reverse transcription-PCR using the TaqMan® EZ
RT-PCR Core Reagents (Applied Biosystems, Foster City, CA, USA). RT-PCR was performed in a total volume of 25 l with the following reagents: 0.02 to 10 l kit-purified MS2 RNA extract, 1× TaqMan® EZ Buffer (Applied Biosystems, Foster City, CA, USA), 3 mM Mn(OAc)2 , 0.3 mM deoxynucleoside triphosphate mixture with 0.6 mM dUTP, 0.2 M primers, 0.1 M probe, 0.25 mg ml−1 bovine serum albumin, 1 U rTth polymerase, 0.25 U uracil-nglycosylase (UNG), and DNase- and RNase-free water (molecular biology grade, MO BIO Laboratories, Carlsbad, CA, USA). The primers were 632F, 5 -GTCGCGGTAATTGGCGC-3 , and 708R, 5 GGCCACGTGTTTTGATCGA-3 , as described for the MS2-specific Assay 1 by O’Connell et al. (2006). The probe was 650P, 5 AGGCGCTCCGCTACCTTGCCCT-3 , labeled on the 5 end with FAM and the 3 end with TAMRA (O’Connell et al., 2006). The RT-PCR was performed on the SmartCycler (Cepheid, Sunnyvale, CA, USA) using the following program: 50 ◦ C for 2 min, 60 ◦ C for 30 min, 95 ◦ C for 5 min, and 40 cycles of 94 ◦ C for 20 s and 62 ◦ C for 1 min (optics on). Reactions were performed in duplicate along with negative and positive controls. The positive control was run using commerciallypurified MS2 RNA that had been purchased (Roche, Indianapolis, IN, USA) and diluted with DNase- and RNase-free water to a concentration of 0.01 ng l−1 and stored in 10 l aliquots at −80 ◦ C. The negative control contained DNase- and RNase-free water. Commercially-purified MS2 RNA was tested to determine the cycle number beyond which non-specific amplification is observed (data not shown). Amplification was determined to be detectable if the cycle threshold (CT ) value was less than 36. 3. Results 3.1. Comparison of RNA extraction kits In order to investigate the ability of each kit to remove PCR inhibitors, the extracts from non-spiked soils were spiked with a known amount of MS2 RNA and evaluated by RT-PCR. Consistent with their high A320 measurements, loam extracts from the PowerSoilTM Total RNA Isolation and FastRNA® Pro Soil-Direct kits showed the highest levels of RT-PCR inhibition. Table 2 shows the amplification results for quadruplicate extracts from soils spiked with MS2 phage at amounts ranging from 107 to 109 PFU g−1 soil. Amplification results were affected by the volume of RNA extract used in the RT-PCR reaction. Some extracts, particularly those from loam, required dilution for amplification to be observed (Fig. 1 and Table 3). Sand and clay extracts were more likely to show amplification when undiluted in the RT-PCR reaction except for the PowerSoilTM Total RNA extracts from sand, which showed amplification when 0.2 l extract was used per reaction (Table 3). Amplification was non-detectable for all samples extracted from non-spiked (0×) soils using each of the five kits. The IT 1-2-3 Platinum PathTM kit yielded detectable RNA from all four replicates of each soil type spiked with the highest amount of MS2 (109 PFU g−1 ) (Table 2). The E.Z.N.A.® Soil RNA kit also showed success with all replicate extracts from sand spiked with the highest amount of MS2 and yielded cycle threshold (CT ) values lower
S.M. Dineen et al. / Journal of Virological Methods 168 (2010) 44–50
47
Table 2 Qualitative RT-PCR amplification results for extracts from spiked soils. Soil type
Concentration of MS2 spiked into soil (PFU g−1 )
No. of successful RT-PCR amplifications for replicate samples (n = 4) extracted using the following kitsa , b FastRNA® Pro Soil-Direct kit
FastRNA® Pro Soil-Indirect kit
PowerSoilTM Total RNA Isolation kit
E.Z.N.A.® Soil RNA kit
IT 1-2-3 Platinum PathTM Sample Purification kit
Sand
0 107 108 109
− − − ++++
− + − ++++
− − − +++
− +++ ++++ ++++
− + + ++++
Clay
0 107 108 109
− − − +
− + + +
− − − −
− − − ++
− + + ++++
Loam
0 107 108 109
− − − ++
− ++++ ++++ ++++
− ++++ ++ ++
− ++ ++ ++++
− ++ ++++ ++++
a
Amplification was non-detectable for all samples extracted from non-spiked soils using each of the five kits. All four replicate extracts showed non-detectable amplification (−); one of four replicate extracts showed detectable amplification (+); two of four replicate extracts showed detectable amplification (++); three of four replicate extracts showed detectable amplification (+++); all four replicate extracts showed detectable amplification (++++). b
than those observed for the positive control (Table 3). When sand was spiked with the lower amounts of MS2 (107 and 108 PFU g−1 ), the E.Z.N.A.® Soil RNA kit yielded more replicate extracts showing detectable RT-PCR amplification than any of the other kits (Table 2). From all replicates of loam spiked with any amount of MS2, the FastRNA® Pro Soil-Indirect kit yielded detectable RNA that produced average extract CT values similar to those obtained for the positive control (Table 3). Extraction results were not dependent on the user or the user’s experience. Amplification results from two experienced users and a novice user were similar (data not shown). 3.2. Evaluation of RNA purification methods Each extract was further purified using one of two methods designed to remove PCR inhibitors. Approximately 15% (7/48) of the clay extracts in which RNA was previously non-detectable showed detectable amplification following cleanup with the RNeasy MinElute Cleanup kit. The RNeasy MinElute Cleanup also resulted in detectable amplification for 32% (10/31) of the previously nondetectable sand extracts. Following RNeasy MinElute cleanup, RNA
Fig. 1. Effect of RNA extract dilution on RT-PCR amplification results. For each soil, the proportion of extracts showing detectable RT-PCR amplification is shown for each volume of extract used per 25 l RT-PCR reaction (0.02, 0.2, 2, and 10 l). For a given soil, each proportion represents extracts from all kits and all MS2 spiking concentrations that had shown detectable amplification with at least one of the four extract volumes in RT-PCR.
Table 3 RT-PCR results for extracts from spiked soils. Soil type
Concentration of MS2 spiked into soil (PFU g-1 )
Volume of RNA extract used in 25 l RT-PCR reaction (l)
FastRNA® Pro Soil-Direct kit
E.Z.N.A.® Soil RNA kit
IT 1-2-3 Platinum PathTM Sample Purification kit
n
CT Ratio ± SD
n
CT Ratio ± SD
n
CT Ratio ± SD
n
CT Ratio ± SD
10 108 109
10 10 0.2–10
0 0 4
NDc ND 1.65 ± 0.07
1 0 4
1.95 ND 1.41 ± 0.22
0 0 3
ND ND 1.25 ± 0.02
3 4 4
1.87 ± 0.12 1.54 ± 0.07 0.88 ± 0.01
1 1 4
1.78 1.85 1.74 ± 0.02
Clay
107 108 109
10 10 10
0 0 1
ND ND 1.87
1 1 1
1.94 1.76 1.80
0 0 0
ND ND ND
0 0 2
ND ND 1.80 ± 0.03
1 1 4
1.87 1.76 1.82 ± 0.14
Loam
107 108 109
0.02–2 0.02–2 0.02–2
0 0 2
ND ND 1.10 ± 0.02
4 4 4
1.19 ± 0.01 1.03 ± 0.07 0.91 ± 0.002
4 2 2
1.29 ± 0.01 1.07 ± 0.01 1.05 ± 0.03
2 2 4
1.36 ± 0.02 1.33 ± 0.05 1.11 ± 0.07
2 4 4
1.58 ± 0.02 1.45 ± 0.05 1.36 ± 0.07
a
CT Ratio ± SDb
PowerSoilTM Total RNA Isolation kit
Sand
7
na
FastRNA® Pro Soil-Indirect kit
Number of replicates (out of four) showing detectable amplification. Average extract CT values were only calculated from replicates that produced detectable amplification results. Each ratio was produced by dividing the average extract CT value by the average positive control CT value (obtained in each RT-PCR assay by running a positive control using the same volume per reaction as the extract). A lower ratio suggests either a higher RNA yield or a lower concentration of humic substances in the extract. c ND, not detected. All replicate extracts showed non-detectable amplification. b
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Table 4 Kit parameters and logistics during RNA extraction. Extraction kit
Cost per extraction ($)a
Soil sample size (g)
Approximate processing time (h)
Consumables/ Additional reagents
Refrigerated reagents
Reaction temperatures required
Additional equipment requiredb
FastRNA® Pro Soil-Direct kit FastRNA® Pro Soil-Indirect kit
7.21
0.5–1
2.5
Yes
RT, 4 ◦ C, −20 ◦ C
FastPrep® bead beater
7.21
1
2.5
Yes
RT, 4 ◦ C, − 20◦ C
FastPrep® bead beater
PowerSoilTM Total RNA Isolation kit
8.84
≤2
3
Yes
RT, 4 ◦ C, −20 ◦ C, 45 ◦ C
Vortex adapter
E.Z.N.A.® Soil RNA kit
7.87
2
3.5
Yes
RT, 65 ◦ C, 4 ◦ C, −20 ◦ C, 50 ◦ C
None
IT 1-2-3 Platinum PathTM Sample Purification kit
11.25
0.5
<1
Tubes, ethanol, isopropanol Tubes, ethanol, chloroform, isopropanol Phenol: chloroform: isoamyl alcohol Tubes, ethanol, phenol, chloroform:isoamyl alcohol None
No
RT
PickPen® 1-M
a b
Approximate free market price. In addition to centrifuge, microcentrifuge, water bath and/or vortexer.
was detected in 100% (10/10) of the PowerSoilTM Total RNA Isolation kit sand extracts in which RNA was previously non-detectable. Of the loam extracts showing non-detectable amplification, only 10% (2/20) showed amplification following cleanup with the OneStepTM PCR Inhibitor Removal kit. 3.3. Time and cost analysis The processing time and cost per sample were determined for each RNA extraction method (Table 4). The PowerSoilTM Total RNA Isolation and the FastRNA® Pro Soil-Direct and -Indirect kits require approximately 2.5–3 h to complete an extraction. The E.Z.N.A.® Soil RNA kit requires slightly more time to complete (3.5 h) whereas the IT 1-2-3 Platinum PathTM Sample Purification kit requires the least amount of time to process (<1 h). The Qiagen RNeasy MinElute Cleanup and Zymo Research OneStepTM PCR Inhibitor Removal kits require approximately 15 and 10 min, respectively, to complete purification. The IT 1-2-3 Platinum PathTM Sample Purification kit is the most expensive, costing approximately $11.25 per sample (Table 4). The other four extraction methods are comparable to each other in cost ($7.21–8.84 per sample). For the Qiagen RNeasy MinElute Cleanup and Zymo Research OneStepTM PCR Inhibitor Removal kits, the cost per sample is $5.20 and $1.90, respectively. As shown in Table 4, only the IT 1-2-3 Platinum PathTM Sample Purification kit does not require low or high temperatures and uses the fewest number of accessories and additional consumables, thus making this kit compatible with field analysis. The other four kits are not all-inclusive, requiring additional consumables and reagents from the user as well as multiple reaction temperatures. In terms of additional instruments, the PowerSoilTM Total RNA Isolation kit requires a vortex adapter for 15 ml conical tubes while the two FastRNA® kit protocols recommend the use of a FastPrep® bead beater (Table 4). The PickPen® 1-M used in the IT 1-2-3 Platinum PathTM Sample Purification kit is a hand-held magnetic tool designed for the transfer of magnetic particles. 4. Discussion Soils are a challenging matrix for PCR analysis because humic substances are often co-extracted with nucleic acids (Schneegurt et al., 2003). Humic substances inhibit the polymerase in RT-PCR, preventing an accurate determination of RNA yield and potentially resulting in false-negative amplification results (Kermekchiev et al., 2009; Tebbe and Vahjen, 1993). In this study, kits were com-
pared via MS2-specific RT-PCR for their ability to extract and purify viral MS2 RNA from soil (O’Connell et al., 2006). Since soils vary widely in their physical and chemical properties, three different soils (sand, clay, and loam) ranging in pH (4.3–5.8) and organic matter (0.1–8.5%) were tested (Table 1). Kits varied in their ability to extract purified RNA from the three soil matrices. The IT 1-2-3 Platinum PathTM kit could reproducibly detect MS2 RNA from all soil types spiked with MS2 at 109 PFU g−1 soil (Table 2). At the lower spiking amounts of MS2 (107 and 108 PFU g−1 ), however, the E.Z.N.A.® Soil RNA kit showed greater success than the IT 1-2-3 Platinum PathTM kit in reproducibly extracting purified RNA from sand. For loam, the results from the IT 1-2-3 Platinum PathTM kit extractions were nearly comparable to those from the FastRNA® Pro Soil-Indirect kit. However, in this qualitative assessment, MS2 RNA could only be detected in two of the four IT 1-2-3 Platinum PathTM kit extracts from loam spiked with the lowest amount of MS2 (107 PFU g−1 ) (Table 2). In comparison, MS2 RNA was detected in all four FastRNA® Pro Soil-Indirect kit extracts from loam spiked with the same amount of MS2. In addition, relative to the CT values obtained for the positive control, loam extract CT values were lower for the FastRNA® Pro Soil-Indirect kit than the IT 1-2-3 Platinum PathTM kit (Table 3). These results suggest that the FastRNA® Pro Soil-Indirect kit yielded extracts that either contained more accessible MS2 RNA and/or fewer PCR-inhibitory substances. Although additional studies are required to establish any significant difference between the two kits, these results may suggest that the FastRNA® Pro Soil-Indirect kit provides greater sensitivity with the loam soil. Of the five kits tested, only the FastRNA® Pro Soil-Indirect kit uses an indirect extraction procedure, likely explaining the detection of MS2 RNA in all four extracts from loam spiked with all three amounts of MS2 (107 –109 PFU g−1 ) (Tables 2 and 3). Viral lysis followed by RNA extraction can either be performed in situ (direct method) or following separation from the soil matrix (indirect method) (Frostegard et al., 1999). While the direct method generally results in higher yields of nucleic acids (due to the adherence of microorganisms to soil particles), the indirect method limits the exposure of released RNA to humic substances and thus improves the purity of the extracted RNA (Steffan et al., 1988). The use of the indirect method is particularly appropriate for soils that contain high levels of humic substances. Inhibitory humic substances are formed during the decomposition of organic matter and include humic acids, which are increasingly released with low pH (Alm et al., 2000; Burgmann et al., 2001; Miller, 2001; Sagova-Mareckova
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et al., 2008). With the highest percentage of organic matter (8.5%) and the lowest pH (4.3), the loam soil likely contained the highest level of humic substances (Table 1) and was thus suited for the indirect method (Table 2). Despite the use of the indirect method, FastRNA® Pro SoilIndirect kit extracts from loam still required dilution prior to RT-PCR amplification. In fact, as illustrated in Fig. 1, only 0.02–2 l of the loam extracts from any of the kits could be added to each 25 l RT-PCR reaction for amplification to be detected (Table 3). By alleviating the inhibition of the polymerase, dilution of the extract was often sufficient for amplification to be detected (Tsai and Olson, 1992; Wilson, 1997). In some cases, amplification remained nondetectable even after dilution, suggesting that either the RNA yield was too low or the level of PCR-inhibiting humic substances was still too high to permit detectable amplification. In these cases, a commercially available kit was used to remove humic substances. However, out of the twenty loam extracts for which dilution was not sufficient and amplification still remained non-detectable, only two showed detectable amplification following cleanup with the OneStepTM PCR Inhibitor Removal kit. Compared to loam, sand and clay extracts tended to show detectable amplification when undiluted in RT-PCR (Fig. 1 and Table 3). The only exceptions were the PowerSoilTM Total RNA Isolation kit extracts from sand, which likely retained inhibitory substances during the isolation process since only 0.2 l could be added to the 25 l RT-PCR reaction for detectable amplification to be observed. In the majority of the sand and clay extracts, however, the level of humic substances was likely low and thus the RT-PCR amplification was dependent on the RNA yield. For clay, many of the extracts showed non-detectable amplification, suggesting low RNA yield (Table 3). Clay content has previously been shown to negatively influence the recovery of RNA or DNA since nucleic acids adsorb to clay particles when released from bacteria or viruses (Frostegard et al., 1999; Goring and Bartholomew, 1952; Paget et al., 1992). Since low RNA yield was a larger concern than the inhibitory effect of humic substances with sand and clay extracts, the Qiagen RNeasy MinElute Cleanup kit was used, with limited success, to purify and concentrate RNA present in those extracts. Of note, however, is that all non-detectable PowerSoilTM Total RNA Isolation kit extracts from sand showed detectable amplification following cleanup. These extracts likely contained inhibitory substances, which were diluted out in some samples but required removal by the Qiagen RNeasy MinElute Cleanup kit in others. For all soils, prior evaluation of the crude extracts (both diluted and undiluted) by RT-PCR helped determine if cleanup was necessary. The selection of an extraction kit largely depends on the soil characteristics. Sample size may also be an important consideration, particularly in cases where the concentration of the target microorganism is suspected to be low and it would be advantageous to process a larger soil sample. However, the five kits evaluated in this study process soil samples comparable in size, ranging from 0.5 g to 2 g (Table 4). To process larger samples of soil, the manufacturers’ instructions suggest processing replicate soil samples in parallel and then pooling the purified RNA. Based on the trends observed in this assessment, the RNA isolation method that yielded the best results, as defined by reproducibility and sensitivity, was the E.Z.N.A.® Soil RNA kit for sand, the IT 1-2-3 Platinum PathTM Sample Purification kit for clay, and the FastRNA® Pro Soil-Indirect kit for loam. However, additional studies are required to establish any significant differences in kit sensitivity and reproducibility. In addition to soil type, other factors such as processing time, extra external equipment, and reaction temperatures required should also be considered in the kit selection. The PowerSoilTM Total RNA Isolation, E.Z.N.A.® Soil RNA, FastRNA® Pro Soil-Direct, and FastRNA® Pro Soil-Indirect kits are
49
comparable in cost ($7.21–$8.84 per extraction), processing time (approximately 2.5–3.5 h), reagent refrigeration, and reaction temperatures required (Table 4). The IT 1-2-3 Platinum PathTM kit, on the other hand, requires the least amount of processing time (<1 h) and does not require any additional reagents, refrigeration, or reaction temperatures (other than room temperature). With its potential for portability and high throughput, the IT 1-2-3 Platinum PathTM kit is appropriate for use in the field, particularly when there is a large number of samples or a constraint on sample testing time. However, if reagents, storage conditions, and processing time are less of a concern, the other kits, at least for the sand and loam soils, may provide greater sensitivity with lower detection thresholds (Tables 2 and 3). The isolation and identification of a microorganism is a critical step in the analysis of an environmental sample. Our results suggest that soil characteristics, such as clay and organic matter content, should be assessed and considered prior to the selection of an appropriate nucleic acid extraction and purification method. These methods vary in their ability to remove humic acids and other PCR-inhibitory substances from the final extracts, thereby influencing the success of real-time PCR identification. Kermekchiev et al. (2009) has recently identified novel mutant Taq and Klentaq enzymes that show resistance to PCR inhibitors. Combining an appropriate nucleic acid extraction method with a real-time PCR assay utilizing a resistant enzyme may significantly facilitate the detection and identification of a microorganism in an environmental sample. Acknowledgements The views and conclusions contained in this document are those of the authors and should not be implied as necessarily representing the official policies, either expressed or implied, of the U.S. government. This is publication 09-35 of the Federal Bureau of Investigation (FBI). Names of commercial manufacturers are provided for identification only, and inclusion does not imply endorsement by the FBI. Thanks to Robert D. Koons, Keith Monson, and Christopher Ehrhardt (FBI CFSRU) for useful discussions. Shauna M. Dineen, Roman Aranda IV, and Marianne E. Dietz participated in this research through the Oak Ridge Institute for Science and Education program. References Alm, E.W., Zheng, D., Raskin, L., 2000. The presence of humic substances and DNA in RNA extracts affects hybridization results. Appl. Environ. Microbiol. 66, 4547–4554. Arbeli, Z., Fuentes, C.L., 2007. Improved purification and PCR amplification of DNA from environmental samples. FEMS Microbiol. Lett. 272, 269–275. Borneman, J., Triplett, E.W., 1997. Molecular microbial diversity in soils from eastern Amazonia: evidence for unusual microorganisms and microbial population shifts associated with deforestation. Appl. Environ. Microbiol. 63, 2647–2653. Braid, M.D., Daniels, L.M., Kitts, C.L., 2003. Removal of PCR inhibitors from soil DNA by chemical flocculation. J. Microbiol. Methods 52, 389–393. Brassard, J., Lamoureux, L., Gagne, M., Poitras, E., Trottier, Y., Houde, A., 2009. Comparison of commercial viral genomic extraction kits for the molecular detection of food borne viruses. Can. J. Microbiol. 55, 1016–1019. Broussard, L.A., 2001. Biological agents: weapons of warfare and bioterrorism. Mol. Diagn. 6, 323–333. Burgmann, H., Pesaro, M., Widmer, F., Zeyer, J., 2001. A strategy for optimizing quality and quantity of DNA extracted from soil. J. Microbiol. Methods 45, 7–20. Dong, D., Yan, A., Liu, H., Zhang, X., Xu, Y., 2006. Removal of humic substances from soil DNA using aluminium sulfate. J. Microbiol. Methods 66, 217– 222. Dreier, J., Stormer, M., Kleesiek, K., 2005. Use of bacteriophage MS2 as an internal control in viral reverse transcription-PCR assays. J. Clin. Microbiol. 43, 4551–4557. Frostegard, A., Courtois, S., Ramisse, V., Clerc, S., Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., Simonet, P., 1999. Quantification of bias related to the extraction of DNA directly from soils. Appl. Environ. Microbiol. 65, 5409–5420. Garnier, L., Gaudin, J.C., Bensadoun, P., Rebillat, I., Morel, Y., 2009. Real-time PCR assay for detection of a new simulant for poxvirus biothreat agents. Appl. Environ. Microbiol. 75, 1614–1620.
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