Evaluation of QIAamp® DNA Stool Mini Kit for ecological studies of gut microbiota

Journal of Microbiological Methods 54 (2003) 13 – 20 www.elsevier.com/locate/jmicmeth

Evaluation of QIAampR DNA Stool Mini Kit for ecological studies of gut microbiota Mei Li a,b, Jianhua Gong a,*, Michael Cottrill a, Hai Yu a, Cornelis de Lange b, Jeremy Burton c, Edward Topp d a Food Research Program, Agriculture and Agri-Food Canada, Guelph, Ontario, Canada N1G 5C9 Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 c Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada N6A 4V2 d Southern Crop Protection Centre, Agriculture and Agri-Food Canada, London, Ontario, Canada N5V 4T3 b

Received 5 November 2002; received in revised form 10 December 2002; accepted 10 December 2002

Abstract Cell lysis efficiency and the quality of DNA extracts from complex bacterial ecosystems are two major concerns in molecular ecological studies of gut microbiota. In this study, we use PCR-denaturing gradient gel electrophoresis (DGGE) DNA profiling, random cloning and sequence analysis of 16S rRNA genes to compare the QIAampR DNA Stool Mini Kit with the bead beating technique in the preparation of DNA extracts from gut microbiota of pigs. We also developed a washing procedure that can release more than 93% of bacterial cells attached to the gut mucosa. Both the QIAamp kit and bead beating method lysed approximately 95% of bacterial cells. PCR-DGGE DNA profiles of ileal and cecal microbiota from both digesta and mucosa that were generated from the DNA extracts using the two methods were nearly identical. Random cloning and sequence analysis also demonstrated the high quality of DNA extracts using the two methods. Two random clone sets of 16S rRNA genes generated from the DNA extracts had a similar degree of bacterial diversity. Different preparations of DNA extract from a single sample using the QIAamp kit consistently produced similar PCR-DGGE DNA profiles with similarity indexes higher than 99%. Our data suggest the appropriateness of the QIAampR DNA Stool Mini Kit for the studies of gut microbial ecology and the effectiveness of the QIAamp kit in processing multiple samples for cell lysis and DNA extraction. Crown Copyright D 2003 Published by Elsevier Science B.V. All rights reserved. Keywords: QIAampR; DNA extraction; 16S rRNA; Microbiota

1. Introduction The gut microbiota in farm animals has long been of research interest because of its impact on the health and well-being of the host animals, which is relevant

* Corresponding author. Tel.: +1-519-829-2400x3107; fax: +1519-829-2600. E-mail address: [email protected] (J. Gong).

to livestock production efficiency, as well as the safety and quality of livestock products. In the past, the gut microbiota has been mainly studied by culture-based methods. These studies have significantly contributed to our understanding on the gut microbiota. However, because of the limits of culture-based methods, such as medial selectivity for readily culturable bacteria and the presence of non-culturable bacteria (Ricke and Pillai, 1999; Theron and Cloete, 2000), our understanding of the gut microbiota based on these methods

0167-7012/03/$ - see front matter. Crown Copyright D 2003 Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-7012(02)00260-9

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may be inaccurate and is certainly incomplete. To overcome the limitations of culture-based techniques, molecular approaches are being used increasingly to characterize the gut microbiota. Cell lysis efficiency and the quality of DNA extracts from complex bacterial ecosystems of the gut are two major concerns in such molecular ecological studies. These factors are crucial in the sensitivity and utility of further analyses, such as PCR approaches and phylogenetic analysis, which determines the accuracy of the ecological studies. In addition, there is increasing interest in how diets and food or feed additives, e.g. probiotic bacteria and prebiotics, influence the gut bacterial populations to improve the host nutrition and health. Studies involving animal or human clinical trials often require analysis of a large number of samples, making the throughput of the DNA isolation technique important. The bead beating method is a widely used technique in molecular ecological studies to lyse bacterial cells followed by phenol/chloroform extraction for DNA isolation (Stahl et al., 1988). To process multiple samples, however, a rapid and efficient technique is required. We, therefore, designed experiments using PCR-denaturing gradient gel electrophoresis (DGGE) DNA profiling techniques, random cloning and sequence analysis of 16S rRNA genes to compare the QIAampR DNA Stool Mini Kit (QIAGEN Canada, Mississauga, ON, Canada) with the traditional method of bead beating for preparation of bacterial chromosomal DNA from pig gut samples (both digesta and mucosa).

2. Materials and methods 2.1. Sample collection and preparation Ileum, cecum and colon samples were obtained from 30-day-old piglets at 2 weeks post-weaning. Digesta was collected from a 40-cm segment of pig ileum beginning at a point 20 cm from the distal end, the entire cecum and the middle part of colon (24 cm in length). All gut samples were kept on ice and processed immediately after dissection. In the experiments where multiple piglets were used, all collected ileal, cecal and colon samples were combined for preparation of bacterial samples. Animal

management and experimental procedures were carried out in accordance with the welfare guidelines of the Animal Care Committee, University of Guelph (AUP01R086). Bacterial samples from digesta were prepared essentially as described previously for chicken gut samples (Apajalahti et al., 1998; Gong et al., 2002a,b). Briefly, 2 g of digesta from the cecum was washed once by suspension in 30 ml of saline containing 0.1% (v/v) Tween 80 and centrifuged (SORVALL SS-34 rotor, 200 rpm) at 4 jC for 5 min. After removal of the pellet, bacterial cells were recovered by centrifugation at 27,000  g for 15 min at 4 jC. To prepare bacterial samples of digesta from the ileum, 10 g of digesta was washed three times in 70 ml of sterile saline containing 0.1% (v/v) Tween 80 in a 350-ml French Push with 50 strokes per wash to separate bacterial cells from feed particles. Bacterial cells were then recovered by centrifugation (27,000  g) from the pooled washing solutions at 4 jC for 20 min. Bacterial samples prepared in this manner were stored at 80 jC until further processing. Ileal, cecal and colonic wall-associated bacterial samples, which we referred to as mucosal bacteria, were prepared as described recently by Gong et al. (2002a,b) with minor modifications. Briefly, collected parts of ileum, cecum and colon were opened longitudinally and briefly washed three times in saline by gentle agitation to remove unattached or loosely attached bacteria from the wall. Bacterial cells were released from the gut wall in three 1-min washes using a 50-ml conical tube containing saline and 0.1% (w/w) Tween 80 by vigorously shaking the tube. The washes were pooled and then centrifuged (27,000  g, 20 min) at 4 jC to pellet the cells. The volume of the saline solution used for each wash was 12.5, 20 and 20 ml for ileal, cecal and colonic samples, respectively. To determine the efficiency of the washing procedure in releasing bacterial cells from the mucosa, bacterial cells remaining on mucosa after washing were collected by scraping with a glass slide. Bacterial cells were counted in washing saline and in scrapings by direct counting under an epifluorescence microscope (Section 2.2). The efficiency of washing to remove bacteria was calculated as the total number of bacterial cells present in washing saline divided by the sum of bacterial cells present in both washing saline and scrapings.

M. Li et al. / Journal of Microbiological Methods 54 (2003) 13–20

2.2. Direct counting of bacterial cells Preparation of bacterial samples for direct counting was performed as described by Leung and Topp (2001). Bacterial samples were diluted 10- or 100fold with sterile saline and fixed with formaldehyde (3.7%, v/v) for 2 min. The fixed cell suspension (5 Al) was smeared in a pre-etched area (113 mm2) of a RITE-ON microscope slide (Gold Seal Products, Highpark, IL, USA). The smears were subsequently air-dried, heat-fixed and stained with 0.02% (w/v) 5[4,6-dichlorotriazin-2-yl]amino-fluorescein (DTAF) (Sigma-Aldrich Canada, Oakville, ON, Canada) at 22 jC for 30 min. Bacterial cells were counted under an epifluorescence microscope equipped with a filter set (Exciter HQ470/40x, Emitter HQ500LP and Dichroic Q495LP) to select blue light. The concentration of bacteria cells was calculated as described by Bloem et al. (1995). 2.3. Cell lysis and DNA extraction Two methods, bead beating and the QIAampR DNA Stool Mini Kit, were used to lyse bacterial cells followed by extraction of chromosomal DNA from cell lysates. Prior to bead beating, 1 g of ileal or 0.5 g each of cecal and colonic bacterial samples in pellets were suspended in 1.2 ml of TE buffer (10 mM Tris
Cl and 5 mM EDTA, pH 8.0) containing hmercaptoethanol (5 Al ml 1) and subjected to five freeze – thaw cycles, alternating between liquid nitrogen and 65 jC for 5 min. After centrifugation at 21,000  g at 4 jC for 10 min to pellet unbroken cells, cell lysates were collected for DNA extraction. The unbroken cells were further treated with bead beating as described previously (Whitford et al., 1998). In brief, the unbroken bacterial cells were suspended in 0.5 ml of lysis buffer (0.2 M NaOH and 1% SDS) and then transferred into a 2-ml screw-capped microcentrifuge tube containing 0.6 ml of phenol, 0.1 ml of 10% (w/v) SDS and 0.5 g each of 0.1- and 0.5-mm zirconium beads. Using a Mini-Beadbeater-8k Cell Disrupter (BioSpec Products, Bartlesville, OK, USA), the bead beating was performed twice for 2 min each at the homogenize level. The tubes were incubated at room temperature for 5 min between bead beatings. The cell lysates generated from the freeze – thaw cycles and bead beadings were combined and ex-

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tracted with phenol/chloroform prior to precipitation of DNA with ethanol (Whitford et al., 1998). To determine the efficiency of cell lysis, bacterial cell counts before and after the freeze – thaw cycles as well as after bead beating were examined by direct counting as described in Section 2.2. QIAampR DNA Stool Mini Kit was purchased from QIAGEN Canada and used according to the manufacturer’s instructions. Briefly, bacterial samples were homogenized in buffer ASL and heated at 95 jC for 5 min to lyse bacterial cells. After removal of potential inhibitors by incubation with an InhibitEx tablet, the lysates were treated with proteinase K and buffer AL at 70 jC for 10 min to remove protein and polysaccharides. DNA was precipitated by ethanol, applied to a column provided in the kit followed by washes with buffers AW1 and AW2, and then dissolved in buffer AE. To determine the efficiency of cell lysis, bacterial cells before and after heating in buffer ASL were examined by direct counting under an epifluorescence microscope as described in Section 2.2. Bacterial samples prepared from cecal digesta were used in the experiment to examine the efficiency of cell lysis by both the bead beating method and use of the QIAamp kit. 2.4. PCR-DGGE analysis The V2 –V3 region of the 16S rRNA genes (positions 339 to 539 in the Escherichia coli gene) of bacteria in the gut samples was amplified by primers HDA1-GC (5V-CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG GAC TCC TAC GGG AGG CAG CAG T-3V; the GC clamp is in boldface) and HDA2 (5V-GTA TTA CCG CGG CTG CTG GCA C-3V) as described by Walter et al. (2000). PCR reaction mixtures and the amplification program were the same as described previously (Gong et al., 2002a,b) except that 30 cycles of amplification were used. Denaturing gradient gel electrophoresis analysis of the amplicons was performed using the Bio-Rad DCode Universal Detection System (BioRad Canada, Mississauga, ON, Canada). The denaturing gradient gel contained a 35 – 65% gradient of urea and formamide increasing in the direction of electrophoresis. A 100% denaturing solution consisted of 7 M urea and 40% (v/v) deionized formamide. The electrophoresis was conducted in 1  TAE buffer

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Table 1 Efficiency of washing to release bacterial cells from mucosaa Sampleb

Experiment 1 Free cells

Ileum Cecum Colon

10

1.12  10 1.59  1010 1.84  1010

Experiment 2 Attached cells 8

2.50  10 1.05  109 8.60  108

Efficiency (%) 98 94 96

Free cells 10

1.52  10 1.66  1010 1.82  1010

Attached cells 8

5.40  10 1.25  109 1.30  109

Efficiency (%) 97 93 93

a

After three washes with 0.1% (v/v) Tween 80 in saline, mucosa was scraped and bacterial cells were examined by direct counting under an epifluorescence microscope. Free cells: the total number of cells released from mucosa by washing. Attached cells: the total number of cells that remained attached to mucosa after washing. The efficiency of washing to release bacterial cells from mucosa expressed as: % = free cells/(free cells + attached cells)  100. b Ileum: 40 cm in length, beginning at the point 20 cm from the distal end. Cecum: entire cecum. Colon: middle part of colon (24 cm in length).

with 100 V at 60 jC for 16 h. DNA bands in gels were visualized by silver staining (van Orsouw et al., 1997). The similarities of PCR-DGGE DNA profiles were analyzed with the software of BioNumerics (Applied Maths BVBA, Belgium) using the Dice function. The degree of similarity was represented by a similarity index. 2.5. Cloning and sequence analysis of 16S rRNA genes 16S rRNA genes were amplified from the bacterial sample prepared from ileal digesta with eubacterial primers, F8 (5V-AGAGTTTGATCCTGGCTCAG-3V) and R1492 (5V-GGTTACCTTGTTACGACTT-3V) (Eden et al., 1991), as described previously (Gong et al., 2002a,b). The amplicons were randomly cloned into the vector, pCRR4-TOPOR, using the TOPO TA cloning Kit (Invitrogen) according to the manufacturer’s instructions. The cloned genes were sequenced using the primer F8 with an ABI PRISMk 377 Automated DNA Sequencer. The partial sequences

were compared directly with nonredundant nucleotides in the GenBank, EMBI and DBJI databases using BLAST.

3. Results and discussion 3.1. Efficiency of washes in preparation of mucosal bacteria Initial attempts to isolate bacterial chromosomal DNA directly from gut mucosa by scraping often gave unsuccessful PCR reactions. Therefore, a series of washes in saline containing 0.1% (v/v) Tween 80 was used to release bacterial cells from the mucosa, improving the quality of DNA preparations for PCR amplification. Table 1 shows the effectiveness of the washing procedure in releasing bacterial cells from mucosa of ileum, cecum and colon. In all tests, more than 93% of mucosa-associated bacterial cells were released from the ileum, cecum and colon (Table 1). The washing procedure, therefore, appears to be appropriate for preparing mucosal bac-

Table 2 Efficiency of cell lysis using QIAampR DNA Stool Mini Kit or bead beating techniquea QIAamp kit

Bead beating

Cells per gram

Cell lysis (%)

Before lysis

After lysis

2.67  1011

1.36  1010

a

95

Cells per gram

Cell lysis (%)

Before lysis

After lysis

2.5  1011

1.04  1010

96

Bacterial samples were from cecal digesta. When using the QIAamp kit, cells were lysed in buffer ASL at 95 jC for 5 min. The bead beating method involved five freeze – thaw cycles, alternating between liquid nitrogen and 65 jC for 5 min in the presence of h-mercaptoethanol (5 Al ml 1), followed by bead beating at room temperature twice (each for 2 min) using a Mini-Beadbeater-8k Cell Disrupter (BioSpec Products, Bartlesville, OK, USA). After lysis: cells remaining intact after the final bead beating. Cell lysis (%) = 100 after lysis/before lysis  100.

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3.3. Effect of cell lysis and DNA extraction methods on PCR-DGGE DNA profiles To further evaluate the appropriateness of the QIAamp kit for ecological studies of gut microbiota, bacterial diversity in the digesta and mucosa of ilea and ceca were examined by PCR-DGGE analysis of DNA samples prepared by the QIAamp kit and by bead beating. No significant differences (similarity indexes >99%) in bacterial diversity were detected in digesta samples from the same gut region of ileum and cecum when prepared using the QIAamp kit or bead-beating method (Fig. 1A). In addition, both

Fig. 1. PCR-DGGE analysis of microbiota from the ileum and cecum of post-weaning piglets. Panel A: bacteria in the digesta. Panel B: bacteria in the mucosa. K: DNA samples were prepared by use of the QIAamp kit. B: DNA samples were prepared by the bead beating method.

terial samples for ecological studies of mucosal gut microbiota. 3.2. Cell lysis efficiency for different lysis methods The efficiency of cell lysis by both the QIAamp kit and bead beating was compared. As shown in Table 2, both methods lysed approximately 95% of bacterial cells, indicating that the QIAamp kit is as effective as the bead beating method in lysis of gut bacterial cells. In addition, the five freeze –thaw cycles prior to the bead beating lysed approximately 50% of bacterial cells (data not shown).

Fig. 2. PCR-DGGE analysis of microbiota from ileal digesta showing the consistency of sample preparation by the bead beating and use of the QIAamp kit, as well as of PCR amplification and DGGE electrophoresis. Panel A: bacterial cell lysis and DNA extraction were conducted using the bead beating method. Panel B: bacterial cell lysis and DNA extraction were performed by use of the QIAamp kit. Prep 1 and 2: two different preparations of DNA extract from the same ileal digesta sample, but prepared on different days. #1 and #2: two replicates of PCR amplification and DGGE electrophoresis from the same DNA extract.

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methods generated PCR-DGGE DNA profiles with similarity indexes greater than 99% from mucosaassociated bacteria in the ileum or cecum (Fig. 1B). In light of these data and the fact that PCR-DGGE technique is widely used for a global assessment of gut bacterial populations (Simpson et al., 1999, 2000; Favier et al., 2002; Heilig et al., 2002; Kocherginskaya et al., 2001; Satokari et al., 2001; Walter et al., 2001), the bacterial populations expressed by the profiles appeared to have a similar degree of bacterial diversity and community structures for both cell lysis/ DNA extraction methods. To examine the consistency of using the QIAamp kit for cell lysis and DNA extraction, as well as our procedures for characterization of gut microbiota (including bacterial sample preparation, DNA extraction, PCR amplification and DGGE analysis), we used a single sample of ileal digesta to repeat the procedures starting from the sample preparation and ending at generation of PCR-DGGE DNA profiles. Fig. 2 demonstrates the PCR-DGGE analysis using bacterial chromosomal template DNA isolated from the same ileal digesta sample on different days using the bead beating and QIAamp kit. As shown in Panel B, the DNA profiles generated from two different DNA

preparations as well as from the two replicates of the same DNA preparation using the QIAamp kit were identical (similarity index >99%). In addition, these DNA profiles exhibited no significant difference (similarity index >99%) from those generated from the DNA extracts prepared by the bead beating method, including different preparations and replicates (Pane A). These data suggest that using the QIAamp kit results in a high degree of consistency in preparation of DNA extracts from gut microbiota. Moreover, our procedures described in this study appear to be consistent and appropriate for characterization of gut microbiota. 3.4. Comparison of bacterial diversity determined by random cloning and sequence analysis As a means to further compare the extraction of bacterial chromosomal DNA from digesta samples using the QIAamp kit and bead beating technique, random cloning and sequence analysis of 16S rRNA genes were also performed. Table 3 documents the bacterial species detected in the two random clone sets of 16S rRNA genes (one from each method) from ileal digesta. There was a total of 12 distinct species

Table 3 Molecular species detected in ileal digestaa Closest relative

Accession #

Escherichia coli

AE005607 AE000406 X80729 M99704 Y17362 AY050173 X97898 AJ002515 AF126738 X76328 AF420311 X96964 X96965 X84007 AF371472 AF371479 AF371695

Lactobacillus acidophilus Lactobacillus crispatus L. delbrueckii subsp. lactis Lactobacillus gallinarum Lactobacillus johnsonii Lactobacillus mucosae Lactobacillus reuteri Lactobacillus salivarius Shigella sonnei Shigella boydii Veillonella atypica Uncultured bacterium

QIAamp kit

Bead beating

Detected

Similarity (%)

Detected

Similarity (%)

+ + + + + + + + + + + +

98 – 99 98 – 99 99 98 – 99 96 – 98 99 98 – 100 98 99 98 – 99 99 98

+ +

99 98 – 99

+ +

98 – 99 97 – 98

+ + + +

98 – 99 97 97 – 100 97 – 99

+ +

92 97

+ + +

98 98 91 – 92

+ +

95 99

a Based on a BLAST analysis that was conducted in April of 2002. The two random clone sets of 16S rRNA genes had 56 and 58 clones, respectively, which were derived from bacteria in the ileal digesta of three piglets. +: detected, : not detected.

M. Li et al. / Journal of Microbiological Methods 54 (2003) 13–20

detected in each clone set, of which 9 species were present in both clone sets. These data indicate that both methods generated good quality of DNA extracts, from which a similar degree of bacterial diversity was derived. In the present study, we have evaluated the use of the QIAampR DNA Stool Mini Kit versus the bead beating method for its efficiency of cell lysis and DNA extraction for molecular ecological studies using two widely used molecular techniques. The data presented here, including cell lysis, PCR-DGGE DNA profiles and sequence analysis of random 16S rRNA gene clones, demonstrated that the QIAamp kit has an efficiency similar to the bead beating method in cell lysis and DNA extraction and is appropriate for ecological studies of gut microbiota. Since the QIAamp kit is simple to use and timeeffective, it offers an efficient means to process multiple samples. During the preparation of this manuscript, McOrist et al. (2002) reported their studies on the comparison of five different methods, including four commercial kits and a guanidium isothiocyanate/silica matrix method, for the efficiency in DNA extraction from human fecal samples. By examining PCR amplification of lactobacillus and bacteroides added into the fecal samples, they found that use of the QIAampR DNA Stool Mini Kit was the most effective method in DNA extraction among the five methods. In the present study, we have evaluated the QIAamp kit, against the widely used bead beating method, for its appropriateness for molecular ecological studies of pig gut microbiota by examining cell lysis, DNA exaction and its influence on the diversity of bacterial populations in the pig guts as assessed by PCR-DGGE and random cloning and sequence analysis of 16S rRNA genes. Therefore, our report appears to have further advanced the observations of McOrist et al. (2002). 3.5. GenBank accession numbers A cloned 16S rRNA gene (PIC29) showing less than 95% homology within the sequenced region to the existing database sequences has the GenBank accession numbers of AY152842. This clone represents a group of eight clones (more than 99% homology) in our random clone sets, which are relatives of

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Veillonella atypica (GenBank accession number X84007).

Acknowledgements The present research was funded by Ontario Pork (0116), and Agriculture and Agri-Food Canada’s Matching Investment Initiative. This paper represents FRP contribution number S130.

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