Comparison of seven RNA extraction methods on stool and shellfish samples prior to hepatitis A virus amplification

Journal of Virological Methods 77 (1999) 17 – 26

Comparison of seven RNA extraction methods on stool and shellfish samples prior to hepatitis A virus amplification C. Arnal a,*, V. Ferre´-Aubineau a, B. Besse a, B. Mignotte b, L. Schwartzbrod b, S. Billaudel a a

Laboratoire de Virologie, Institut de Biologie, CHRU de Nantes, 9, quai Moncousu, 44093 Nantes Cedex 01, France b Laboratoire de Virologie, Faculte´ de Pharmacie, BP 403, F-54001 Nancy Cedex, France Received 19 February 1998; accepted 19 May 1998

Abstract When choosing an extraction method, two parameters have to be considered: recovery of the viral material and elimination or inactivation of inhibitory substances. Seven techniques for extracting hepatitis A virus (HAV) from stool and shellfish samples were compared, in order to identify the protocol most suited to both types of sample and with the best extraction yield. The protocols tested were either techniques for the recovery and purification of total RNA, such as RNAzol, PEG-CETAB, GTC-silica and Chelex, or techniques for isolating specifically HAV using a nucleotide probe or a monoclonal antibody. For stool samples, RNAzol, PEG-CETAB, and magnetic beads with antibody allowed detection of the virus in 11/12 and 12/12 of samples. For shellfish samples, three protocols allowed RNA to be extracted in 90% of cases, RNAzol, PEG-CETAB, and GTC-silica. Their rapidity and low cost make RNAzol and GTC-silica the most suitable for routine diagnostic testing. © 1999 Elsevier Science B.V. All rights reserved. Keywords: RT-PCR; Inhibitors; Extraction; HAV; Shellfish; Stool

1. Introduction Gene amplification methods are being used increasingly for virological diagnostic tests as a * Corresponding author. Tel.: +33 2 40084105; fax: + 33 2 40084114; e-mail: [email protected]

result of their great sensitivity, specificity and rapidity (Clementi et al., 1993). The use of RTPCR to detect viral genome in samples of complex composition such as stool and shellfish is rendered difficult by the low concentrations of virus and the presence of substances liable to interfere with the enzyme system used for amplifi-

0166-0934/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII S0166-0934(98)00083-4

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cation (Jiang et al., 1992, Wilde et al., 1990, Shieh et al., 1995). Amplification techniques are well standardized, but the methods used to prepare samples usually depend on the nature of the specimen to be tested. The different extraction techniques available are of variable complexity, ranging from simple boiling to more elaborate methods involving lysis in a saline buffer in the presence of detergents and proteinase K, followed by organic extraction and ethanol precipitation. When choosing an extraction method, two parameters have to be considered: recovery of the viral material and elimination or inactivation of inhibitory substances. The latter are substances of a diverse nature. Inhibition has been described in samples rich in proteins or rich in genomic material (Gouvea et al., 1990). The presence of salts, traces of detergent (SDS) or phenol (Beutler et al., 1990) following extraction can also interfere with amplification. Finally, certain more specific substances, such as bilirubin and bile salts in stool specimens or acid polysaccharides in shellfish, have been identified as inhibitors (Wilson, 1997). Numerous protocols for use with stool and shellfish samples have been developed, with a view to eliminating such substances with varying degrees of specificity. Methods for purifying the genomic material involving media composed of Sephadex (De Leon et al., 1992; Straub et al., 1994), cellulose (Wilde et al., 1990), or Chelex (Straub et al., 1994; Hale et al., 1996), for instance, allow salts and small proteins to be effectively eliminated. Likewise, chemical processes based on cetyltrimetylammonium bromide (CTAB) (Jiang et al., 1992; Straub et al., 1994) have made it possible to eliminate certain polysaccharides. In general, such processes only deal with a single category of inhibitors, while a combination of several such processes leads to a loss of material which, in samples that are only lightly contaminated, is unacceptably high. Viral immunocapture methods, however, are more effective, since they specifically isolate the virus from the whole range of inhibitory substances in the sample. Moreover, the use of a solid medium makes it possible to concentrate the sample (Jansen et al., 1990; Schwab et al., 1993).

The present study was designed to compare the efficiency of seven different extraction techniques on two specimen types known to contain numerous inhibitors (stool and shellfish) for the detection of hepatitis A virus by RT-PCR. Extraction protocols falling into two main categories were chosen: on the one hand, protocols that recover and purify total RNA, such as RNAzol, PEGCTAB, silica in the presence of a guanidinium thiocyanate (GTC) buffer, and Chelex; and on the other, protocols that specifically isolate HAV, using a specific probe for the HAV genome (beads with probe) or a monoclonal antibody directed against the viral capsid (beads with antibody and antigen capture). The purpose was to identify an HAV RNA extraction protocol that would be suitable for different specimen types and could be applied in routine diagnostic testing.

2. Materials and methods

2.1. Virus Hepatitis A strain CF-53 was supplied by Dr Crance, Centre de Recherche du Service de Sante´ des Arme´es, Grenoble, France. A quantified cell culture supernatant containing 107 TCID50 per ml of HAV was extracted as a positive control.

2.2. En6ironmental and clinical samples Samples of mussel (Mytilus edulis) used for the experiments were from areas of cultivation on the Atlantic coast of France. Shellfish were artificially contaminated by immersing them for 1 h in reconstituted seawater (Reef Crystal, Aquarium System, Sarrebourg, France) containing 9× 103 TCID50 of HAV per ml as described by Mignotte et al. (1997). Sixteen stool samples were analyzed for their HAV content: 15 were collected from patients with hepatitis A during an outbreak that occurred in France between December 1991 and March 1992 (Apaire-Marchais et al., 1994). The remaining sample was a fecal specimen isolated from a patient not infected with HAV.

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2.3. Preparation of samples prior to RNA extraction 2.3.1. Stool samples The 10% stool suspensions were prepared in phosphate-buffered saline (pH 7.4) and clarified by centrifugation for 15 min at 4800×g to eliminate larger fecal debris. Clarified supernatants were frozen in 200 ml portions until use. 2.3.2. Shellfish samples After contamination, the mussels were rinsed, opened and the intervalvar fluid discarded. Tissue was extracted aseptically and stored at −20°C in 60-g aliquots before further processing. For the extraction-concentration of virus from contaminated mussels, an experimental design combining four extraction procedures, three concentration techniques and one detoxification was developed. Fourteen of the 24 possible combinations of extraction and concentration were tested. The four extraction protocols have previously been described in detail (Mignotte et al., 1997). The first extraction technique was based on the elusion of virus from ground-up mussel tissue with a solution of 0.1 M borate/3% beef extract at pH 9 as described by Boher and Schwartzbrod (1993). For the second, a solution of 0.05 M glycine/0.15 M NaCl at pH 8.5 was used as described by Van der Veen (1995). For the third, virus was extracted using a solution of NaCl/beef extract at pH 7.5 as described by Alouini and Sobsey (1995). The fourth was a secondary freon extraction of the products from the previous NaCl/beef extract as described by Alouini and Sobsey (1995). The three concentration methods have been described fully by Mignotte et al. (1997). The step common to all the concentration techniques was resuspension of the final pellet in 12 ml Na2HPO4 at pH 9. The first concentration technique was based on the precipitation of proteins at an acid pH. Flocculation of viruses contained in the mussel extracts was obtained by lowering the pH to 3.5 as described by Katzenelson et al. (1976). For the second, precipitation with 10% polyethylene glycol 6000 (PEG 6000) was used as described by Lewis and Metcalf (1988). For the last, precipitation with 12% PEG 8000 was used as described by

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Alouini and Sobsey (1995). Half of each concentrate (6 ml) was detoxified using a method based on filtration through a Sephadex LH20 gel (Beril et al., 1991). Concentrates and detoxified concentrates were frozen in 200 ml portions until analyzed.

2.4. RNA extraction methods Seven RNA extraction methods were evaluated on the different samples (shellfish concentrates, culture supernatant and 10% stool suspensions). Extractions were carried out under the same conditions, regardless of the type of sample. For all these methods, the initial volume of sample extracted was the same (200 ml). Volumes in which the RNA pellet was resuspended for each technique were adjusted to provide the optimal conditions for each protocol, and ranged from 10 to 40 ml depending on the method used. Negative controls were included in the course of each extraction. Extraction 1. RNAzol extraction was based on the method of Chomczynski and Sacchi (1987) and used a commercially available mixture of acid guanidinium thiocyanate and phenol chloroform (RNAzol® Bioprobe Systems, Montreuil-sousBois, France). Isolation of total RNA was performed according to the manufacturer’s recommendations. Briefly, samples were mixed with 2 vols RNAzol® and extracted with 200 ml chloroform. The RNA-containing aqueous phase was precipitated with 500 ml isopropanol, 100 ml 7.5 M ammonium acetate and 20 mg glycogene (Boehringer, Mannheim, Germany). The RNA pellet was washed with 75% ethanol and then resuspended in 40 ml sterile deionized water. Extraction 2. For the PEG-CTAB method, total RNA was extracted as described by Jiang et al. (1992). At the end of the procedure the final pellet was resuspended in 40 ml sterile deionized water. Extraction 3. For the GTC-silica method, the RNA was extracted by a modified version of the technique described by Boom et al. (1990). Thirtymicroliter size-fractionated silica was added to 200 ml sample and the RNA was released from the virus capsid by addition of 1.8 ml guani-

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dinium thiocyanate (GTC) lysis buffer. The silica/ RNA complexes were then washed in turn with GTC buffer (two washes), 75% ethanol (two washes) and acetone. The silica particles were dried and RNA was eluted with 40 ml sterile deionized water. Extraction 4. For the Antigen-Capture method, we used the procedure described by Jansen et al. (1990) and modified by Deng et al. (1994). The technique was applied to HAV clone 1009 antihepatitis A antibody (Argene, Biosoft, Varilhes, France). Polypropylene tubes were coated with 200 ml of an 0.05 mg/ml antibody solution, saturated with BSA and then loaded overnight at 4°C with 200 ml sample. After the last wash, 24.5 ml RT-PCR reaction mixture without enzyme was added. Following denaturation of viruses for 10 min at 95°C and rapid cooling on ice, 0.5 ml enzyme blend (Titan system, Boehringer, Mannheim, Germany) was added and RT-PCR was carried out. Extraction 5. HAV was extracted using magnetic beads coated with a labeled biotinylated oligonucleotide originally developed for enteroviruses, following the modified lysis method of Beaulieux et al. (1997) based on the procedure described by Muir et al. (1993). Nucleic acids were released from the virus (200 ml) using RNAzol® solution in a 1:1 volume ratio. Then, 1 mg M-280 streptavidine beads (Dynal, Oslo, Norway) were coated with 60 ml biotinylated D hepatitis A antisense primer at 100 pmol/ml, washed three times with 6× SSPE to eliminate unbound probes, and redissolved in 10 ml 12× SSPE. Then, 400 ml coated beads were added to 400 ml RNAzol lysate and incubated for 20 min at 4°C with continuous stirring. After three washes with 6 × SSPE, beads were resuspended in 20 ml sterile deionized water. Extraction 6. For the Chelex method, 100 ml 30% w/v Chelex 100 resin (Bio-Rad, Richmond, CA) was added to the sample and after vortexing for 10 s the sample was incubated at 56°C for 30 min, vortexed again, and then incubated at 100°C for 5 min. The samples were vortexed a third time and centrifuged at 14000× g for 3 min. To adjust its final volume to the same range as the final volumes for all RNA extraction methods (10 – 40

ml), the Chelex supernatant (250 ml) was precipitated with 250 ml isopropanol, 100 ml 7.5 M ammonium acetate, and 20 mg glycogene, and washed with 75% ethanol. The final pellet was resuspended in 20 ml sterile deionized water. Extraction 7. For the magnetic immunocapture method, a modified version of the procedure described by Monceyron and Grinde (1994) was used. Dynabeads precoated with sheep antimouse IgG1 were purchased from Dynal (Oslo, Norway). HAV clone 1009 antibody (Argene, Biosoft, Varilhes, France) directed against HAV surface epitope was used. The antibody was coated on to the dynabeads according to the manufacturer’s recommendations, i.e. by overnight incubation of 10 mg antibodies per mg beads at 4°C with continuous stirring. For antigen capture, 50 mg beads was added to each of the 200 ml samples, which were then incubated for 2 h on rollers to maintain the beads in suspension. After two washes with 400 ml 10 mM Tris–HCl and 50 mM KCl at pH 8.3, the beads were resuspended in 10 ml sterile deionized water. The virus was then denatured for 5 min at 95°C and cooled rapidly on ice.

2.5. Re6erse transcription and PCR (RT-PCR) RT-PCR primers were derived from HAV conserved DNA sequences encoding for VP1-VP3 capsid proteins (Cohen et al., 1987, Genbank accession No M14707). The 39-nucleotide primers, E: 5%-GTTTTGCTCCTCTTTATCATGCTATGGATGTTACTACAC-3% (2167–2205), and D: 5%-GGAAATGTCTCAGGTACTTTCTTTGCTAAAACTGGATCC-3% (2389–2413), yielded a predicted 248-bp RT-PCR product (Arnal et al., 1998). The Titan one step RT-PCR system (Boehringer, Mannheim, Germany) was developed for the detection of HAV. RT-PCR was carried out in a volume of 25 ml including 5 ml RNA extract (previously denatured for 10 min at 65°C), 0.5 mM each of primers D and E, 5 mM DTT, 10 U RNasine, 5 ml 5X RT-PCR buffer containing 1.5 mM MgCl2, and 0.5 ml enzyme blend containing AMV reverse transcriptase and expand high fidelity enzyme mix (Taq and Pwo DNA polymerases). The amplification mixture was kept

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Table 1 Comparative sensitivities of RT-PCR detection after seven different RNA extraction methods RNA extraction methodb RNAzol

Limit of detectiona 3.2 (TCID50/ml)

PEG-CTAB

GTC-silica

Chelex

Beads with probe

Beads with Ab

Antigen capture

0.64

3.2

0.64

0.13

0.025

3.2

PEG, polyethylene glycol; CTAB, cetyltrimethylammonium bromide; GTC, guanidinium thiocyanate. a Limit of detection: concentration of the lowest dilution of culture supernatant with a positive RT-PCR signal after PAGE and ethidium bromide staining. b See Section 2 for details.

on ice during the preparation to avoid inappropriate conforming of long primers. Amplification conditions were provided by 30 min reverse transcription at 42°C, denaturation for 5 min at 94°C and then the first ten amplification cycles (denaturation at 94°C for 30 s, annealing at 62°C for 30 s and extension at 68°C for 45 s), followed by 25 further amplification cycles (denaturation at 94°C for 30 s, annealing at 62°C for 30 s, and extension at 68°C for 45 s in the first cycle and increasing by 5-s increments in each subsequent cycle). Finally, a 7-min extension cycle at 68°C was performed on a Hybaid Omnigene thermocycler. To avoid false positive results due to contamination, rigorous experimental procedures were employed. RNA extraction, preparation of the amplification mixture and PCR were carried out in separate positive-pressure rooms using pipettes with sterile filter-plugged disposable tips. Appropriate negative and positive controls were included in each batch of experiments. Positive controls were essential to show that neither RNase nor any other ingredients were inhibiting amplification (false negative results). Negative controls were to show that amplification was not the result of cross-contamination (false positive results).

2.6. Detection of amplified products Amplified products (10 ml) were visualized by electrophoresis on a 9% polyacrylamide gel (Bioprobe Systems, Montreuil-sous-Bois, France) in Tris-Borate-EDTA buffer followed by staining with ethidium bromide. The intensity of each

electrophoresis band was graded by comparison with a positive control, the results being recorded as grades 0= no discernible product to 3= intensity equal to that of the positive control. The DNA enzyme immunoassay (DEIA) described in a previous study (Arnal et al., 1998) was applied to mussel and stool samples to confirm the specificity of observed PCR products.

3. Results

3.1. Efficiency of RNA reco6ery from culture supernatants Starting from a supernatant with an HAV titer of 107 TCID50/ml, a series of 5-fold dilutions with viral concentrations ranging between 400 TCID50/ ml and 0.025 TCID50/ml were prepared. Seven different extraction techniques were then used on these samples prior to RT-PCR, and the amplified products obtained were detected by electrophoresis on polyacrylamide gel (PAGE). The experiments for each protocol were repeated three times. Thresholds of detection, corresponding to the lowest concentrations of virus (TCID50/ml) for which positive amplification signals were observed, are set out in Table 1. The observed limits of detection varied, according to technique, from 0.025 to 3.2 TCID50/ml—that is, by a factor of 100. The extraction protocols using magnetic beads (beads with antibody and beads with probe) were the most sensitive.

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Table 2 Results of RT-PCR detection in 12 positive stool specimens using seven RNA extraction methods Intensity of PCR productsa

RNA extraction methods

RNAzol

PEG-CTAB

GTC-silica

Chelex

Beads with probe

Beads with Ab

Antigen capture

Grade 3 Grade 2 Grade 1 Grace 0

1 5 5 1

2 4 6 0

4 2 2 4

1 1 3 7

2 3 2 5

7 2 2 1

0 3 5 4

Total number of positive samples

11/12

12/12

8/12

5/12

7/12

11/12

8/12

a

Intensity grade of the PCR product observed after PAGE and ethidium bromide staining (0: no discernible product, 1: weak band, 2: intensity weaker than positive control, 3: intensity equal to that of positive control sample).

3.2. E6aluation of RNA extraction techniques on stool samples The extraction protocols tested, followed by amplification, were carried out on the following stool specimens: four negative samples and 12 samples known to be positive for the HAV genome. The four specimens expected to test negatively remained negative regardless of the extraction protocol used. The results for the 12 remaining specimens are set out in Table 2. They are expressed as the number of samples testing positive with each protocol as a function of the intensity of the amplification signal observed after PAGE. All samples tested positive with at least one of the three different extraction techniques used. For three of the extraction protocols, RNAzol, PEG-CTAB and Ab-beads, positivity scores of 11/12 or 12/12 were observed. Extraction using Chelex was the only technique to show less than six positive samples.

3.3. E6aluation of RNA extraction techniques on shellfish samples The extraction protocols were carried out on concentrates of shellfish specimens that had been deliberately contaminated by immersion in seawater containing 9× 103 TCID50/ml HAV. Different procedures for virus extraction and concentration found in the literature were carried out on these

mussel samples in order to study the efficiency of virus recovery and elimination of amplification inhibitors. The extraction protocol using Chelex did not yield any positive results. Three of the protocols showed results of less than 50% positivity; and three others allowed positive detection of almost 90% to be obtained (Table 3). These were RNAzol, PEG-CTAB, and GTC-silica. The intensity of the PCR signals varied as a function of the viral extraction–concentration procedure used. The protocols based on glycine and borate, which after electrophoresis yielded PCR product intensities of grade 2, are more efficient than those using NaCl/beef extract buffers with or without freon, which yielded lower intensities (grade 1).

4. Discussion To detect viruses using PCR, it is important that the samples of nucleic acids from the original specimens be as pure as possible. The purification stage is particularly important when the virus concerned is an RNA virus, for which a reverse transcription (RT) stage is necessary. Indeed, the high susceptibility of reverse transcriptase to interfering or inhibitory substances is a major limiting factor in amplification reactions (Wilde et al., 1990). With this in mind, we compared seven RNA extraction techniques falling into two main groups: those that specifically extract the virus,

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Table 3 Results of RT-PCR detection in 14 different shellfish concentrates using seven RNA extraction methods Type of shellfish concentrates

RNA extraction methods RNAzol

PEG-CTAB

GTC-silica

Chelex

Beads with probe

Beads with Ab

Antigen capture

glycine/PEG 6000 glycine/OF glycine/OF/LH20 Borate/PEG 6000 Borate/OF Borate/PEG 6000/LH20 Borate/OF/LH20 Saline beef/PEG 6000 Saline beef/PEG 8000 Saline beef/OF Saline beef/PEG 6000/LH20 Saline beef/PEG 8000/LH20 Saline beef freon/PEG 8000 Saline beef freon/PEG 8000/LH20

2 2 2 2 2 2 2 1 1 0 1 1 2 0

2 2 3 ND 2 2 3 1 1 1 1 1 2 0

2 1 2 2 0 1 2 1 1 0 1 1 1 1

0 0 0 0 0 0 0 0 ND 0 0 0 0 0

0 1 0 0 0 1 1 0 ND 0 0 0 0 ND

1 1 3 0 0 1 3 0 0 0 0 0 1 0

0 0 1 1 ND 1 1 ND ND 0 0 0 1 0

Total number of positive samples

12/14 (86%)12/13 (92%)

12/14 (86%)

0/14 (0%) 3/12 (25%)

6/14 (43%) 5/11 (45%)

Intensity grade of the PCR product observed after PAGE and ethidium bromide staining (0: no discernible product, 1: weak band, 2: intensity weaker than positive control, 3: intensity equal to that of positive control sample, ND: not done). Extraction–concentration methods used for shellfish samples: see Section 2 for details. OF, organic flocculation concentration; PEG 6000, polyethylene glycol 6000 concentration; PEG 8000, polyethylene glycol 8000 concentration; LH20, gel filtration on Sephadex LH20.

and those that extract and purify RNA as a whole. The protocols were chosen, according to available data in the literature, as methods in current use for the detection of HAV or other enteroviruses in stool or shellfish specimens. Initially, we evaluated the efficiency of each technique using serial dilutions of a quantified HAV culture supernatant. The results obtained after RT-PCR and PAGE showed that the techniques using magnetic beads (beads with antibody, beads with probe) were the most sensitive and were able to detect 0.13 and 0.025 TCID50/ml, respectively, as compared to 3.2 TCID50/ml for RNAzol and silica and 0.64 TCID50/ml for PEGCTAB. These specific capture systems using antibody-antigen or probe-sequence links are at an advantage in such dilute media, since there are very few interfering substances. The type of physical support, however, also makes a difference. Beads provide the best physical support, and our results are of the same order as those obtained by

Monceyron and Grinde (1994), despite the fact that they used an antibody with a different specificity. Using tubes as a physical support yields results that vary widely between different research teams. While Jansen et al. (1990) and Deng et al. (1994) obtained thresholds of 0.050 pfu and Graff et al. (1993) of 0.006 TCID50 per unit volume of the analyzed sample, the sensitivity observed by Shen et al. (1997) when testing enterovirus culture supernatants was quite low (1 pfu/ml), like our own (3.2 TCID50/ml). Indeed, Shen et al. (1997) recommends the use of beads to increase the area of surface contact between antibody and antigen. Techniques based on purification of RNA (PEG-CTAB, GTC-silica, RNAzol), on the other hand—being designed for complex media—are less sensitive because they involve losses of genomic material that in these simpler media are readily apparent. The next step was to apply the different extraction techniques to fecal specimens obtained dur-

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ing an epidemic of hepatitis A (Apaire-Marchais et al., 1994), and shellfish specimens artificially contaminated under conditions close to those encountered in a natural environment. With the exception of the Chelex procedure for shellfish samples, the extraction methods tested here were compatible with virological testing of natural specimens containing low virus concentrations. The beads with antibody protocol gave good detection results in stool samples (11/12). The grade of intensity yielded by the PCR products was particularly high (seven samples with intensities of grade 3). This technique, therefore, effectively eliminates any inhibitors contained in feces (Monceyron and Grinde, 1994). When applied to shellfish specimens, however, the level of detection fell sharply (six positive samples out of 14). In mussel concentrates, tissue fragments may be adsorbed on to the magnetic beads in a non-specific way and prevent their recovery by the magnets, causing considerable loss of virus. A solution to this problem might perhaps be to clarify the mussel concentrates by centrifugation. The beads with probe protocol yielded disappointing results with these complex specimens, despite the use of a relatively long probe (40 nucleotides) located in a quite well preserved region of the HAV genome (Jansen et al., 1990). This technique, which has been validated for detecting enteroviruses close to HAV (Muir et al., 1993; Beaulieux et al., 1997) only allowed HAV to be detected in 3/12 shellfish and 7/12 stool samples. PEG-CTAB, as for other researchers (Jiang et al., 1992; Hale et al., 1996), was shown to be a very effective extraction method in eliminating inhibitors from fecal samples. It can also be applied to shellfish specimens, with a 92% success rate. CTAB, in fact, makes it possible to eliminate the polysaccharide compounds present in shellfish (Jaykus et al., 1993). The results obtained with RNAzol confirm that this technique can be applied to shellfish samples (Cromeans et al., 1997), for which we observed positive detection in 12/14, and to stool specimens (11/12 positive). Tests using GTC-silica extraction followed detection of HAV were effective both in stool speci-

mens (8/12) and in shellfish samples (12/14). These are methods frequently used for virological testing of waste water samples (Puig et al., 1994; Pallin et al., 1997), serum (Boom et al., 1990), and stool specimens (Green et al., 1993; Hale et al., 1996). Lees et al. (1994) has also used a related protocol based on ground glass in the presence of isothiocyanate buffer to detect enteroviruses in shellfish concentrates. Chelex seemed to be the least useful method. HAV was only detected in five stool samples, and was not detected in any of the shellfish specimens. Hale et al. (1996) showed that this protocol did not allow all the inhibitors found in feces to be eliminated when testing for SRSV. Three extraction procedures, with detection rates of over 90% (PEG-CTAB, RNAzol, GTCsilica), are therefore suitable for testing these two categories of complex samples for HAV. In such samples, the purification of RNA with regard to different inhibitory substances is necessary if a good amplification yield is to be obtained. In shellfish specimens, more particularly, where the concentration of virus is low, the intensity of the signals found in these three procedures depends on the extraction-concentration technique used. Extraction–concentration methods based on glycine or borate give detection rates (intensity grade 2 or 3) that are higher than those using a buffer containing beef extract (average intensity grade 1–2). The inhibitory effect of beef extract with regard to RT-PCR reactions has, in fact, already been described by other authors (Schwab et al., 1995). The choice of an extraction technique that can be used for routine testing of a large number of samples must also take into account simplicity of use (with the possibility of automatization), rapidity of execution, and cost. The seven protocols tested here include between four and nine different stages. The average time required to process a series of 15 specimens varied from 90 min for the beads with probe protocol, to 5 h for the PEGCTAB protocol. The majority of the techniques used—RNAzol, GTC-silica, beads with antibody, and Chelex can be completed within 120–150 min. Costs associated with the most expensive extraction techniques are twice those of the cheap-

C. Arnal et al. / Journal of Virological Methods 77 (1999) 17–26

est. The techniques using magnetic beads (beads with antibody and beads with probe), or requiring many stages (PEG-CTAB), are the most expensive. The protocols based on Chelex or on antigen capture are the most economical. In conclusion, none of the techniques which we studied can be applied to both complex and simple specimens. To test samples that are simple in composition or relatively dilute, such as culture supernatants, techniques based on specific capture of the virus using magnetic beads (beads with probe, beads with antibody) are the most efficient. But for more complex specimens (stool, shellfish), purification of RNA using more classic techniques (PEG-CTAB, RNAzol, GTC-silica) is necessary. RNAzol and GTC-silica are the most suitable for routine diagnostic use, in view of their rapidity and lower cost.

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