JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2004, p. 1605–1608 0095-1137/04/$08.00⫹0 DOI: 10.1128/JCM.42.4.1605–1608.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 42, No. 4
Chromatography Paper Strip Method for Collection, Transportation, and Storage of Rotavirus RNA in Stool Samples Mustafizur Rahman,1,2 Truus Goegebuer,1 Karolien De Leener,1 Piet Maes,1 Jelle Matthijnssens,1 Goutam Podder,2 Tasnim Azim,2 and Marc Van Ranst1,3* Laboratory of Clinical and Epidemiological Virology, Rega Institute for Medical Research, University of Leuven,1 and Department of Laboratory Medicine, University Hospital Gasthuisberg3, Leuven, Belgium, and Laboratory of Virology, ICDDR,B: Centre for Health and Population Research, Dhaka, Bangladesh2 Received 21 November 2002/Returned for modification 3 March 2003/Accepted 9 December 2003
We developed a novel method that uses sodium dodecyl sulfate-EDTA-treated chromatography paper strips to collect unconcentrated fresh stool samples. After the paper strips were stored for 4 months at room temperature, rotavirus RNA could be successfully amplified by using reverse transcriptase PCR. The use of filter paper strips as a specimen support allows (self-)collection of stool samples by untrained persons. Diarrheal stool samples from remote areas can be stored and transported to a central diagnostic laboratory without the need for freezers or special shipping conditions. This convenient and inexpensive rotavirus sample collection system can be of use in epidemiological surveillance studies and vaccine trials. infectious bursal disease virus, hemorrhagic enteritis virus, Enterocytozoon bieneusi, and Plasmodium vivax, etc., have been successfully stored on filter paper for several weeks to years (1, 2, 4, 5, 7, 8, 13, 17, 20, 21, 23). Thus far, the use of filter paper strips for the transportation and storage of RNA from stool samples has not been described. This report details the use of specially formulated chromatography paper strips in the collection, transportation, storage and reverse transcriptase PCR (RT-PCR) of rotavirus RNA from stool samples.
Human group A rotaviruses are the major etiological agent of severe diarrhea in infants and young children worldwide, leading to significant morbidity and mortality. Extensive genetic variation and reassortment of the 11 double-stranded RNA rotaviral genome segments has resulted in the presence of a large spectrum of different rotavirus genotypes in humans and animals (14). In recent years, numerous novel emerging genotypes have been isolated in many countries (6, 9, 11). Currently, considerable research efforts are being devoted to the development of a rotavirus vaccine. Since immunity to rotaviruses is largely homotypic, it is very important to understand the geographical distribution of different rotavirus genotypes. Large vaccine trials involving thousands of volunteers are being conducted in both industrialized and developing countries to investigate the safety and efficacy of the vaccines under different environmental and socioeconomic circumstances (3, 12, 16). Because future rotavirus vaccines will need to be updated regularly to reflect temporal and spatial genotype fluctuations, such vaccine trials are likely to continue for years. In many rotavirus field studies or vaccine trials, stool samples need to be transported to reference laboratories for genetic characterization. Often this involves shipping fecesfilled containers or soiled diapers under cooled and biohazardous conditions. Even the simple acts of sample collection, aliquoting, and freezing might be difficult in field situations in some areas in developing countries where no electricity or skilled laboratory technicians are available. Many investigators have described the use of filter paper for the collection, transportation, and storage of eukaryotic or microbial DNA or RNA from blood samples. The genetic materials of human herpesviruses 6 and 7, human immunodeficiency virus, hepatitis C virus, hepatitis B virus, measles virus,
MATERIALS AND METHODS Preparation of the chromatography paper strips. Whatman-grade 17chr paper (Whatman, Kent, United Kingdom), a 0.92-mm-thick and highly absorbent (870 g of water/m2) pure cellulose chromatography paper with a flow rate of 190 mm/30 min, was cut into strips of 80 by 4 mm. Disposable gloves were used when handling the filter paper strips. The chromatography strips were soaked for 1 min in a solution of 2% (wt/vol) sodium dodecyl sulfate (SDS), 10 mM EDTA, and 60 mM Tris-HCl and allowed to dry overnight. Rotavirus samples. A diarrheal stool sample that was positive for rotavirus antigen by the Premier Rotaclone solid-phase sandwich enzyme immunoassay (Meridian Bioscience, Cincinnati, Ohio) was used for this study. The undiluted feces sample contained 1.14 ⫻ 1010 G1P[8] rotavirus particles per ml of stool, as calculated from a standard curve supplied with the antigen enzyme immunoassay kit. To evaluate the sensitivity of the assay, the stool sample was diluted in saline. The dilutions used were 1:150 (7.6 ⫻ 107 particles/ml; dilution a), 1:1,500 (7.6 ⫻ 106 particles/ml; dilution b), 1:15,000 (7.6 ⫻ 105 particles/ml; dilution c), and 1:150,000 (7.6 ⫻ 104 particles/ml; dilution d). The Meridian enzyme immunoassay with a sensitivity limit of 5.0 ⫻ 105 particles/ml (cutoff optical density [OD] ⫽ 0.100) could detect rotavirus antigen in dilutions a (OD ⫽ 2.696), b (OD ⫽ 1.895), and c (OD ⫽ 0.164), but not in dilution d (OD ⫽ 0.008). A stool sample negative for rotavirus antigen was also included as a negative control. Sample loading on the chromatography paper strips. The pretreated chromatography strips were immersed for 30 s into the different dilutions of the stool sample. Approximately 0.3 ml of diluted stool sample saturated each strip. The strips were allowed to air dry overnight at room temperature. After complete drying, each strip was kept in a separate envelope and stored. Storage of the rotavirus strips. The rotavirus strips were stored under four different temperature conditions: ⫺20°C, 4°C, room temperature (20 to 25°C), and 37°C. RNA extraction and RT-PCR. RNA extraction from the chromatography paper strips was performed at different time intervals (1, 14, 30, 60, and 120 days) after loading of the rotavirus sample onto the strips and storage under different
* Corresponding author. Mailing address: Laboratory of Clinical and Epidemiological Virology, Department of Microbiology and Immunology, Rega Institute for Medical Research, Minderbroedersstraat 10, BE-3000 Leuven, Belgium. Phone: 32-16-347908. Fax: 32-16332131. E-mail:
[email protected]. 1605
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environmental conditions. Half of the strip (160 mm2) was used for the RNA extraction. The filter paper was inserted into an Eppendorf tube with 500 l of ultrapure water and thoroughly squeezed out. An aliquot of 140 l of the squeezed eluate was used for RNA extraction using the QIAamp Viral RNA mini kit (Qiagen/Westburg, Leusden, The Netherlands) according to the manufacturer’s instructions. The extracted RNA was denatured at 97°C for 5 min. RT-PCR was carried out by using the Qiagen OneStep RT-PCR Kit (Qiagen/Westburg) as previously described by Gouvea et al. (10). A 1,062-bp fragment of the VP7 gene was amplified with the forward primer Beg9 (5⬘-GGCTTTAAAAGAGAGAATTT CCGTCTGG-3⬘; nucleotides 1 to 28) and the reverse primer End9 (5⬘-GGTC ACATCATACAATTCTAATCTAAG-3⬘; nucleotides 1062 to 1036). The reaction was carried out with an initial reverse transcription step at 45°C for 30 min, followed by PCR activation at 95°C for 15 min, 35 cycles of amplification (30 s at 94°C, 45 s at 53°C, 1 min at 72°C), and a final extension of 7 min at 72°C in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Foster City, Calif.). PCR products were run on a polyacrylamide gel, stained with ethidium bromide, and visualized under UV light. Nucleotide sequencing. The PCR amplicons were purified with the QIAquick PCR purification kit (Qiagen/Westburg) and sequenced in both directions using the dideoxy-nucleotide chain termination method with the ABI PRISM BigDye Terminator Cycle Sequencing Reaction kit (Applied Biosystems) on an automated sequencer (ABI PRISM 3100). The Beg9 and End9 RT-PCR primers for the VP7 gene were used as sequencing primers. Biosafety test for rotavirus. A biosafety assay was carried out to check if any rotavirus could survive on our SDS-EDTA-treated chromatography paper strips. First, strips were loaded with cell culture-adapted rotavirus SA11 (5 ⫻ 107 particles/ml) and allowed to dry at room temperature for 60 min. The strips were then placed into an Eppendorf tube containing 500 l of Dulbecco’s modified Eagle medium (DMEM) (Invitrogen, Merelbeke, Belgium) supplemented with 100 U of penicillin-streptomycin (Sigma-Aldrich, Bornem, Belgium) and 200 mM L-glutamine (Sigma-Aldrich). The strips were thoroughly squeezed in the medium and the eluate was dialyzed using 3,500-Da Slide-A-Lyzer dialysis cassettes (Pierce Biotechnology, Rockford, Ill.) to remove the cytotoxic SDS. The eluate was inoculated on fetal monkey kidney cells (MA104) as described by Urasawa et al. (18) with a minor modification. In brief, the eluate was first activated for 30 min at 37°C with 1⫻ trypsin-DMEM (100 g/ml; Invitrogen) to cleave the VP4 gene which is necessary for rotavirus activation. The activated eluate was then sonicated for 3 min and incubated on a confluent monolayer of MA104 cells at 37°C. After 60 min the monolayer was washed three times with DMEM and incubated with DMEM supplemented with penicillin-streptomycin (5,000 g/ml) and 200 mM L-glutamine in a humidified incubator with a 5% CO2 environment. Untreated strips loaded with SA11 and SDS-treated strips without SA11 were used as positive and negative controls, respectively. The MA104 cell line was regularly checked for cytopathic effect during seven days. The presence of cytopathic effect indicated the presence of live replicating virus on the strip. The tissue culture supernatant was also tested for rotavirus antigen using the Premier Rotaclone solid phase sandwich enzyme immunoassay (Meridian Bioscience). Cytopathic effects were monitored up to the third passage of the tissue culture supernatant. Biosafety test for pathogenic bacteria. Five different bacterial pathogens commonly found in diarrheal stool samples were chosen: Vibrio cholerae O1, enterotoxigenic E. coli, enteropathogenic E. coli, Salmonella enterica serovar Typhimurium, and Shigella dysenteriae type 1. Approximately 108 CFU of each pathogen/ml (comparable to a turbidity of 3 on the McFarland scale) was prepared in broth culture medium (Trypticase soy broth containing 0.3% yeast extract). Three media were used for culturing the pathogens, TCBS (thiosulfate citrate bile salts sucrose agar) for V. cholerae, MacConkey agar for E. coli, and SS agar (Salmonella-Shigella agar) for S. enterica serovar Typhimurium and S. dysenteriae. SDS-treated strips were used for checking if any of those pathogens could survive on the strips. The presence of bacteria was observed by growing them on selective media by using standard microbiological methods (22). Briefly, the SDS-treated strips were immersed into the tubes containing different bacterial pathogens in broth culture medium for 30 s. They were allowed to dry at room temperature for 90 min and were incubated overnight in broth culture medium at 37°C to facilitate the growth of each pathogen if present on the strips. After overnight incubation they were transferred to selective culture medium plates. The plates were incubated in 37°C and were observed three days for the presence of characteristic colonies. Untreated strips loaded with pathogens were used as positive controls. An untreated strip without pathogen was also included as a negative control. The presence of characteristic colonies in the culture plates indicated the presence of live bacteria on the strips.
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RESULTS AND DISCUSSION We describe the development of a novel method that allows the collection, transport, and storage of rotavirus samples on a solid chromatography paper support. The chromatography strips were pretreated with SDS and EDTA. The strips contained SDS, a surfactant with protein denaturing ability, in order to inactivate the rotaviruses and other microorganisms upon contact with the chromatography strip. The loss of microbial infectivity allows transport of the strips without extensive biohazard precautions. Since viral RNA is sensitive to rapid degradation by endogenous ribonucleases (RNase), we also preincubated the chromatography strips with EDTA, a strong chelating agent, and with Tris-HCl, a weak organic base that ensures the proper action of the chelating agents in binding the divalent cations that are necessary for nuclease activity. A diarrheal stool sample containing 1.14 ⫻ 1010 rotavirus particles per ml was serially diluted 1:150 (dilution a), 1:1,500 (dilution b), 1:15,000 (dilution c), and 1:150,000 (dilution d). An RT-PCR that amplified a 1,062-bp fragment in the VP7 rotavirus outer capsid gene worked successfully on all four tested feces dilutions, including the highest 1:150,000 dilution (7.6 ⫻ 104 rotavirus particles/ml). The commercial Meridian antigen ELISA kit, with a detection limit of 5.0 ⫻ 105 particles/ml failed to detect rotavirus antigen in this dilution. The SDS-EDTA-pretreated chromatography filter paper strips were inserted into the four feces dilutions and stored at ⫺20°C, 4°C, room temperature (20 to 25°C), and 37°C. RTPCR analysis was carried out after storage of the sample for 1 day, 14 days, 30 days, 60 days and 120 days (Fig. 1). The presence of amplifiable rotaviral RNA on the chromatography strips was inversely correlated to the duration of storage and the storage temperature. At ⫺20°C, rotaviral RNA could be detected by RT-PCR in the 1:150,000 dilution after 120 days. At 4°C and at room temperature, the 1:150,000 dilution was detectable up to 60 days. At 37°C, only the 1:150 dilution remained detectable after 60 days. These data are in agreement with similar results demonstrating the maintenance of mammalian and plant RNA on filter paper for 2 to 3 months (15). Vilcek et al. (19) reported no difference in the yield of PCR products of bovine diarrheal virus after storage of dried blood samples at ⫺20°C, 4°C, and room temperature after 180 days of storage. Our findings indicate that rotaviral RNA will be stable for a sufficient amount of time at room temperature or even in warmer climatic conditions to allow transportation of the samples from a field site to a reference laboratory using regular postal mail. Further long-term storage of rotaviral strips should preferably occur at ⫺20°C. The PCR product from the chromatography strip that had been stored for 120 days at ⫺20°C was successfully sequenced. The nucleotide sequence of the VP7 gene was identical to the G1 sequence of the original stool sample, indicating that samples stored on the SDS-EDTA-pretreated chromatography filter paper strips can be used for further genetic characterization. Using the chromatography paper strips for transport of rotavirus samples could especially be of interest for investigators and vaccine companies that are planning large field trials in areas with elevated temperatures where electricity or freezers
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FIG. 1. Agarose gel electrophoresis of RT-PCR products of rotaviral RNA extracted from chromatographic paper strips that had been stored for various lengths of time at four different temperatures. Four tenfold dilutions of a titrated feces sample were used. Lane a, 1:150 (7.6 ⫻ 107 particles/ml); lane b, 1:1,500 (7.6 ⫻ 106 particles/ml); lane c, 1:15,000 (7.6 ⫻ 105 particles/ml); lane d, 1:150,000 (7.6 ⫻ 104 particles/ml).
are not available. Cooled transportation to a reference laboratory can be avoided as the rotaviral RNA was shown to be stable for several weeks at room temperature as well as at 37°C. We performed biosafety tests to find out if any virus or bacteria could survive on our SDS-treated strips. We found that pathogenic bacteria such as V. cholerae O1, enterotoxigenic E. coli, enteropathogenic E. coli, S. enterica serovar Typhimurium, and S. dysenteriae type 1 could survive on the paper strips without SDS-EDTA but could not survive on the SDS-EDTA-treated strips. No bacterial colonies were detected on the culture plates that were inoculated with the SDSEDTA-treated strips loaded with pathogenic bacteria. We detected colonies in the culture plates inoculated with the untreated pathogen-loaded strips (positive controls). We also proved that rotavirus could not survive on the strips. In the MA104 cell line no cytopathic effect was observed for the strips loaded with rotavirus SA11. The untreated strips loaded with SA11 rotavirus showed cytopathic effect. We also performed a rotavirus-specific ELISA to confirm the presence or absence of rotavirus antigen in tissue culture supernatant. The tissue culture supernatant corresponding to the untreated strip gave a positive rotavirus ELISA test, whereas no rotavirus antigen was detected in tissue culture supernatant inoculated with SDS-EDTA-treated strips. It can be concluded from the biosafety tests that the strips can be used for the collection of stool samples in a safe way. For the preparation of the strips, minute amounts of inexpensive ingredients (SDS, Tris-HCl, and EDTA) are used. The transportation of the strips loaded with samples can be achieved by regular postal services. Because no cooling apparatus is necessary, this also lessens the costs of storage and transportation of the samples. In conclusion, the SDS-EDTA-pretreated chromatography filter paper strips are a convenient, safe, and inexpensive method to collect, store, and transport rotavirus samples for further genetic characterization. It is likely that this technique will also be applicable to other enteric viral agents such as enteroviruses, adenoviruses, astroviruses. and Norwalk-like caliciviruses.
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