Two-hundnd and twenty stooi specimens were collected from infants and young childrenwithdianfum, md l&W6 faecal suspensions were made. Sever81 methods of rotwkurda8NAeWacUonwomaesayed.Electr@uxeticanaIyakofviralRNAwascarriedouton 10% wi gals followed by sikver stahing. RTPCR was performed using oliiuckoti& primers specifk for both 3' and 5' ends of the rotavirus gene encodingVP7whichuehighlyamaerk& among goup A rotaviruses.
Fobwing RNA extracth whh phenol-and ethanol precipitatiin, RT/PCR could~rabvinlePNAkronlyllof25~knormtoco~nratavinr~by comwrtwwul~~~nofR)JA*nblrcbbyCFll~~mdtheappli-cationoftheMAlDmMhodvrrnnequ&lyeReMveinextmcWgMUAand/orremoving inhibitory#rbdrro#fmm~forulsampks.RTIPCf?ledtothedetecWnof6cposi-tivesat@esfmmim ~tested(30%6),whilst64specimenswerepo&iveby EUSA U9%), 58 @M%) by PWE ad 56 (25.1%) by EM. In our study, RT/PCR was 100tiAnr~~thn~EUSAt~in~rotrrir~~rlly~in afwcJYllYMLYlAkhOUI(IWfCRitttwatWy much more sensitive than EUSA, PAOEwwlEMfor~drotrvinrsgt,~ursmwtbe~~to~einhibi-torysulBtmufromtbe~Nactions.wedonot~tbatRT/Pa?shoukl replace~wfthhlghsaMvityandspacWtyforrotavirustestinginfae---WhrlthoyhtNsWkrS-.W Hcethesearchforrotavirus indiRemMdkMrpldrrmrtwncc#sbrorpmd'Ruii,rapimtqsecm&ns,etc.)and in~rmgkr.aswellasthetypkgafviralstrainsusing~ific primcm. Key-words: Rotavirus, RT/PCR; ELISA, PAGE, Electron microscopy, Viral analysis, Sensitivity. Submitted June 18, 1996 , accepted August 14, 1996 (*) Corresponding author.
Group A rotaviruses cause an estimated 140 million cases of gastroenteritis in infants and children and one million deaths worldwide each year (Kapikian et aZ., 1986; Glass et al, 1994) . Rapid diagnosis of this infection is of great value in the formulation of the prognosis for children with acute diarrhoea and in decisions concerning management and control measures. Since the cultivation of rotaviruses from clinical specimens is difficult to achieve, the most widely used diagnostic techniques for rotavirus detection in faecal samples include immunoassays such as ELISA and latex agglutination, electron microscopy (EM) and immune electron microscopy, and polyacrylamide gel electrophoresis (PAGE) of viral double-stranded RNA (Kapikian and Chaneck, 1996) . Nucleic acid hybridization techniques in the format of "dot blots" have also been applied to the detection of rotaviral RNA @'lores et al., 1983) . However, RNA detection assays have generally not proven to be substantially more sensitive than optimized immunoassay procedures (Wilde et aZ., 1990) .
The reverse transcription/polymerase chain reaction (RT/PCR) should enable the detection of rotaviruses at concentrations substantially lower (1,000 times less) than those required for detection by means of immunoassays and electron microscopic studies (Wilde et al., 199 1) . RTZPCR has been applied by several authors to the detection of rotaviral RNA (Gouvea et al., 1990 ; Wilde et aZ., 1990 Wilde et aZ., , 1991 Xu et al., 1990; Ushijima et aZ., 1992 , Husain et aZ., 1995 and for typing virus strains, characterizing both G and P serotypes (Gouvea et al., 1990; Ushijima et al., 1992 ; Gentsch et aZ., 1992) .
We have evaluated the application of RTZPCR in the detection of group A rotavirus RNA in faecal specimens as a routine diagnostic procedure compared to the enzyme-linked immunosorbent assay (ELISA) currently employed in our laboratory, and to EM and PAGE. Special efforts have been made in the search for an efficient, simple and fast procedure to extract viral nucleic acids and to avoid interference with the enzymatic reactions by inhibitory substances present in faecal samples.
During the period October 1994 to March 1996, two-hundred and twenty faecal specimens were obtained from infants and young children with signs of gastroenteritis who were followed at the Pediatric Departments of the Hospital Clinico Universitario of Valencia, Spain, and at the Hospital of Sagunto, Spain. Faecal suspensions (lo-20%) were made in Tris-NaCl-CaCl, buffer (0.05 M Tris-HCl pH 7.5, 0.15 M sodium chloride, 0.01 M calcium chloride) and stored at -70°C. Samples were assayed by ELISA for rotavirus antigens, by EM for viral particles and by PAGE and RT/PCR for rotavirus RNA.
ELISA for the detection of rotavirus antigens Solid phase enzyme immunoassays were carried out on all samples. The "Rotaclone" rotavirus diagnostic kit (Cambridge Biotech, Worcester, MA, USA) was applied to detect rotavirus antigens in faecal specimens following the procedure described by the manufacturer. Results were read spectrophotometrically at 450 nm. The Rotaclone test utilizes a monoclonal antibody directed against the rotavirus VP6 protein in a solid-phase sandwich-type ELISA. According to the manufacturer, the sensitivity limit of the test is 5 x lo5 viral particles per ml.
Previously prepared lo-20 % faecal suspensions were centrifuged at 50,000 rpm for 90 min (LS-70M negatively stained with 2% phosphotungstic acid, pH 4.5, as described by Nakata et al. (1987) . The preparations were examined under an electron microscope (Zeiss EMlOC/CR, voltage 60 KV) at a magnification of 31,500x. Samples were considered negative when no virus particles were found during 15 min of observation.
The following methods to extract viral dsRNA from faecal suspensions were assayed: (i) phenolchloroform extraction and ethanol precipitation ; (ii) "RNAID" method (Bio 101, Inc., La Jolla, CA, USA); (iii) "GeneReleaser protocol (BioVentures, Inc., Murfreesboro, TN, USA); and (iv) phenolchloroform extraction and purification through CFl 1 cellulose.
Rotavirus dsRNA was extracted from stool suspensions by a modification of the procedure originally described by Herring et al. (1982) and commonly used in our laboratory for subsequent RNA electrophoresis in polyacrylamide gels (PAGE). Briefly, 400~pl stool suspensions were adjusted to contain 1% SDS and 0.1 M sodium acetate (pH 5.0), and incubated for 15 min at 37°C. The suspension was extracted with phenol-chloroform (1: 1). The RNA in the aqueous phase was ethanol-precipitated at -70°C and dissolved in 50 p.l of water.
The RNAID kit (Bio 101, Inc., La Jolla, CA, USA) was also applied to extract viral dsRNA from stool suspensions following the instructions of the manufacturer with the modifications described by Gentsch et al. (1992) . Briefly, 250 pl of 6 M guanidinium thiocyanate (FIuka) were mixed with an equal volume of stool suspension. A volume of 12 pl of the RNAID glass powder was added and the mixture was vortexed and rotated at room temperature for 10 min. After centrifugation for 30 s at 650 g in a Beckman microcentrifuge, the supernatant was removed, the pellet washed twice with 400 p.l of the RNAID kit wash buffer and recentrifuged at 850 g. After a third wash in the same buffer, the sample was finally centrifuged at 10,000 g for 60 s, dried under vaccum, redissolved in 25 pl of deionized water and incubated for 10 min at 65°C. The samples were centrifuged at 10,000 g for 60 s, and the supernatants stored at -2O'C until use for the reverse transcriptase reaction and PCR. Just before use, the samples were heated at 56°C for 5 min and centrifuged at 10 000 g for 10 s to pellet any residual RNAID matrix from the sample.
The Gene Releaser protocol (BioVentures Inc., Murfreesboro, TN, USA) was also tested. This commercial polymeric matrix was originally described to release DNA from heterogeneous biological samples and sequester cell lysis products which might inhibit polymerases. Levy et al. (1994) have described the use of this method for PCR and RT/PCR detection and identification of viruses and viroids in plant tissue extracts. Both thermocycler and microwave protocols for sample preparation described by the manufacturer were assayed. In the microwave procedure, 2-5 pl of faecal suspension was placed in a 0.5~ml thermocycling tube together with 16-19 ~1 of GeneReleaser matrix, yielding a total volume of 21 pl. The mixture was vortexed for 30 s and the tube was placed in a microwave oven and heated at the maximal power setting for 5 min. The tube was then heated in a pm-warmed thermocycler at 90°C incubated on ice for 5 min and then microfuged at maximal speed at 4°C. An aliquot of the supematant was used to perform RT/PCR reactions as described in this paper.
RNAs extracted from faecal samples with phenol-chloroform were purified using CFll cellulose fibre powder as described by Wilde et al. (1990) . After phenoI&loroform/isoamyl alcohol extraction (25:24: l), the aqueous phase was mixed with 95 % ethanol to bring the final solution to 15% ethanol (vol/vol). To this mixture, 30 mg of CFll cellulose (fibrous medium cellulose, Sigma) was added, vortexed and rotated for 90 min at 4°C. After centrifugation at 6 000 g for 60 s, the supernatant was removed and the pellet washed three times with STE buffer (O.lM NaCl, O.OOlM EDTA, 0.5M Tris-HCl pH 7.0) containing 15% ethanol (vol/vol). After a final wash in STE without ethanol to elute the RNA from the cellulose, the sample was centrifuged for 5 min to pellet the CFll cellulose. RNA in the supematant was precipitated with ethanol in the presence of 0.3 M sodium acetate pH 5.2. The RNA pellet was redissolved in 50 p1 of deionized water and stored at -20°C.
In the preliminary phase of our study, viral RNA was extracted by the methods noted above from 25 faecal samples which were previously found to be positive for rotaviruses by ELISA, PAGE and EM.
The PAGE technique for rotavirus RNA detection and analysis was performed on 10 % polyacrylamide gel slabs lacking SDS and silver staining as previously described (Herring et al., 1982 ; Buesa et al., 1987) .
The oligonucleotide primers used, Beg9 and End9, were described previously (Gouvea et al., 1990) . These primers are specific for the gene cod-ing for VP7 which may be either RNA segment 7,8 or 9, depending on the viral strain. They produce full-length copies of the gene from any group A rotavirus strain.
The following RT/PCR method was used: 1 pl of dimethyl sulphoxide was added to 9 pl of the extracted RNA and heated at 97°C for 5 min to denature nucleic acids, cooled on ice, and the reaction buffer (6 pl 5 x AMV buffer [Promega]), 0.25 ul RNasin @omega), 3 p.l of 10 mM solutions of each of the deoxynucleoside triphosphates (dATP, dCTP, dGTP and dTTP), 2.5 pM of each primer, Beg9 and End9, and 10 units AMV reverse transcriptase (Promega) were added; reactions were incubated at room temperature for 10 min and subsequently at 42°C for 35 min. ending with 92°C for 2 min to inactivate the revem transcriptase.
The cDNA amplification reactions were carried out in 50 p.1 1 x PCR buffer (10 mM Tris-HCl pH 8.8) containing 1 pl of the RT reaction, 1.5mM MgCl , 50mM KCl, 0.1% Triton X-100, 2 mM of each c&HP, 1 pM each primer and 2 units of "Dynazyme" DNA polymerase (Finnzymes Oy, Finland). Reaction mixtures were overlaid with 50 pl of light mineral oil (Sigma) and PCR was conducted in a thermal cycler (GenE, Techne) for 40 cycles (94°C for 1 min, 47°C for 2 min and 72°C for 2 miu) with a final 7-miu incubation at 72°C. Negative and positive controls were included in every RT/PCR assay. The positive control was viral RNA from SAl 1 rotavirus cultivated in MA-104 cells and extracted by the method of Herriug er al. (1982) . PCR products were analysed by electrophoresis on 1.2 % agarose gels with ethidium bromide at 100 V for 30 min and photographed under UV light.
The sensitivity of the RT/PCR in detecting rotaviruses was determined by using ten-fold serial dilutions in a negative faecal suspension of a SAl 1 rotavirus preparation containing 5 x 10 7 PFU/ml, as titrated by plaque assay. RNA was extracted by the RNAID method and RT/PCR was done as previously described.
In a preliminary set of assays, 25 faecal samples previously found to be rotavirus-positive by ELISA, PAGE and EM were tested using differ-ent methods of RNA extraction in order to determine the most reliable and efficient procedure. The results of these assays are shown in table I. Phenol-chloroform extraction followed by binding of RNA to CFl 1 cellulose in ST"&ethanol and elution with STE, as described by Wilde et al. (1990) , was an efficient method to recover rotaviral RNA from stool samples free of inhibitory activity. All 25 samples analysed by RT/FCR were positive when phenol-extracted and purified by CFl 1 cellulose, whereas only 11 (44%) were positive when purification with CFll was omitted. Equally effective appeared to be the RNAID method, which also allowed the detection of viral RNA in all 25 samples tested. However, the GeneReleaser method was, in our experience, inefficient in purifying rotaviral RNA or removing inhibitors from the faecal samples.
Having established the efficacy of both CFll cellulose purification and the RNAlD method in extracting viral RNA and/or removing inhibitory substances from faecal samples, we used the RNAID me&d with all the specimens tested.
Sensitivity of the RT/PCR assay The limit of detection of the RWPCR using an SAl 1 rotavirus suspension serially diluted in a 10% faecal extract was 5 x lo3 PFU/ml ( fig. 1 ). In comparison, the ELISA test used in this study could detect 5 x lo5 PFU/ml. The results obtained from analyses by electron microscopy, ELISA, PAGE and RT/PCR are presented in table II. Sixty-eight samples (30.9%) were found to be positive by one or more of the techniques applied. In two specimens, the result of RT/PCR was negative, whereas the ELISA test showed positive results. These were considered to be false-negative PCR results. Conversely, four samples were positive by RT/l?CR but negative by ELISA.
Rotavirus particles were detected by EM in 56 specimens (25.4%). Other viruses were also detected among the 220 faecal samples studied, mainly adenovirus (8.1%), astrovirus (2.2 %), calicivirus (1.8%) and coronavirus-like particles (1.3%).
PAGE analysis was positive with 59 faecal samples (26.8 %). Most rotavirus strains showed "long" electrophoresis pattern, with the exception of 5 strains (8.4%) which were found to have "short" electrophoretypes. Ten different electrophoretypes were detected during the course of this study (results not shown).
PCR amplification products were easily detected by agarose gel electrophoresis (figs. 2 and 3). A clear and distinctive fragment of about are RT/PCR products from faecal samples extracted with phenol-chloroform and purified using CFll cellulose. Lanes 3 and 4 are from faecal samples extracted with the RNAID method as described in "Materials and Methods". Lanes 5 and 6 are positive (RNA from simian rotavirus SAll) and negative controls, respectively. K = 1-kb DNA ladder. The oligonucleotide primers used are specific for the viral gene encoding VP7 from any group A rotavirus strain.
1 kb was consi&mxl to he the product of a specific anplikdon of the rotavirus gene 9 (or gene 8).
Many authors have applied RT/PCR to the detection of rotaviruses in clinical specimens (Gouvea et tad., 1990; Wilde et aZ., 1990; Xu et al., 1990 ; Ushijima et al., 1992 ; Husain et al., 1995) . Moreover, direct "serotyping" of rotavirus strains (VW, G types ; VP4, P types) is feasible by means of RT/PCR using type-specific primers derived from distinct regions of the genes (Gouvea et al., 1990; Ushijima et al., 1992 ; Gentsch et al., 1992) . However, not many surveys have been performed with the purpose of making a comparison between RT/PCR and other methods currently in use in diagnostic laboratories for the detection of rotaviruses in stool specimens.
We have taken advantage of the previous work performed by Gouvea et al. (1990), and Ushijima et al. (1992) to establish the optimal conditions (MgC12, dimethyl sulphoxide and template RNA concentrations) for the RT/PCR procedure. Our first attempts to amplify rotavirus RNA from faecal extracts were sometimes hampered by an apparent inhibition of the reactions. This faecal inhibition of the RT/PCR has been reported by others (Wilde et al., 1990 ) and may constitute a significant limitation on the detection of RNA viruses in faecal specimens. A variety of extraction procedures have been proposed to remove inhibitory substances from faecal extracts as well Fii. 3. Agarose gel electrophoresis of rotavirus RTPCR products from stool specimens. Viral RNAs were extracted by the RNAID method. M= I-kb DNA ladder; lane 1 =positive control (RNA from simian rotavirus SAI 1) ; lanes 2 to 9=RT/PC!R products obtained from faecal samples; lane lO=negative control. A clear and distinctive band of ahout 1 kb was considered to be the product of a specific amplification of rotavirus dsRNA. as from environmental samples (Le Guyader et al., 1994) . Our results suggest that both CFll purification and the RNAID procedure for extracting viral RNA may be used prior to performing RTKR.
According to our data, RTBCR is not substantially more sensitive for detecting rotavirus than other techniques such as the ELBA test used in our study, in agreement with the results obtained by Gouvea et al. (1990) and Ushijima et al. (1992) . The use of a double amplification system, nested or semi-nested PCR, has been reported by some authors to improve the sensitivity of rotavirus detection (Gouvea et al., 1990 ; Gentsch et al., 1992 ; Le Guyader et al., 1994) , though others did not observe an increase in sensitivity using these procedures (Ushijima et az., 1992) .
It has been reported that the RlYPCR assay can be approximately 100 times (Husain et aI., 1995) and even 1,000 times (Wilde et al., 1990 ) more sensitive than ELISA tests, as determined by serial dilutions of faecal specimens. Although this increase in sensitivity may be feasible, in practice it may be very occult to achieve. In our study, four samples were positive by RT/PCR but negative by ELBA, probably due to the higher sensitivity of the fust technique. Moreover, great care must be taken to avoid i~ibi~~ substances in the enzymatic reactions. PAGE can detect as little as 10 ng rotavirus RNA (Herring et al., 1982) and in aur hands, appears to be a very reproducible technique. Gouvea et al. (1990) reported the requirement of at least 2 ng (lo* cop ies) of viral RNA in the faecal specimen in order to be detected as rotavirus-positive by RT/ER. Xu et al. (1990) were able to detect 8 x lo3 virus particles by RT/PCR under experimental conditions. However, we do not consider that RT/FCR should replace the more conveutional immunoassays in the diagnosis of rotavirus infections performed in clinical virology laboratories. Obviously, RTECR is a very sensitive and specific assay, but it is more expensive due to the costs of enzymes and more time-consuming, and the results are obtained later than with other techniques such as immunor~ys and/or EM. Both G and P typing of rotavirus strains with specific primers is a main application of RWFCR (Gouvea et ab, 1990; Ushijima et & 1992; Gentsch et d, 1992) as are other studies on the genomic variability of these viruses (Wnsover et d, 1993) .
RT/PCR has been applied to the detection of rotaviral RNA not only in faecal samples but also in other types of clinical specimens (throat swabs, sera and cerebrospinal fluids) with some intriguing results. Ushijima et al. (1994) reported the detection by RT/PCR of human rotavirus RNA in throat swabs from seven children with rotavirus gastroenteritis and in CSF from seven other patients with gastoenteritis and convulsions, as well as in two sera samples. Such studies will lead to a better understanding of the pathogenesis of rotavirus infections and may confirm the detection of rotavirus antigens in respiratory secretions reported by others (Fragosa et al., 1986; Zheng et al, 1991) . In addition, RT/PCR is also a powerful technique to further investigate the mechanisms of transmission of these viruses and their presence in the environment (Wilde et ul., 1992; Le Guyader et d., 1994) .
EM continues to be a mainstay in the diagnosis of rotaviral diseases and has been used in the past as the final arbiter when discrepancies occurred with other techniques (Kapikian and Chanock, 1996) . It remains a very useful tool, especially in the detection of unexpected viral agents for which suitable probes may not be available with immunological or nucleic acidbased techniques. We wish to thank to A.P. MacCabe for critical reading of the manuscript.
lbhu~tion de la RTD'CR pour la ddteetloa des rotavirus: applications de cette technique La RT/PCR a Cd Cvalute pour la detection des rotavirus dans les selles des enfants ayant une infection due B ces virus. Les rkwltats ont ttC cornpar& B ceux de la technique ELISA, de la microscopic &ctronique (ME) et de l'klectrophorbse de 1'ARN viral bicatknaire en gel de polyacrylamide (PAGE).
Nous awns pn5pax~5 des suspensions f&ales g lo-20% en Tris-NaCl-CaClz g partir de 220 Cchantillons provenant d'enfants souffrant de gastroentdrite aiguZ et utilisd diffkrentes mkthodes d'extraction de I'ARN viral. Dans la RT/PCR, des oligonucl&ides spkcifiques des extr&nids 3' et 5' du g&e codant la glycoprotiine VP7 ont Ctk utilids. La purification des extraits d' ARN avec la CFl 1 cellulose et le mkthode "RNAID" ont CtB kgalement effkaces pour l'obtention de 1'ARN viral.
L'amplification par la RT/PCR a permis de dktecter 66 Cchantillons positifs (30 %), pendant que 64 ont ttC positifs par I'ELISA (29%), 59 (26.8%) par l'klectropho&se de 1'ARN viral et 56 (25.4%) par la ME.
La RT/PCR est plus sensible que I'ELISA, la PAGE et la ME pour la d&e&on des rotavirus. Cependant il faut veiller & Climiner les possibles inhibiteurs des rtactions enzymatiques. Malgrt cette efficacitb, nous consid&ons que la RT/PCR ne doit pas remplacer les techniques immunoenzymatiques ayant une grande sensibilitd et une haute spkifkiti pour la d&e&on des rotavirus dans les selles. Mais cette technique est utilisable pour la recherche des rotavirus dans d'autres prCl&vements (s&urn, LCR, s&r&ions respiratoires etc.) ou dans des &hantillons provenant de l'environnement; elle peut, de plus, permettre de caractb riser les strotypes viraux.
Mots-clts : Rotavirus, RT/PCR ; ELISA, PAGE, Microscopic klectronique, Analyse virale, Sensibilitt.