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1 181 Appendix 21 Evaluation of RT-PCR procedures for diagnosis of clinical samples of foot-and-mouth disease virus (serotypes O, A, C and Asia 1) under the European Union Concerted Action Group Project Pl Scott M. Reid, Geoffrey H. Hutchings and Nigel P. Ferris Institute for Animal Health, Pirbright laboratory, Ash Road, Pirbright, Woking, Surrey, GU24 ONF, UK Summary A collaborative study initiated from the first annual European Union (EU) Concerted Action Group meeting (project PL , 5-7 May 1999, Federal Research Centre for Virus Diseases of Animals, Tübingen, Germany) was organised between the Veterinary and Agrochemical Research Centre in Brussels, the Federal Research Centre in Tübingen and the OIE/FAO World Reference Laboratory for Foot-and-Mouth Disease (WRL for FMD), Pirbright. Each laboratory was supplied with 40 epithelium samples of FMD virus positive clinical material (ten each of the serotypes O, A, C and Asia 1) and the supernatant fluids resulting from their passage in cell culture to evaluate the sensitivity and specificity of locally employed RT-PCR procedures. The 40 samples were supplied unlabelled with respect to FMD virus serotype. In the WRL, FMD virus was detected (but not serotyped) in 36 of the 40 epithelial suspensions and in 39 of the 40 cell culture supernatant fluids by a diagnostic RT-PCR protocol using a universal O/A/C/Asia 1 primer set (1F/1R). These primers also detected 36 of the 40 epithelial suspensions in a prototype antigen capture (immunocapture) RT-PCR but variable results were achieved in a diagnostic RT- PCR using serotype-specific primers. The latter primers were sensitive on the FMD virus type Asia 1 samples but less sensitive on virus samples of the other serotypes. The sensitivities of the universal O/A/C/Asia 1 primer set and the serotype-specific primers in the diagnostic RT-PCR mirrored results previously achieved on other virus samples while the antigen capture format was suitable for diagnosis of FMD virus and could be further developed for serotype-specific detection. FMD virus in all of the cell culture supernatants was detected by a universal primer/probe set for FMD using a quantitative PCR machine. The results will be compared with those from the other laboratories to evaluate and improve existing PCR protocols designed for FMD virus diagnosis. Keywords: RT-PCR, FMDV serotyping, Diagnosis, Antigen capture, Immunocapture Introduction Diagnosis of foot-and-mouth disease (FMD) virus by reverse transcription polymerase chain reaction (RT-PCR) procedures have been widely documented and have involved universal primers for the detection of all seven serotypes of the virus (Amaral-Doel et al., 1993) and specific primers for the detection of each serotype (Vangrysperre and De Clercq, 1996: Callens

2 182 and De Clercq, 1997). Although these published RT-PCR methods, were successful in the detection or serotype-specific diagnosis of FMD virus, none have been extensively evaluated on enough clinical samples to cover the sequence variation within each serotype and their suitability for primary diagnosis of FMD virus using epithelial suspensions (ES) or original vesicular fluids of clinical samples have not been fully determined. This was addressed in three studies by Reid et al. (1998; 1999; 2000, in press) at the OIE/FAO World Reference Laboratory for Foot-and- Mouth disease (WRL for FMD), Pirbright where universal or serotype-specific primer sets were used in RT-PCR procedures on ES and cell culture supernatant fluids prepared from large panels of positive FMD virus specimens of each serotype. The study carried out by Reid et al. (1999) had used the same specific primer sets as had been used in the earlier work of Vangysperre and De Clercq (1996) and Callens and De Clercq (1997). However, the RT-PCR protocols used at the WRL differed from those in the earlier work in that significantly lower concentrations of primer were used which could have accounted for differences in the performance of the RT-PCR procedures between the laboratories. The higher concentrations of primer used in the earlier studies would not have been practical at the WRL given the number of RT-PCR=s routinely performed there. In order to achieve a higher sensitivity for diagnosis of FMD virus by RT-PCR, alternative formats have been developed such as the nested PCR format of Moss and Haas (1999) and PCR-ELISA (Callens and De Clercq, 1999). These formats have a high sensitivity for the diagnosis of field samples but also have a high risk of contamination and are expensive to carry out. Many laboratories use RT-PCR procedures for the diagnosis or serotyping of FMD virus but the protocols are not standardised and the inter-laboratory variation has not been investigated. A collaborative study initiated from the first annual European Union (EU) Concerted Action Group meeting (project PL , 5-7 May 1999, Federal Research Centre for Virus Diseases of Animals [BFAV], Tübingen, Germany) was organised between the Veterinary and Agrochemical Research Centre in Brussels, BFAV Tübingen and the OIE/FAO World Reference Laboratory for Foot-and-Mouth Disease (WRL for FMD), Pirbright. This blind trial would provide an assessment of the sensitivity and specificity of the RT-PCR procedures employed by the laboratories within the EU and should lead to improvements in the diagnosis of FMD virus by RT-PCR. The results from the RT-PCR procedures on the clinical samples obtained at the WRL for FMD are presented and discussed here. Materials and methods Preparation of the unlabelled samples for the blind trial in the collaborating laboratories Ten epithelium samples each of the FMD virus serotypes O, A, C and Asia 1 were chosen from the WRL reference bank of positive submitted specimens along with the supernatant fluids prepared from the propagation of epithelial suspensions (ES) of these samples in cell culture at the time of receipt (and stored in PBS/glycerol at C since then). Fresh cell culture supernatant fluids were prepared in calf thyroid and/or IB-RS-2 cell culture for a minority of the 40 samples

3 183 where the original cell culture supernatants were not available. These were aliquotted for distribution to the collaborating laboratories (including the WRL for FMD) but were not labelled in terms of the strain or serotype of the sample. At the WRL for FMD, suspensions were prepared from the epithelium tissues in phosphate buffer (Ferris and Dawson, 1988) and stored at C until use. An ES was similarly prepared from bovine epithelial tissue from a non-infected animal to act as a negative control. The identity of the samples was not revealed until all the testing had been carried out so that a truly blind trial was performed. RNA extraction and reverse transcription Total RNA was extracted from the samples, negative control ES and non-infected cell culture with TRIzol reagent 7 (Gibco Life Technologies; Simms et al., 1993) according to the manufacturer=s protocol. Five Φl of RNA was subjected to reverse transcription by following the longer of the two protocols (final reaction volume of 20 Φl) as previously described (Reid et al., 1999). Synthetic oligonucleotide primers and RT-PCR The universal O/A/C/Asia 1 primer set (1F/1R) was designed for the intended diagnosis of all seven FMD virus serotypes. In a previous evaluation this set had been found to be sensitive in RT-PCR for the primary diagnosis of the serotypes O, A, C and Asia 1 but could also detect the SAT serotypes (Reid et al., 2000, in press). The specific were designed for the diagnosis of the serotypes O, A, C and Asia 1 (Vangrysperre and De Clercq, 1996; Callens and De Clercq, 1997) and were evaluated in RT-PCR procedures (Reid et al., 1999; Reid et al., 2000, in press). A forward primer, reverse primer and probe were also designed from conserved sequences of the FMD viral genome for use in a quantitative PCR machine. The RT product from each sample was tested with the 1F/1R primer pair as before in a diagnostic RT-PCR procedure (Reid et al., 2000, in press). Similarly, the RT product from each sample was tested with a cocktail of the specific in RT-PCR procedures as before (Reid et al., 1999) using both thermocycler amplification programmes, namely : (1) 94 0 C for 5 min, 1 cycle; 94 0 C for 1min, 58 0 C for 1 min, 72 0 C for 2 min, 20 cycles; 72 0 C for 7 min, 1 cycle and (2) 94 0 C for 5 min, 1 cycle; 94 0 C for 1min, 59 0 C for 1 min, 72 0 C for 2 min, 20 cycles; 72 0 C for 7 min, 1 cycle. Antigen capture RT-PCR (Immunocapture RT-PCR) Each sample was tested in a prototype antigen capture RT-PCR method for FMD virus diagnosis involving PCR amplification with the 1F/1R primer set after the samples had been captured in a microtitre plate well coated with a cocktail of guinea pig anti-fmd immune sera against all seven serotypes. A prototype antigen capture RT-PCR for serotype-specific diagnosis was also used to test ten of the epithelial suspensions after the samples were added to wells coated

4 184 individually with serotype-specific guinea pig anti-fmd immune serum and the specific primer sets P33/P38, P33/P87-P92, P33/P40 and P33/P74-P77 (for the diagnosis of serotypes O, A, C and Asia 1 respectively) were used in separate PCR amplifications so that the serotype specificity of the primer matched that of the coating antibody. The procedures for the antigen capture RT- PCR are given elsewhere in these proceedings. TaqMan 7 RT-PCR All of the cell culture supernatant fluids were tested in a prototype TaqMan 7 RT-PCR in a GeneAmp thermocycler machine (PE Biosystems, UK) which can be used for quantitative studies but was used in this trial to give a positive or negative result for FMD viral RNA in each sample. Briefly, one Φl of RT product was added to 24 Φl of a PCR mix containing forward primer, reverse primer and probe in a 96-well optical reaction plate (PE Biosystems, UK) and PCR amplification for 50 cycles was carried out in the thermocycler machine. Results RT-PCR The results from the RT-PCR on the 40 epithelial suspensions and cell culture supernatant fluids with the1f/1r primer set and the cocktail of specific P92//P40/P74-P77 are summarised for each serotype in Table 1. Only one sample was not detected either as an ES or cell culture supernatant with the 1F/1R primer set which detected every other cell culture supernatant and 36 of the 40 ES samples including all of the serotype O and Asia 1 samples. This mirrored the results from a previous evaluation with this primer set (Reid et al., 2000; in press) on other FMD virus samples where better results were also achieved on type O, A and Asia 1 samples than on type C samples. The specific primers successfully detected both ES and cell culture supernatant fluids of type Asia 1 samples using both thermocycler programmes but were less successful for detection of the other serotypes which again mirrored previous results with these primers (Reid et al., 1999). The annealing temperature used in the thermocycler programme was particularly significant in the performance of the specific primers for detection of the serotype A strains as the sensitivity of the primers dropped as the annealing temperature was raised from 58 0 C to 59 0 C (Table 1). Antigen capture RT-PCR The performance of the 1F/1R primer set on the 40 ES samples in the antigen capture RT-PCR were identical to those achieved by this primer set in the (diagnostic) RT-PCR (Table 1). These methods were therefore of equal sensitivity for primary diagnosis of the O, A, C and Asia 1 serotypes with this primer set. Table 1 also shows the results of the antigen capture RT-PCR with the specific primers on ten of the ES samples. These results were again similar to those achieved with the same primers in the (diagnostic) RT-PCR but the serotype A strains were not detected in the antigen capture format. TaqMan 7 RT-PCR

5 185 All of the cell culture supernatant fluids were detected (but not serotyped) by the TaqMan 7 RT- PCR (Table 1). This methodology is at an early stage of development and is currently subject to patent considerations. Conclusion When the 1F/1R primers were used in the prototype antigen capture RT-PCR format for FMD virus detection on the 40 ES samples, the results were identical to those from the diagnostic RT-PCR with the same primer set. FMD virus was detected in all but one of the 40 samples so that disease would have been reported had the samples been submitted for diagnosis from a suspected outbreak. Attempts to specifically detect the strains of the serotypes O, A and C were less successful which suggested a lack of sensitivity of the primers for the detection of the homologous serotype. The prototype antigen capture format for serotype-specific diagnosis was unable to detect the type A strains but otherwise had similar sensitivity to the diagnostic RT-PCR with the specific primers. An evaluation of these specific primers by Reid et al. (1999) concluded that additional or alternative primers were required to improve the detection of the serotypes O and C. However, the sensitivity of the RT-PCR for the detection or serotyping of FMD virus in field samples may be improved to greater extent by development of alternative formats such as the antigen capture RT-PCR and TaqMan 7 RT-PCR. Highly promising results were achieved with the TaqMan 7 RT-PCR which detected all 40 cell culture supernatant fluids with a universal primer and probe set designed from conserved sequences in the FMD virus genome. The sensitivity of this system was 100% on the cell culture supernatant fluids of the EU samples but the ES of the samples will have to be tested to provide a more thorough evaluation of this method for FMD virus diagnosis. However, this method is no more time-consuming than the diagnostic or antigen capture RT-PCR methods and only one microlitre of RT product is required for the testing of each sample (cf. five micolitres with the diagnostic RT-PCR) so that the tests can be performed in duplicate or triplicate. This study was a true blind trial but it was known beforehand that all 40 samples were positive for FMD virus which therefore imparted a bias to the way in which the testing was performed. With this prior knowledge, more tests can be performed on the samples in order to >achieve= the positive result. Such a luxury is not normally available. AcknowledgementsThe authors would like to thank Professor Soren Alexandersen (Institute for Animal Health, Pirbright) for the design of the primers and probe and for technical assistance with the TaqMan 7 RT-PCR. This work was supported financially by the Ministry of Agriculture, Fisheries and Food, UK. References Amaral-Doel, C. M. F., Owen, N. E., Ferris, N. P., Kitching, R. P. and Doel, T. R. (1993) Detection of foot-and-mouth disease viral sequences in clinical specimens and ethyleneimineinactivated preparations by the polymerase chain reaction. Vaccine 11,

6 186 Callens, M. and De Clercq, K. (1997) Differentiation of the seven serotypes of foot-and-mouth disease virus by reverse transcriptase polymerase chain reaction. J. Virol. Methods 67, Callens, M. and De Clercq, K. (1999) Highly sensitive detection of swine vesicular disease based on a single tube RT-PCR system and DIG-ELISA detection. J. Virol. Methods 77, Ferris, N. P. and Dawson, M. (1988) Routine application of enzyme-linked immunosorbent assay in comparison with complement fixation for the diagnosis of foot-and-mouth and swine vesicular diseases. Vet. Microbiol. 16, Moss, A. and Haas, B.(1999) Comparison of the plaque test and reverse transcription nested PCR for the detection of FMDV in nasal swabs and probang samples. J. Virol. Methods 80, Reid, S. M., Forsyth, M. A., Hutchings, G. H. and Ferris, N. P. (1998) Comparison of reverse transcription polymerase chain reaction, enzyme linked immunosorbent assay and virus isolation for the routine diagnosis of foot-and-mouth disaease. J. Virol. Methods 70, Reid, S. M., Hutchings, G. H., Ferris, N. P. and De Clercq, K. (1999) Diagnosis of foot-andmouth disease by RT-PCR: evaluation of primers for serotypic characterisation of viral RNA in clinical samples. J. Virol. Methods 83, Reid, S. M., Ferris, N. P., Hutchings, G. H., Samuel, A. R. and Knowles, N. J. (2000) Primary diagnosis of foot-and-mouth disease by reverse transcription polymerase chain reaction. J. Virol. Methods, in press. Simms, D., Cizdziel, P. E.and Chomczynski, P. (1993) TRIzol TM : a new reagent for optimal single-step isolation of RNA. Focus 15, Vangrysperre, W. and De Clercq, K. (1996) Rapid and sensitive polymerase chain reaction based detection and typing of foot-and-mouth disease virus in clinical samples and cell culture isolates, combined with a simultaneous differentiation with other and/or symptomatically related viruses. Arch. Virol. 141, Table 1 Summary of the results from the blind trial using the diagnostic RT-PCR, antigen capture RT- PCR and TaqMan 7 RT-PCR methods Ratio of number of samples tested positive to number of samples tested for each FMD virus serotype

7 Primer set/ RT-PCR format 187 O A C Asia 1 ES a ES cc ES cc ES cc cc b Diagnostic RT-PCR 1F/1R 10/10 10/10 9/10 10/10 7/10 9/10 10/10 10/10 5/10 c 2/10 c 4/10 c 3/10 c 2/10 c 4/10 c 9/10 c 8/10 c 5/10 d 4/10 d 1/10 d 1/10 d 1/10 d 3/10 d 8/10 d 8/10 d Antigen capture RT- PCR 1F/1R 10/10 NT e 9/10 NT 7/10 NT 10/10 NT 1/1 c NT c 0/2 c NT c 0/1 c NT c 2/2 c NT c 0/1 d NT d 0/1 d NT d NT d NT d 2/2 d NT d TaqMan 7 RT-PCR NT 10/10 NT 10/10 NT 10/10 NT 10/10 a ES, epithelial suspension. b cc, cell culture supernatant fluid. c PCR amplification with themocycler programme : 94 0 C for 5 min, 1 cycle; 94 0 C for 1min, 58 0 C for 1 min, 72 0 C for 2 min, 20 cycles; 72 0 C for 7 min, 1 cycle. d PCR amplification with themocycler programme : 94 0 C for 5 min, 1 cycle; 94 0 C for 1min, 59 0 C for 1 min, 72 0 C for 2 min, 20 cycles; 72 0 C for 7 min, 1 cycle. e NT, not tested.

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