Scott M. Reid, Nigel P. Ferris and Geoffrey H. Hutchings

Transcription

1 Appendix 26 Comparison 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, Nigel P. Ferris and Geoffrey H. Hutchings Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, UK Abstract Results compiled from the reverse transcription polymerase chain reaction (RT-PCR) collaborative study under the European Union (EU) Concerted Action Group Project PL involving the laboratories in Pirbright, Brussels, Valdeolmos, Brescia, Tübingen, Lelystad and Lindholm were presented in order to evaluate the sensitivity and specificity of the locally employed RT-PCR procedures. All 7 laboratories were supplied with 40 unlabelled epithelial suspensions (ES) of foot-and-mouth disease (FMD) virus positive clinical material (10 each of the serotypes O, A, C and Asia 1) and 5 of the laboratories supplied with the supernatant fluids (cc) following the inoculation of the ES in cell culture. At the Pirbright Laboratory virus was detected (but not serotyped) in 36 of the 40 ES and in 39/40 cc by conventional (not nested and non-quantitative) RT-PCR and in 36/40 ES by a prototype immunocapture method. Similar sensitivity with universal primer sets was achieved in RT-PCR assays on the ES and cc in Brescia but 39/40 ES were detected by a novel RT-PCR in Valdeolmos. A nested PCR protocol in Tübingen also detected 39/40 ES but a positive result on all 40 ES samples was obtained by quantitative RT-PCR for the universal detection of FMD virus (TaqMan assay at Pirbright, LightCycler protocol at Lelystad and SYBR Green assay at Lindholm). Serotype-specific primers were used only in Pirbright and Brussels but a more thorough comparison of the sensitivity and specificity of the RT-PCR procedures can be obtained only after all of the experimental details employed by the laboratories (particularly amplification programmes, cutoff criteria and the use of positive and negative controls) are considered in greater detail. 1. Introduction RT-PCR procedures are becoming more important for diagnosis of FMD virus and three studies by Reid et al. (1998; 1999; 2000a) at the OIE/FAO World Reference Laboratory for Foot-and- Mouth Disease (WRL for FMD), Pirbright, reported the use of conventional RT-PCR formats involving universal or serotype-specific primer sets for the testing of ES and cc prepared from large panels of positive FMD virus specimens of all 7 serotypes. Since the beginning of the United Kingdom (UK) 2001 FMD epidemic, new RT-PCR methodologies for FMD virus diagnosis have been developed and evaluated at the WRL for FMD to supplement the routine diagnostic procedures of ELISA and virus isolation in cell culture (Ferris and Dawson, 1988) and which have involved non-automated and automated fluorogenic assays (Reid et al., 2001a; Reid et al., 2001b; Reid et al., 2002a; Reid et al., 2002b, in press). Results currently suggest that a definitive diagnostic result might be achieved by RT-PCR at the end of one cell culture passage (Reid et al., 2002b, in press). This would obviate the necessity of passaging samples in cell 210

2 culture so that laboratory results could be issued much faster. At the first annual European Union (EU) Concerted Action PL meeting during 5-7 May 1999 at the Federal Research Centre for Virus Diseases of Animals [BFAV] in Tübingen, a collaborative study was organised between the Veterinary and Agrochemical Research Centre (VARC) in Brussels, BFAV Tübingen and the WRL for FMD, Pirbright, to provide an assessment of the sensitivity and specificity of the RT-PCR procedures employed by these laboratories. The RT-PCR protocols used by the EU laboratories are not standardised and inter-laboratory variation has not been investigated so the study could suggest improvements in the diagnosis of FMD virus by RT- PCR. At the EU Concerted Action meeting in Pirbright the following year, the Center of INIA (CISA-INIA) in Valdeolmos (Madrid) and the Istituto Zooprofilattico Sperimentale in Brescia joined the study and the number of participating laboratories rose to 7 at the next meeting in Brussels in 2001 when the laboratories of CIDC-Lelystad (Central Institute for Animal Disease Control Lelystad) and the Danish Veterinary Institute for Virus Research in Lindholm requested to participate. This study will investigate the FMD diagnostic potential within the EU of some of the more recently introduced RT-PCR methodologies such as quantitative (Taqman and SYBR Green) RT-PCR as well as previously described conventional or nested RT-PCR formats such as those of Vangrysperre and De Clercq (1996) and Moss and Haas (1999). Some of the results obtained by the WRL for FMD were reported previously (Reid et al., 2000b). 2. Materials and methods 2.1. Preparation of the unlabelled samples for the blind trial between the collaborating laboratories The 10 ES each of the FMD virus serotypes O, A, C and Asia 1 and the cc resulting from the propagation of the ES in cell culture were prepared and aliquotted at the WRL for FMD and distributed (unlabelled) to the laboratories of BFAV Tübingen, VARC in Brussels, Center of INIA (CISA-INIA) in Valdeolmos and to the Istituto Zooprofilattico Sperimentale in Brescia (as well as to the WRL for FMD) as previously described (Reid et al., 2000b). The 40 ES but not the cc were also sent to the laboratories in Lelystad and Lindholm Summary of the RT-PCR methods used in each laboratory WRL for FMD, Pirbright Conventional RT-PCR Total RNA was extracted from the trial samples, uninfected negative control ES and cc and subjected to RT as described previously (Reid et al., 2000b). The universal O/A/C/Asia 1 primer set (1F/1R) designed for the intended diagnosis of all 7 FMD virus serotypes was used as it was 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., 2000a). The specific primers P33/P38/P87-P92/P40/P74- P77 designed for the diagnosis of the serotypes O, A, C and Asia 1 (Vangrysperre and De Clercq, 1996) and evaluated in RT-PCR procedures by Reid et al. (1999; 2000a) were also used. The RT product from each sample was tested with the 1F/1R primer pair and with the cocktail of the specific primers P33/P38/P87-P92/P40/P74-P77 in RT-PCR procedures as described previously 211

3 (Reid et al., 2000b). Immunocapture (antigen capture) RT-PCR Each ES was tested in a prototype immunocapture RT-PCR method for FMD virus diagnosis involving PCR amplification with the 1F/1R primer set as described previously (Reid et al., 2000b; 2000c). Ten ES samples were tested by another prototype immunocapture protocol involving 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) in separate PCR amplifications as described previously (Reid et al., 2000b; 2000c). Quantitative (fluorogenic or TaqMan ) RT-PCR A forward primer, reverse primer and probe were designed from conserved sequences of the FMD viral genome for use in a quantitative PCR machine. All ES and cc were tested in a TaqMan RT-PCR using a GeneAmp 5700 Sequence Detection System (Applied Biosystems, UK) to give a positive or negative result for FMD virus in each sample based on quantitative values (Reid et al., 2002a) VARC, Brussels Twenty eight samples each of the ES and cc were tested by RT-PCR using serotype-specific primers designed in-house from the 1D region of the virus genome (Vangrysperre and De Clercq, 1996) CISA-INIA, Valdeolmos All ES and cc were tested by the novel RT-PCR protocol of Sáiz et al. (2001) using the primer set A1 designed from the 3D1/3D2 region of the genome for the detection of the FMD virus serotypes O, A, C and Asia 1 as a group (Rodríguez et al., 1992; Núñez et al., 1998) and the primer set U (Sáiz et al., 2001) for the universal diagnosis of all 7 serotypes of FMD virus Istituto Zooprofilattico Sperimentale, Brescia The cc was re-passaged in order to produce larger volumes for testing. Thirty nine of the 40 ES and all 40 re-passaged cc were tested by an RT-PCR using a primer set F17/F21 which is the primer set A1 (Rodríguez, et al., 1992; Núñez et al., 1998) and which was used in this study by the laboratory of CISA-INIA in Valdeolmos. Thirty nine of the 40 ES and 16 cc were also tested by an RT-PCR with a primer set LD2/LR2 (Lomakina, unpublished) designed from the 3D region of the FMD virus genome for the intended universal detection of all 7 serotypes BFAV Tübingen All ES and cc were tested by nested PCR in combination with a plaque test as described by Moss and Haas (1999) with the slight modification of using TRIzol Reagent for RNA extraction Danish Veterinary Institute for Virus Research, Lindholm RNA was purified from 90 µl of each ES sample by a QIAamp Viral RNA Mini Spin Kit 212

4 (QIAGEN) and RT performed on 5 µl of the extracted RNA by a RETROscript First Strand Synthesis Kit (Ambion). PCR amplification was carried out by SYBR Green assay on 5 µl cdna using the universal O/A/C/Asia 1 primer set (1F/1R) of Reid et al. (2000a) in a 7700 Sequence Detection System (Applied Biosystems). The 1F/1R primer set was used by the WRL for FMD in conventional and immunocapture RT-PCR methodologies as previously described (2.2.1.). Further testing of the samples will be performed using a QuantiTect SYBR Green RT- PCR Kit (QIAGEN) CIDC-Lelystad The LightCycler RT-PCR protocol of Moonen et al. (2001) using labelled probes was used to test the 40 ES samples. 3. Results 3.1. WRL for FMD, Pirbright RT-PCR results obtained on the 40 ES and cc samples obtained by each laboratory are shown in Table 1. At the WRL for FMD, one sample was not detected by conventional RT-PCR with the primer set 1F/1R either as an ES or cc but all other cc samples were detected as were 36 of the 40 ES samples including all of the serotype O and Asia 1 viruses. This mirrored the results from a previous evaluation with this primer set in conventional RT-PCR (Reid et al., 2000a) 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 cc of type Asia 1 viruses using both thermocycler programmes but were less successful for detection of the other serotypes which again mirrored previous results (Reid et al., 1999). The specific primers did however detect the serotype C virus which was negative with the primer set 1F/1R. Annealing temperature significantly influenced the performance of the specific primers for detection of serotype A strains as the performance of the primers dropped when this was 59 o C rather than 58 o C (Reid et al., 2000b). The performance of the 1F/1R primer set on the 40 ES samples in the immunocapture RT-PCR were identical to those achieved by the same primers in the conventional RT-PCR (Reid et al., 2000b). The results of the immunocapture RT-PCR on the 10 ES samples with the specific primers were also similar to those achieved by the conventional RT-PCR but the serotype A strains were not detected (Reid et al., 2000b, data not presented here). All 40 samples of ES and cc were positive for FMD virus by the TaqMan RT-PCR VARC, Brussels The specific primers correctly serotyped some of the trial samples but a selection of the ES and cc produced results corresponding to more than one serotype rather than to the single FMD virus serotype. The results did indicate that FMD virus was present in 26 of the 28 ES and/or cc samples tested CISA-INIA, Valdeolmos Primer set A1 detected 34/40 ES and 37/40 cc samples. Three viruses were negative on both ES and cc. However, the primer set U detected 39/40 ES and all 40 cc. The ES of one serotype C 213

5 virus was negative with both primer sets Istituto Zooprofilattico Sperimentale, Brescia RT-PCR with primer set F17/F21 detected 35/39 ES and 39/40 cc viruses (compared to 34/40 ES and 37/40 cc detected using the same primer set at CISA-INIA) and all 40 virus strains were detected either as an ES and/or cc. The primer set LD2/LR2 detected 33/39 ES and 15/16 cc in an RT-PCR BFAV Tübingen Nested PCR detected 39 out of 40 ES, of which 30 were already positive after first amplification. All 40 cc were detected by nested PCR and of these, 36 were positive after first amplification. The ES sample testing negative by nested PCR was positive in the other laboratories CIDC-Lelystad and Danish Veterinary Institute for Virus Research, Lindholm All 40 samples of ES were positive by RT-PCR at Lelystad and Lindholm by the LightCycler and SYBR Green assays respectively. 4. Discussion The laboratories of the WRL for FMD, CISA-INIA, Istituto Zooprofilattico Sperimentale, BFAV Tübingen, CIDC-Lelystad and Lindholm correctly achieved a positive diagnostic result for FMD virus on either the ES or cc (and in most cases on both ES and cc) of all 40 trial samples by their RT-PCR procedures although positive results were not achieved solely on the ES of all samples by the RT-PCR procedures at CISA-INIA, Istituto Zooprofilattico Sperimentale and BFAV Tübingen. The conventional RT-PCR at the WRL for FMD also failed to detect FMD virus in all ES and had an equivalent sensitivity to a prototype immunocapture assay, which may have a potential role in 'on-site' diagnosis of FMD by RT-PCR, with the universal primer set 1F/1R. Nested and quantitative methodologies were used for universal FMD virus diagnosis. Nested PCR at BFAV Tübingen was positive on all samples except one ES but primary diagnosis of FMD virus in all 40 ES was achieved by the quantitative TaqMan, LightCycler and SYBR Green RT-PCR assays at the WRL for FMD, Lelystad and Lindholm respectively. The TaqMan assay was also positive on all 40 cc. This highlighted the FMD diagnostic potential of these assays given that the test results can be obtained within a few hours of sample receipt and in a much shorter time-scale than virus isolation in cell culture. These quantitative RT-PCR formats also avoid the necessity of analysing PCR products by gel electrophoresis, which is laborious, time-consuming and relatively insensitive. Interestingly, the primer set 1F/1R was used in the SYBR Green RT-PCR assay (Lindholm) and in conventional and immunocapture RT-PCR formats at the WRL for FMD and while all 40 ES samples were detected by the SYBR Green assay, 36/40 ES were detected by the other methodologies. This may have been due to the positioning of the positive to negative cut-off in the respective assays. To achieve a more substantial evaluation of the sensitivity and specificity of the described RT-PCR procedures it will be necessary to compare aspects like this and other key features of the assays such as the amplification programme and the role played by positive and negative control samples in the validation of the test. It would also be interesting to investigate the cause of negative results. 214

6 The primer and primer/probe sets used in the study were predominantly designed for the universal diagnosis of FMD virus in clinical samples rather than for serotype-specific diagnosis. Such an approach is clearly useful in a circumstance similar to that of the UK 2001 epidemic in order to establish the presence of FMD virus in clinical samples so that control measures could be implemented quickly. However, serotype-specific, semi-specific or strain-specific primer (or primer/probe) sets could also extend the scope of the RT-PCR formats by detecting emerging FMD virus strains. Serotype-specific primers were used only in non-quantitative RT-PCR formats at the WRL for FMD and in Brussels and while more positive results were achieved at the WRL for FMD with the universal primer set in conventional RT-PCR, a positive result was produced with serotype-specific primers on the cc of a type C strain which was negative with the universal primer set (sample no. 19). This study was a true blind trial in that the samples were tested unseen in the laboratories but it was known beforehand that the ES and cc of all 40 samples were positive for FMD virus. A bias could therefore be imparted to the testing approach as more tests can be performed on any given sample in order to achieve the positive result. Such a luxury would not normally be available in the diagnostic laboratory. Furthermore, it would have been interesting to have included samples which did not contain FMD virus. This would have provided a greater test of the specificity of the diagnostic RT-PCR methodologies. Acknowledgements The authors thank Kris De Clercq (VARC, Brussels), Esther Blanco (CISA-INIA), Emiliana Brocchi (Istituto Zooprofilattico Sperimentale), Bernd Haas (BFAV Tübingen), Aldo Dekker (CIDC-Lelystad), Karin de Stricker (Danish Veterinary Institute, Lindholm) and their co-workers for participating in this study and Soren Alexandersen (Institute for Animal Health, Pirbright) for the design of the primers and probe and for technical assistance with the TaqMan RT-PCR. The RT-PCR assays carried out at the WRL for FMD, Pirbright, were supported financially by the Department for Environment, Food & Rural Affairs (DEFRA), UK. References Reid, S. M., Forsyth, M. A., Hutchings, G. H., Ferris, N. P., Comparison of reverse transcription polymerase chain reaction, enzyme linked immunosorbent assay and virus isolation for the routine diagnosis of foot-and-mouth disease. J. Virol. Methods 70, Reid, S. M., Hutchings, G. H., Ferris, N. P., De Clercq, K., Diagnosis of foot-and-mouth 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., Knowles, N. J., 2000a. Primary diagnosis of foot-and-mouth disease by reverse transcription polymerase chain reaction. J. Virol. Methods 89, Ferris, N. P., Dawson, M., 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, Reid, S. M., Ferris, N. P., Hutchings, G. H., Zhang, Z., Belsham, G. J., Alexandersen, S., 2001a. Diagnosis of foot-and-mouth disease by real-time fluorogenic PCR assay. Vet. Record 149,

7 Reid, S. M., Ferris, N. P., Hutchings, G. H., Alexandersen, S., 2001b. Evaluation of automated RT-PCR systems to accelerate FMD diagnosis. Report of the Session of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Island of Moen, Denmark, September, Rome: FAO 2001 Appendix 25, pp Reid, S. M., Ferris, N. P., Hutchings, G. H., Zhang, Z., Belsham, G. J., Alexandersen, S., 2002a. Detection of all seven serotypes of foot-and-mouth disease virus by real-time, fluorogenic reverse transcription polymerase chain reaction assay. J. Virol. Methods 105, Reid, S. M., Grierson, S. S., Ferris, N. P., Hutchings, G. H., Alexandersen, S., 2002b. Evaluation of automated RT-PCR to accelerate the laboratory diagnosis of FMD virus. J. Virol. Methods, in press. Vangrysperre, W., De Clercq, K., 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 genomically and/or symptomatically related viruses. Arch. Virol. 141, Moss, A., Haas, B., 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., Hutchings, G. H., Ferris, N. P., 2000b. 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 Report of the Session of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Borovets, Bulgaria, 5-8 September, Rome: FAO 2000 Appendix 21, pp Reid, S. M., Hutchings, G. H., Ferris, N. P., 2000c. The development of an antigen capture reverse transcription polymerase chain reaction method for foot-and-mouth disease virus antigen detection. Report of the Session of the Standing Technical Committee of the European Commission for the Control of Footand-Mouth Disease, Borovets, Bulgaria, 5-8 September, Rome: FAO 2000 Appendix 19, pp Sáiz, M., de la Morena, D. B., Blanco, E., Núñez, J. I., Fernández, R., Sánchez-Vizcaíno, J. M., A novel method for detection of FMDV from culture and clinical samples by RT-PCR and restriction enzyme analysis. Report of the Session of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Island of Moen, Denmark, September, Rome: FAO 2001 Appendix 26, pp Rodríguez, A., Martínez-Salas, E., Dopazo, J., Dávila, M., Sáiz, J. C., Sobrino, F., Primer design for specific diagnosis by PCR of highly variable RNA viruses: typing of foot-and-mouth disease virus. Virology 189, Núñez, J. I., Blanco, E., Hernandez, T., Gómez-Tejedor, C., Martín, M. J., Dopazo, J., Sobrino, F., A RT-PCR assay for the differential diagnosis of vesicular diseases of swine. J. Virol. Methods 72, Moonen, P., Boonstra, J., Hakze-van der Honing, R., Boonstra-Leendertse, C., Jacobs, L., Dekker, A., Validation of a LightCycler based RT-PCR for the detection of foot-and-mouth disease. Report of the Session of the Standing Technical Committee of the European Commission for the Control of Footand-Mouth Disease, Island of Moen, Denmark, September, Rome: FAO 2001 Appendix 27, pp

8 Table 1. RT-PCR results obtained on epithelial suspensions (ES) and virus preparations in cell culture (cc) following inoculation with ES No. of sample and FMD virus serotype O/A/C/Asia 1 (1F/1R) (all) WRL for FMD, Pirbright a VARC, Brussels b CISA-INIA, Valdeolmos c Istituto Zooprolilattico, Brescia d BFAV Tübingen e CIDC-Lelystad f Lindholm g O/A/C/Asia 1 (specific) TaqMan (all) O/A/C/Asia 1 (specific) A1 (O/A/ C/Asia1) U (all) F17-F21 (3D) LD2-LR2 (3D) PCR nested PCR Labelled probes O/A/C Asia 1 (1F/1R) (all) ES cc ES cc ES and cc ES cc ES Cc ES cc ES cc ES cc ES cc ES cc ES ES 1 A + + A A + A A NT h Asia Asia 1 Asia O NT O + + O O + O O NT Asia Asia 1 Asia 1 + Asia 1 NT NT C O A + + A A NT Asia Asia 1 Asia 1 + O, A, Asia 1 O, A, C, Asia NT A A A C + + C C + - C NT O O O NT A O O NT C C + - A, C NT + NT NT A A Asia Asia 1 Asia O NT O O + O O A A Asia Asia 1 Asia 1 + O O NT C C C C + C C NT A + + A O NT Asia Asia 1 Asia 1 + O, Asia 1 O, A, C, Asia NT O + + O - + O O NT

9 23 Asia Asia 1 Asia 1 + O, Asia 1 O, Asia NT A O, A O, A C C C NT O + + O - + O O NT C + + C C + C C NT A O + + O O + NT NT NT Asia NT NT NT O NT NT NT Asia Asia 1 Asia 1 + NT NT NT A + + A A + NT NT O NT NT C NT NT O NT NT C C + NT NT Asia Asia 1 Asia 1 + NT NT O + + O O + NT NT (?) C NT NT a Conventional and immunocapture RT-PCR procedures using the universal O/A/C/Asia 1(1F/1R) primer set for detection of all 7 serotypes. Conventional RT-PCR also performed with the specific primer sets used in Brussels for detection of the serotypes O, A, C and Asia 1. The conventional and immunocapture RT-PCRs produced identical results on the ES of all 40 samples with the 1F/1R primer set. The TaqMan assay used a universal primer set for the intended detection of all 7 serotypes. b RT-PCR procedure using specific primers for the detection of individual serotypes O, A, C and Asia 1. c Novel RT-PCR procedure using primer set A1 for the detection of serotypes O, A, C and Asia 1 as a group and the primer set U for the universal detection of all 7 serotypes. d RT-PCR procedure using primer set F17/F21 (same as primer set A1 used in Valdeolmos) and primer set LD2/LR2 for the universal detection of all 7 serotypes. e PCR (first amplification) followed by nested PCR. f LightCycler RT-PCR using labelled probes. g SYBR Green assay using the 1F/1R primer set as used by the WRL for FMD, Pirbright, in conventional and immunocapture RT-PCR. h NT, not tested. +, positive result. -, negative result 218