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S1 - O - 04 A DUPLEX ONE-STEP REAL TIME RT-PCR FOR DIAGNOSIS OF FOOT AND MOUTH DISEASE Kamila Górna, Romey Aurore, Anthony Relmy, Aude Allmandou, Sandra-Blaise Boisseau, Stephan Zientara, Labib Bakkali Kassimi ANSES, Laboratoire de Santé Animale, UMR1161 ANSES INRA ENVA, Maisons-Alfort 94706 France Foot- and –Mouth Disease; Diagnosis; Real Time RT-PCR Introduction Foot-and-mouth disease (FMD) is a highly contagious transboundary disease of wild and domesticated cloven-hooved ruminants. It is one of the most economically devastating diseases in livestock, due to measures employed to control the outbreak and trade loss resulting from embargos on the country. The causative agent, the FMD virus (FMDV), is a single stranded RNA virus belonging to the Picornaviridae family, genus Aphtovirus. There are seven immunologically distinct serotypes of the virus, namely types O, A, C, Asia 1, SAT 1, SAT 2 and SAT 3. Quick diagnosis of FMD is critical for the rapid implementation of control measures to limit the spread of the virus and eradicate the disease. The recommended real-time RT-PCR method (rtRT- PCR) for detection of FMDV is a two-step simplex method targeting conserved regions in the internal ribosomal entry site within the 5’ untranslated region (IRES) and in the RNA polymerase gene (3D)(1). In parallel, a rtRT-PCR targeting a cellular gene (endogenous control) is performed to allow normalization of results. This method has the disadvantage of being performed in two separate steps (RT and PCR) and detects only one target at a time. It is therefore time-consuming, and since several pipetting steps are required, this method can be subject to possible errors and contamination. Multiplex rtRT- PCR is a modification of the basic rtRT-PCR method in which pairs of primers targeting different targets are used in the same reaction. In one-step multiplex rtRT-PCR, the reverse transcription and subsequent amplification take place in a single tube, thereby reducing the reaction time and number of reaction mixtures, and also diminishing the risk of contamination. In this study, duplex one-step rtRT-PCRs were established to detect and type FMDV. These methods are simple, specific, and sensitive for early diagnosis of FMDV infection. Material & methods Samples: Artificially FMDV spiked samples were prepared by making 10 fold serial dilutions of virus stock (strains O1 Manisa, A22 Iraq or Asia 1) in FMDV-free bovine tongue epithelial tissue suspension. Field bovine epithelium samples (n:19) were collected from suspected cases in West Africa. Each sample was subjected to virus isolation, Ag-ELISA, Chromatographic test (Svanodip) and rtRT-PCR. Virus Isolation: Samples were prediluted 5 and 10 times in cell culture medium and inoculated into goat tongue cell cultures (ZZ-R). Chromatographic test (lateral flow device): Svanodip (SVANOVA Biotech AB) test was performed according to the manufacturer’s instructions. Antigen Capture Elisa was performed as described in the OIE manual (1). rtRT-PCR: RNA was extracted from samples by using the QIAamp Viral RNA mini-kit (QIAGEN). Two step simplex rtRT- PCRs for IRES, 3D and -actin targets were performed using TaqMan Reverse Transcription Reagents kit and TaqMan PCR Core Reagents kit (Life technologies) following protocols described previously (2, 3, 4). One Step duplex rtRT- PCRs were performed using the AgPath-IDOne-Step RT-PCR Reagents (Life Technologies). The thermal conditions were as follows: 10 min at 45°C (RT), 10 min at 95°C (denaturation) and 45 cycles of 15s at 95°C and 1 min at 60°C. The final concentration of primers and probes in duplex assay was optimized for IRES, 3D and - actin targets and was as previously described for the typing protocol (5). Each probe for FMDV specific targets, marked by FAM dye, was coupled in the same test tube with β-actin probe labelled with VIC dye. with two other vesicular disease viruses, swine vesicular disease and vesicular stomatitis viruses. Artificially FMDV spiked samples containing different amounts of virus were used to compare the sensitivity of FMDV diagnostic methods. Both one-step and twostep rtRT-PCR methods were 1000 to 10,000 fold more sensitive than the antigen capture Elisa and Svanodip test. VI and all rtRT- PCRs displayed quite similar sensitivity. The 3D/-actin duplex real-time one-step method gave better results than the two-step method. The serotype-specific one-step duplex rtRT-PCR was slightly less sensitive than the pan-FMDV one-step duplex rtRT- PCR method. A better detection of the internal β-actin control was observed with the one-step protocol. All rtRT-PCR methods were evaluated with 19 field samples. FMDV was isolated from all samples. The one-step duplex rtRT-PCR for both IRES and 3D targets was more sensitive than the two-step simplex rtRT- PCR. Moreover, four samples found negative for IRES by a twostep method were positive using the one-step method. It was not possible to type the 19 field samples by rtRT-PCR, despite the presence of FMDV type O and A in these samples, as revealed by VP1 sequencing. Discussion & conclusion This study reports the development of a one-step duplex rtRT- PCR method for detection of FMDV and characterization of the three most commonly occurring serotypes worldwide. This method is more sensitive than other recommended FMDV detection methods tested. Simultaneous detection of FMDV and -actin within the same reaction permits the exclusion of false negatives that may result from improper extraction or degradation of the RNA, and permits normalization of the results. Thus, this one-step duplex rtRT-PCR method could be of interest for FMD diagnostic laboratories. We were unable to type FMDV from the field samples by rtRT-PCR. Analysis of the FMDV VP1 sequence from these samples revealed multiple mismatches in the primer and probe sequences for type O and type A. This is not surprising since the primers and probes were designed from sequences of FMDV strains belonging to lineages not present in West Africa (5). New primers and probes specific to WA lineages will be designed and evaluated. The development of a multiplex one-step rtRT-PCR could provide an improved method for rapid detection of FMDV, as it should allow detection of more targets in the same test and in less time. Acknowledgements The author thanks Jennifer Richardson for editing the English version of the manuscript. References 1-Manuel of Diagnostic Test and Vaccines for Terrestrial Animals, chapter 2.1.5.1C, 2009, OIE. 2- M. Reid et al. Detection of all seven serotypes of foot-and-mouth disease virus by real-time, fluorogenic reverse transcription polymerase chain reaction assay. J. Virol. Methods, 2002, 105 : 67-80 3- JD Callaham et al. Use of a portable real-time reverse transcriptasepolymerase chain reaction assay for rapid detection of foot-and-mouth disease virus. YAVMA, 2002, 220, 11:1636-42. 4- J.F. Toussaint et al. Bluetongue virus detection by two real-time RTqPCR targeting two different genomic segments. J. Virol. Methods, 2007, 140 : 115-128 5- Reid et al. Detection of FMDV serotypes O, A and Asia1 by real-time RT-PCR. EuFMD 2008 Meeting. The global control of FMD – Tools, ideas and ideals – Erice, Italy 14-17 October 2008. Results One step duplex rtRT-PCR methods for pan-FMDV (IRES and 3D) successfully detected all 7 serotypes of FMDV. The typing rtRT-PCRs detected exclusively the specific serotype and did not cross react with other serotypes. All tests gave negative results
S1 - O -05 VALIDATION OF A COMPETITIVE ELISA FOR THE DETECTION OF ANTIBODIES ANTI-p26 OF EQUINE INFECTIOUS ANEMIA VIRUS IN EQUINE SERA R. Nardini 1 , G.L. Autorino 1 , R.Lorenzetti 1 , P. Cordioli 2 , 1 A. Caprioli, M.T. Scicluna 1 1 National Reference Centre for equine infectious diseases, Istituto Zooprofilattico Sperimentale Lazio e Toscana, 00178 Roma Italia. 2 Istituto Zooprofilattico Sperimentale Emilia Romagna e Lombardia, Via A. Bianchi 9, 25124 Keywords: Equine infectious anemia, competitive ELISA, validation Introduction Equine infection anemia (EIA) is a viral infection of equidae, caused by a virus (EIAV) of the Lentivirus genus, Retroviridae family. Hematophagous insects belonging to the Tabanidae, Hippoboscidae and Muscinae families are involved as passive vectors in viral transmission. The infection is usually persistent, and can occur as an asymptomatic form or with recurrent febrile episodes. Since 2006, Italy has adopted a National Surveillance Plan (NSP) (1) consisting in a serological screening of all animals except for meat horses. According to the Italian Regulations, the official confirmatory test is the Agar Gel Immunodiffusion (AGID) test which detects antibodies anti-p26, a highly conserved capsidic protein of the virus. Since this technique has a low detection limit, development of new and more sensitive tests has become necessary. WOAH Manual (2) indicates ELISA, immunoblotting and PCR as confirmatory tests for the serological diagnosis of EIA. The ELISA, for its characteristics, is the most suitable for screening purposes. The aim of this paper is to present the results of validation of a competitive ELISA (c-ELISA) for the detection of antibodies anti-p26 of EIAV in equine sera. Materials and methods The procedure of the c-ELISA, object of this validation, is briefly described as following. A 96-well microplate is pre-adsorbed overnight at 4°C with a monoclonal antibody (Mab) anti-p26. Serum samples and a recombinant antigen p26, produced in E. coli, are mixed on a separate plate and incubated at 37°C for 75 minutes. At the end of the incubation, the serum-antigen mix is transferred onto the pre-absorbed plate and a second Mab conjugated with horseradish peroxidase is added. After another incubation at 37°C for 75 minutes, ortophenyl-diamine substrate is added and the plate is incubated at room temperature for 15 minutes in the dark. The reaction is stopped by the addition of sulphuric acid and the optical density (OD) is read with a 492nm filter. Sera are categorized as positive, negative or equivocal according to the percentage inhibition (PI), calculated as the ratio between the sample and the internal negative control. Validation of the c-Elisa was performed according to WOAH Manual guidelines (3) and the aim of this test was for screening purposes. For its validation the following parameters were evaluated. Analytical specificity was estimated at three different levels: 1. Selectivity, defined as the capability to detect the target analyte in the presence of other interferences, was evaluated by changing the composition of the wash solution and processing positive and negative International Reference Serum (IRS). 2. Exclusivity, considered as the capacity to discriminate target analyte from other crossreactive analytes, was evaluated processing ten sera positive for each of the following virus: Feline Immunodeficiency Virus, Feline Leukaemia Virus and Visna Maedi Virus. Each sample was repeated ten times. 3. Inclusivity which is how a test can differentiate between different serovars. p26 protein is highly conserved in this virus and (4) thus evaluation was not necessary. Analytical sensitivity is represented by limit of detectability (LOD) and in this validation, the LOD of ELISA was compared with the AGID LOD, analysing progressive dilutions of a positive IRS in both methods. Repeatability was evaluated estimating the standard deviation (S r ) of the OD values of 30 repetitions of a negative IRS; this value was compared with another set of 30 values, processed by the same person during the same session. Reproducibility was estimated taking into account both qualitative and quantitative characteristics of ELISA test. a) To analyse qualitative reproducibility a Ring Test was performed by eleven laboratories and the statistic K of Cohen was calculated. b)Quantitative reproducibility was evaluated in seven sessions examining 30 replica of negative IRS in each, and calculating S R of OD values. Diagnostic performances were evaluated from sensitivity (Dse) and specificity (Dsp). Minimum number of samples to evaluate a sensitivity of 90% and a specificity of 80% with confidence interval of 99% and a precision of 5% was calculated. 1095 sera from field samples and from European Union Reference Laboratory, consisting in 857 positive and 238 negative samples were analysed with both ELISA and AGID, using the latter as Gold standard (GS). Dse, Dsp, positive and negative predictive values, efficacy and test Bias were calculated (5). For statistical analysis XL-Stat 2011 ® was used. Results 1.ELISAs performed changing the washing solution did not correctly recognise positive and negative sera. 2.All sera positive for other Lentivirus were classified as negative by the ELISA. 3.LOD of ELISA test is at least 1 Log 10 more sensitive than AGID. 4.Comparison between two data sets for evaluation of repeatability did not show significant differences with test F (p=0.42) and S r resulted to be equal to 0.184. 5.Statistic K value was equal to 0.967. 6.Comparison between seven data sets for evaluation of reproducibility did not show significant differences with test F (p=0,092) and S r was equal to 0.129. 7.Dse and Dsp resulted respectively 100 and 80,3%. 8.Positive and negative predictive values resulted respectively 94.8% and 100% 9.Efficacy of ELISA which is the percentage of samples correctly identified (positive and negative together) compared with the GS resulted equal to 95.7% 10.Test Bias which is the ratio between apparent and real prevalence resulted 1.05. Discussion and conclusions The results of the parameters evaluated for the validation of the c-ELISA are all satisfactory. In particular, the lower Elisa LOD would allow not only the detection of “poor responders” then when using the AGID only, but also the possibility of detecting earlier, new cases. Although the Dsp is 80%, this value is still acceptable for a sreening test compared to the advantages that it presents to the AGID, i.e the possibility of its standardization, the objectivity of the results reading, the lower amount of reagents used and the higher processing of samples per unit time. The use of this diagnostic test is an added value, together with other measures, in a control programme for AIE. ReferenceS 1.Ordinanza 8 agosto 2010 Piano di sorveglianza nazionale per l'anemia infettiva degli equidi. G.U. Serie Generale n. 219 del 18 settembre 2010 2.Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2010, Chapter 2 . 5 . 6 .Equine infectious anaemia 3.Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2010,Chapter 1.1.4/5. Principles of validation of diagnostic assays for infectious diseases. 4.Charles J. Issel, R. Frank Cook (1993)REVIEW ARTICLE A review of techniques for the serologic diagnosis of equine infectious anemia J Vet Diagn Invest 5: 137-141 5.Signorelli C. Elementi di metodologia epidemiologica SEU, 2005
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