Detection of antibodies to porcine reproductive and respiratory syndrome virus (PRRSV) in blended sera or in blood samples collected on filter papers

The detection of serum antibodies against PRRSV is easily done with commercial ELISA kits. These diagnostic tools are reliable and now routinely used. The kits are, however, generally designed for individual sera. Our objective was to validate the assays for blended sera and blood samples collected on filter papers in order to reduce diagnostic costs. Two ELISA kits, commercialised in France, were used. In a first step, the feasibility of detecting specific antibodies in blood eluted from filter papers was assessed. Blood was collected on filter papers from small skin scarifications of the ear or the tail. When dried, the filter papers were sent to the laboratory, and there, eluted in the sample buffer provided with the ELISA kits. The best protocol for testing blood eluted from the filter papers was defined for each kit. Then, by testing around 100 filter papers collected from non-infected pigs and 5 filter papers collected from 5 low sero-positive pigs (as assessed in the immunoperoxidase monolayer assay, IPMA), we determined the positive thresholds for the filter papers. In a second step, the amount of antibodies detectable by the two ELISAs was evaluated for the sera and filter papers by titrations of paired samples collected from around 70 infected pigs. For both kits, the geometric mean titres of specific antibodies in sera or filter papers were nearly identical. From these titre determinations, the probability for detecting one positive sample (one serum or one filter paper) mixed with increasing number of negative samples was determined. The results indicated that blends of up to 5 samples still gave a good probability of detection. Sensitivity and specificity of the detection of PRRS infection with kit 1 were then determined on 200 paired serum-paper samples, using randomly blended samples in comparison to individual sera. With blends of 5 sera, the sensitivity and specificity compared to the analysis of individual sera were 71% and 100%, respectively, whereas, the sensitivity and specificity of blends of 5 filter papers were 79% and 86%, respectively. For kit 2, the sensitivity and specificity of PRRS detection were only determined on individual sera and individual filter papers in comparison with the results of kit 1: the results were very similar, thus validating the performances of kit 2. The specificities of the two kits were subsequently estimated under routine use in a Regional Veterinary Laboratory. Around 120 blended filter papers were tested. From these tests, the specificity was statistically estimated: for both kits, specificity was above 97.5% (p = 0.05). The validation of the two kits with blended filter papers was finally achieved by a running interlaboratory test which involved 8 Regional Veterinary Laboratories and a set of 10 blends of five filter papers (1 negative, 2 low positive, 3 high positive blends and 4 repetitions). All but one laboratory obtained results in agreement with the expected results. After analysis, it turned out that the laboratory which had failed did not correctly store the samples before testing. The results of this study allow to propose a test using three random blends of 5 filter papers by ELISA, in order to diagnose PRRSV infection on farms. This protocol has been evaluated as sensitive as 10 individual sera for PRRSV infection detection. This study was granted by the Fédération Régionale des Groupements de Défense Sanitaire des Pays de la Loire and the French Ministry of Agriculture

Validation of two commercial real-time RT-PCR kits for rapid and specific diagnosis of classical swine fever virus.

&lt;p&gt;Two real-time RT-PCR kits, developed by LSI (TaqVet CSF) and ADIAGENE (Adiavet CSF), obtained an agreement to be commercialised in France, subject to conditions, defined by the French Classical Swine Fever (CSF) National Reference Laboratory. The producers were asked to introduce an internal control to check the RNA extraction efficacy. The different criteria assessed were sensitivity, &quot;pestivirus specificity&quot;, reproducibility and ease of handling, using 189 different samples. These samples were either CSFV inactivated strains or blood/serum/organs collected from CSFV experimentally infected pigs or naturally infected wild boars. The reproducibility of the assays was confirmed by the analysis of a batch-to-batch panel control that was used for inter-laboratory tests involving nine laboratories. The two kits were also tested for the use in mass diagnostics and the results proved the kits to be suited using pools of blood, serum and tonsils. Moreover, a field evaluation, carried out on spleen samples collected from the CSF surveillance of wild boars in an area known to be infected and from domestic pigs at a slaughterhouse, confirmed the high sensitivity and specificity of the two kits. This step-by-step evaluation procedure confirmed that the two commercial CSF real-time RT-PCR kits have a higher predictive value than the current diagnostic standard, Virus Isolation.&lt;/p&gt;</p

Development and inter-laboratory validation study of an improved new real-time PCR assay with internal control for detection and laboratory diagnosis of African swine fever virus

A real-time polymerase chain reaction (PCR) assay for the rapid detection of African swine fever virus (ASFV), multiplexed for simultaneous detection of swine beta-actin as an endogenous control, has been developed and validated by four National Reference Laboratories of the European Union for African swine fever (ASF) including the European Union Reference Laboratory. Primers and a TaqMan probe specific for ASFV were selected from conserved regions of the p72 gene. The limit of detection of the new real-time PCR assay is 5.7 to 57copies of the ASFV genome. High accuracy, reproducibility and robustness of the PCR assay (CV ranging from 0.7 to 5.4%) were demonstrated both within and between laboratories using different real-time PCR equipments. The specificity of virus detection was validated using a panel of 44 isolates collected over many years in various geographical locations in Europe, Africa and America, including recent isolates from the Caucasus region, Sardinia, East and West Africa. Compared to the OIE-prescribed conventional and real-time PCR assays, the sensitivity of the new assay with internal control was improved, as demonstrated by testing 281 field samples collected in recent outbreaks and surveillance areas in Europe and Africa (170 samples) together with samples obtained through experimental infections (111 samples). This is particularly evident in the early days following experimental infection and during the course of the disease in pigs sub-clinically infected with strains of low virulence (from 35 up to 70 dpi). The specificity of the assay was also confirmed on 150 samples from uninfected pigs and wild boar from ASF-free areas. Measured on the total of 431 tested samples, the positive deviation of the new assay reaches 21% or 26% compared to PCR and real-time PCR methods recommended by OIE. This improved and rigorously validated real-time PCR assay with internal control will provide a rapid, sensitive and reliable molecular tool for ASFV detection in pigs in newly-infected areas, control in endemic areas and surveillance in ASF-free areas