Development And Application Of Porcine Circovirus Type2Diagnostic Kit

PCVD is caused by PCV2infection that has the main disease feature for PMWS,PDNS, PRDC, CT and reproductive failure. With the PCV2epidemic characteristicschange constantly that maked the PCVD epidemic and spread more and morecomplex. The worldwide occurrence of PCVD resulted in serious losses to the pigindusty. A variety of other pathogenic to mixed or secondary infection has a veryserious threat to the pig industry in condition of herd immunity that caused by PCV2infection. Early diagnosis of PCV2infection has a great signigicance to thecomprehensive prevention and control for PCVD. The study focused on the epidemicand genotype characteristics of PCV2that PCV2PCR dentection kit and PCV2real-time fluorescence quantitative PCR detection reagent and indirect ELISA PCV2antidody detection kit and PCV2antibody colloidal gold strip for rapid detection weredeveloped. According to the gene characteristics of epidemic PCV2strains inShandong area that the PCR assay method was established to identify PCV2genotyoes. The genotype characteristics and genetic variation of Shandong isolatedstrains were analysed in this study. The study had provided the first hand data for theearly diagnosis of PCV2and molecular epidemiology and comprehensive preventionand control.Study1: Establishment of PCR detection method for PCV2and development ofkitThe method was established and rapid PCR kit was developed for detection ofPCV2in the optima reaction conditions. The sensitivity, specificity, repeatability andthe preservation period performances were evaluated for the PCR detection kit. The208clinical and pathological samples in Shandong area were detected by this PCR kit.The results showed that the amplified specific fragment was490bp by this PCR kit forPCR gene. The minimum detectable amount was1×10-6ng/mL nucleic acid in thisPCR detection method.The variation coefficient was3.2%and5.7%in intra and interbatch for this kit that were no cross reaction with PRRSV, PPV, PRV, JEV, CSFV andPCV1by gene PCR reaction.The preservation period test showed that the kit has12moths validity period under the condition of4℃. The208clinical samples detectionresults showed that PCV2positive samples were158and the positive rate was67.3%.The specific PCR method was established for PCV2gene detection that the kitshowed good specificity, sentsitivity, reproducibility and a long preservation period.The study showed that the PCR kit could be used for the rapid detection andmolecular epidemiology of PCV2infection in clinic samples. Study2: Development and application of a Real-time Fluorescent QuantitativePCR Method for detection of porcine circivirus type2The study developed a method of real-time quantitative PCR in the SYBR GreenⅠwhich can detect porcine circovirus type2specificity. The q-PCR reactionconditions were optimized and the repeatability, sensitivity and specificity of q-PCRmethod performance were detected in the study.In order to studying for relation ofPCV2DNA load and clinical pathogenicity that the clinical samples with differentdegree of pathology were detected by the developed q-PCR method.The11strains ofPCV2virus in cell culture conditions were detected for DNA load in order toscreening of high titer virus cell culture condition. The results showed that thestandard curve of the quantitative fluorescence PCR method has a good linearrelationship between Ct value and the standard template DNA amounts in3.21×100-4.16×108copies/μL range, and the correlation coefficient is0.9988and slope is-3.2865.The fluorescence quantitative PCR was no cross-reactions with PRRSV、PCV1、CSFV、PRV、PPV and Escherichia coli genome. The sensitivity of thismethod was proved to be3.21×100copies/μL, which was1000fold better thantraditional PCR, and the coefficient of variation value was less than5.0%.The positiverate was68.94%of suspected PCV2infection material that were detected byfluorescence quantitative PCR that has significantly higer than that of the traditionalPCR. The results of detection for clinical samples showed that PCV2DNA load ofconfirmed PMWS pathological samples was higher than that of106cop/μL and theDNA load of sub clinical infection and healthy samples nucleic acid was lower thanthat of104cop/μL. The results of quantitative detection of isolated strains in cellcultured showed that the2strains were selected high titer virus culture that DNA loadreached3.62×108cop/μL-6.47×107cop/μL respectively. A SYBR Green1fluorescentquantitative PCR for detecting ORF2gene of PCV2was developed for the basis ofthe early diagnosis and the quantitative analysis the infection degree of PCV2.Study3: Identification of genotype and analysis of genetic variation of PCV2strains in Shandong areaORF2gene of PCV2strains were amplified by PCR that was sequenced by DNAsequencing which for analysis of genetic variation of PCV2strains in Shandongregion. The PCR method was established for PCV2genotyping and the genotype of158PCV2positive clinical samples were detected by the genotype PCR method. Theresults showed that the nucleotide homology was99.5%-99.8%in between28strainsof PCV2clinical isolates and major domestic and foreign isolates strains in which thehomology of amino cadi was88.0%-99.1%. In encoding Cap protein that theexistence of3hypervariable regions in53-91,121-151and190-210amino acdis. Thegenotyoe identification showed that3strains were PCV2a genotype and18strainswere PCV2b genotype and4strains had PCV2a/PCV2b genotype recombinant and3 strains were tentatively new genotype PCV2d. The PCR method was established fordetecting genotype identification that amplified fragment sizes were341bp and177bp.The minimum detection of virus DNA was5ng/μL for the estbblished PCRmethod. The test showed that the reproducibility and specificity of the PCR methodwers good for genotype detection. The detection result of158PCV2positive samplesshowed that23strains were PCV2a genotypes and96strains were PCV2b genotypesand28strains were the presence of PCV2a and PCV2b co-infection. The resultshowed that PCV2b genotype was the dominant genotype in Shandong area popularPCV2strains.Study4: Development and application of antibody in direction kit for Porcinecircovirus type2The expression vector was constructed for the recombinant Cap that removed thenuclear localization signal peptide (NSL). The recombinant Cap was inducedexpression and was purified by affinity chromatography. With purified recombinantprotein as the coated antigen, methods of ELISA diagnosis and diagnostic kit wereestablished for PCV2antibody that the condition of ELISA was optimized by thetest.The ELISA reaction conditions were cured and shandaded that ELISA kit wasassembled in optimal conditions. The results showed that the optimal coatingconcentration of antigen was determined by checkerboard titration and amounted to6.25μg/mL of fusion protein perwell, the serum sample for testing was diluted to1:100for detection, and the cutoff was determined to0.371. Compared with BeijinCentury Yan Heng company kit, the specificity, sensitivity, accuracy wererespectively90.0%,93.3%and91.4%. The assay showed no cross-reactivity with thereference sera of other swine virus: PCV1、PPV、PRV、CSFV、PRRSV、JEV. Thevariant coefficient of ELISA test kit in a batch and between batches varied from1.44%to2.94%and from2.26%to4.88%, respectively. The storage phase dentectionshowed that the storage period of up to months at4℃contion. The ELISA kit clinicaldetection showed that the correlation between the antibody positive rate withpathological degree and herd growth stage. The results show that the level of antibodyin pigs has significantly differences in different immune status. Results show that thedeveloped ELISA diagnostic kits with good specificity, sensitivity, fully meet thequality of similar products at home and abroad can be applied to large-scale clinicaltesting. This study indicated that an indirect ELISA method for detection PCV2inserum was successfully established, and could be used for the rapid clinic diagnosis ofPCV2.Study5: Development and application of Colloidal Gold Strip in detecting theantibody of Porcine Circovirus type2An immunochromatograpic strip was developed for the detection of antibody ofporcine circovirus type2(PCV2). Staphyloccal protein A (SPA) was labled with colloidal gold as a detection reagent, and recombinant ORF2protein of PCV2wasblotted on the test line, while pig IgG was used as the control line of the nitrocellulosemembrane.The results showed that the optimal pH value is8.0and the bestcombination is26μg/mL for the optimized marker SPA. The results showed that Capoptimal coating concentration was1.5mg/mL and the IgG package was aconcentration of0.8mg/mL in the condition. The test results could be observed in5-10min with the naked eye and the test operation was conveninent and fast fordetection of PCV2antidody. Compared with the indirect ELISA antibody PCV2kit,the sensitivity was95.2%and specificity was97.4%and coincidence rate was92.6%in the strip. There has no cross reaction for PCV1, CSFV, PRV, PRRSV, PPV positiveserum in detection of strip. The colloidal gold immunochromatographic stip has someadvantanges in sensibility and specificity and stability that has opetating simple andrapid.The results showed that the strip was suitable for rapid detection for manyclinical PCV2antibody samples in the work sites.