Development And Application Of Real-time RT-PCR For Detection Porcinecircovirus Type2

Porcine circovirus type2(porcine circovirus2, PCV2) is a main pathogen of postweaningmultisystemic wasting syndrome(PMWS), Procine dermatitis and nephropathysyndrome(PDNS), swine reproductive disorder svndmme (SRDS), Congenital tremors ofpiglets(CT), Porcine respiratory complex(PRBC) and other diseases.It is one of theimportant pathogens threatening the world’s pig industry now. This study established areal-time SYBR Green quantitative PCR method to detect PCV2DNA and a TaqManreal-time One-Step RT-PCR method to detect PCV2RNA.These methods can befoundational research tools for dynamic detection of PCV2virus load, identificat detectionof PCV2clinical and subclinical infection pigs, evaluation of virual tissue tropism andprevention. The main contents and results are as follows:1PVC2ORF2gene cloning: The specific primers were designed according to thenucleotide sequence of porcine circovirus type2(PCV2) available in GenBank, geneamplified by PCR, connection, transformation, positive clone selection and identification,determination of nucleotide sequence. cloned into the vector,screened positive plasmidstandards.2Establishment and application of SYBR Green fluorescence quantitative PCR fordetection of porcine circovirus type2: The specific primers were designed according to thenucleotide sequence of porcine circovirus type2(PCV2) and porcine circovirus type1(PCV1) available in GenBank,gene amplified by PCR and cloned into the pMD18-Tvector,screened positive plasmid standards.By quantitative PCR optimization of reactionconditions,establishment of a real-time detection of PCV2SYBR Green quantitative PCRmethod. The results indicated that the method was highly specificial The detection limit ofthe assay was10copies/μ L of plasmid DNA,1000times higher than that of the routinePCR.The standard curve displayed a linear range from1×102to1×107copies and a goodreproducibility.3Establishment of Taq Man fluorescence quantitative PCR for detection of porcinecircovirus type2: A TaqMan real-time One-Step RT-PCR assay was developed for detectionof RNA transcripts produced by replicating PCV2. A pair of primers and a TaqMan probetargeting the spliced Cap were designed. A synthetic p-32T-Cap DNA fragment wasconstructed to mimic the spliced mRNA by PCR-based gene assembly and was used for generation of standard RNAs. The detection limit of the assay was10RNA copies/μ L andstandard curve displayed a linear range from1×100to1×109copies and a goodreproducibility.4The newly developed real-time RT-PCR assay was applied to determine the viralmRNA loads in different tissues of PCV2naturally infected pigs and to evaluate the load ofPCV2mRNA and DNA of supernatants and criolysates were taken separately post-infectionfrom the PK-15cells. Results indicate that intracellular PCV2mRNA and DNA was detectedas early as24h with significant increase from48～96h. A parallel increase in the number ofviral DNA copies was detected in the supernatants. But PCV2mRNA load detected in thesupernatants collection did not increase significantly from48～96h. The clinical results showedthat PCV2mRNA was demonstrated in a variety of tissues, showing a wide distribution ofthe infectious virus in the organism. However,viral mRNA loads were not strictly correlatedwith the counterpart DNA titres.