| In recent years, there are many emerging pathogens in swine herds, especially smallDNA viruses,which directly or indirectly cause enormous economic losses for swineindustries at home and broad. Porcine circovirus type2(PCV2), porcine torqueteno susvirus (TTSuV) and porcine bocavirus (PBoV) are all small DNA viruses, the length of theirgenome varies from1.7kb to5.9kb. In this study, we mainly described their molecularepidemiology as follows. According to the principle of design of PCR primers, by modernmolecular biology techniques, The purpose of the experiment is establishingsimultaneously to detect PCV2and PBoV of multiple PCR detection methods and TTSuV1and TTSuV2multiple nested PCR test method. Constantly optimize the single PCR primerconcentration and the annealing temperature conditions, to establish an optimal detectionsystem for testing samples.The121samples collected from lungs, spleen, lymph nodes and small intestine ofsuspected infection of PCV2virus at the various stages of infected pigs from23regions ofJiangxi in2013were detected PCV2and PBoV by multiple PCR. PCV2positive simpleswere detected in the number of79copies of the material, the positive rate was65.29%,PBoV-positive number is68, the positive rate of56.2%, the number of PCV2and PBoVmixed infection is59, the infection rate was48.67%.67copies of the blood samples with corresponding tissue samples collected from9regions of Jiangxi are tested, the results showed that infected TTSuV1and TTSuV2positive rate was55.22%and65.67%, especially mixed infection rate was41.79%.34samples showed a mixed infection of PCV2and TTSuV1, accounting for50.75%of thetotal sample;42samples showed mixed infection of PCV2and TTSuV2, accounting for62.69%of the total sample; Addition, there are28samples of triple infection, accountingfor41.79%of the total sample.Meanwhile, the rapid detection of PCV2method,loop-mediated isothermalamplification (Loop-mediated isothermal amplification, LAMP), is established in text, thesensitivity of method is higher100times than conventional PCR, and has a high specificityfor PCV2.By using conventional PCR and LAMP,the121samples of suspected infectionof PCV2virus are respectively detected. the results showed that: the LAMP detection rateis65.29%, that is same rate by PCR.By PCR and LAMP method,the positive rate of resultis exactly same.According PCV2genome sequence published on GenBank designed a pair ofback-to-back primers for amplification of PCV2whole genome sequence.Afteramplification by PCR and sequencing, we got the whole genome sequence of9strains inJiangxi PCV2isolates.The results showed that the complete genome of eight out of the9strain all1767bp in length and one strains were1768bp,by analyzing the whole genomesequences of the nucleotide and protein sequences and producting phylogenetic tree. Thehomology of nucleotide sequences of the9strains was94.7%-99.8%. Compared with others whole genome sequences of PCV2in Genbank, the homology was94.3%-99.8%,Phylogenetic tree analysis showed that the9strains could be divided into3genotypes,5strains(JJ-JX-CN,GA-JX-CN, XJ-JX-CN, NC-JX-CNand YC-JX-CN)forming a branch were closed to the representative strain of UK (AF055394)and belong toPCV2b;3strains(JA-JX-CN,FZ-JX-CN and SR-JX-CN)were close to the representativestrains of China(AY181946) belong to PCV2d. GZ-JX-CN were closed to therepresentative strains of Japan(AB426905)belong to PCV2a. The homology of nucleotidesequences identity of the ORF2,ORF1and ORF3of the9strain had91.2%-99.9%,96.9%-100.0%and97.1%-100%,and the homology of amino acid identity of the ORF2,ORF1and ORF3of the9strain had80.3%-100%,97.8%-99.7%and96.2%-100%. Theseconclusions providing a theoretical basis and reference value for a better understanding ofPCV2in the prevalence and evolution of the Jiangxi region. |
