非典型猪瘟病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立
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摘要
参照GenBank上登录的非典型猪瘟病毒(atypical porcine pestivirus virus,APPV)全基因组序列设计特异性引物成功从感染APPV阳性样品中扩增出270 bp的APPV基因片段,并将其克隆到pEASY-Bluent Zero Cloning Kit载体,以纯化的重组质粒为模板优化反应条件,建立了检测APPV的特异性荧光定量PCR方法。结果显示,建立的荧光定量PCR方法C_t值与标准品在1.49×10~1～1.49×10~(10) copies/μL呈现良好的线性关系,相关系数为R~20.999,斜率为-3.442,扩增效率为95.207%。该方法与PCV2、PRV、PRRSV、PPV及CSFV基因组均无交叉反应,敏感性比常规PCR高出1 000倍,组内和组间重复试验变异系数均小于2%。对临床采集的20份疑似感染APPV的样品进行检测,结果显示应用所建立的荧光定量PCR成功检测到3份APPV阳性样品,而常规PCR仅检测到1份,表明所建立的荧光定量PCR比常规PCR灵敏度更高。本试验所建立的APPV SYBR GreenⅠ实时荧光定量PCR检测方法,可实现对APPV早期诊断及感染进行定量分析检测。
A 270 bp conserved region of atypical porcine pestivirus virus(APPV) gene was amplified using specific primers by conventional PCR from the positive samples which infected with APPV and cloned into pEASY-Bluent Zero Cloning Kit vector and sequenced.The plasmid DNA was used as standard DNA temple to optimize assay condition of developing a SYBR GreenⅠreal-time quantitative PCR for specificity detection of APPV.The standard curve generated a wide dynamic range from 1.49×10~1 to 1.49×10~9 copies/μL with a linear correlation(R~2) of 0.999 and efficiency of 95.207% between the C_(t )value and the logarithm of the plasmid copy number.No amplification was detected by this method in unrelated DNA or RNA samples,including porcine circovirus type 2,pseudorabies virus,porcine reproductive and respiratory syndrome virus,porcine parvovirus virus and classical swine fever virus.Good reproducibility was obtained for detecting constructed positive plasmid DNA with intra-assay variation of 0.090% to 0.683% and inter-assay variation of 0.367%to 1.331%.After detected 20 samples of suspected APPV positive samples,3 samples were detected for APPV using real-time fluorescence quantitative PCR and 1 positive samples were detected by conventional PCR,indicating that the real-time fluorescence quantitative PCR was more sensitive than conventional PCR.The SYBR GreenⅠreal-time fluorescence quantitative PCR detection method established in this research can realize the quantitative analysis and detection of APPV early diagnosis and infection of APPV.
A 270 bp conserved region of atypical porcine pestivirus virus(APPV) gene was amplified using specific primers by conventional PCR from the positive samples which infected with APPV and cloned into pEASY-Bluent Zero Cloning Kit vector and sequenced.The plasmid DNA was used as standard DNA temple to optimize assay condition of developing a SYBR GreenⅠreal-time quantitative PCR for specificity detection of APPV.The standard curve generated a wide dynamic range from 1.49×10~1 to 1.49×10~9 copies/μL with a linear correlation(R~2) of 0.999 and efficiency of 95.207% between the C_(t )value and the logarithm of the plasmid copy number.No amplification was detected by this method in unrelated DNA or RNA samples,including porcine circovirus type 2,pseudorabies virus,porcine reproductive and respiratory syndrome virus,porcine parvovirus virus and classical swine fever virus.Good reproducibility was obtained for detecting constructed positive plasmid DNA with intra-assay variation of 0.090% to 0.683% and inter-assay variation of 0.367%to 1.331%.After detected 20 samples of suspected APPV positive samples,3 samples were detected for APPV using real-time fluorescence quantitative PCR and 1 positive samples were detected by conventional PCR,indicating that the real-time fluorescence quantitative PCR was more sensitive than conventional PCR.The SYBR GreenⅠreal-time fluorescence quantitative PCR detection method established in this research can realize the quantitative analysis and detection of APPV early diagnosis and infection of APPV.
引文
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[2] WU Z,REN X,YANG L,et al.Virome analysis for identification of novel mammalian viruses in bat species from Chinese provinces[J].J Virol,2012,86(20):10999-11012.
[3] FIRTH C,BHAT M,FIRTH M A,et al.Detection of zoonotic pathogens and characterization of novel viruses carried by commensal rattus norvegicus in New York city[J].MBio,2014,5(5):e01933-01914.
[4] HAUSE B M,COLLIN E A,PEDDIREDDI L,et al.Discovery of a novel putative atypical porcine pestivirus in pigs in the USA[J].J Gen Virol,2015,96(10):2994-2998.
[5] ARRUDA B L,ARRUDA P H,MAGSTADT D R,et al.Identification of a divergent lineage porcine pestivirus in Nursing piglets with congenital tremors and reproduction of disease following experimental inoculation[J].PLoS One,2016,11(2):e0150104.
[6] YUAN J,HAN Z,LI J,et al.Atypical porcine pestivirus as a novel type of pestivirus in pigs in China[J].Front Microbiol,2017(8):862.
[7] KNOX B,ASKAA J,BASSE A,et al.Congenital ataxia and tremor with cerebellar hypoplasia in piglets borne by sows treated with Neguvon vet(metrifonate,trichlorfon) during pregnancy[J].Nord Vet Med,1978,30(12):538-545.
[8] BEER M,WERNIKE K,DRAGER C,et al.Highprevalence of highly variable atypical porcine pestiviruses found in Germany[J].Transbound Emerg Dis,2017,64(5):e22-e26.
[9] DE GROOF A,DEIJS M,GUELEN L,et al.Atypical porcine pestivirus:a possible cause of congenital tremor type A-Ⅱ in newborn piglets[J].Viruses,2016,8(10):E271.