猪圆环病毒2型快速直接PCR检测方法的建立与流行病学调查
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摘要
为了解河南省猪圆环病毒2型(PCV2)的流行病学情况,参考Gen Bank的PCV2 ORF2的序列,设计1对引物,建立PCV2的快速直接PCR检测方法,并进行特异性和敏感性试验。运用该方法对2016年5月至2017年5月采集到的河南省共125份疑似猪圆环病毒2型的病料进行PCV2检测,阳性样品测序,分析河南省PCV2的遗传变异情况。结果显示,建立的PCR检测方法快速、特异、敏感,适用于临床检测或流行病学调查;所检测的125份样品中,55份样品为PCV2阳性,阳性率高达44%。阳性结果序列分析显示,核苷酸序列同源性在88.7%~99.8%,42份在PCV2b基因群。结果表明,河南省PCV2感染率较高,PCV2b为流行血清亚型,病毒变异严重。
To gain insight into molecular epidemiology of porcine circovirus type 2(PCV2) in Henan, a pairs of primers were designed with reference to the nucleotide sequence of PCV2 ORF2 in Gen Bank, the direct PCR detection method of PCV2 was established and the specificity and sensitivity test were carried out. 125 suspected Porcine Circovirus type 2 samples were collected and detected with PCV2 PCR in Henan from May 2016 to May 2017. Positive samples were sequenced and genetic variation of PCV2 in Henan was analyzed. The results showed that the established PCR method was rapid, specific, sensitive, and suitable for clinical testing or epidemiological investigation. 55 of the 125 samples were PCV2 positive, with positive rate reaching as high as 44%. The PCV2 ORF2 nucleotide sequence homology was 88.7%-99.8%, 42 samples were found in the PCV2 b gene cluster. The results showed that the rate of PCV2 infection in Henan was high, PCV2 b was the epidemic subtype, and the virus variation was serious.
To gain insight into molecular epidemiology of porcine circovirus type 2(PCV2) in Henan, a pairs of primers were designed with reference to the nucleotide sequence of PCV2 ORF2 in Gen Bank, the direct PCR detection method of PCV2 was established and the specificity and sensitivity test were carried out. 125 suspected Porcine Circovirus type 2 samples were collected and detected with PCV2 PCR in Henan from May 2016 to May 2017. Positive samples were sequenced and genetic variation of PCV2 in Henan was analyzed. The results showed that the established PCR method was rapid, specific, sensitive, and suitable for clinical testing or epidemiological investigation. 55 of the 125 samples were PCV2 positive, with positive rate reaching as high as 44%. The PCV2 ORF2 nucleotide sequence homology was 88.7%-99.8%, 42 samples were found in the PCV2 b gene cluster. The results showed that the rate of PCV2 infection in Henan was high, PCV2 b was the epidemic subtype, and the virus variation was serious.
引文
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[2]ALLAN G M,ELLIS J A.Porcine circoviruses:a review[J].Vet Diagn Invest,2000,12(1):3-14.
[3]ELLIS J,HASSARD L,CLARK E.Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome[J].Can Vet J,1998,39(1):44-51.
[4]张蕾,代松宝,张丽琳,等.PCV2逃逸宿主天然免疫的分子机制研究[J].华北农学报,2018,33(2):149-156.
[5]李富强,王利丽,李秀丽.猪圆环病毒2型分子生物学研究进展[J].天津农业科学,2015,21(9):57-60.
[6]高歌,孙荡,茅翔.猪Ⅱ型圆环病毒江苏株全基因进化及ORF3蛋白的生物信息学分析[J].山西农业科学,2012,40(8):881-885.
[7]刘建波.猪圆环病毒与猪圆环病毒2型分子生物学特性研究[D].北京:中国农业科学院,2014.
[8]OPRIESSNIG T,XIAO C T,GERBER P F.Identification of recently described porcine parvoviruses in archived North American samples from 1996 and association with porcine circovirus associated disease[J].Vet Microbiol,2014,173(1/2):9-16.
[9]HUYNH T M,NGUYEN B H,NGUYEN V G,et al.Phylogenetic and phylogeographic analyses of porcine circovirus type 2 among pig farms in Vietnam[J].Transbound Emerg Dis,2014,61(6):e25-34.
[10]WELTI S,SYDLER T,WIEDERKEHR D,et al.Postweaning multisystemic wasting syndrome(PMWS)and porcine dermatitis and nephropathy syndrome(PDNS)in Switzerland in the years2003-2006[J].Schweiz Arch Tierheilkd,2012,154(10):417-427.
[11]郎洪武,王丽,王琴,等.猪圆环病毒分离鉴定及猪断奶多系统衰弱综合征的诊断[J].中国兽医科技,2001,31(3):3-5.
[12]FAN S,KU X,CHEN F,et al.Complete genome sequence of a novel Porcine Circovirus type 3 strain,PCV3/Hubei-618/2016,isolated from China[J].Genome Announc,2017,5(15):e00100-00117.
[13]ZHENG S,WU X,ZHANG L,et al.The occurrence of porcine circovirus 3 without clinical infection signs in Shandong Province[J].Transbound Emerg Dis,2017,64(5):1337-1341.
[14]李鹏,郭军庆,金前跃,等.猪圆环病毒2型荧光定量PCR检测方法的建立[J].华北农学报,2014,29(2):66-70.
[15]樊振华,王娟萍,孟帆,等.猪细小病毒和猪圆环病毒2型多重PCR检测方法的建立与应用[J].山西农业科学,2012,40(10):1102-1106.
[16]ZHAI S L,CHEN S N,XU Z H,et al.Porcine circovirus type 2 in China:an update on and insights to its prevalence and control[J].Virol J,2014,14(11):88.
[17]SHI J,XU S,Fu F,et al.Phylogenetic analysis of 32 porcine circovirus type 2 isolates from Shandong,China[J].Arch Virol,2015(2):465-468.
[18]WEI C,ZHANG M,CHEN Y,et al.Genetic evolution and phylogenetic analysis of porcine circovirus type 2 infections in southern China from 2011 to 2012[J].Infect Genet Evol,2013,17:87-92.
[19]MU C,YANG Q,ZHANG Y,et al.Genetic variation and phylogenetic analysis of porcine circovirus type 2 infections in central China[J].Virus Genes,2012,45(3):463-473.
[20]焦文强,王治方,王克领,等.河南省及周边地区猪圆环病毒2型分子流行病学调查[J].河南农业科学,2017,46(5):125-129.