方法检测猪瘟病毒的 比较及应用

中国动物传染病学报　2010,18(3):66-72

Chinese Journal of Ani m al I nfecti ous D iseases

・简报・

两种荧光定量PCR 方法检测猪瘟病毒的

比较及应用

袁雪梅

1,2

,陈　宁2,陈　宇3,王　萍1,童　超2,李得江2,万　婧2,李肖梁2,方维焕

2

(1.江西农业大学动物科学技术学院,南昌330045;2.浙江大学动物预防医学研究所

浙江省动物预防医学重点实验室,杭州310029;3.农标普瑞纳(嘉兴)饲料有限公司,嘉兴314002)

摘　要:根据猪瘟病毒(Classical s wine fever virus,CSF V )5θ端非编码区保守区域,设计猪瘟病毒特异性的引物和探针,建立了基于SY BR Green 和Taq M an 探针的两种荧光定量PCR 检测方法。灵敏性和特异性试验结果表明,两种方法能够特异性地检测出不同型的猪瘟病毒,而对同属的牛病毒性腹泻病病毒以及其它一些主要猪病病原检测结果均为阴性。两种方法对猪瘟病毒C 株的检测下限均为101T C I D 50,均高于常规的套式PCR 。

用携带该基因片段的重组质粒为模板进行检测,SY BR Green 方法的检测下限为3×100cop ies,比Taq Man 探针

方法更加灵敏(3×101

cop ies )。应用建立的SY BR Green 方法对2009年采集于浙江地区不同猪场的20份病猪

样品进行CSF V 检测,有8份样品为阳性,与套式PCR 方法检测结果一致。进一步应用SY BR Green 方法对不同感染阶段细胞内病毒RNA 复制水平进行检测,与病毒滴度的结果基本一致。因此,本研究建立的SY BR

Green 荧光定量PCR 检测方法能够应用于组织和培养细胞中猪瘟病毒的检测。

关键词:猪瘟病毒;5θ非编码区;荧光定量PCR 中图分类号:S852.659.6 文献标识码:A

文章编号:1674-6422(2010)03-0066-07

收稿日期:2010204222

基金项目:杭州市科技创新服务平台项目(20082332Y03)作者简介:袁雪梅,女,硕士研究生,预防兽医学专业通讯作者:方维焕,E 2mail:whfang@zju .edu .on

COM PAR I S O N AND APPL I CAT I O N O F S Y BR GREEN AN D TAQ M AN

BASED REAL 2T IM E PCR ASSAY S FO R D ETECT I O N O F

CLASS I CAL S W INE FEVER V I RUS

YUAN X ue 2m ei

1,2

,CH EN N ing 2,CH EN Yu 3,W AN G Ping 1,TON G C hao 2

,

L I D e 2jiang 2

,W AN J ing 2

,L I X iao 2liang 2

,FAN G W ei 2huan

2

(1.College of A ni m al Science,J iangxi A gricultural U niversity,N anchang 330045,China; 2.Zhejiang Provincial Key

L aboratory of Preventive V eterinary M edicine,Institute of Preventive V eterinary M edcine,Zhejiang U niversity,

Hangzhou 310029,China; 3.A gribrands Purina Feedm ill Co .,L td,J iaxing 314002,China )

Abstract:SY BR Green and Taq Man p r obe based quantitative real 2ti m e PCR (qRT 2PCR )assays targeting the conserved 5θ2UTR regi on were established for detecti on of classical s wine fever virus (CSF V ).The t w o assays were capable of s pecifically detecting different gr oup s of CSF V,whereas bovine viral diarrhea virus and a nu mber of other porcine viruses were tested negative .The detecti on li m it was 101

TC I D 50f or both assays on the vaccine C 2strain,more sensitive than that of conventi onal RT 2nested PCR.U sing the recombinant p las m id containing the 5θ2UT R gene as te mp late,the detecti on li m it was 3.0×100

and 3.0×101

cop ies for SY BR Green and Taq Man based qRT 2PCR,res pectively .Out of 20sa mp les of diseased p igs collected fr om Zhejiang p r ovince in 2009,8sa mp les were positive f or CSF V by SY BR Green based qRT 2PCR,which was consistent with those using RT 2nested PCR.The SY BR Green based qRT 2PCR was further app lied t o detecti on of viral RNA in PK 215cells infected with CSF V.The curve of accu mulated viral RNA was in agree ment with the infectivity titer at different ti m es post 2infecti on .Theref ore,the SY BR