猪轮状病毒(G9型)TaqMan荧光定量RT-PCR检测方法的建立  

Development of Taq Man probe-based RT-PCR of porcine rotavirus(G9-genotype)

作　　者：王宇飞[1] 时洪艳[2] 朱向东[2] 陈建飞[2] 袁婧[2] 张鑫[2] 石达[2] 刘建波[2] 王潇博[2] 高晶[3] 冯力[2] 

机构地区：[1]东北农业大学生命科学学院,黑龙江哈尔滨150001 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/猪消化道团队,黑龙江哈尔滨150001 [3]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《中国预防兽医学报》2015年第11期846-849,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省科研机构创新能力提升专项(YC 2015 D011)

摘　　要：为建立灵敏、特异的G9型猪轮状病毒(Po RV)Taq Man的荧光定量RT-PCR检测方法,本研究根据Gen Bank中登录的G9型Po RV NMTL株的VP7基因保守区域设计一条特异性引物和探针。以104TCID50的病毒液所提取的总RNA进行10倍系列稀释作为标准品,通过对反应条件进行优化,建立了检测G9型Po RV的Taq Man荧光定量RT-PCR检测方法。结果显示,该方法的相关系数R2=0.991,对其它常见的Po RV(G5)、猪传染性胃肠炎、猪流行性腹泻、猪繁殖与呼吸综合征和猪圆环病毒2型等猪病病原检测均为阴性,具有良好的特异性;该方法最低可以检测到的病毒滴度为10-3TCID50,比普通RT-PCR方法灵敏度高约1 000倍;组内和组间变异系数均小于0.87%,稳定性高,重复性好。对疑似感染Po RV的45份临床病料样品进行检测,结果表明,该方法阳性率(64.4%)高于普通RT-PCR(44.4%),两者总符合率为80%。本研究建立的方法为Po RV的流行病学调查和早期诊断奠定基础。In order to establish a specific and sensitive method for detection of G9-genotype porcine rotavirus （PoRV）, a pair of probe and primers was designed based on the conserved region of VP7 gene of PoRV（G9） strain NMTL. Under the optimized conditions, the standard curve was set up by a serious 10-fold dilutions of the total RNA from cell-adapted virus of 104 TCID50 which showed the linear correlation coefficient of R2=-0.991. This assay was specific for G9-genotype PoRV detection, but had no cross-reactions with other related porcine pathogens, such as, PoRV （G5-genotype）, transmissible gastroenteritis virus, porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus and porcine cirovirus type 2; Moreover, the detection limit of the assay was 10-3 TCID50, which was 1,000 times more sensitive than that of conventional RT-PCR. The repetitive tests of intra- and inter-coefficient of variation showed that the Ct values were both less than 0.87%. The positive ratio of this method （64.4%） was higher than conventional RT-PCR （44.4%） when detecting 45 clinical samples, indicating that this method was much more sensitive.

关 键 词：轮状病毒 G9基因型 VP7基因 TaqMan荧光定量RT—PCR 

分 类 号：S852.65[农业科学—基础兽医学]