蓝舌病毒RT-PCR检测方法的建立及对云南省库蠓蓝舌病流行病学检测
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摘要
建立蓝舌病毒(bluetongue virus,BTV)RT-PCR检测方法,用于2018年云南省库蠓的蓝舌病(BT)流行病学检测。设计2对保守引物,优化RT-PCR方法的退火温度、循环次数、退火时间和延伸时间,并进行灵敏性和特异性评价。采集了昭通市3个县及普洱市澜沧县的BT传播媒介库蠓共25万余只,进行研磨后,用建立的RT-PCR方法检测样本。筛选出1对引物BTVS10-F和BTVS10-R作为蓝舌病毒RT-PCR检测引物,敏感度达到10~(2.5) TCID_(50)/0.1 mL,通过对BTV16型的阳性病毒检测,证实该方法的可行性。
The method of RT-PCR detection of bluetongue virus(BTV) was established and used for epidemiological detection of bluetongue disease carried in Cangwu,Yunnan province in 2018.Two pairs of more conservative primers were designed to establish RT-PCR detection.The annealing temperature,cycle number,annealing time and extension time of RT-PCR method were optimized to evaluate its sensitivity and specificity.On the basis of establishing the RT-PCR detection method,a total of more than 250 000 aphids were collected from the bluetongue transmission media(Cangyu) in three counties of Zhaotong city and the county of Pu?er city.After grinding,the established RT-PCR was used to detect bluetongue virus in the sample.A pair of primers BTVS10-F and BTVS10-R were screened as primers for blue-tongue virus RT-PCR.The sensitivity of this method can reach 10~(2.5)TCID_(50)/0.1 mL.The feasibility of the method was confirmed by detecting the positive virus of BTV16.The detection method of blue-tongue disease by RT-PCR in this experiment can detect blue tongue virus;the established RT-PCR method was used to detect 250 000 samples from Yunnan province,and no blue tongue virus was detected.
The method of RT-PCR detection of bluetongue virus(BTV) was established and used for epidemiological detection of bluetongue disease carried in Cangwu,Yunnan province in 2018.Two pairs of more conservative primers were designed to establish RT-PCR detection.The annealing temperature,cycle number,annealing time and extension time of RT-PCR method were optimized to evaluate its sensitivity and specificity.On the basis of establishing the RT-PCR detection method,a total of more than 250 000 aphids were collected from the bluetongue transmission media(Cangyu) in three counties of Zhaotong city and the county of Pu?er city.After grinding,the established RT-PCR was used to detect bluetongue virus in the sample.A pair of primers BTVS10-F and BTVS10-R were screened as primers for blue-tongue virus RT-PCR.The sensitivity of this method can reach 10~(2.5)TCID_(50)/0.1 mL.The feasibility of the method was confirmed by detecting the positive virus of BTV16.The detection method of blue-tongue disease by RT-PCR in this experiment can detect blue tongue virus;the established RT-PCR method was used to detect 250 000 samples from Yunnan province,and no blue tongue virus was detected.
引文
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[12] 史喜菊,马贵平,柏亚铎,等.多重连接探针扩增对5种牛病毒同步检测方法的建立[J].中国兽医学报,2017,37(2):224-230.
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[2] 宋红梅,杨涛,马健男,等.蓝舌病毒VP7基因的原核表达[J].中国预防兽医学报,2009,31(12):925-928.
[3] 耿宏伟,秦永丽,李俊平,等,蓝舌病病毒重组VP7蛋白单克隆抗体制备及竞争ELISA 检测方法的建立[J].中国预防兽医学报,2012,34(5):388-392.
[4] CARPI G,HOLMES E C,KITCHEN A.The evolutionary dynamics of bluetongue virus[J].J Mol Evol,2010,70(6):583-592.
[5] GERDESG H.A south African overview of the virus,vectors,surveillance and unique features of bluetongue[J].Vet Ital,2004,40(3):39-42.
[6] 张胜男,吕建华,常建华,等.2013年内蒙古地区蓝舌病流行情况调查[J].中国预防兽医学报,2014,36(10):75-762.
[7] 张念祖,李志华,张开礼,等.绵羊蓝舌病的调查研究报告[J].云南畜牧兽医,1989(4):3-12.
[8] 李志华,张开礼,皱福中,等.绵羊蓝舌病(湖北毒株)的分离鉴定报告[J].云南畜牧兽医,1989(4):49-51.
[9] 周维翰.安徽省蓝舌病流行病学调查[J].畜牧与兽医,1995,27(1):3-5.
[10] 林理惠,黄巧玲,康长风,等.四川省蓝舌病调查和病原分离报告[J].云南畜牧兽医,1989(4):57-60.
[11] 许建民,雷怀朋,李文昭.绵羊蓝舌病的诊断[J].中国兽医杂志,1993,19(1):13-14.
[12] 史喜菊,马贵平,柏亚铎,等.多重连接探针扩增对5种牛病毒同步检测方法的建立[J].中国兽医学报,2017,37(2):224-230.
[13] YANG T,LIU N,XU Q,et al.Complete genomic sequence of bluetongue virus serotype 1 from China[J].J Virol,2012,86(2):1288-1289.
[14] BATTEN C A,HENSTOCK M R,STEEDMAN H M,et al.Bluetongue virus serotype 26:infection kinetics,pathogenesis and possible contact transmission in goats[J].Vet Microbiol,2013,162(1):62-67.