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

Establishment of RT-PCR diagnosis for bluetongue virus and epidemiological detection of blue tongue disease in cucoidopsis chinensis in Yunnan province

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 102.5TCID50/0.1 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.