摘要
山羊痘和绵羊痘是由羊痘病毒引起的一种急性、热性、接触性传染病。为了更快、更准确的诊断该病,本研究根据Gen Bank已经发表的GTPV和SPPV参考毒株(登录号分别为AY077836和AY077832)A29L基因序列,设计合成了1对特异性引物和2条Taq Man探针,同时制备重组质粒标准品。经过反应条件的优化,建立了羊痘病毒实时荧光定量PCR检测方法,对所建立的实时荧光定量PCR方法反应体系的特异性、灵敏性和重复性进行了评价,并用此方法对19份临床样品进行了检测。结果显示:该诊断方法与4种非羊痘病毒不发生交叉反应,并且山羊痘病毒和绵羊痘病毒之间也不发生交叉反应。该方法的重复性试验变异系数均低于2%,并且双重实时荧光定量PCR最低浓度检测限分别为470和440 fg。对标准阳性模板进行定量检测,生成的标准曲线相关系数分别为0.995和0.997。在检测的临床样品中,GTPV和SPPV阳性分别有14和4份,混合感染有1份。结果表明所建立的方法具有良好的特异性,灵敏性、稳定性,可以对GTPV和SPPV进行准确的定量检测。
Goat pox and sheep pox are a kind of acute, febrile, contagious infectious disease caused by capripoxvirus. The purpose of this study is to develop a faster and more accurate diagnostic method. Based on the published reference strains of GTPV and SPPV （the accession number is re- spectively AY077836 and AY077832） by GenBank and A29L gene sequence, the study designed and synthesized a pair of specific primers and two Taq Man probes, and prepared and regrouped plasmid standards at the same time. After the optimization of reaction condition,a real-time quan- titative PCR method for detection of capripoxvirus was established and an evaluation on the speci- ficity, sensitivity and repeatability of reaction system for this method was conducted with detecting ! 9 clinical samples by this method. The result indicates that this diagnostic method did not gener- ate cross reaction with four kinds of non-capripoxvirus,and neither does the situation between goat pox and sheep pox. The repeatable test variable coefficients of this method are all under 2% ,and the minimum concentration LOD of double real-time quantitative PCR is 470 and 440 fg,respectively. The quantitative determination on the standard positive template generates standard curve correlation coefficients of 0. 995 and 0. 997 relatively. In the clinical samples detec- ted,the numbers of positive GTPV and SPPV are relatively 14 and 4,and 1 mixed infection. The result shows that the method is in good specificity, sensitivity and stability,which can be used to accurately conduct a quantitative determination for GTPV and SPPV.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2016年第4期572-578,共7页
Chinese Journal of Veterinary Science
基金
兽用疫苗国家工程实验室资助项目
