山羊痘病毒LAMP检测方法的建立  

Establishment of LAMP Method for Detecting Goat Pox Virus

作　　者：徐滢 张琪[1] 付明哲[1] 许信刚[1] XU Ying;ZHANG Qi;FU Ming-zhe;XU Xin-gang(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi,712100,China)

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《动物医学进展》2020年第3期12-16,共5页Progress In Veterinary Medicine

基　　金：2018年省级农业专项资金支持科技创新驱动项目(K3360218035);西北农林科技大学试验示范站(基地)科技成果推广项目(TGZX2015-32)。

摘　　要：为了建立适用于基层养殖场快速、可视化的山羊痘病毒(GTPV)环介导等温扩增技术(LAMP)检测方法,根据GenBank公布的GTPV ORF103保守区核苷酸序列为靶序列,设计并合成一组特异性LAMP引物,优化反应条件(时间、温度)和反应体系(dNTP、内外引物浓度比),并进行特异性和灵敏度试验。结果表明,所建立的LAMP方法在65℃时反应50 min即可获得清晰的梯状条带;扩增物经10×SYBR GreenⅠ荧光染料染色,紫外线照射或日光下即可直接判定结果;灵敏度试验结果表明,该方法能够检出的GTPV核酸最低浓度为1.287 ng/mL,与常规PCR方法相比,灵敏度增高100倍;该方法特异性良好,与小反刍兽疫病毒(PPRV)、羊支原体(Mccp)、羊口疮病毒(ORFV)均无交叉反应;对10份临床样品检测结果显示,该方法与传统PCR检测结果一致。建立的LAMP检测方法可用于临床山羊痘病毒的检测。In order to establish a quick and visual LAMP method that can be used for grassroots farm,regarding GTPV ORF103 gene conserved sequence region published in GenBank as the target,a set of specific LAMP primers were designed and synthesized.The method was established by optimizing the reaction condition(such as time and temperature),reaction system(such as dNTP and primer concentration),specificity and sensitivity tests.The results showed that clear ladder bands can be obtained at 65℃ for 50 min using this method.Stained with 10×SYBR GreenⅠfluorescent dye,the amplified products were determined directly under UV irradiation or sunlight.The sensitivity test showed that the lowest GTPV nucleic acid concentration was 1.287 ng/mL,which was 100 times more sensitive than that of polymerase chain reaction.The results of specificity assay demonstrated that there was no cross reaction between peste des petits ruminants virus(PPRV),Mycoplasma capricolum subsp.capripneumonia(Mccp)and orf virus(ORFV),which came to a conclusion that the method was specific.10 cases of clinical samples were used for detection,it showed concordant results with polymerase chain reaction.This method can be applied to clinical detection of GTPV because of good specificity and sensitivity.

关 键 词：环介导等温扩增 山羊痘病毒 ORF103基因 检测方法 

分 类 号：S852.65[农业科学—基础兽医学]