毒害艾美耳球虫和产气荚膜梭菌双重PCR检测方法的建立  

Establishment of Duplex PCR Methods for Detection of Eimeria necatrix and Clostridium perfringens

作　　者：陈曦[1] 王一 王佳丽 杨新 宋军科[1] 赵光辉[1] CHEN Xi;WANG Yi;WANG Jiali;YANG Xin;SONG Junke;ZHAO Guanghui(College of Veterinary Medicine,Northwest A&F University,Yangling 712100,China)

机构地区：[1]西北农林科技大学动物医学院,杨凌712100

出　　处：《畜牧兽医学报》2023年第9期3985-3990,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：陕西省重点研发计划项目(2020NY-006);陕西省创新能力支撑计划项目(2021TD-31)。

摘　　要：旨在初步建立可同时检测毒害艾美耳球虫(Eimeria necatrix)和产气荚膜梭菌(Clostridium perfringens)的双重PCR及纳米PCR检测方法。基于E.necatrix的ITS-2基因和C.perfringens的α毒素基因分别设计、筛选靶基因位点的特异性扩增引物,通过反应体系和退火温度的优化,建立适用于两种病原的特异性双重PCR和双重纳米PCR检测方法;用所建立的方法对6种其它常见寄生性原虫和3种其他细菌进行PCR扩增以验证其特异性;构建两种病原的重组质粒pMD19-T-ITS2和pMD19-T-cpa,将其按10倍倍比稀释成不同浓度梯度的质粒模板用于所建方法的敏感性试验。结果表明:两种方法均能特异性扩增出E.necatrix约150 bp和C.perfringens约400 bp的目的片段,且所建立的两种检测方法对其他6种其他原虫和3种其他细菌的检测结果均为阴性;双重PCR扩增E.necatrix和C.perfringens的最低模板检出量分别是181和1050 copies,而双重纳米PCR的最低模板检出量分别是1.81和105 copies;临床检测结果显示,所建立的2种方法与临床病原学检测结果一致。成功建立了用于E.necatrix和C.perfringens双重PCR和双重纳米PCR检测方法,且敏感性好、特异性高,可为临床上毒害艾美耳球虫和产气荚膜梭菌感染的检测提供技术支持。The aim of this study was to establish a duplex PCR and a duplex nano-PCR method for simultaneous detection of Eimeria necatrix and Clostridium perfringens.Primers for target gene loci were designed and screened based on the E.necatrix ITS-2 gene and the C.perfringensαtoxin gene,respectively,and specific duplex PCR and nano-PCR detection methods for two pathogens were established by optimizing reaction systems and annealing temperatures.PCR amplifications of six other protozoan parasites and three other bacteria were used to verify their specificities.The recombinant plasmids pMD19-T-ITS2 and pMD19-T-CPA were further constructed and diluted into different concentration gradients with 10 times ratio for sensitivity tests of the proposed methods.The results showed that the target fragments of E.necatrix and C.perfringens were about 150 bp and 400 bp,respectively.The PCR amplifications using the two detection methods were negative for other six protozoan parasites and three other bacteria.The minimum detectable limits for E.necatrix and C.perfringens by using dual PCR were 181 copies and 1050 copies,respectively,while the minimum detectable limits for E.necatrix and C.perfringens by using dual nano-PCR were 1.81 and 105 copies,respectively.Clinical detection showed that the detection results of two established methods were consistent with clinical pathogenic detection.The present study successfully established duplex PCR and duplex nano-PCR methods for detection of E.necatrix and C.Perfringens,with high sensitivity and specificity,providing technical supports for clinical detection of E.necatrix and C.perfringens infections.

关 键 词：毒害艾美耳球虫 产气荚膜梭菌 PCR 纳米PCR 

分 类 号：S854.43[农业科学—临床兽医学]