3种核酸提取方法在病媒生物携带肠道致病菌检测中的应用比较  

Comparison of Three Nucleic Acid Extraction Methods in the Detection of Intestinal Pathogenic Bacteria Carried by Vectors

作　　者：张思祺 何建安 赵纯中 闫俊宇 刘国平[1] Zhang Siqi;He Jian'an;Zhao Chunzhong;Yan Junyu;Liu Guoping(College of Animal Science and Technology,Yangtze University,Jingzhou 434025,Hubei,China;Shenzhen International Travel Health Care Center,Out-Patient Department of Shenzhen Customs Port,Shenzhen 518000,Guangdong,China)

机构地区：[1]长江大学动物科学技术学院,湖北荆州434025 [2]深圳国际旅行卫生保健中心(深圳海关口岸门诊部),广东深圳518000

出　　处：《中国动物检疫》2024年第12期95-102,共8页China Animal Health Inspection

基　　金：国家重点研发计划项目(2022YFC2302804);口岸重要消化道病原体传播风险预警技术研究项目。

摘　　要：为比较并筛选出简便、经济、高效的提取病媒生物携带肠道致病菌核酸的方法,采用3种代表性核酸提取方法一一柱式提取法、磁珠吸附法、盐析法,从模拟感染沙门菌、志贺菌及大肠杆菌的苍蝇和样品中提取核酸,并系统比较核酸质量浓度、荧光定量PCR(qPCR)检测Ct值和细菌回收率,来全面评估各种方法的提取效率。结果显示:3种核酸提取方法中柱式提取法及盐析法均能成功从模拟感染样品中提取核酸,磁珠吸附法未能全部成功提取;通过比较核酸质量浓度、qPCR检测Ct值及回收率,柱式核酸提取法的提取效率最高。结果表明,在检测病毒载量较低的样品,如病媒生物样品时,可选择性价比高、操作简便且结果准确的核酸提取方法(如柱式提取法),以提升检测结果的准确性。In order to compare and select a simple,economic and efficient method for extracting nucleic acids of intestinal pathogenic bacteria carried by vectors,nucleic acids were extracted from flies and cockroaches simulated to be infected with salmonella,Shigella and Escherichia coli using three representative methods including column extraction,magnetic bead adsorption and salt precipitation,and the mass concentration of nucleic acids,Ct value measured by quantitative fluorescence PCR(qPCR)and bacterial recovery rate were systematacially compared to fully assess the extraction efficiency of the above methods.The results showed that nucleic acids were successfully extracted from the simulated samples by both column extraction and salt precipitation,and partially by magnetic bead adsorption;and the extraction efficiency of the column extraction was the highest through comparison of mass concentration of nucleic acids,the Ct value and bacterial recovery rate.In conclusion,the affordable,accessible and accurate method,such as column extraction,could be used to detect samples with low viral load,such as vector samples,in order to improve the accuracy of detection results.

关 键 词：肠道致病菌 核酸提取方法 病媒生物 口岸检疫 

分 类 号：S851.3[农业科学—预防兽医学]