弓形虫529重复序列检测方法的比较  

Comparison of methods for detection of the repetitive sequence fragments of Toxoplasma gondii

作　　者：殷位刚 朱银银 杨佩才 张洪英[1,2] YIN Weigang;ZHU Yinyin;YANG Peicai;ZHANG Hongying(Nanjing Center for Disease Control and Prevention Affiliated to Nanjing Medical University,Nanjing 210003,China;School of Public Health,Nanjing Medical University,Nanjing 211166,China)

机构地区：[1]南京医科大学附属南京疾病预防控制中心,江苏南京210003 [2]南京医科大学公共卫生学院,江苏南京211166

出　　处：《畜牧与兽医》2023年第12期111-116,共6页Animal Husbandry & Veterinary Medicine

基　　金：江苏省地病协会项目(X202130)。

摘　　要：旨在通过比较针对弓形虫529 bp重复序列片段(Rep-529)的环介导等温扩增(LAMP)和荧光定量PCR(qPCR),筛选出较优的弓形虫DNA检测方法,并初步应用于宠物猫弓形虫感染检测。试验以含有弓形虫Rep-529的质粒作为模板,比较文献报道和自主设计的引物组的最低检测限,并用其他病原DNA进行交叉反应性测试,用筛选出的最优引物组对宠物猫粪中弓形虫DNA进行检测。结果表明:以包含Rep-529的质粒为模板比较检出限,LAMP 529-11引物组检测限最低,为10^(3) copies/μL;其次为文献报道的LAMP RE引物组和qPCR引物组,检测限均为10^(4) copies/μL;LAMP 529-3和2021引物组的检测限最高,均为10^(5) copies/μL;各引物组对隐孢子虫、贾第鞭毛虫、旋毛虫、大肠杆菌、鼠伤寒沙门菌、细小病毒DNA均无扩增;用LAMP 529-11引物组,对39只宠物猫弓形虫总检出率为33.33%(13/39),其中29份粪便中检出11份阳性(37.93%,11/29),10份肛拭子中检出2份阳性(20.00%,2/10)。总之,以弓形虫Rep-529重复序列为靶基因,本研究筛选获得的LAMP 529-11引物组快速、敏感且特异,在30 min内对弓形虫DNA进行有效扩增;用该法检测发现,33.33%的宠物猫正在排出卵囊,对环境和人类具有较大的潜在污染和传染风险。In this study,the loop mediated isothermal amplification(LAMP) and fluorescence quantitative PCR(qPCR) targeting the repetitive sequence fragments of Toxoplasma gondii(Rep-529) were compared,and the optimal DNA detection method was screened out and preliminarily applied to molecular detection of Toxoplasma gondii infection in pet cats.A plasmid containing a 529 bp repetitive sequence fragment(Rep-529) of Toxoplasma gondii was synthesized as a template,and the minimum detection limit of the reported and self-designed primer groups was compared.Next,cross-reactivity was tested using DNA extracts from other pathogens;and the selected optimal primer set was used to detect Toxoplasma gondii DNA in pet cat feces.The results showed that,when the detection limit was compared with the plasmid containing Rep-529 as a template,the detection limit was the lowest in the primer group 529-11 in LAMP.This was followed by the LAMP RE primer set and qPCR,both of which had a detection limit of 10^(4) copies/μL.The LAMP 529-3 and 2021 primer sets had the highest detection limit,both at 10^(5) copies/μL.However,none of the primer sets amplified DNA from Cryptosporidium,Giardia,Trichinella spiralis,Escherichia coli,salmonella typhimurium or feline panleukopenia viruses.Using isothermal amplification of the LAMP 529-11 primer group,11 out of 29 pet cat feces were detected positive with a total detection rate of 37.93%(11/29);and 2 out of 10 anal swabs were positive(20.00%,2/10),with a total detection rate of 33.33%(13/39).Using the Rep-529 repeat sequence of Toxoplasma gondii as the target gene,this study screened the 529-11 LAMP primer group as a rapid,sensitive,and specific isothermal amplification primer group,which effectively amplifiedToxoplasma gondiiDNA within 30 minutes.This method caused 33.33% of pet cats to ovulate their oocysts,whichposed a huge potential risk of pollution and infection to the environment and humans.

关 键 词：弓形虫 感染 分子监测 LAMP qPCR 

分 类 号：S855[农业科学—临床兽医学]