【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。更多还原

【Abstract】 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.更多还原