Establishment and application of a method for detecting Toxoplasma gondii based on recombinant polymerase amplification technology
10.3969/j.issn.1002-2694.2025.00.018
- VernacularTitle:基于重组聚合酶扩增技术检测弓形虫方法的建立及应用
- Author:
Shao-zheng SONG
1
;
Le-ying GU
;
Ying-chao WU
;
Ya-qin MENG
;
Kang-ying YU
;
Xiao-hua HUANG
Author Information
1. 无锡太湖学院健康与护理学院基础医学系,无锡 214000
- Publication Type:Journal Article
- Keywords:
recombinant polymerase amplification;
Toxoplasma gondii;
detecting;
sensitivity;
specificity
- From:
Chinese Journal of Zoonoses
2025;41(2):107-112
- CountryChina
- Language:Chinese
-
Abstract:
To establish a method for detecting Toxoplasma gondii based on recombinant polymerase amplification(RPA)technology and apply it to clinical sample validation of pet cats.Using the 529 repeat sequence of the Toxoplasma gondii gene as the target gene sequence,primers and probes were designed,and the Rep-529 recombinant plasmid was constructed as the standard.A fluorescent RPA reaction system was established.Dilute the plasmid standard 10 times to different concentrations as the detection template for sensitivity testing;Specific testing was conducted using genomic DNA from several parasitic spe-cies,including Toxoplasma gondii,Cryptosporidium,Neosporidium,Trichinella spiralis,Giardia flagellata,Babesia bo-vis and Theileria annulata as templates;Simultaneously,fluorescence RPA and RT-PCR were used to detect 52 positive and 40 negative cats clinical samples,and the coincidence rate of the detection results of the two methods were compared and ana-lyzed.The RPA reaction system was successfully established using PTRep recombinant plasmid as the standard,ToxD-F/ToxD-R as the primer,and RepD-P as the fluorescent probe.The reaction temperature was constant at 39 ℃,the reaction time was 30 minutes,and the detection sensitivity was 1 copy/μL.There is no significant cross reaction with parasites such as Cryptosporidium,Neosporidium,Trichinella spiralis,Giardia,Babesia bovis and Theileria annulata,and the specificity is good.A total of 92 clinical fecal samples from cats were tested,and the positive coincidence rate of fluorescence RPA detection method was higher than that of conventional RT-PCR method(98.08％vs.82.69％),and the difference of the positive rate was not statistically significant(X2=1.392,P＞0.05).The fluorescence RPA detection method for Toxoplasma gondii suc-cessfully established in this study has the characteristics of being fast,sensitive,specific,accurate,and reliable.It can be used as a rapid clinical detection kit for Toxoplasma gondii in cats and other animals,providing new technical support for the subsequent epidemiological monitoring and precise clinical diagnosis of toxoplasmosis in cats,other animals,and humans in the future.
