Establishment of a fluorescence quantitative PCR detection method for Strepto-coccus equi subspecies zooepidemicus and its application in retrospective detection of imported horses
10.16303/j.cnki.1005-4545.2024.08.21
- VernacularTitle:马链球菌兽疫亚种荧光定量PCR检测方法的建立及在入境马匹回顾性检测分析中的应用
- Author:
Yutong HU
1
;
Xuehui ZHOU
;
Mengru ZHAO
;
Xiang CHEN
;
Xiaowei WU
;
Zhiguo ZHAO
;
Yan WANG
;
Guangwei ZHAO
Author Information
1. 西南大学动物医学院,重庆荣昌 402460
- Keywords:
Streptococcus equi subspecies zooepidemicus;
fluorescence quantitative PCR;
SYBR Green Ⅰ;
horse
- From:
Chinese Journal of Veterinary Science
2024;44(8):1735-1742
- CountryChina
- Language:Chinese
-
Abstract:
In order to establish a rapid,specific and sensitive detection method for Streptococcus equi subspecies zooepidemicus(SEZ)and to understand the infection status of SEZ in horses ente-ring China,specific primers were designed and synthesized based on the conserved gene comB of standard strain SEZ(ATCC 43079)in this work.Then,the pMD19-T-comB recombinant plasmid was constructed and used as a standard positive template.After that,the fluorescence-based quantitative PCR(qPCR)detection method based on SYBR Green Ⅰ dye was established.Totally,477 equine entry serum samples from 6 countries,including Netherlands,Belgium,Japan,Germa-ny,Argentina and New Zealand,during 2018 to 2023,were randomly selected and detected for SEZ by the qPCR method.Results showed that the established qPCR method had specific amplification for only SEZ,which illustrated a good specificity.Sensitivity test of the method showed that the limited detection amount was 4.58 X101 copies/μL.And the repeatability test showed that the coef-ficient of variation of intra-batch repeatability was less than 0.5％,while the inter-batch repeat-ability was less than 3.0％,which indicated good repeatability and high stability.Retrospective a-nalysis showed that totally 11 of 477 positive samples were detected,with a relatively low positive rate of 2.31％(11/477).Among them,all the 40 samples from Netherlands in 2018 were negative(0/40).In the samples of 2019,one positive was detected from Belgium(1/20),while all other 36 samples which form Japan and Germany were negative.In the samples of 2021,three samples(3/34)from Japan and one sample(1/20)from Argentina were positive,and all the other 40 samples from the Netherlands were negative.In the samples of 2022,76 samples from Netherlands were all negative.While in the 2023,5(5/126)of 126 samples from Netherlands and one(1/88)of 88 from New Zealand were found positive with SEZ.To summarize,The SYBR Green Ⅰ qPCR method for the diagnosis of SEZ was successfully established,and it could provide necessary technical support for the rapid quarantine of China's entry-exit and port departments,as well as the epidemiological investigation of the disease.
