Establishment and application of TaqMan fluorescent quantitative PCR detection method of Nocardia in dairy cows
10.16303/j.cnki.1005-4545.2025.01.06
- VernacularTitle:牛源诺卡菌TaqMan荧光定量PCR检测方法的建立及应用
- Author:
Yan ZHAO
1
;
Meiyi REN
;
Jingdi TONG
;
Yalan SU
;
Deyuan SONG
;
Guojun JIANG
;
Jia CHENG
;
Jian GAO
;
Mingchao LIU
Author Information
1. 河北农业大学动物医学院,河北保定 071001
- Publication Type:Journal Article
- Keywords:
Nocardia;
mastitis;
TaqMan real-time PCR;
dairy cow
- From:
Chinese Journal of Veterinary Science
2025;45(1):39-45
- CountryChina
- Language:Chinese
-
Abstract:
Nocardia is a Gram-positive pathogen responsible for causing dairy mastitis,which leads to purulent granulomatous lesions in mammary tissue and can significantly impact the dairy indus-try,resulting in substantial economic losses.To develop a rapid and accurate diagnostic method for detecting Nocardia of bovine origin,a conserved sequence of the 16S rRNA gene from Nocardia was selected from the NCBI database.Based on this sequence,a pair of primers and a TaqMan fluo-rescent quantitative probe were designed.The validation of the TaqMan fluorescence quantitative PCR(qPCR)method found in this study showed that the Ctvalue had a good linear relationship with recombinant plasmid concentrations ranging from 1×1010 to 1×102 copies/μL,with a regres-sion equation of y=-3.536x+43.78,a correlation coefficient(R2)of 0.997 5,a slope of-3.536,and an amplification efficiency(E)of 91％(where 90％＜E＜110％).The specificity was strong,with no cross-reactions with other pathogens.The standard curve had a high sensitivity with a low-er detection limit of 1 × 102 copies/μL,it was 100-fold higher than conventional PCR.The repeatability of the standard curve was also good.Both intra-and inter-group coefficients of varia-tion were below 2％.Using this method,234 milk samples and 80 environmental samples were tested using this method,respectively,with a positive detection rate of 27.07％,whereas conven-tional PCR had a positive detection rate of 19.43％,indicating that this method was more sensitive compared to conventional PCR.The fluorescent quantitative PCR detection method established in this study provides an effective means for the clinical detection of Nocardia in dairy cows.
