Establishment of multiple TaqMan qPCR assay for Pasteurella multocida in cat-tle and sheep
10.16303/j.cnki.1005-4545.2024.11.08
- VernacularTitle:牛羊多杀性巴氏杆菌多重TaqMan qPCR检测方法的建立
- Author:
Yanan GUO
1
;
Zhenggang ZHANG
;
Jiandong WANG
;
Jingsong WANG
;
Ke LI
;
Jidong LI
;
Xiaojun LIANG
Author Information
1. 宁夏农林科学院动物科学研究所,宁夏银川 750002
- Keywords:
cattle and sheep;
Pasteurella multocida;
capsule typing;
multiple TaqMan qPCR;
detec-tion method
- From:
Chinese Journal of Veterinary Science
2024;44(11):2363-2370
- CountryChina
- Language:Chinese
-
Abstract:
This study aims to establish a multiplex TaqMan fluorescence quantitative PCR(qPCR)assay for Pasteurella multocida(P.multocida).Specific primers and fluorescent labeling probes were designed based on the sequences of five podoplanar genes of P.multocida hyaC-hyaD,bcbD,dcbF,ecbJ,and fcbD in the NCBI database.We adjusted the annealing temperature by gradient setting,optimized the primer and probe concentrations by matrix method,constructed standard curves,and performed specificity,sensitivity and reproducibility tests,and finally established mul-tiplexed TaqMan qPCR assays for these five genes.The results showed that the established assay had a good linear relationship between the amplification curves.The sensitivity of this method was high,10-100 times higher than that of ordinary PCR;the specificity was strong,and there was no amplification curve in the DNA detection of eight pathogenic bacteria such as Bacillus,Proteus mirabilis,Staphylococcus aureus,and Rhizoctonia rad iodurans.This assay had a good linear rela-tionship,and the coefficients of variation for Ct values of the inter-and intra-group reproducibility tests were all less than 3％,and the detection rate of this assay was 11.25％higher than the con-ventional PCR assay through the detection of 90 clinical samples.The method established in this study is able to detect P.multocida rapidly and sensitively,which is important for its rapid clinical and laboratory diagnosis.
