Establishment of detection method for Chlamydophila abortus by using SYBR green real-time PCR and determination of bacterial load in mice

VernacularTitle:流产衣原体荧光定量PCR方法的建立及小鼠感染菌体载量测定
Author: Zhi-Jun ZHANG ¹ ; Zhao-Cai LI ; Zhong-Zi LOU ; Ji-Zhang ZHOU
Author Information

1. 中国农业科学院兰州兽医研究所
Keywords: Balb/c mouse; Chlamydophila abortus; real-time PCR; SYBR Green dye
From: Chinese Journal of Zoonoses 2018;34(3):224-229
CountryChina
Language:Chinese
Abstract: We established a fluorescent quantitative PCR (qPCR)method for the detection of Chlamydophila abortus (C. abortus),and replaced the method of smear staining which has subjective influence on the detection of C.abortus inactivated vaccine titer.According to the conserved sequence of the large cysteine-rich periplasmic protein(envB)of C.abortus,a specific primer was designed and the EnvB-PMD19T positive plasmid was used as the reference standard,optimization condition,sensi-tivity assay,specificity assay,repeatability assay and the bacteria loads of organs from mouse have been done.The results showed that the standard curve established with positive plasmid had a liner response from 1×102copies/μL to 1×106copies/μL with the correlation coefficient of 97%,sensitive for detecting C.abortus with the detection limit of 10 copies/μL,and re-peatable and stable with the coefficients of variation less than 2%.According to the result,the established method can detect the bacteria loads in organ of mouse,which provide a reliable method for evaluation of inactivated C.abortus vaccine.