Application research on quantitative real-time fluorescence quantitative PCR assay for Brucella
10.3760/cma.j.issn.1000-4955.2009.05.002
- VernacularTitle:实时荧光定量PCR检测布鲁杆菌方法的应用
- Author:
Hong-xia, ZHANG
;
Wen-xue, QI
;
Wen-xing, LIU
;
Sen, HU
;
Zhan-shui, YU
;
Mei-lan, DU
;
Wei-guo, XUE
;
Zhi-gao, BU
- Publication Type:Journal Article
- Keywords:
Brucella;
Polymerase chain reaction;
Detection
- From:Chinese Journal of Endemiology
2009;28(5):476-479
- CountryChina
- Language:Chinese
-
Abstract:
Objective To discuss a real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) wether if can be used to detect Brucella. Methods According to the BCSP31 gene sequences specific for Brucella, one pair of primers and one TaqMan probe were designed. A real-time PCR was developed with the BCSP31 fragments cloned into PMD18-T vector. The standard cure was established and the sensitivity, the species specificity and the stability of the assay were evaluated. The clinical blood specimens were detected by QT-PCR and compared with clinical diagnosis. Results The standard curve was established with the standard template and the relationship between the Ct and the DNA copy number was linear(r=0.999). The sensitivity of the real-time PCR was 5 copies/μl. The sensitivity of the common PCR was 5×102 copies/μl. The sensitivity was about 100 times higher than common PCR. Species specificity of this FQ-PCR assay evaluated using genomic DNA from 6 Bmcella strains and 5 non-Brucella strains and strong fluorescence was detected in all Brucella strains. The CV of intra-assay and inter-assay reproducibility were 0.71%,7.23%, reprectively. Twenty-four specimens from clinical brucellosis cases, 19 showed positive, the positive coincident rate was 79%(19/24). The negative results were obtained for all 31 negative control, and the negative coincident rate was 100%(31/31). Two were positive from all 30 specimens clinically suspected. Conclusions Highly specific, sensitive, repeatable and coincidental with clinic, this FQ-PCR is quite useful for rapid detection of tiny DNA of Brucella in various samples and laboratory diagnosis.
