Brucella real-time quantitative PCR kit for rapid detection of clinical samples

Abstract

Objective To establish a real-time fluorescent quantitative polymerase chain reaction assay system for rapid and accurate detection of Brucella and to provide a guidance for clinical doctors in making diagnosis of Brucella infection. Methods A real-time polymerase chain reaction assay was developed for detection of all species and bivors of Brucella. According to the sequences of Brucella surface protein gene 31, a probe and two primers were designed. The specificity (Brucella standard strains 16M, 544A, 1330S, vaccine strains 104M, reference strains Yersinia enterocolitica O: 9, E. coli O: 157) and repeatability (4 different concentrations of standard) were verified and the lowest limit of detection of the reaction system was analyzed. Results The established method can be used for specific amplification of Brucella, the different substrate concentrations of the reaction system showed good linearity with the Ct value (Y=-2.77X + 43.19, R2 = 0.994 8), the lowest limit of detection of reaction system was 1 × 103 copies/ml, the test results of Brucella standard strains 16M, 544A, 1330S and 104M were positive, test results of Yersinia enterocolitica O: 9 and E. coli O: 157 were negative, 4 different concentrations of standard and negative quality control were tested 4 times, and the variation coefficients were 0.15%, 0.16%, 0.23% and 0.18%, respectively. Conclusion A Brucella real-time fluorescent quantitative polymerase chain reaction assay system is established successfully, which could be used to identify Brucella rapidly and accurately. Key words: Brucella; Infection; Fluorescence; Quantitative real-time PCR