Develonment Of Real-Time Fluorescence Quantitative PCR For Rapid Detection Of Bartonella

Objective:(1) Bartonella spp. are the major etiological agents of CSD, trench fever, IE, and Carrion’s disease, as well as Bh and Bq are the major pathogens of IE, which pose a serious threat to human health. To facilitate the detection of Bartonella spp., this study aimed to develop rapid, highly sensitive and specific assays, based on HRM analysis for identifying the Bartonella spp. and duplex HRM analysis and TaqMan probe real-time PCR assay for simultaneously identifying Bh and Bq.Methods:Primers were designed to amplify partial internal ssrA、BH 14560、 BQ11520 and BQ01180. The amplification products were cloned into pEASY(?)-T5 Zero vector for preparation of the standard. The annealing temperature, final concentration of primers and probe, and the melting rate were optimized. Specificity, sensitivity and reproducibility of the PCR system were assessed by 10×dilution series of the reference plasmids. Amplification efficiency and linearity were analyzed.Results:The optimized annealing temperature is 60 ℃, the primer concentration is 300 nmol/L, the probe oncentration is 200 nmoL/L and the melting rate is 0.2 ℃/s. (1) The HRM assay showed a excellent specificity by detecting Bartonella with the Tm value of 81.05 ℃±0.31, and showed no cross reactivity with the non-Barlonella bacteria and the other animals. The sensitivity of detection was 3.82 × 101 copies per 20 μL PCR reaction, and the Ct value of the assay depicted a strong linear relationship, with a correlation coefficient of 0.999 and efficiency of 98.4%. The CV from the intra-and inter-groups were 0.30%-0.62% and 0.29%-0.36%, respectively, which were acceptable. (2) The duplex HRM assay showed a good specificity by detecting Bh and Bq. The sensitivity of detection of both single and duplex HRM assay was 3.64×101 copies and 5.62×101 copies per 20 μL PCR reaction, respectively, and also reflects a good linearity and high efficiency, which the correlation coefficient R2 were 0.998 and 0.997, respectively, and E-value were 102.8% and 104.7%, respectively. The CV from the intra-and inter-groups were 0.22%-0.50% and 0.38%-1.20%, respectively. (3) The duplex TaqMan probe real-time PCR assay showed a good specificity by detecting Bh and Bq. The sensitivity of detection of both single and duplex TaqMan probe assay was 3.64X 101 copies and 3.28X 101 copies per 20 p.L PCR reaction, respectively, and the correlation coefficient R2 were 0.994 and 0.998, respectively, and E-value were 100.0% and 103.4%, respectively, which reflects a good linearity and high efficiency. The CV from the intra-and inter-groups were 0.19%-0.53% and 0.49%-1.66%, respectively, which were acceptable. The established three assays was 100 times more sensitive than that of conventional PCR.Conclusion:(1) The established HRM assay has sufficient specificity, high sensitivity and good reproducibility for the detecting Bartonella in the clinical diagnosis, epidemiological survey and disease surveillance. (2) The duplex HRM and TaqMan probe real-time PCR assays have sufficient specificity, high sensitivity, rapid and reliable for detecting IE associated Bartonella (Bq and Bh).