Establishment of a simple and rapid identification method for Bartonella spp. using high-resolution melting analysis
LIU Yun-yan1, 2, SONG Xiu-ping1, LIU Qi-yong1, CHEN Zhong-ke2, LI Dong-mei1
1. State Key Laboratory for Infectious Diseases Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; 2.State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention / China CDC Key Laboratory of Surveillance and Early Warningon Infectious Disease, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases / WHO Collaborating Center for Vector Surveillance and Management, Beijing 102206, China
Abstract:In this study, we aimed to develop a rapid, highly sensitive and specific assay, based on real-time PCR with high resolution melting (HRM) analysis, for identifying the Bartonella. Without the need of any probe, the products were identified by HRM analysis after real-time PCR amplification using the specific primers of ssrA. The amplification products were cloned into pEASY -T5 Zero vector for preparation of the B. henselae standard. The annealing temperature and the final concentration of primers were optimized. Specificity, sensitivity and reproducibility of the PCR system were assessed by 10 dilution series of the B. henselae reference plasmids. Results showed that the optimized annealing temperature was 60 ℃ and the primer concentration was 300 nmol/L. The HRM real-time fluorescence quantitative PCR assay showed a excellent specificity by detecting Bartonella spp. with the Tm value of 81.05 ℃±0.31, and showed no cross reactivity with the non-Bartonella bacteria and the other animals. The sensitivity of detection of HRM real-time PCR was 3.82×101 copies, per 20 μL PCR reaction, which was 100 times more sensitive than that of conventional PCR, and also reflected a good linearity and high efficiency, in which the correlation coefficient R2 and E-value were 0.999 and 98.4%, respectively. The coefficients of variations (CV) from the intra- and inter-groups were in the range of 0.30%-0.62% and 0.29%-0.36%, respectively, which were acceptable. The established HRM real-time PCR assay has sufficient specificity, high sensitivity and good reproducibility for the detection of Bartonella spp. in the clinical diagnosis, epidemiological survey and disease surveillance.
刘云彦, 宋秀平, 刘起勇, 陈忠科, 栗冬梅. 应用实时高分辨率熔解曲线技术检测巴尔通体[J]. 中国人兽共患病学报, 2015, 31(11): 1027-1032.
LIU Yun-yan, SONG Xiu-ping, LIU Qi-yong, CHEN Zhong-ke, LI Dong-mei. Establishment of a simple and rapid identification method for Bartonella spp. using high-resolution melting analysis. Chinese Journal of Zoonoses, 2015, 31(11): 1027-1032.
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