动物造模,荧光定量PCR z-bio 2022-03-03 574

　　Objective: To establish a rapid, sensitive and specific TaqMan probe real-time quantitative PCR detection method for Helicobacter bilis to quantitatively detect Helicobacter bilis.

　　Methods: The full-length ORF435 bp of the conserved gene P17 sequence of H.bilis was amplified by PCR and cloned by TA to construct the plasmid standard pMD-HBP17. Through the quantitative analysis of pMD-HBP17 standard, the reaction system was optimized, and the sensitivity, specificity and repeatability of the TaqMan probe real-time quantitative PCR method were detected; 77 clinical samples were detected by the established qPCR method, and the results were compared with those of ordinary PCR. Compare the test results.

　　Results: The established qPCR detection method showed good linearity and correlation between the plasmid DNA concentration between 108 and 101 copies, the slope of the obtained standard curve was -3.46, the correlation coefficient was 0.999, and the detection sensitivity reached 20 copies. The detection rate of 77 clinical samples was 14.3%, which was higher than that of ordinary PCR (7.8%).

　　Conclusion: The established H.bilis qPCR detection method has good specificity, high sensitivity and strong stability, and can be used for the quantitative and qualitative detection of Helicobacter bilis.