关键词: 多重聚合酶链反应, 熔解曲线分析, 感染性腹泻, 病原菌

Abstract:

Objective To establish a rapid，accurate and highly sensitive diagnostic method for simultaneous identification of 6 common acute infectious diarrhea pathogens based on multiple polymerase chain reaction（PCR） and probe melting curve analysis，and to verify its determination performance. Methods The melting temperature（Tm） values of 6 common acute infectious diarrhea pathogens were analyzed by probe melting curve analysis，and the multiple PCR combined probe melting curve analysis system was established. The sensitivity，specificity and repeatability were evaluated. The method was used to determine fecal samples from 175 patients with suspected infectious diarrhea. The difference between the method results and the results of bacterial culture and fluorescent PCR was compared. The inconsistent results were confirmed by sequencing. Results The established method could determine 6 kinds of pathogens simultaneously by single PCR，the determination limit was 1×10³-1×10⁴ copies/mL，and there was no cross-reaction among different pathogens. The specificity was high. A total of 91（52%） fecal samples from 175 diarrhea patients were positive. The coefficients of variation（CV） within determination for 6 pathogens were <0.1%. The consistency rates of Salmonella，Shigella and Clostridium difficile were 94.85%（kappa=0.894），100.00%（kappa=1.000） and 98.28%（kappa=0.900），respectively. Conclusions The established multiple PCR based on probe melting curve analysis has high sensitivity and specificity for determining 6 kinds of diarrhea pathogens，which can provide a reference for clinical diagnosis of acute infectious diarrhea in time.

Key words: Multiple polymerase chain reaction, Melting curve analysis, Infectious diarrhea, Pathogen