建立多色探针熔解曲线技术单管同步检测6种呼吸道病原体方法OA

Objective A multi-color probe melting curve analysis technique was established to simultaneously detect six respiratory pathogens of influenza A virus(IAV),influenza B virus(IBV),novel coronavirus(SARS-CoV-2),enterovirus(EV),human rhinovirus(HRV),and respiratory syncytial virus(RSV)in a sin-gle tube.Methods Primers and probes were tailored to the conserved regions of these pathogens,and a reaction system and procedure were optimized.The optimized conditions were used to evaluate the sensitivity,specificity and preliminary application of clinical samples.Results The lower limit of detection of IAV,EV,HRV and RSV was 30 copies/ml,and the lower limit of detection IBV virus and SARS-CoV-2 was 300 copies/ml.The coeffi-cient of variation of repeated experiments was less than 0.5％,and non-target pathogens were not detected in cross experiments.The negative coincidence rate of 6 pathogens was 100％,the positive coincidence rate of IAV,IBV and HRV was 100％(Kappa=1),the positive coincidence rate of SARS-CoV-2 was 93.33％(Kappa=0.97),the positive coincidence rate of EV was 96.00％(Kappa=0.98),and the positive coincidence rate of RSV was 94.12％(Kappa=0.98),indicating that there was a high consistency between the two detection meth-ods.Conclusion A single-tube assay for the simultaneous identification of six respiratory pathogens was prelimi-narily constructed.This method uses the difference of melting temperature(Tm)of different fluorescent labeled probes to achieve multiple discrimination,simultaneous detection of six pathogens in a single tube and single re-action was achieved,significantly improves the detection efficiency and reduces the cost of reagents.It has the characteristics of high sensitivity,strong specificity and good repeatability,and can be used for simultaneous de-tection of 6 pathogens in respiratory tract.