Fluorescence-Based Quantitative PCR For Rapid Nucleic Acid Detection Of Respiratory Pathogens

BackgroundAcute respiratory disease(ARD)accounts for a large proportion of acute morbidity and mortality worldwide.As one of the common respiratory pathogens,influenza virus and Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),which cause fever,cough,and viral pneumonia,seriously affect and threaten human life and health worldwide.Real-time quantitative PCR(qPCR)is the gold standard for the detection of influenza virus and SARS-CoV-2 at home and abroad,but the testing environment is demanding and time-consuming,so the establishment of rapid and efficient point-of-care testing(POCT)methods is an important tool for the prevention and control of acute respiratory diseases.Methods1.To construct plasmid standards for influenza A virus(IAV)and influenza B virus(IBV),to collect clinical samples of influenza viruses,to establish an ultra-fast thermocycling reaction system with a reaction time of 27 min based on a fluorescence quantitative PCR method,and to perform a methodological evaluation of the ultra-fast thermocycling fluorescence quantitative PCR for the detection of SARS-CoV-2.The methodological evaluation was performed for the detection of IAV and IB V.2.To establish a method for the rapid detection of SARS-CoV-2 in the field based on an ultra-fast thermocycling fluorescence quantitative PCR with a reaction time of 32 min.3.To establish a fluorescence quantification method based on a convective PCR(CPCR)system for the detection of IAV.Results1.The plasmid standards for IAV and IBV were successfully cloned,and an ultra-fast thermocycling fluorescence quantitative PCR was established.The method has good linearity in the range of 1 × 100～1 ×105copies/μL for the detection of IAV and IBV,and the minimum detection limit was 1 ×100 copies/μL,and the coefficient of variation(CV)for reproducibility evaluation was 4.74%(IAV)and 1.94%(IBV),which were less than 5%.The CV was 4.74%(IAV)and 1.94%(IBV),which were less than 5%,and the detection performance was stable.There was no cross-reactivity with other respiratory pathogens.In the clinical evaluation,the sensitivity was 92.57%,the specificity was 100%,and the negative and positive predictive values were 88.79%and 100%,respectively,compared with the detection results of qPCR,and the total detection time was 27 min.2.Ultra-fast thermocycling fluorescence quantitative PCR for SARS-CoV-2,according to The primers and probes were designed based on the ORF lab and N genes of SARS-CoV-2,the qRnaseP internal reference gene was introduced,and the reaction conditions were optimized.The detection limit of this method for SARS-CoV-2 was 1×100 copies/μL,CV were less than 5%,no cross-reactivity with other respiratory pathogens.The detection results were in accordance with the instructions of SARS-CoV-2 negative reference product,with 100%positive compliance and 100%negative compliance,and the total detection time was 32 min.3.The fluorescence quantification method of IAV detection by CPCR system uses the difference between the peak area of the emission spectrum before and after the amplification of IAV by CPCR and fluorescence probe,and the logarithm of the virus copy number to calculate the quantification standard curve,thus quantifying IAV.The detection limit of this method was 1 × 101 copies/μL with good reproducibility,CV=1.42%and positive compliance rate of 88.89%(8/9)The total detection time was 19 min.ConclusionIn this paper,we successfully achieved the ultra-fast detection of IAV and IBV based on fluorescence quantitative PCR taking 27 min,conducted the methodological evaluation of the ultra-fast thermocycling fluorescence quantitative PCR system for the detection of IAV and IBV,and successfully established the method for the detection of SARS-CoV-2 based on the ultra-fast thermocycling fluorescence quantitative PCR system and the fluorescence quantitative method for the detection of IAV by the CPCR system.These two efficient,rapid,and low-cost on-site rapid detection systems for nucleic acids of respiratory pathogens can be used in a wide range of pathogen detection applications,providing technical support for the development of pathogen molecular POCT system construction.