关键词: 新型冠状病毒肺炎, 新型冠状病毒, PCR-飞行质谱系统, 基质辅助激光解吸电离飞行时间质谱, 核酸检测

Abstract: Background　To contain the COVID-19 pandemic，laboratory test results are an important reference for making diagnosis whose accuracy is closely associated with the sensitivity and specificity of the detection method.RT-PCR is a general detection method for SARS-CoV-2，but its sensitivity is limited and depends on the content of virus.Optimizing PCR-Time-of-flight mass spectrometry （TOFMS） may improve the sensitivity of low virus content detection，which may be an effective supplement to the existing methods.Objective　To develop a PCR-TOFMS method for detecting SARS-CoV-2 nucleic acid based on Clin-ToF-II TOFMS system using matrix-assisted laser desorption/ionization（MALDI）principle，and evaluate its practical application value.Methods　186 samples were used as the data for developing a system of PCR-TOFMS，including 121 external quality assessment samples〔47（40 were SARS-CoV-2 positive and 7 were negative）from Shanghai Center for Clinical Laboratory，and 74（41 were SARS-CoV-2 positive and 33 were negative）from National Health Commission of the People's Republic of China〕，and 65（32 positive plasmids and diluted samples，28 samples of healthy people and 5 of ultrapure water as blank control） bought by our laboratory.Another 100 samples collected during January to February，2020，were used as the data for testing the performance of our system of PCR-TOFMS， including 80 samples of upper respiratory tract secretions（55 throat swab samples， and 25 sputum samples） of suspected COVID-19 cases collected clinically by Anhui's hospitals and sent to Anhui Provincial Center for Disease Control and Prevention for detection，and 20 throat swab samples of healthy people.The detection procedure for the system was developed， including PCR amplification， SAP digestion， single-base extension and mass spectrometry detection. Based on the positive plasmid，the template quantity was optimized.The diagnostic criterion for SARS-CoV-2 was determined according to the peak signal-to-noise ratio of 186 samples' results. The samples of 80 suspected COVID-19 cases and 20 healthy people were analyzed，and the results were compared with those by RT-PCR.Results　By optimizing the template conditions of PCR-TOF MS，the typical mass spectra of PCR-TOFMS was obtained when the template amount was 6 μl.The established RT-PCR reaction system could effectively detect the amplification of ORF1ab gene and nucleocapsid protein （N） gene of SARS-CoV-2 nucleic acid. In this study，the detection rate of PCR-TOFMS for clinically suspected cases was 90.00%，which was higher than 75.00% of RT-PCR. Meanwhile，the results showed that 25.00% of suspicious results were detected by RT-PCR，but no suspicious results were found by PCR-TOFMS. PCR-TOFMS had higher rates of detecting SARS-CoV-2 in sputum samples（100.00% vs 64.00%）and throat swab samples（85.45% vs 43.64%）of suspected COVID-19 cases than RT-PCR，but both of them did not find SARS-CoV-2 in negative samples.Conclusion　The combination of PCR and TOFMS technology makes the detection of SARS-CoV-2 target fragment expand exponentially，thereby improving the detection sensitivity of samples.In addition，MALDI-TOF MS technology has high accuracy and specificity through the detection and analysis of ion molecular weight，which also confirms that sputum sample is better than throat swab sample for detection.This method can be used as a supplement and collaboration of RT-PCR，which can make up for the false negative problem to a certain extent，thus reducing the clinical missed diagnosis rate，improving the detection efficiency，and shortening the time for pandemic situation research and judgment.

Key words: COVID-19, SARS-CoV-2, PCR-TOF MS, MALDI-TOF MS, Nucleic acid testing