【摘要】 目的研究和建立一种数字PCR检测方法,用于检测新型冠状病毒(SARS-CoV-2)的核酸RNA。方法建立使用一步法逆转录聚合酶链扩增法(RT-PCR)的数字PCR检测方法。然后使用假病毒质粒参考品对检测方法的准确性、灵敏度进行评价;使用其他病毒和健康人核酸RNA样本,进行特异性评价;临床新型冠状病毒肺炎患者的核酸RNA进行临床适用性评价,同时与荧光定量PCR方法进行比较。结果建立了新型冠状病毒的数字PCR检测方法,假病毒质粒参考品均能准确检出,灵敏度为50 copies/mL;其他病毒和健康人核酸RNA样本均为阴性;临床新型冠状病毒肺炎患者的核酸RNA均能准确检出。经过比较,数字PCR检测方法检测灵敏度为50 copies/mL,荧光定量PCR法的灵敏度为100copies/mL。结论本研究建立的新型冠状病毒数字PCR检测方法具有较高准确性、灵敏度、特异性和临床适用性,能够改善临床新型冠状病毒核酸检测的假阴性问题。更多还原

【Abstract】 Objective To develop a digital PCR method for the detection of nucleic acid RNA of a novel coronavirus(SARS-CoV-2). Methods A digital PCR detection method using one-step reverse transcription polymerase chain amplification(RT-PCR)was developed. Pseudoviral plasmid reference materials were applied to evaluate the accuracy and sensitivity of the method. The specificity was also analyzed using other viral and healthy human nucleic acid RNA samples. The clinical application was evaluated using COVID-19 patient nucleic acid RNA samples and compared with the fluorescence quantitative PCR method. Results The pseudoviral plasmid reference materials can be accurately detected with a sensitivity of 50 copies/mL. All nucleic acid RNA samples from COVID-19 patients are positive,and negative from other viruses and healthy people as controls. The sensitivity of the digital PCR detection method is 50 copies/mL,much higher than that of compared with the fluorescent quantitative PCR method(100 copies/mL). Conclusion The digital PCR method for the detection of SARS-CoV-2 has high accuracy,sensitivity,specificity and clinical applicability,and can improve the problem of false negatives in clinical detection of novel coronavirus nucleic acids.更多还原