微滴式数字PCR和实时荧光定量PCR对新型冠状病毒肺炎患者不同时间血便尿中核酸检测结果初步探讨

目的分析微滴式数字PCR(droplet digital PCR,ddPCR)和实时荧光定量PCR(quantitative real-time PCR,qPCR)的核酸检测结果,比较两种方法检测各类样本的差异性,为改进新型冠状病毒核酸检测方案提供数据支持。方法利用ddPCR和qPCR技术对已经确诊的3例新型冠状病毒肺炎患者发病不同时间的全血、尿液、粪便共22份标本进行新型冠状病毒核酸检测。结果两种方法对人保守区域基因扩增结果一致:全血标本信号最强,尿液次之,粪便最少;ddPCR在1份全血,1份尿液,5份粪便中检出ORF-1ab和N基因的阳性微滴,qPCR仅在3份粪便中检出上述基因,漏检的3个标本基因拷贝数平均浓度为128 copies/ml;ddPCR在发病<5、5~15、>15 d的各类标本中都有检出,qPCR检出以中晚期为主;重症病例用ddPCR均可测到阳性微滴,qPCR检测的各类标本均为阴性;轻症病例的各类标本中qPCR只有粪便核酸检测阳性,ddPCR检出率高于qPCR。结论ddPCR可以有效克服qPCR灵敏度不足的难题,是对qPCR的有益补充,尤其是针对病毒载量比较低的血液、尿液和可疑的粪便或肛拭子标本,适用于早期感染的判断及患者治愈后出院诊断。

Preliminary discussion on results of droplet digital PCR and real-time fluorescence quantitative PCR in nucleic acid detection of 2019-nCoV

Objective To analyze the 2019 novel coronavirus (2019-nCoV) nucleic acid results detected by droplet digital PCR (ddPCR) and real-time fluorescence quantitative PCR (qPCR), to compare the differences in detecting various specimens by the two assays, and to provide data support for improving detection scheme of 2019-nCoV nucleic acid. Methods ddPCR and qPCR were used to detect 2019-nCoV nucleic acid in 22 blood, urine and feces specimens collected at different times in 3 clinically-confirmed COVID-19 patients. Results The gene amplification results of a conserved region from the human cells were consistent with the two methods: whole blood samples had the strongest signal, followed by urine, and the least feces. The positive microdrops of ORF-1ab and N genes were detected in 1 whole blood specimen, 1 urine specimen and 5 feces specimens, respectively by ddPCR, while only 3 feces specimens were detected positive by qPCR. The average concentration of 2019-nCoV genes in the 3 missed samples was 128 copies/ml. ddPCR could screen the positive specimens less than 5 days, within 5-15 days and more than 15 days after the onset of the disease, whilepositive specimens from COVID-19 patients at middle-late stage were mainly detected by qPCR.Positive droplets were detected in three kinds of specimens of severe cases by ddPCR, but all specimens were negative by qPCR. Only fecal specimens of mild cases were detected 2019-nCoV positive, and the positive rate of fecal detection by ddPCR in mild cases was higher than that of qPCR. Conclusions ddPCR can effectively overcome the lack of sensitivity of qPCR, and is a useful supplement to qPCR, especially for the blood, urine and suspected fecal or anal swab samples with low viral load, which is suitable for the early and discharge diagnosis of 2019-nCoV.