Study on the detection of SARS-CoV-2 in the urine of infected persons by droplet digital PCR. [J]. Journal of Southwest Medical University 45(4):301-307(2022)
DOI:
Study on the detection of SARS-CoV-2 in the urine of infected persons by droplet digital PCR. [J]. Journal of Southwest Medical University 45(4):301-307(2022) DOI: 10.3969/j.issn.2096-3351.2022.04.005.
Study on the detection of SARS-CoV-2 in the urine of infected persons by droplet digital PCR
Objective The objective of the present study is to establish a sensitive,accurate and reliable polymerase chain reaction (PCR) platform,to develop a simple and effective analytical method to measure the SARS-CoV-2 clinically,and to provide solution of rapid diagnosis in case of outbreaks of infectious diseases through increasing the sensibility of detection limit. Methods The total RNA was extracted from throat swabs and urine of 12 infected people,respectively. The cDNA was used as templates and the PCR program was carried out after the generation of the droplets. The concentration of positive fragments were calculated by the droplet analyzer. Integrated with a 2 983 bp fragment of SARS-CoV-2,the pUC57 plasmid was diluted by multiples of 10 and used as templates for RT-qPCR. The standard curve was made according to the results of the amplification of the standard plasmid. The original concentration in specimen was calculated according to the standard curve. The veracity of the measurements of both ddPCR and RT-qPCR were compared,respectively. The quantity of the original copies in urine was measured through ddPCR method. Results The detection rate of 12 positive urine samples was 75%,and the detection limit was 5.4 copies/μL,the detection sensitivity of that was obviously higher in comparison with RT-qPCR method. Conclusions ddPCR is accurate,rapid and sensitive,which can meet the requirements of rapid detection of low copy nucleic acid in urine samples.