Establishment of a nested PCR assay for the detection of 2019 novel coronavirus and its preliminary application
10.3760/cma.j.cn112866-20211215-00211
- VernacularTitle:新型冠状病毒巢式PCR检测方法的建立与初步应用
- Author:
Weixian SHI
1
;
Zhaomin FENG
;
Shujuan CUI
;
Yang PAN
;
Cheng QIAN
;
Ruolei XIN
;
Peng YANG
;
Quanyi WANG
;
Daitao ZHANG
;
Zhiyong GAO
Author Information
1. 北京市疾病预防控制中心传染病地方病控制所，北京 100013
- Keywords:
2019 novel coronavirus;
Real-time fluorescent PCR;
Nested PCR
- From:
Chinese Journal of Experimental and Clinical Virology
2022;36(2):214-218
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a nested PCR method to detect the 2019 novel coronavirus (2019-nCoV), as a supplement to the real-time fluorescent PCR method, and discuss the preliminary application value of this method in clinical diagnosis.Methods:According to the conservative sequences of the 2019-nCoV gene, the nested PCR primers including N gene and S gene, were designed on line. By optimizing the nested PCR reaction systems, the qualitative detection was established by testing N gene and sequencing its PCR product while the preliminary type identification was established by testing S gene and sequencing its PCR product. The sensitivity was evaluated by the gradient dilution of 2019-nCoV positive samples’ nucleic acid and the specificity was evaluated by detecting the human coronavirus OC43, 229E, HKU1, NL63, influenza virus positive samples. The established method was applied to 15 samples with Ct >33 and 15 samples with Ct <33 screened by real-time fluorescent PCR, and the positive amplification result were sequenced and analyzed to verify the result. Results:The established nested PCR method could amplify specific bands of 355 bp N gene fragment and 449 bp S gene fragment. No amplifications occurred in other human coronaviruses samples including 229E、OC43、HKU1、NL63 or in influenza virus samples including H3N2, H1N1(pdm) and B. The minimum detection limit of the N gene fragment could reach Ct value about 37.21. Among the 30 COVID-19 positive samples, the N gene positive coincidence rate detected by nested PCR was 100% (30/30); the S gene positive coincidence rate reached 60% (18/30). 28 samples’ sequences of N gene fragment were completely consistent with 2019-nCoV by BLAST, and the characteristic result of site mutations of 12 samples’ S gene was obtained. Conclusions:A nested PCR method for the specific detection of 2019-nCoV was established, and some characteristic mutations on S gene could be analyzed by sequencing the PCR amplified products. It could be used as a supplement to the real-time fluorescent PCR method.
