Establishment of real-time fluorescence quantitative PCR method for detecting the N subgenome of SARS-CoV-2
10.3760/cma.j.cn112866-20240904-00134
- VernacularTitle:新型冠状病毒N_sgRNA荧光定量PCR检测方法的建立
- Author:
Taoli HAN
1
;
Zhi ZHANG
;
Jiaxin ZHAO
;
Pan LU
;
Yang JIAO
;
Jianhong ZHAO
;
Yan GAO
;
Shiyao ZHANG
;
Kuankuan LIU
;
Yujie LIU
;
Ru FAN
;
Wenjing LI
;
Lingli SUN
Author Information
1. 北京市朝阳区疾病预防控制中心微生物检验科，北京 100021
- Publication Type:Journal Article
- Keywords:
Severe acute respiratory syndrome coronavirus 2;
Subgenomic RNA;
Fluorescence quantitative PCR
- From:
Chinese Journal of Experimental and Clinical Virology
2025;39(1):96-101
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a fluorescent quantitative RT-PCR assay based on N_sgRNA of SARS-CoV-2 and preliminarily apply it on real samples.Methods:Recombinant plasmid, specific primers and probes of N_sgRNA were designed and synthesized based on Wuhan-Hu-1/2019_MN908947 and synthesis mechanism of subgenomic RNA (sgRNA). Using recombinant plasmid as amplification templates, the optimal reaction conditions and reaction system were screened according to the Ct value, fluorescence intensity, and shape of amplification curve and was evaluated for sensitivity, reproducibility, and specificity. Meanwhile, the possibility of practical application of the method was explored by testing 172 clinical samples and 256 municipal wastewater samples. Results:A qRT-PCR assay for N_sgRNA in SARS-CoV-2 was initially established. The detection limit of the assay was 20 copies/mL, and the variation coefficients of in-batch (0.002%~0.767%) and batch to batch repetition (0.016%~0.752%) were less than 1%. Only N_sgRNA recombinant plasmid was detected in the specificity assay. So the method is more highly sensitive, specific and reproducible. The RatiosgRNA/ gRNA of clinical samples HK.3, EG.5.1, JN.1 and their sub-lineages and their corresponding urban sewage samples in epidemic period were significantly different ( P<0.05). There is a strong correlation between the median of RatiosgRNA/ gRNA in clinical samples and sewage samples in the same period (correlation coefficient r=1.000, P=0.010). Conclusions:In this study, a qRT-PCR method for detecting N_sgRNA of SARS-CoV-2 was established and the method has the characteristics of higher sensitivity, stronger specificity and better repeatability, and it can be used to detect SARS-CoV-2 infectivity.
