Construction and application of quantitative detection method for Norovirus GⅡ copy number in fecal specimens

Abstract:

Objective To construct the standard plasmid and detection system for copy number of Norovirus GⅡ (NoV GⅡ). Methods The synthesized highly conserved NoV GⅡ gene sequence was cloned into pUC57 vector for constructing NoV GⅡ standard plasmid, then plasmid was verified by polymerase chain reaction (PCR). The standard curve of Ct value and copy number of NoV GⅡ was drawn according to real-time fluorescence quantitative PCR amplification curve, and corresponding standard curve equation was obtained. Four clinical fecal specimens obtained from the Children's Hospital of Kunming in December 2018 were detected by the constructed NoV GⅡ standard plasmid and detection system. Results PCR proved that the standard plasmid for detecting copy number of NoV GⅡ was constructed correctly. Standard curve equation of Ct value and viral copy number based on amplification curve of real-time fluorescence quantitative PCR was obtained as follows:Y=-3.972X+39.03, R²=0.991. Viral copy numbers in 4 fecal specimens were 30 443.45, 9 468.40, 53 176.69, and 4 493.12 copies/μL respectively. Conclusion The standard plasmid and its detection system for detecting the copy number of NoV GⅡ virus in fecal specimens are successfully established, which can provide an effective quantitative detection method for disease prevention and control and related experimental research.