Establishment Of Multiple Detection Methods For SARS-CoV-2 N Protein

The SARS-CoV-2 epidemic has had a great impact on people’s physical and mental health and life and work around the world.The efficient detection and prevention of SARS-CoV-2 is a hot spot in clinical medicine and life science research.Nucleocapsid protein N protein has the advantages of rich content,conserved sequence and few mutations in the process of SARS-CoV-2 virus infection,and is an excellent early detection target for the new coronavirus compared with other structural proteins of SARS-CoV-2.In this paper,we intend to establish a variety of detection methods for SARS-CoV-2 N protein,in order to provide a variety of detection methods adapted to different actual detection scenarios for the clinical rapid detection of SARS-CoV-2.In this project,immunological methods were established for N protein of SARS-CoV-2:enzyme-linked immunosorbent method and magnetic microsphere chemiluminescence immunodetection method,and molecular biology detection method:RT-q PCR detection method and RT-LAMP isothermal amplification detection method,and the four methods were constructed and experimental conditions were optimized,and the specificity,sensitivity and repeatability of the established four detection methods were evaluated.The experimental results show that the linear regression equation of the ELISA detection method for SARS-CoV-2 was established in this study:Y=0.2323X+0.1532,R~2 is0.9967.Good specificity and repeatability,common pathogenic bacteria will not cause false positives,and the minimum detection concentration can reach 400 pg/m L;The linear regression equation for the magnetic microsphere chemiluminescence immunodetection method is:Y=61.45X+10714,and R~2 is 0.9925.Good specificity and repeatability,with a minimum detection concentration of 200 pg/m L;The average recovery rate was between97.44%and 103.35%,and the accuracy was good.The standard curve of RT-q PCR detection method was established,in which the linear regression equation of the internal reference gene RPP30 was:Y=-3.111X+40.11,the correlation coefficient R~2 was 0.9967,the linear regression equation of N gene was Y=-3.302X+41.53,and the correlation coefficient was0.9956.The lowest sample concentration that can be detected by this assay is 24 copies/m L and the sensitivity(limit of quantification)is 47 copies/m L;Good specificity and reproducibility;The established RT-LAMP detection method can produce results at 65°C for 30 minutes,with a sensitivity of up to 25 copies,and uses p H method,dye method and probe method to make the detection results visible,and the three methods are also consistent in this detection result,with good specificity and repeatability.This study provides ideas,experiences and methods for the establishment of detection methods for SARS-CoV-2 and other pathogenic substances that may occur in the future.