Establishment Of A Reverse Transcriptase Mediated Visualrapid Detection Method For The Novel Coronavirus

Objective: In 2019,the emergence of a novel coronavirus,known as SARS-COV-2,triggered a rare outbreak of viral pneumonia.The COVID-19 outbreak poses a serious threat to the international health ecosystem,disrupting the global economy and severely affecting human physical and mental health.The rapid detection of Novel Coronavirus is of great importance for the early diagnosis,timely and effective treatment of novel Coronavirus infection and rapid control of the spread of the epidemic.It is also of great significance to increase the stockpile of novel coronavirus detection methods.This study aims to establish a visual and rapid detection technique for Novel Coronavirus nucleic acid using isothermal amplification.Methods: SARS-COV-2 RT-RAA primers and probes were designed using novel Coronavirus nucleocapsid protein（N）gene,spike protein（S）gene and ORF1 ab conserved region sequence genes as templates,and the design principles of RT-RAA probe primers were applied.Reverse transcriptase mediated isothermal amplification fluorescence assay（fluorescence RT-RAA method）and colloidal gold lateral flow immunochromatographic strip（LFD）were respectively established to detect novel Coronavirus nucleic acid expressed by plasmids.The sensitivity and specificity of primers and probes were optimized.The novel coronavirus N,S and ORF1 ab plasmids were used as templates to evaluate the sensitivity and repeatability of the two detection methods.The novel coronavirus N,S and ORF1 ab plasmids were constructed and the novel coronavirus N,S and ORF1 ab plasmids were constructed.The three novel coronavirus plasmid templates with a concentration of 105copies/μL were used as the positive control group and the other coronavirus plasmid templates（such as HCo VOC43,HCo V-229 E,HCo V-HKU1,HCo V-NL63,MERS-Co V,SARS）as the experimental group.DEPC water as negative control group,to verify the specificity of the two coronaviruses detection methods.Results: Both novel Coronavirus detection methods could complete the detection within 12 min at 39 ℃.In the fluorescence RTRAA method,the amplification curves of 101-1010 copies samples of SARS-Co V-2 N and S genes were observed for 12 min,while no amplification curves were observed for 100 copies samples and negative control,indicating that the N and S gene sequences could be detected at the concentration of 101 copies.All samples with 100-1010 copies of SARS-COV-2 ORF1 ab gene had an amplification curve,but only the negative control had no amplification curve,indicating that the minimum detection limit of SARS-Co V-2 ORF1 ab gene was 100 copies.In RT-RAA-LFD method,N,S and ORF1 ab genes showed red detection line and blue quality control line at 103-1010 copies,while blank control（NC）and 100-2 copies showed no red detection line,but only blue quality control line.The lower limit of RT-RAA-LFD detection was 103 copies.In the specificity evaluation test,fluorescence RT-RAA method was used to detect SARS-COV-2,and only ARS-COV-2 was positive.Other samples such as HCOV-OC43,HCOV-229 E,HCOV-HKU1,HCOV-NL63,MERS-COV,SARS and the negative control group were all negative.The results of RT-RAA-LFD method were similar to those of fluorescence RT-RAA method.Only SARS-COV-2 showed positive results,and the samples showed a positive red detection line.Other samples（HCOVOC43,HCOV-229 E,HCOV-HKU1,HCOV-NL63,MERS-COV,SARS）and negative controls were all negative.The fluorescence RT-RAA method of SARS-COV-2 showed good repeatability.The peak time of amplification reaction was basically the same in the three replicate groups,and the curve shape was similar.The results of RT-RAALFD method were the same as those of fluorescence RT-RAA method,and the positive red detection lines were found in the repeat groups with the same color depth.Conclusions: In this study,novel Coronavirus fluorescent RT-RAA method and RTRAA-LFD method were established in combination with reverse transcriptase mediated isothermal amplification technology.Both methods can detect Novel Coronavirus in constant temperature at 39 ℃ for 12 minutes,showing good sensitivity,specificity and repeatability.It provides a new means for rapid detection of novel Coronavirus.It provides the possibility for rapid detection of COVID-19 in areas with poor medical conditions.The two methods in this study also make up for the shortage of novel Coronavirus isothermal detection technology in our country.Rapid isothermal detection of other pathogens is expected.More importantly,the rapid and accurate detection of Novel Coronavirus infection is conducive to the early diagnosis,timely treatment and rapid control of the spread of novel Coronavirus infection.