Establishment And Optimization Of Novel Coronavirus Assay By Reverse Transcription-loop Mediated Isothermal Amplification

The outbreak of a series of respiratory infections called Novel Coronavirus Pneumonia 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus type 2(SARS-Co V-2)has had a significant impact on the health and economy of the world.The virus can spread rapidly from person to person through droplets,contact and pollutants,so it is vitol to detect the virus genome or virus antigen quickly and accurately to prevent the virus from spreading on a large scale.The current detection methods for COVID-19 are divided into three categories:pathology testing,nucleic acid testing or serology.Pathology examination mainly depends on clinical manifestations,the incubation period is generally long,patient serum conversion usually occurs in 5 to 10 days after the initial symptom onset,however,the use of nucleic acid diagnostic methods-based real-time RT-PCR methods to detect new coronaviruses takes a long time and is difficult to detect,so it cannot meet the current needs for testing a large number of suspected patients,asymptomatic patients,and close contacts.It is urgent to create a nucleic acid detection method with fast,simple,sensitive,specific,economical and suitable for large-scale detection of SARS-Co V-2 at the grassroots level.Reverse Transcription Loop-Mediated Isothermal Amplification(Reverse Transcription Loop-Mediated Isothermal Amplification,RT-LAMP)is a new type of nucleic acid amplification technology.The RT-LAMP reaction can identify pathogenic microorganisms with high efficiency,celerity,well accuracy and convenience,because of its simplicity,speed and specificity.High sensitivity and other characteristics have been successfully applied to the detection of some pathogens,viruses,and fungi.This study aimed at the conserved sequences in the SARS-Co V-2 gene,designed primers,established an RT-LAMP detection method,and provided an effective technical methods for the early identification of SARS-Co V-2 infection.The main research results were as follows:(1)The reaction temperature,magnesium ion concentration,BST enzyme dosage,internal and external primer concentration ratio,SYBR GreenⅠfluorescent dye concentration and other conditions were optimized,and a d UTP/UDG anti-pollution system was established to overcome the false positive shortcomings of the LAMP method.And through screening a series of PCR enhancers,it is concluded that adding DMSO and BSA to the reaction system can further improved the reaction rate and stability,and finally determined the optimized RT-LAMP reaction system.(2)The established RT-LAMP method was used to detect the three coronaviruses and target sequences,and the results showed that the test for target sequences were positive,and the test results for other coronaviruses were all negative,indicating that this method showed good specificity.(3)Use the established RT-LAMP method to detect the sensitivity of the designed template nucleic acid,and the minimum detection limit was 9.2×10~1 copies/μL.In this study,a new RT-LAMP method for rapid detection of SARS-Co V-2 was successfully established.This method has the advantages of simple operation,high sensitivity,strong specificity,simple and quick,etc.which provides technical support for on-site detection of SARS-Co V-2 at grass-roots level and has a good development prospect.