Rapid Identify Of Characteristic Point Mutation In Major Mutants Of SARS-CoV-2 Using Ligase Detection Reaction

A novel coronavirus outbreak in 2019 was named SARS-COV-2 by the The International Committee on Taxonomy of Viruses(ICTV).SARS-COV-2 has shown a higher infectivity and concealment level than previous coronavirus,such as Severe Acute Respiratory Syndrome,SARS and Middle East Respiratory syndrome,MERS.The disease caused by SARS-COV-2infection has been declared a global pandemic by the World Health Organization(WHO).SARS-COV-2 is a single-stranded RNA virus,which can easily accumulate natural mutations in the process of copying represented,finally these natural mutations over time and with the human host choice,cause the mutant strains of various kinds are springing up constantly,compared to the original strains some mutant strains have different characteristics,such as infectious and toxicity of change.The Delta mutant containing the D614 mutation is one of the most infectious and virulent mutants so far.Developing rapid and effective nucleic acid detection to identify high-risk mutants of novel coronavirus and their characteristic point mutation sites is an important means to block the epidemic of the disease.In this study,the ligase detection reaction(LDR)technology was used as the core to establish a nucleic acid detection system that could quickly identify the specific mutation sites of the main mutants of SARS-COV-2 at constant temperature.We first established the basic reaction system of LDR,and the results show that the method has good discrimination against the point mutations carried by the viral RNA template of more than 5 pmol/L and longer than 40nt.In order to further improve the sensitivity of the method,the original ligase probe was optimized to lock probe.After screening the optimal reaction conditions,the sensitivity of the ligase detection system was increased to 5 fmol/L,and the sensitivity was increased 1000 times.In order to further improve the sensitivity of the method,we optimized the original connecting probe into a lock probe,and screened the optimum reaction conditions to improve the sensitivity of the ligase detection system by 10~3times;In order to further shorten the detection time and sensitivity,we combined the lock probe ligase detection system with CRISPR-Cas12b system,and realized the result readout by using fluorescent signal.The final detection time was shortened to 45 minute,and the sensitivity was finally improved to 0.5 fmol/L.In conclusion,this study has established a nucleic acid detection system based on ligase.After a series of optimization of conditions,a nucleic acid detection system that does not rely on variable temperature instruments to quickly identify the characteristic point mutations of novel coronavirus mutants has been preliminarily formed,and the system can detect fragmented RNA templates,with the shortest template length as low as 50 nt.There is poor quality of RNA templates and other important SNP detection fields,which also have certain application potential.