Study Of SARS-CoV-2 Nucleic Acid Detection

Coronavirus disease 2019(COVID-19)is one of the most dangerous infectious diseases in the world.As of April 5,2022,it has infected about 6.39%of the global population and the number of infections is still increasing rapidly.Diagnosis and isolation remain the first and most efficient step in epidemic prevention and control.Genome sequencing is the most accurate detection method among SARS-CoV-2 detection methods,but its high requirements for laboratory safety,equipment,and operators limit its application in clinical detection.Antigen and antibody detection can provide results within 30 minutes,but the fatal flaw is that the detection window period is lengthy,and only the virus content in the patient is high enough,can be accurately detected.Reverse transcriptionpolymerase chain reaction(RT-PCR)detection has become the gold standard for diagnosing COVID-19 because of its higher throughput,lower cost and more sophisticated technology compared to isothermal amplification technologies(IAT).However,the following shortcomings of RT-PCR detection still need to be improved in practice.Low virus content at the early stage of infection and low sample utilization rate result in the detection sensitivity not reaching the expected level.Mutations in Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)genome may lead to false-negative detection results.The nucleic acid detection is time-consuming and cannot be detected immediately.So it is urgent to improve the detection efficiency.In this thesis,the system and process of SARS-CoV-2 nucleic acid detection were explored,the existing detection technology was improved and the goal of more rapid and accurate detection was achieved.The contents of this research are organized as follows:(1)Quantitatively analyze the loss of sensitivity in the process of SARS-CoV-2 nucleic acid detection,and provide support for optimizing the detection process and solving falsenegative problems.In this part,the factors that may affect the diagnostic sensitivity of SARS-CoV-2,such as pH value,volume of preservation solution,nucleic acid extraction method and sample utilization rate,were analyzed systematically and quantitatively,and an optimized detection scheme was proposed.The results showed that the loss of sensitivity could be avoided as much as possible using acidic sample preservation solution and extracting nucleic acid by centrifugal column method.For a single sample,the release of pseudovirus was more sufficient with the increase of preservation solution volume,and when the volume reached 3 mL,the pseudovirus were nearly completely eluted.The optimized detection scheme(nucleic acid was extracted from all the preservation solution by centrifugal column and 12 μL RNA was added to the PCR system)was 10 times more sensitive than the conventional detection scheme(nucleic acid was extracted from 200 μL preservation solution and 4 μL RNA was added to the PCR system).(2)Explore the application of mixed detection in the diagnosis of COVID-19,design a new nucleic acid extraction method for large volume samples,and provide a more efficient and effective detection scheme for large-scale nucleic acid screening.In this part,the mixed detection of SARS-CoV-2 was studied and a method for nucleic acid extraction from large samples was designed using a syringe purification column.For positive samples with Ct value of around 30,the Ct value will increase by about 3 in the case of 10 mixed samples according to the dilution mixing test guidelines issued by The State Council;the Ct value will increase by about 1 in the case of 10 mixed samples according to the n-in-1 test guidelines issued by The State Council.Using the method designed in this thesis to extract nucleic acid from all the preservation solutions of the mixed samples,no matter whether the dilution mixing test or the n-in-1 test is used,when the mixed sample ratio reaches 1:29,the detected Ct value will not increase compared with "single sample and single test".This meant that the detection rate will not decrease and the occurrence of false-negative phenomenon was avoided.(3)An innovative primer design strategy was proposed based on protein three-dimensional structure and genetic code,which reduced the false-negative phenomenon of nucleic acid detection caused by SARS-CoV-2 genome mutation.In this part,the effect of mismatch of target sequences with primers or probes on PCR detection was investigated.Consistent with previous reports,the effect of mispairing at the 3’end of primers was the greatest,while mismatch at other positions did not significantly affect PCR detection.This suggested that placing the 3’ end of the primers on the nucleotides encoding amino acids which never mutate may reduce the risk of PCR failure due to SARS-CoV-2 genome mutations.Tryptophan is a particular amino acid.It has only one coding codon,and it is also the largest amino acid,with special side chains,which plays an important role in the stability of the protein structure core.Mutations in the tryptophan codon commonly affect the activity of protein.Based on the above analysis,two pairs of detection primers(NAml and NAm2)targeting N gene of SARS-CoV-2 were designed in this thesis.The results showed that the thermal stability of the corresponding domain of the four amino acid single-base mutant protein at the 3’ end of the two pairs of primers decreased significantly,especially that of the tryptophan mutant protein,whose thermal denaturation temperature was lower than 37℃ of normal human body temperature.At the same time,when the mutant protein was expressed at 37℃,the yield of the mutant protein decreased significantly,which meant that viruses mutated at this site cannot survive in humans.The results of the detection performance validation showed that NAml and NAm2 had excellent repeatability and had the same detection specificity and sensitivity as the primers designed by the Chinese and US Centers for Disease Control and Prevention(CDC).(4)A rapid RT-PCR detection system was designed and optimized to shorten the time of SARS-CoV-2 nucleic acid detection.The current nucleic acid detection kits all adopt a two-step method,which reduces the elongation temperature of polymerase to the annealing temperature of primers(55-60℃),resulting in low amplification rate of polymerase and long detection time.In this thesis,the primers and probes were extended and optimized so that their annealing temperature meets the optimal elongation temperature of Taq DNA polymerase(70-74℃),which ensures the fastest extension rate of Taq DNA polymerase.By using FQS probe(the 5’ end was labeled with fluorescence group,the base T near the 5’ end was labeled with quenching group,and the 3’ end was blocked with Spacer C3),the decrease in quenching efficiency caused by the prolongation of the probe was eliminated,and the background fluorescence of the reaction system was reduced.The results showed that the annealing and elongation steps of PCR reaction could be shortened from the conventional 20-40 s to 13 s by using the primer pair.In addition,this thesis optimized the time of other links in the PCR reaction.Under the premise that the detection performance was not degraded,the whole nucleic acid detection time was shortened from 74 min to 37 min.In summary,the defects of SARS-CoV-2 nucleic acid detection were studied and improved in this thesis.A detection scheme to reduce sensitivity loss and a nucleic acid purification method for large-volume samples were designed,and a novel primer design strategy was proposed to reduce the risk of false-negative nucleic acid detection due to SARS-CoV-2 genome mutation.On this basis,primers and probes in RT-PCR reaction were optimized to shorten the nucleic acid detection time to 50%of the original.