Research On Rapid Amplification Detection Of Highly Sensitive Gene Mutation

Nucleotide substitutions and deletions derived from the genome may alter the function and properties of the original protein after transcription and translation,resulting in changes in the organism’s traits.Base substitutions and deletions occurring in pathogens genomes will affect the diagnosis and treatment of diseases.For example,single nucleotide mutations at locus A2142 C,A2142G,and A2143 G in the 23 S r RNA gene of Helicobacter pylori lead to clarithromycin resistance,which affects the eradication effect of conventional treatment.Besides,base substitutions and deletions of the SARS-Co V-2will cause the mutation of antigen recognition sites and reduce the sensitivity to antibody neutralization,resulting in the appearance of new variant strains with higher infection rates,which would increase the transmissibility of this virus.Therefore,the realization of rapid and sensitive detection of base substitutions and deletions in pathogens genomes is of great significance for disease prevention and treatment.At present,the most used detection method for nucleotide substitutions and deletions is next generation sequencing.However,this method involves cumbersome steps,high equipment requirements,and time-consuming.In our previous work,We have built the method of narrow-thermal cycling amplification,which only requires an ordinary fluorescent real-time PCR instrument and could shorten the detection time to 30 minutes.Moreover,the results of which could be reported in real time.To shorten the detection time and improve the detection efficiency,a detection method for rapid nucleic acid amplification,accelerated circulation polymerase chain reaction(AC-PCR),was developed in this study by combining the above narrow-thermal cycling amplification method and sequence specific amplification technology,which was employed for the detection of nucleotide substitutions and deletions.This method has excellent sensitivity and specificity,whose detection limit is as low as 10 and 100 copies/reaction for single nucleotide mutations of Helicobacter pylori A2143 G and A2142G/C detection,while this method could detect 0.1% of the A2143 G mutation in samples.In addition,this method could detect as low as 10 copies of spike protein expression gene with base deletions in each reaction for SARS-Co V-2 Lambda and Delta varieties identification.The test results of simulated throat swab samples showed that the detection limit of this method had a separation coefficient of 3.3% for ten replicates,verifying the favorable stability of the method.Based on the AC-PCR,a method for clarithromycin resistant Helicobacter pylori detection and analysis in gastric mucosa samples was established in this study,which is proved to have the potential for providing clinical medication guidance and reducing the failure of H.pylori eradication caused by antibiotic resistance.Besides,a method for SARS-Co V-2 Lambda and Delta varieties detection was also established,which is expected to be used in COVID-19 prevention and control in developing countries.