Department of Clinical Laboratory,General Hospital of the Southern Theater Command of the People’s LiberationArmy,Guangzhou 510010 在期刊界中查找 在百度中查找 在本站中查找
Department of Clinical Laboratory,General Hospital of the Southern Theater Command of the People’s LiberationArmy,Guangzhou 510010 在期刊界中查找 在百度中查找 在本站中查找
Objective：To establish a CRISPR/Cas13a based method for detecting TP53 R248W mutant molecules. Methods：TP53 R248W mutant plasmid was constructed by Over⁃lap PCR using TP53 wild⁃type plasmid as template. The CRISPR/Cas13a method for the detection of TP53 R248W variant was initially established by optimizing the amplification product size，amplification technology， the length and concentration of crRNA. The sensitivity of CRISPR/Cas13a method was evaluated by TP53 R248W variants with different mutation rates and simulated plasma ctDNA. Results：The size of the amplified product detected by CRISPR/Cas13a was about 368 bp. The concentration of crRNA had influence upon the detection intensity of CRISPR/Cas13a. In the range of 0.05~0.25μmol/L， the higher the concentration of crRNA，the higher the detection intensity of CRISPR/Cas13a was detected. The sensitivity of CRISPR/ Cas13a in detecting TP53 R248W variants was 104 copies/μL，and the minimum mutation rate was 0.01% . Conclusion：The established CRISPR/Cas13a method has the advantages of rapid，simple，sensitive and specific，and provides a new technology for the detection of TP53 R248W variant in tissues and plasma.
