Establishment of isothermal detection method for human parainfluenza virus type 1-4 nucleic acid based on CRISPR/Cas12a system
10.3760/cma.j.cn112866-20230725-00006
- VernacularTitle:基于CRISPR/Cas12a系统的人副流感病毒1-4型核酸等温检测方法的建立
- Author:
Zhengtao ZHANG
1
;
Libin YOU
;
Yuwei WENG
Author Information
1. 福建医科大学公共卫生学院，福州 350122
- Keywords:
Human parainfluenza viruses;
Reverse transcription recombinase-aid amplification;
CRISPR;
Cas12a
- From:
Chinese Journal of Experimental and Clinical Virology
2023;37(5):524-529
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a nucleic acid detection method for human parainfluenza virus (HPIV) based on reverse transcription recombinase-aid amplification (RT-RAA) combined with CRISPR/Cas12a.Methods:The type-specific primer pairs of RT-RAA and CRISPR RNA (crRNA) targeted on conserved sequence of nucleocapsid protein (N) gene of HPIV were designed. Fluorescence intensities from cleavage of fluorophore labeled probes mediated by Cas12a were measured for screening of crRNA and concentration optimization of crRNA, Cas12a as well as the probe. With the RNA transcribed in vitro and clinical specimen, the lower limit of detection and specificity of RT-RAA combined CRISPR/Cas12a detection were evaluated. Results:The crRNA specific to each type of HPIV 1-4, with strongest cleavage activity, were screened out. With the optimal concentration of crRNA, Cas12a as well as the probe, the lower limit of detection could reach 10 copies of target gene per reaction on fluorescence intensity measurement. No cross-reaction was found in clinical samples of eight other respiratory viruses detected by this method .Conclusions:The established HPIV1-4 fluorescence CRISPR nucleic acid detection method is rapid, specific, and does not require professional nucleic acid detection equipment.
