Isothermal amplification combined with a CRISPR system for detection of human parainfluenza virus
10.3969/j.issn.1002-2694.2024.00.173
- VernacularTitle:等温扩增联合CRISPR系统检测人副流感病毒方法的建立
- Author:
Zheng-tao ZHANG
1
;
Da-feng ZHENG
;
Zi-rong LIU
;
Li-bin YOU
;
Yu-wei WENG
Author Information
1. 福建医科大学附属南平第一医院,南平 353000;福建医科大学公共卫生学院,福州 350108
- Publication Type:Journal Article
- Keywords:
human parainfluenza virus;
RT-RAA;
CRISPR;
Cas12a;
Cas13a
- From:
Chinese Journal of Zoonoses
2024;40(12):1122-1127
- CountryChina
- Language:Chinese
-
Abstract:
This study was aimed at establishing a nucleic acid detection method for human parainfluenza viruses(HPIV)based on reverse transcription recombinase-aided amplification(RT-RAA)combined with a CRISPR system.Type-specific primer pairs for RT-RAA and CRISPR RNA(crRNA)targeting conserved sequences of the nucleocapsid(N)gene of HPIV-1 were designed.Fluorescence intensities from the cleavage of fluorophore labeled probes mediated by Cas13a were measured to optimize the crRNA and Cas13a concentrations.We evaluated the lower limit of detection,sensitivity,and specificity of RT-RAA combined with CRISPR/Cas13a detection in both in vitro transcribed RNAs and clinical specimens.The limit of detection of RT-RAA combined with CRISPR/Cas13a for HPIV-1 reached 1 copy/reaction.The method was subsequently used to detect clinical samples containing HPIV-2～4 and eight other respiratory viruses,and no cross-reaction was observed.This method had high sensitivity(100％)and specificity(97.8％),and high detection consistency with commercial kits(Kappa=0.88).Thus,our HPIV-1 nucleic acid detection method based on RT-RAA combined with the CRISPR/Cas13a system was success-fully established.This method is rapid,sensitive,and specific,and does not require specialized nucleic acid detection equip-ment.
