Establishment of a method for detecting Helicobacter pylori based on recombinase-aided isothermal amplification and CRISPR-Cas13a
10.3760/cma.j.cn114452-20240102-00001
- VernacularTitle:基于重组酶介导的等温扩增和CRISPR-Cas13a检测幽门螺杆菌的方法的建立
- Author:
Yaxuan WANG
1
;
Xiaochuan LIU
;
Zixiao ZHU
;
Jihong HU
Author Information
1. 北京医院 国家老年医学中心 国家卫生健康委临床检验中心 中国医学科学院老年医学研究院，北京 100730
- Keywords:
Helicobacter pylori;
Nucleic acid testing;
Infection;
Genes
- From:
Chinese Journal of Laboratory Medicine
2024;47(6):686-692
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To develop a nucleic acid detection system for Helicobacter pylori ( H. pylori) based on recombinase-aided isothermal amplification (RAA) and clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas13a). Methods:Thirty strains of H. pylori, as well as two strains each of Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Enterobacter cloacae, and Klebsiella pneumoniae were collected from the Emergency General Hospital between 2021 and 2022. The specific primers and CRISPR RNA (crRNA) required for this newly established method were designed based on the conserved region of the ureC gene of H. pylori. Then, the primer pair that produced the least non-specific products was screened out using agarose gel electrophoresis, and the crRNA sequence with the highest cleavage efficiency was screened according to the fluorescence intensity produced by Cas13a cutting fluorescence probe. The RAA-Cas13a nucleic acid detection system was developed, and the limit of detection and the specificity of which were evaluated by detecting gradient dilutions of H. pylori ATCC 43504 genomic DNA and 5 different clinically common pathogens′ genomic DNA. The consistency with quantitative real-time PCR(qPCR) method was obtained by simultaneously detecting clinical strains using this method and established qPCR method. Two-tailed paired t-test was used to compare the fluorescence results between the two groups, and a P value less than 0.05 indicates a statistically significant difference. Results:The established RAA-Cas13a nucleic acid detection system could detect target DNA as low as 10 copies/μl. within 1 hour ( t=11.05, P<0.01), without cross-reaction with the other 5 clinically common strains. That method also showed good consistency compared to the qPCR method, the kappa coefficient=1. Conclusions:A method combining RAA with CRISPR-Cas13a for detecting H. pylori has been established, which can be used for rapid and sensitive identification of H. pylori infection.
