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A rapid and high-throughput Helicobacterpylori RPA-CRISPR/Cas12a-based nucleic acid detection system

文献类型： 外文期刊

作者： Liu, Hua 1 ; Wang, Jinbin 2 ; Hu, Xiuwen 2 ; Tang, Xueming 3 ; Zhang, Chao 1 ;

作者机构： 1.Tongji Univ, Shanghai YangZhi Rehabil Hosp, Fundamental Res Ctr, Sch Life Sci & Technol,Shanghai Sunshine Rehabil C, Shanghai 201619, Peoples R China

2.Shanghai Acad Agr Sci, Inst Biotechnol Res, Key Lab Agr Genet & Breeding, Shanghai 201106, Peoples R China

3.Shanghai Jiao Tong Univ, Sch Agr & Biol, Shanghai 200240, Peoples R China

关键词： Helicobacter pylori; UreB gene; RPA-CRISPR; Cas12a; Rapid high-throughput detection

期刊名称：CLINICA CHIMICA ACTA （ 影响因子：5.0； 五年影响因子：4.2 ）

ISSN： 0009-8981

年卷期： 2023 年 540 卷

页码：

收录情况： SCI

摘要： Background: Helicobacter pylori lives in the human stomach and causes gastric cancer and other gastric diseases. The development of molecular technology has facilitated low-cost, rapid, and high-throughput detection of H. pylori.Materials and methods: The combination of isothermal recombinase polymerase amplification (RPA) and CRISPR-Cas12a was used for early diagnosis and monitoring of H. pylori in clinical settings. The UreB genes from 242 H. pylori strains were subjected to cluster analysis, and we designed corresponding RPA primers and screened 2 sets of CRISPR-derived RNAs (crRNAs) for accurate H. pylori recognition. We then performed specificity and sensitivity validation of seven strains using this RPA-CRISPR/Cas12a method. In addition, the cut-off values of this RPA-CRISPR/Cas12a method based on fluorescence values (i.e., RPA-CRISPR/Cas12a-FT) were determined by comparison with quantitative PCR (qPCR), and further experiments comparing different methods were per -formed using clinical samples.Results: We developed a rapid detection system based on the combination of RPA and CRISPR-Cas12a, which was applied to the early diagnosis and monitoring of H. pylori in clinical settings. The RPA-CRISPR/Cas12a system was used to detect the UreB gene. We found that the limit of detection (LOD) for the CRISPR/Cas12a method based on the lateral flow dipstick result (i.e., CRISPR/Cas12a-LFD) was 100 copies, the cut-off value was 1.4; and for CRISPR/Cas12a-FT the LOD was 50 copies. This system was used to assess clinical samples and showed high reproducibility with proof-of-concept sensitivity, and the whole detection process was completed within 40 min.Conclusion: As a diagnostic method that can detect the UreB gene of H. pylori in gastric tissue samples rapidly, sensitively, visually, and in a high throughput manner, our method provides a new diagnostic option for clini-cians. This system is ideal for hospitals or testing sites with limited medical resources.

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