期刊
FRONTIERS IN MICROBIOLOGY
卷 12, 期 -, 页码 -出版社
FRONTIERS MEDIA SA
DOI: 10.3389/fmicb.2021.732426
关键词
Salmonella; foodborne disease; RPA; CRISPR-Cas13a; molecular detection
类别
资金
- Key Research and Development Project of Jiangsu Province [BE2019761]
- Natural Science Foundation of Jiangsu Province [BK20211373]
- Jiangsu Provincial Key Medical Discipline of Epidemiology [ZDXKA2016008]
- 333Projects of Jiangsu Province [BRA2017552]
The study developed a simple, rapid, and specific detection method for Salmonella spp. using one-tube RPA-Cas13a, while the two-step assay was more sensitive, particularly for samples at low abundance.
Salmonella spp. is one of the most common foodborne disease-causing pathogens that can cause severe diseases in very low infectious doses. Rapid and sensitive detecting Salmonella spp. is advantageous to the control of its spread. In this study, a conserved short fragment of the Salmonella invA gene was selected and used to design primers and specific crRNA (CRISPR RNA) for establishing a one-tube and two-step reaction system for Salmonella spp. detection, by combining recombinase polymerase amplification (RPA) with CRISPR-Cas13a (Clustered Regularly Interspaced Short Palindromic Repeats associated protein 13a) cleavage. The established one-tube RPA-Cas13a method can complete the detection within 20 min and the two-step RPA-Cas13a method detection time within 45 min. The designed primers were highly specific to Salmonella spp. and had no cross-reaction with the other nine diarrheal bacteria. The one-tube RPA-Cas13a could detect the Salmonella genome with the limit of 10(2) copies, which was the same as real-time polymerase chain reaction (PCR), but less sensitive than two-step RPA-Cas13a (10(0) copies). The detection results of one-tube or two-step RPA-Cas13a and real-time PCR were highly consistent in clinical samples. One-tube RPA-Cas13a developed in this study provides a simple, rapid, and specific detection method for Salmonella spp. While two-step assay was more sensitive and suitable for samples at low abundance.
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