Rapid detection of in based on CRISPR-Cas13a coupled with recombinase polymerase amplification.
Xiao-Xuan Zhu, Ying-Si Wang, Su-Juan Li, Ru-Qun Peng, Xia Wen, Hong Peng, Qing-Shan Shi, Gang Zhou, Xiao-Bao Xie, Jie Wang
Author Information
Xiao-Xuan Zhu: Guangdong Provincial Key Laboratory of Nutraceuticals and Functional Foods, College of Food Science, South China Agricultural University, Guangzhou, Guangdong, China.
Ying-Si Wang: Guangdong Provincial Key Laboratory of Nutraceuticals and Functional Foods, College of Food Science, South China Agricultural University, Guangzhou, Guangdong, China.
Su-Juan Li: Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, Guangdong, China.
Ru-Qun Peng: Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, Guangdong, China.
Xia Wen: Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, Guangdong, China.
Hong Peng: Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, Guangdong, China.
Qing-Shan Shi: Guangdong Provincial Key Laboratory of Nutraceuticals and Functional Foods, College of Food Science, South China Agricultural University, Guangzhou, Guangdong, China.
Gang Zhou: Guangdong Provincial Key Laboratory of Nutraceuticals and Functional Foods, College of Food Science, South China Agricultural University, Guangzhou, Guangdong, China.
Xiao-Bao Xie: Guangdong Provincial Key Laboratory of Nutraceuticals and Functional Foods, College of Food Science, South China Agricultural University, Guangzhou, Guangdong, China.
Jie Wang: Guangdong Provincial Key Laboratory of Nutraceuticals and Functional Foods, College of Food Science, South China Agricultural University, Guangzhou, Guangdong, China.
The principal pathogen responsible for chronic urinary tract infections, immunocompromised hosts, and cystic fibrosispatients is , which is difficult to eradicate. Due to the extensive use of antibiotics, multidrug-resistant has evolved, complicating clinical therapy. Therefore, a rapid and efficient approach for detecting strains and their resistance genes is necessary for early clinical diagnosis and appropriate treatment. This study combines recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats-association protein 13a (CRISPR-Cas13a) to establish a one-tube and two-step reaction systems for detecting the gene in . The test times for one-tube and two-step RPA-Cas13a methods were 5 and 40 min (including a 30 min RPA amplification reaction), respectively. Both methods outperform Quantitative Real-time Polymerase Chain Reactions (qRT-PCR) and traditional PCR. The limit of detection (LoD) of genome in one-tube and two-step RPA-Cas13a is 10 aM and 1 aM, respectively. Meanwhile, the designed primers have a high specificity for gene. These two methods were also verified with actual samples isolated from industrial settings and demonstrated great accuracy. Furthermore, the results of the two-step RPA-Cas13a assay could also be visualized using a commercial lateral flow dipstick with a LoD of 10 fM, which is a useful adjunt to the gold-standard qRT-PCR assay in field detection. Taken together, the procedure developed in this study using RPA and CRISPR-Cas13a provides a simple and fast way for detecting resistance genes.
