論文 - 詳細
| RRC ID | 74613 |
|---|---|
| 著者 | Qiu X, Xu S, Liu X, Han L, Zhao B, Che Y, Han L, Hou X, Li D, Yue Y, Chen S, Kang Y, Sun L, Li Z. |
| タイトル | A CRISPR-based nucleic acid detection platform (CRISPR-CPA): Application for detection of Nocardia farcinica. |
| ジャーナル | J Appl Microbiol |
| Abstract |
AIMS:To establish a CRISPR-based nucleic acid detection platform and apply it to the detection of Nocardia farcinica. METHODS AND RESULTS:A CRISPR-based nucleic acid detection platform, termed CRISPR-CPA (CRISPR/Cas12a combined with PCR amplification), which employed PCR for pre-amplification of target sequences and CRISPR-Cas12a-based detection for decoding of the PCR amplicons, was developed. To demonstrate its feasibility, CRISPR-CPA was applied to the detection of N. farcinica. A pair of PCR primers and a crRNA, which targeting the conservative and specific part of gyrA of N. farcinica reference strain IFM 10152, were designed according to the principle of CRISPR-CPA. The whole detection process of N. farcinica CRISPR-CPA assay, including sample pre-treatment and DNA extraction (~20 min), PCR pre-amplification (60 min), CRISPR-based detection (10 min), can be completed within 90 min. A total of 62 isolates were used to evaluate the specificity of N. farcinica CRISPR-CPA assay. Clinical specimens were employed to determine the feasibility of the method in practical application. The limit of detection of the N. farcinica CRISPR-CPA assay is 1 pg DNA per reaction in pure cultures and 105 CFU/ml in sputum specimens, which is similar with culture but significantly more timesaving. CONCLUSIONS:The N. farcinica CRISPR-CPA assay is an economic and specific method to detect N. farcinica and provides a high-efficiency tool for screening of pathogens especially of some hard-to-culture and slow-growth infectious agents. SIGNIFICANCE AND IMPACT OF THE STUDY:In CRISPR-CPA system, the PCR primers are engineered with a protospacer adjacent motif (PAM) site of Cas12a effector and an additional base A was added at the 5' end of the engineered PCR primer for protecting PAM site, thus the CRISPR-CPA can detect any sequence. Also, we applied CRISPR-CPA to rapidly detect N. farcinica, which is slow-growing bacteria and is firstly detected by a CRISPR-based method. |
| 巻・号 | 132(5) |
| ページ | 3685-3693 |
| 公開日 | 2022-5-1 |
| DOI | 10.1111/jam.15424 |
| PMID | 34936163 |
| MeSH | CRISPR-Cas Systems* DNA Nocardia* / genetics Nucleic Acid Amplification Techniques / methods |
| IF | 3.066 |
| リソース情報 | |
| 病原真核微生物 | Nocardia farcinica IFM 10152 |