期刊
VIROLOGY JOURNAL
卷 19, 期 1, 页码 -出版社
BMC
DOI: 10.1186/s12985-022-01878-z
关键词
CRISPR-Cas12a; RT-RAA; Diagnostics; Human norovirus; GII; 4 or GII; 17
类别
资金
- Natural Science Foundation of China [81973531]
- Fundamental Research Project of Shenzhen Science and Technology Innovation Commission [20200812211704001]
- Medical Scientific Research Foundation of Guangdong Province [A2019502]
- Scientific Research Program by Shaanxi Provincial Education Department [22JC010]
- Science and Technology Program of State Administration of Market Supervision and Administration [2021MK107]
This study developed a low-cost, disposable, and ultrasensitive assay for diagnosing human pathogens through the integration of CRISPR-Cas12a sensors with isothermal signal amplification. The assay successfully detected the GII.4 and GII.17 genotypes of norovirus (NOV) with high specificity and a low limit of detection. The method provided visual results and a faster alternative to real-time RT-PCR, with reliable accuracy.
Background Integrating CRISPR-Cas12a sensors with isothermal signal amplification can be exploited to develop low-cost, disposable, and ultrasensitive assays for the diagnostics of human pathogens. Methods RT-RAA-Cas12a-mediated real-time or end-point fluorescent and lateral flow strip (LFS) assays for direct detection of norovirus (NOV) genotype GII.4 or GII.17 were explored. Results The results showed that our RT-RAA-Cas12a-mediated fluorescent and LFS assay could detect NOV GII.4 or GII.17 by targeting the viral protein 1 gene. Our RT-RAA-Cas12a-mediated fluorescent and LFS assay can specifically detect NOV GII.4 or GII.17 with no cross-reactivity for other related viruses. The low limit of detection could reach 0.1 copies/mu L within approximately 30-40 min, and the results were visualized using an ultraviolet light illuminator or on a LFS without complex equipment. In addition, our RT-RAA-Cas12a-mediated fluorescent and LFS assay provided a visual and faster alternative to real-time RT-PCR assay, with 95.7% and 94.3% positive predictive agreement and 100% negative predictive agreement. Conclusions Together, our RT-RAA-Cas12a-mediated approach would have a great potential for point-of-care diagnostics of NOV GII.4 and/or GII.17 in resource-limited settings.
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