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To combine reverse transcription recombinase-aided amplification (RT-RAA) technology with the CRISPR/Cas13a detection system to establish a rapid, sensitive, and specific detection method for arthropod-borne viruses (RT-RAA-CRISPR/Cas13a).
We designed and synthesized RT-RAA isothermal amplification primers and specific crRNAs for four arthropod-borne viruses, namely, Zika (ZIKV), dengue (DENV), Japanese encephalitis (JEV), and chikungunya (CHIKV), and screened for the optimal combinations of amplification primers and crRNAs to establish the detection system.Viral nucleic acids were detected by fluorescence and lateral flow assay to determine the sensitivity and specificity. A TCEP/EDTA-based heat lysis protocol was optimized to enable virus detection without nucleic acid extraction.
Highly efficient amplification primers and crRNAs for ZIKV, DENV, JEV and CHIKV were screened, and an RT-RAA-CRISPR/Cas13a fluorescence and lateral flow assay was established for detection of arthropod-borne viruses. The method can complete the detection in 40 min, and the lowest detection limit was 102 copies/μl (ZIKV, DENV and CHIKV) to 101 copies/μl (JEV), and the sensitivity was comparable to that of RT-qPCR. There was no cross-reactivity among the four pathogens, and the specificity was high. Meanwhile, this method combined with nucleic acid extraction-free method can directly detect virus particles in liquid samples, which has the potential for field site application.
The CRISPR/Cas13a-assisted RT-RAA method established in this study demonstrates notable advantages, including simple operation, rapid reaction, high specificity, superior sensitivity, and low cost, and does not rely on specialized nucleic acid detection equipment, but only requires a thermostatic heating instrument to complete the detection. The method is suitable for the immediate detection of arthropod-borne viruses, offering a novel technical platform for arbovirus identification.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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