关键词: 疟原虫, 蚊种属, 登革病毒, 快速检测

Abstract:

Objective To develop an assay for rapid detection of mosquito species and their transmitted pathogens based on the clustered regularly interspaced short palindromic repeats-associated protein 12a (CRISPR/Cas12a) system, so as to improve the surveillance and control efficiency of mosquito-borne disease. Methods Genomic DNA was extracted from 30 first-instar Aedes albopictus larvae and 10 one-day-old adult Anopheles stephensi. Species-specific genomic sequences were amplified using recombinase polymerase amplification (RPA). Ae. albopictus genomic DNA was subjected to RPA with specific RPA primers of An. stephensi to assess the specificity of RPA. The CRISPR/Cas12a system was employed for fluorescence detection, and An. stephensi and Ae. albopictus-derived CRISPR RNA (crRNA) was employed for cross-reactivity evaluation to assess the specificity of the CRISPR/Cas12a system. Following recovery of cryopreserved Plasmodium yoelii, three mice were intravenously injected with 1 × 10⁶ P. yoelii each via tail vein. On day 3 post-infection, 100 An. stephensi mosquitoes were allowed to engorge on infected mice. Mosquitoes were dissected 1, 2, 3, 4, 5 and 10 days following blood meals and their midguts were sampled and observed for Plasmodium oocysts under a microscope. An. stephensi mosquitoes were infected with Plasmodium, and genomic DNA was extracted from mosquito pools 1, 2, 3, 4, 5 and 10 days post-infection. Plasmodium 18S rRNA gene fragment was amplified using RPA and CRISPR/Cas12a-based fluorescence detection. CRISPR/Cas12a-based fluorescence detection was performed with Plasmodium genomic DNA at serial dilutions of 10^-9, 10^-10, 10^-11, 10^-12, 10^-13, 10^-14, 10^-15, 10^-16, 10^-17 and 10^-18 mol/L to evaluate the lowest limit of detection of the system. Dengue virus serotypes Ⅰ, Ⅱ, ⅢandⅣ-specific sequences were synthesized, and cloned into the pUC18 plasmid vector. The nonstructural protein 5 (NS5) gene fragment was amplified using RPA, and then detected using CRISPR/Cas12a-based fluorescence assay. Dengue virus serotypesⅠ, Ⅱ, Ⅲ and Ⅳ cRNA were mixed with other dengue virus plasmids to evaluate the specificity of the CRISPR/Cas12a system, and CRISPR/Cas12a-based fluorescence detection was performed with Plasmodium genomic DNA at serial dilutions of 10^-9, 10^-10, 10^-11, 10^-12, 10^-13, 10^-14, 10^-15, 10^-16, 10^-17, 10^-18 and 10^-19 mol/L to evaluate the lowest limit of detection of the system. In addition, Cas12-based specific nucleic acid test strips were employed for visualized detection of An. stephensi, Ae. albopictus, Plasmodium, and dengue virus. Results RPA assay yielded the An. stephensi cytochrome c oxidase subunit 1 (cox1) gene fragment with 89 bp in length and Ae. albopictus cox1 gene fragment with 87 bp in length. CRISPR/Cas12a system detected robust activation of LbCas12a trans-cleavage activity by both Ae. albopictus and An. stephensi cox1 gene fragments, with detectable fluorescence signals emerging within 5 minutes of reaction initiation. The maximal signal intensity was observed for adult An. stephensi samples, reaching equilibrium by 13 minutes. RPA assay of P. yoelii 18S rRNA gene produced a fragment with 142 bp in length. CRISPR/Cas12a detection confirmed P. yoelii infection in An. stephensi samples 1, 2, 3, 4, 5, 10 days post-infection, with the lowest limit of detection of 3.3 × 10^-17 mol/L. Microscopic examinations revealed no oocyst formation on days 1 to 4 following blood meals, two observable oocysts on day 5, and progressive increased in both oocyst burdens and sizes on day 10. RPA assay of dengue virus serotypesⅠ, Ⅱ, Ⅲ and Ⅳ NS5 gene fragments generated serotype-specific products of 109, 93, 147, and 170 bp, respectively. CRISPR/Cas12a system detected dengue virus sequences in samples with detectable fluorescence signals within 5 minutes and peak intensity attained by 17 minutes. In addition, no inter-serotypic cross-reactivity was observed, with the lowest limit of detection of 2.5 × 10^-18 mol/L, and the Cas12-based nucleic acid test strips achieved visualized detection of target virus sequences within 30 minutes. Conclusion A rapid, highly sensitive and specific, visualized CRISPR/Cas12a-based assay has been established for identification of mosquito species and detection of pathogens, which provides an effective tool for early warning and transmission interruption of mosquito-borne diseases.

Key words: Plasmodium, Mosquito species, Dengue virus, Rapid detection