关键词: 登革病毒, 蚊虫, 环介导等温扩增, 微流控芯片, 现场检测技术

Abstract:

Objective To establish a loop-mediated isothermal amplification (LAMP) based microfluidic chip technology for the rapid detection of dengue virus in mosquitoes. Methods According to the gene fragments encoding dengue virus capsid protein (C), pro-membrane protein (prM) and non-structural protein 2A (NS2A), plasmids pUC-GW-Amp-CprM and pUC-GW-Amp-NS2A were constructed, and specific primer sets for multi-group loop-mediated isothermal amplification were designed. The LAMP microfluidic chip was prepared for amplification with the target gene plasmids and dengue virus nucleic acid used as templates. Optimal primer combinations were screened according to the peak time, fluorescence intensity, false positivity, reproducibility, and sensitivity of the assay. The optimal primer combinations were used to detect the cDNAs of major dengue vectors, including Aedes aegypti and A. albopictus and commonly seen Culex quinquefasciatus, Anopheles sinensis, and Chironomus. The combinations were also used for cross-examination with common mosquito-borne pathogens, such as Zika virus, epidemic B encephalitis virus, Plasmodium falciparum, and Yellow fever virus to evaluate the specificity of the LAMP assay. The LAMP assay sensitivity for detection of pUC-GW-Amp-CprM and pUC-GW-Amp-NS2A plasmids was evaluated by comparing with the results of PCR and qPCR, respectively. Dengue virus RNA was mixed with their vector A. aegypti and A. albopictus RNA at 1 ∶ 10, 1 ∶ 100, and 1 ∶ 1 000, respectively, to simulate samples of vector carrying dengue viruses for LAMP microfluidic microarray detection. The A. albopictus cDNA samples collected from five locations, including Sandu Island in Ningde City, Xi’an City, Zhujiajiao, Changxing Island and Wujiaochang in Shanghai, were examined. Results The primer screening results suggested that the CprMG2 and NS2AG1 primers were identified as the optimal primer combinations because of their early peak onset, high relative fluorescence unit and highest sensitivity. Among them, CprMG2 can detect pUC-GW-Amp-CprM plasmid templates at concentrations as low as 10^-6 ng/μl, with a minimum detection limit of 1.3 × 10³ copies, and NS2AG1 can detect pUC-GW-Amp-NS2A plasmid templates at concentrations as low as 10^-9 ng/μl, with a minimum detection limit of 1 copy. The optimal primer combinations CprMG2 and NS2AG1 showed no unspecifical amplification of arthropod cDNAs of A. aegypti and A. albopictus and other common arthropods, including C. quinquefasciatus, An. sinensis, and Chironomus. They did not cross-react with the nucleic acids of the Zika virus, encephalitis B virus, P. falciparum and Yellow fever virus. The lowest detection limit of CprM target gene detected by LAMP microfluidic chip was 1.3 × 10³ copies, which was ten folds more sensitive than that of PCR (1.3 × 10⁴ copies); the lowest detection limits of CprM and NS2A target genes detected by LAMP microfluidic chip were 1.3 × 10³ and 1 copies, which were equivalent to those of qPCR (1.3 × 10³ and 1 copies). The LAMP microfluidic chip testing results on the simulated field samples showed that CprMG2 could detect 1 ∶ 10, 1 ∶ 100 and 1 ∶ 1 000 dilutions of the virus, and NS2AG1 could detect a minimum of 1 ∶ 100 dilutions of the virus, with a specificity of 100%. LAMP microfluidic chip testing of A. albopictus collected in the five field sites yielded negative results. Conclusion A LAMP microfluidic chip method for dengue virus detection was established. The method shows high sensitivity and specificity, short detection time, and can be used for field detection of mosquito vectors cattying dengue virus.

Key words: Dengue virus, Mosquitoes, Loop-mediated isothermal amplification, Microfluidic chip, Field testing technology