目的 建立一种基于环介导等温扩增（LAMP）微流控芯片技术的快速检测蚊媒携带登革病毒的方法。方法 根据登革病毒衣壳蛋白（C）、前膜蛋白（prM）和非结构蛋白2A（NS2A）基因片段构建pUC-GW-Amp-CprM和pUC-GW-Amp-NS2A质粒，设计多组环介导等温扩增特异性引物组。制备LAMP微流控芯片，以靶基因质粒、登革病毒核酸为模板进行检测，根据起峰时间、相对荧光单位、假阳性、重复实验稳定性、检测灵敏度等指标筛选最优引物组。用最优引物组分别检测埃及伊蚊、白纹伊蚊等主要登革热传播媒介和常见的致倦库蚊、中华按蚊、摇蚊的cDNA，并与寨卡病毒、流行性乙型脑炎病毒、恶性疟原虫、黄热病毒等常见蚊媒传播病原体进行交叉检测，评价LAMP法的特异性。评价LAMP法检测CprM和NS2A靶基因质粒的灵敏度，分别与PCR、实时荧光定量PCR（qPCR）检测结果进行比较。用登革病毒RNA与传播媒介埃及伊蚊、白纹伊蚊RNA分别以1∶10、1∶100和1∶1 000混合，模拟媒介携带登革病毒的样品进行LAMP微流控芯片检测。对采集自宁德市三都岛、西安市、上海朱家角、长兴岛和五角场等5地的白纹伊蚊的cDNA进行检测。结果 引物组筛选结果显示，CprMG2和NS2AG1引物组起峰时间早，相对荧光单位高，灵敏度最高，为最优引物组。CprMG2引物组可检测浓度低至10^-6 ng/μl的pUC-GW-Amp-CprM质粒，最低检测限为1.3 × 10³拷贝；NS2AG1引物组可检测浓度低至10^-9 ng/μl的pUC-GW-Amp-NS2A质粒，最低检测限为1拷贝。最优引物组CprMG2、NS2AG1对埃及伊蚊、白纹伊蚊、中华按蚊、致倦库蚊、摇蚊等蚊虫cDNA无非特异性扩增，与寨卡病毒、流行性乙型脑炎病毒、恶性疟原虫、黄热病毒核酸无交叉反应。LAMP微流控芯片检测CprM靶基因的最低检测限为1.3 × 10³拷贝，较PCR检测的最低检测限（1.3 × 10⁴拷贝）高1个数量级；LAMP微流控芯片检测CprM和NS2A靶基因的最低检测限分别为1.3 × 10³拷贝、1拷贝，与qPCR检测的最低检测限（1.3 × 10³拷贝、1拷贝）相当。LAMP微流控芯片检测模拟现场样品的结果显示，CprMG2可检出与埃及伊蚊、白纹伊蚊RNA 1∶10、1∶100和1∶1 000混合的登革病毒RNA，NS2AG1最低可检出1∶100混合的登革病毒，特异性均为100%。LAMP微流控芯片检测5地现场采集白纹伊蚊的结果均为阴性。结论 建立了基于LAMP微流控芯片检测登革病毒的方法，该法敏感性和特异性高，检测时间短，可用于现场蚊媒携带登革病毒的检测。

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.