Screening of mosquitoes using SYBR Green I-based real-time RT-PCR with group-specific primers for detection of Flaviviruses and Alphaviruses in Taiwan
Screening of mosquitoes using SYBR Green I-based real-time RT-PCR with group-specific primers for detection of Flaviviruses and Alphaviruses in Taiwan
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DOI：
10.1016/j.jviromet.2010.05.006
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发表时间：
2010-09-01
影响因子：
3.1
通讯作者：
Wu, Ho-Sheng
中科院分区：
文献类型：
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作者：
Yang, Cheng-Fen;Chen, Chien-Fu;Wu, Ho-Sheng
监测蚊子携带的传染媒介对预测病媒传染病的风险很重要。在这项研究中，建立了一种方法来大规模筛选蚊子的病毒感染。该检测方法在36.64-43.45个循环中检测了4种登革病毒（DENV）血清型（DENV-1、DENV-2、DENV-3和DENV-4）、日本脑炎病毒（JEV）、辛德毕斯病毒和基孔肯雅病毒的病毒载量（1 PFU/mL，通过实时RT-PCR测定）。将该方法应用于75，364只现场捕获的蚊子，这些蚊子被分成10，343个池。从25个三带喙库蚊库（906只）和1个库（44只）中检出乙型脑炎病毒。fuscocephala雌性。这些病毒从一半的阳性样本池中分离出来。在2个43只雌性埃及伊蚊池中检测到登革病毒。此外，在实验室中经口感染登革热病毒的蚊子也被用来验证该测试。个体蚊子在喂食病毒污染的血液后的最佳检测时间是在第0天和第10天。每个样本池检测到的蚊子数量最多为1只受感染的蚊子加上59只未受感染的蚊子;在24小时内保存样本的适当储存物质包括冰块和干冰。该方法结合稳健的自动化RNA提取方法和96孔实时RT-PCR机器，允许一次处理大量样品，使其成为同时监测黄病毒和甲病毒的本地和新出现的媒介传播传染病的有力工具。这项研究是第一个量化的病毒载量在个别蚊子超过16天的外源性潜伏期的过程。(C)2010爱思唯尔有限公司版权所有。
Surveillance for infectious agents carried by mosquitoes is important for predicting the risk of vector-borne infectious diseases. In this study, a method was established to mass-screen mosquitoes for viral infections. The assay detected the viral load of 4 dengue virus (DENV) serotypes (DENV-1, DENV-2, DENV-3, and DENV-4), the Japanese encephalitis virus (JEV), the Sindbis virus and the Chikungunya virus at 1 PFU/mL (determined by real-time RT-PCR) in 36.64-43.45 cycles. This method was applied to 75,364 field-captured mosquitoes that were grouped into 10,343 pools. Japanese encephalitis viruses were detected in 25 pools of 906 Culex tritaeniorhynchus females and a single pool of 44 Cx. fuscocephala females. These viruses were isolated from half of the positive pools. Dengue viruses were detected in 2 pools of 43 Aedes aegypti females. Additionally, mosquitoes that were infected orally with dengue viruses in the laboratory were also used to verify the test. The best detection times for individual mosquitoes after being fed virally-contaminated blood were at day 0 and day 10. The number of mosquitoes detected per pool was up to one infected mosquito plus 59 non-infected mosquitoes; the appropriate storage substances for holding samples within 24 h included ice cubes and dry ice. This method, combined with a robust and automated RNA-extraction method and a 96 well real-time RT-PCR machine, allows the processing of a large number of samples at once, making it a powerful tool for monitoring simultaneously local and emerging vector-borne infectious diseases of Flaviviruses and Alphaviruses. This study is the first to quantify the viral load in individual mosquitoes over the course of a 16-day extrinsic incubation period. (C) 2010 Elsevier B.V. All rights reserved.