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首页> 外文期刊>Journal of Medical Entomology >Identification of Mosquito Bloodmeal Source by Terminal RestrictionFragment Length Polymorphism Profile Analysis of the Cytochrome BGene
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Identification of Mosquito Bloodmeal Source by Terminal RestrictionFragment Length Polymorphism Profile Analysis of the Cytochrome BGene

机译：通过细胞色素B基因的末端限制性片段长度多态性谱分析鉴定蚊子血粉来源

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Previous studies have shown that polymerase chain reaction (PCR) heteroduplex analysis (HDA) of the cytochrome B (cytb) gene is useful in identifying mosquito bloodmeals derived from avian hosts. However, interpretation of PCR-HDA gels is performed visually, which can make it difficult to analyze large numbers of specimens and to compare results between laboratories. We investigated the utility of a terminal restriction fragment length polymorphism (T-RFLP) assay to analyze cytb PCR products. PCR was performed on 123 blood or tissue samples from 55 avian, 13 mammalian, and one amphibian species by using end-labeled primers to amplify a 358-bp segment of cytb. Each PCR product was sequenced to determine predicted terminal restriction fragment (TRF) profiles. Additionally, experimental TRFs were determined by sizing fragments from restriction endonuclease digests with capillary electrophoresis. A Web-based searchable database was created to compare unknown mosquito bloodmeal TRF profiles against sequence-predicted and experimentally derived terminal fragment lengths of known vertebrates. The predictive value of experimental profiles was found to be accurate to the species level for 67 of 69 species (97%). Fifty-nine field-collected mosquitoes were tested to determine the bloodmeal source using the T-RFLP method. The bloodmeal source from 50 of these mosquitoes was identified by comparing the TRF profile of the unknown source against the cytochrome B database. The bloodmeal source from the remaining nine mosquitoes was not identified as no known profile matched the experimentally derived profile. T-RFLP analysis is a highly reproducible technique and the searchable TRF database is continually being expanded to include additional species from diverse geographic areas.
机译：先前的研究表明，细胞色素B（cytb）基因的聚合酶链反应（PCR）异源双链分析（HDA）可用于鉴定源自禽类宿主的蚊子血粉。然而，PCR-HDA凝胶的解释是目视进行的，这可能使得难以分析大量标本以及比较实验室之间的结果变得困难。我们调查了终端限制性片段长度多态性（T-RFLP）分析法来分析cytb PCR产物的效用。通过使用末端标记的引物扩增Cytb的358-bp片段，对来自55个禽类，13个哺乳动物和一种两栖动物的123个血液或组织样本进行PCR。对每种PCR产物进行测序以确定预测的末端限制性片段（TRF）图谱。另外，通过用毛细管电泳确定限制性核酸内切酶消化片段的大小来确定实验性TRF。创建了一个基于Web的可搜索数据库，以将未知蚊子的血TRF谱与已知脊椎动物的序列预测和实验得出的末端片段长度进行比较。发现实验概况的预测值精确到69种物种中的67种（97％）的物种水平。使用T-RFLP方法测试了59个野外采集的蚊子以确定血粉来源。通过将未知来源的TRF图谱与细胞色素B数据库进行比较，确定了其中50种蚊子的血粉来源。其余九个蚊子的血粉来源尚未确定，因为没有已知的分布与实验得出的分布相符。 T-RFLP分析是一种高度可重复的技术，可搜索的TRF数据库正在不断扩展，以包括来自不同地理区域的其他物种。

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