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Abstract
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Protocol
Results
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Biology

一种多检测试验为疟疾蚊子发射

Published: February 28, 2015
doi:
10.3791/52385
, , , ,
1Department of Pathology, Microbiology and Immunology,School of Veterinary Medicine, University of California – Davis, 2Veterinary Genetics Laboratory,School of Veterinary Medicine, University of California, Davis

Summary

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

Abstract

在冈比亚按蚊种类复杂，包括主要的疟疾在非洲发射蚊子。由于这些品种都是这样的医疗重要性，一些特质的典型特点采用分子检测的流行病学研究提供帮助。这些特征包括物种鉴定，抗药性，寄生虫感染状态，和主机偏好。由于冈比亚按蚊复杂的群体在形态上没有区别，聚合酶链式反应（PCR）是传统上用于识别物种。一旦种是已知的，一些下游的测定是进行例行阐明进一步的特点。例如，在一对位基因被称为KDR突变赋予对DDT和拟除虫菊酯类杀虫剂抗性。此外，酶联免疫测定（ELISA）或疟原虫寄生虫的DNA检测的PCR测定法是用于检测存在于蚊子组织寄生虫。最后，一​​个combinatioÑPCR和限制酶消化的可用于阐明宿主偏好（ 例如 ，人类与动物的血液）通过筛选蚊子吸血寄宿特异性DNA。我们已经开发了在一个时刻将所有的上述分析成单个多重反应的基因分型33SNPs为96或384个样品的多检测分析（MDA）。因为MDA包括多个标记的物种， 疟原虫的检测，和主机血液鉴定，产生假阳性或阴性的可能性大大从以前的测定法，其中包括每性状只有一个标记降低。这个强大而简单的分析可以经济高效地在现有的实验的一小部分时间检测到这些关键的蚊子特点。

Introduction

阿拉伯按蚊，按蚊coluzzii和冈比亚按蚊负责非洲1疟疾传播的主要媒介。这三种物种在形态区分2，并且只能通过分子测定法3-9进行区分。此外，还有许多下游实验经常进行，以帮助流行病学和群体遗传学的研究。这些包括（1）为形态岛屿10-12基因分型测定法中，（2）一种基因分型测定法识别在1014的非同义的SNP 个 段基因的氨基酸的密码子的位置（ 击倒抗性或KDR，SNP）13 -18，（3）的寄生虫检测的PCR 19-23，和（4）筛选在蚊子中肠24,25宿主特异的DNA。

我们开发了将所有这些测定与一个目标的单个多重反应的多检测分析（MDA）的alyzing在非洲疟疾向量的流行病学重要特征。 MDA的测定法包括：多个标记，用于检测（1）种（A.阿拉伯按蚊，冈比亚按蚊，A coluzzii，或其它（所有的3）的），（2）杀虫剂中KDR的SNP表示电阻（两者L1014F 和L1014S） ，（3）存在的两个主要的疟原虫，恶性疟原虫和P.间日疟原虫 ，和（4）从一禽和六个哺乳动物宿主血液来源。

传统上，这些测定法在分开的聚合酶链式反应进行的。当使用常规PCR平台完成所有这些测定中，它需要进行8-10 PCR反应检测和伴随凝胶电泳步骤。从准备每个PCR反应，记录结果需要4-5小时，而MDA方法，这里介绍需要大约5小时的总和。这相当于节省了单独的劳动力成本的90％。在MDA这里介绍的价格为5美元每个样品基因型的所有33个SNP。这是相当便宜比单琼脂糖凝胶为基础的分析，其中的成本约为1.50美元样本。测定法检测所涵盖的所有的MDA的特性将需要至少8-10分开琼脂糖凝胶为基础的测定以$ 12-15每一个样品的成本。此外，MDA的大大减少产生假阳性或阴性通过利用至少三个标记为每个寄生虫或主机的源检测的机会。

我们所使用的平台不限于疟疾的载 ​​体，但可以在各种各样的应用，如医药，兽药和基础生物学26-28中使用。深入涉及的样品大（为了100S）的数量关联研究或群体遗传学研究需要经济高效的检测筛选同时为多个标记。利用两个或更多个单独的PCR测定大多数研究可以实现MDA的用于以较低的成本更快的结果。 </p>

Protocol

1. PCR扩增混合所有的PCR引物（见补充表S1）的1.5ml离心管。每个SNP具有两个引物（正向和反向）。确保原料浓度每种PCR引物被保持在100微米。使足够引物混合物为500-1000的反应，以减少在工作台上移液误差和时间（参见补充表S2）。如果需要的话，制备等分试样，每管并储存100-200反应在-20℃。 制备的PCR鸡尾酒如生产商的方案（表1）中所描述。 96反应的体积包括20％的突出端。涡包含P…

Representative Results

物种鉴定： 以下5个SNP一起识别3种（A.阿拉伯按蚊，A coluzzii和冈比亚按蚊 ）（ 见表4）。如果样品是不是3种之一，三个单核苷酸多态性（01073-213，04679-157和10313 -052）无法放大。 KDR基因型推断抗药性： 对位电压-门控钠通道的1014 个密码子对应于KDR。两个单核苷酸?…

Discussion

该MDA是由五个主要步骤：PCR扩增，虾碱性磷酸酶（SAP）的反应，SNP延伸，延伸产品的调理和基质辅助激光解吸/电离-飞行（MALDI-TOF）质谱33-37的时间。第一次PCR扩增步骤扩增的DNA的侧翼每个SNP，使得足够的模板DNA将是可利用的SNP的延伸步骤。在SAP反应中和未使用的dNTP其可以用下面的SNP延伸步骤干涉。 SNP扩展涉及每个​​SNP位点和大众修饰的核苷酸终止一个延伸引物。 3'-末端修饰的核苷…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢博士。安东尼山茱萸和劳拉·诺里斯在加州大学戴维斯分校和卡塔琳娜Kreppel博士在格拉斯哥大学从坦桑尼亚提供蚊子标本。我们感谢Smita达斯女士和道格拉斯·诺里斯博士公共卫生约翰霍普金斯学院分享蚊虫样本赞比亚。我们也感谢李五米永先生在兽医遗传学实验室对实验设计培训。这项工作是支持由美国国立卫生研究院授予R01AI 078183和R21AI062929。

Materials

Name of Material/ Equipment Company Catalog Number Comments/Description
MassARRAY Analyzer Compact Sequenom MT9 MALDI-TOF mass spectrometry for genomic applications to analyze nucleic acids.
MassARRAY Nanodispenser Sequenom RS1000 Transfers completed iPLEX reaction products to the SpectroCHIP
iPLEX Gold Genotyping Reagent Set Sequenom 10158 Reagents used for iPLEX assay including SAP kit.
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Tags

Multi-detection AssayAnopheles GambiaeMalariaMosquitoesSpecies IdentificationInsecticide ResistancePlasmodium DetectionHost PreferencePCRELISAMultiplex Genotyping
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Lee, Y., Weakley, A. M., Nieman, C. C., Malvick, J., Lanzaro, G. C. A Multi-detection Assay for Malaria Transmitting Mosquitoes. J. Vis. Exp. (96), e52385, doi:10.3791/52385 (2015).
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