一种用于鉴定致倦库蚊 2Rc 倒位的 PCR-RFLP 方法。

A PCR-RFLP method for genotyping of inversion 2Rc in Anopheles coluzzii.

机构信息

Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, 46556, USA.

Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN, 46556, USA.

出版信息

Parasit Vectors. 2021 Mar 22;14(1):174. doi: 10.1186/s13071-021-04657-x.

DOI:10.1186/s13071-021-04657-x

PMID:33752733

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7983089/

Abstract

BACKGROUND

Genotyping of polymorphic chromosomal inversions in malaria vectors such as An. coluzzii Coetzee & Wilkerson is important, both because they cause cryptic population structure that can mislead vector analysis and control and because they influence epidemiologically relevant eco-phenotypes. The conventional cytogenetic method of genotyping is an impediment because it is labor intensive, requires specialized training, and can be applied only to one gender and developmental stage. Here, we circumvent these limitations by developing a simple and rapid molecular method of genotyping inversion 2Rc in An. coluzzii that is both economical and field-friendly. This inversion is strongly implicated in temporal and spatial adaptations to climatic and ecological variation, particularly aridity.

METHODS

Using a set of tag single-nucleotide polymorphisms (SNPs) strongly correlated with inversion orientation, we identified those that overlapped restriction enzyme recognition sites and developed four polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) assays that distinguish alternative allelic states at the tag SNPs. We assessed the performance of these assays using mosquito population samples from Burkina Faso that had been cytogenetically karyotyped as well as genotyped, using two complementary high-throughput molecular methods based on tag SNPs. Further validation was performed using mosquito population samples from additional West African (Benin, Mali, Senegal) and Central African (Cameroon) countries.

RESULTS

Of four assays tested, two were concordant with the 2Rc cytogenetic karyotype > 90% of the time in all samples. We recommend that these two assays be employed in tandem for reliable genotyping. By accepting only those genotypic assignments where both assays agree, > 99% of assignments are expected to be accurate.

CONCLUSIONS

We have developed tandem PCR-RFLP assays for the accurate genotyping of inversion 2Rc in An. coluzzii. Because this approach is simple, inexpensive, and requires only basic molecular biology equipment, it is widely accessible. These provide a crucial tool for probing the molecular basis of eco-phenotypes relevant to malaria epidemiology and vector control.

摘要

背景

对疟疾病媒如 An. coluzzii Coetzee & Wilkerson 中的多态性染色体倒位进行基因分型非常重要，这既是因为它们会导致潜在的种群结构，从而误导媒介分析和控制，也是因为它们会影响具有流行病学意义的生态表型。传统的细胞遗传学基因分型方法存在障碍，因为它劳动强度大，需要专门的培训，并且只能应用于一种性别和发育阶段。在这里，我们通过开发一种简单、快速的分子方法来规避这些限制，该方法可以对 An. coluzzii 中的 2Rc 倒位进行基因分型，既经济又适用于野外。该倒位强烈暗示了对气候和生态变化，特别是干旱的时空适应。

方法

我们使用一组与倒位方向强烈相关的标记单核苷酸多态性（SNP），确定了那些与限制性内切酶识别位点重叠的标记 SNP，并开发了四种聚合酶链反应（PCR）限制性片段长度多态性（RFLP）分析，可区分标记 SNP 的替代等位基因状态。我们使用来自布基纳法索的蚊种群样本评估了这些分析的性能，这些样本已经通过细胞遗传学核型分析进行了基因分型，同时还使用了两种基于标记 SNP 的互补高通量分子方法进行了基因分型。在来自其他西非（贝宁、马里、塞内加尔）和中非（喀麦隆）国家的蚊种群样本中进行了进一步验证。