迪尔斯复合种鉴定 PCR（DiCSIP）提高了大湄公河次区域按蚊复合种的鉴定。

Dirus complex species identification PCR (DiCSIP) improves the identification of Anopheles dirus complex from the Greater Mekong Subregion.

机构信息

Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand.

HSM, Univ. Montpellier, CNRS, IRD, Montpellier, France.

出版信息

Parasit Vectors. 2024 Jun 16;17(1):260. doi: 10.1186/s13071-024-06321-6.

DOI:10.1186/s13071-024-06321-6

PMID:38880909

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11181648/

Abstract

BACKGROUND

The Anopheles dirus complex plays a significant role as a malaria vector in the Greater Mekong Subregion (GMS), with varying degrees of vector competence among species. Accurate identification of sibling species in this complex is essential for understanding malaria transmission dynamics and deploying effective vector control measures. However, the original molecular identification assay, Dirus allele-specific polymerase chain reaction (AS-PCR), targeting the ITS2 region, has pronounced nonspecific amplifications leading to ambiguous results and misidentification of the sibling species. This study investigates the underlying causes of these inconsistencies and develops new primers to accurately identify species within the Anopheles dirus complex.

METHODS

The AS-PCR reaction and thermal cycling conditions were modified to improve specificity for An. dirus member species identification. In silico analyses with Benchling and Primer-BLAST were conducted to identify problematic primers and design a new set for Dirus complex species identification PCR (DiCSIP). DiCSIP was then validated with laboratory and field samples of the An. dirus complex.

RESULTS

Despite several optimizations by reducing primer concentration, decreasing thermal cycling time, and increasing annealing temperature, the Dirus AS-PCR continued to produce inaccurate identifications for Anopheles dirus, Anopheles scanloni, and Anopheles nemophilous. Subsequently, in silico analyses pinpointed problematic primers with high Guanine-Cytosine (GC) content and multiple off-target binding sites. Through a series of in silico analyses and laboratory validation, a new set of primers for Dirus complex species identification PCR (DiCSIP) has been developed. DiCSIP primers improve specificity, operational range, and sensitivity to identify five complex member species in the GMS accurately. Validation with laboratory and field An. dirus complex specimens demonstrated that DiCSIP could correctly identify all samples while the original Dirus AS-PCR misidentified An. dirus as other species when used with different thermocyclers.

CONCLUSIONS

The DiCSIP assay offers a significant improvement in An. dirus complex identification, addressing challenges in specificity and efficiency of the previous ITS2-based assay. This new primer set provides a valuable tool for accurate entomological surveys, supporting effective vector control strategies to reduce transmission and prevent malaria re-introducing in the GMS.

摘要

背景

在大湄公河次区域（GMS），按蚊致倦库蚊复合体作为疟疾媒介发挥着重要作用，其种间媒介效能存在差异。准确鉴定该复合体中的姊妹种对于了解疟疾传播动态和部署有效的病媒控制措施至关重要。然而，针对 ITS2 区域的原始分子鉴定检测方法，即致倦库蚊等位基因特异性聚合酶链反应（AS-PCR），具有明显的非特异性扩增，导致结果不确定和姊妹种的错误鉴定。本研究旨在探讨这些不一致的根本原因，并开发新的引物来准确鉴定按蚊致倦库蚊复合体中的种。

方法

修改了 AS-PCR 反应和热循环条件，以提高鉴定致倦库蚊成员种的特异性。使用 Benchling 和 Primer-BLAST 进行了计算机分析，以确定有问题的引物并设计一组新的致倦库蚊复合体种鉴定 PCR（DiCSIP）引物。然后用实验室和现场采集的按蚊致倦库蚊复合体样本对 DiCSIP 进行验证。

结果

尽管通过降低引物浓度、减少热循环时间和提高退火温度进行了多次优化，但 Dirus AS-PCR 仍继续对致倦库蚊、扫描按蚊和嗜人按蚊的鉴定产生不准确的结果。随后，计算机分析确定了具有高鸟嘌呤-胞嘧啶（GC）含量和多个非靶标结合位点的有问题引物。通过一系列计算机分析和实验室验证，开发了一组新的致倦库蚊复合体种鉴定 PCR（DiCSIP）引物。DiCSIP 引物提高了特异性、操作范围和灵敏度，可准确鉴定 GMS 中的五个复合体成员种。用实验室和现场采集的按蚊致倦库蚊复合体标本对 DiCSIP 进行验证，结果表明 DiCSIP 可以正确鉴定所有样本，而原始 Dirus AS-PCR 在使用不同热循环仪时则错误地将致倦库蚊鉴定为其他种。