背景:按蚊复合体作为大湄公河次区域(GMS)的疟疾媒介发挥着重要作用,物种之间具有不同程度的媒介能力。准确识别该综合体中的同胞物种对于了解疟疾传播动态和部署有效的媒介控制措施至关重要。然而,原始的分子鉴定试验,Dirus等位基因特异性聚合酶链反应(AS-PCR),以ITS2区域为目标,具有明显的非特异性扩增,导致结果不明确和同胞物种的错误识别。这项研究调查了这些不一致的根本原因,并开发新的引物以准确识别按蚊中的物种。
方法:修改AS-PCR反应和热循环条件以提高对An的特异性。dirus成员物种鉴定。使用Benchling和Primer-BLAST进行计算机模拟分析以鉴定有问题的引物并设计用于Dirus复合物物种鉴定PCR(DiCSIP)的新集合。然后用An的实验室和现场样品验证了DiCSIP。Dirus情结.
结果:尽管通过降低引物浓度进行了多次优化,减少热循环时间,提高退火温度,DirusAS-PCR继续对按蚊进行不准确的鉴定,Scanloni按蚊,和嗜线虫按蚊。随后,计算机模拟分析确定了具有高鸟嘌呤-胞嘧啶(GC)含量和多个脱靶结合位点的有问题的引物。通过一系列的计算机模拟分析和实验室验证,已开发出一套用于Dirus复杂物种鉴定PCR(DiCSIP)的新引物。DiCSIP引物提高特异性,操作范围,和灵敏度,准确识别GMS中的五个复杂成员物种。通过实验室和现场进行验证。dirus复杂标本表明DiCSIP可以正确识别所有样品,而原始的DirusAS-PCR错误识别了An。当与不同的热循环仪一起使用时,与其他物种一样。
结论:DiCSIP测定法在An中提供了显着改善。dirus复杂的识别,解决先前基于ITS2的检测在特异性和效率方面的挑战。这种新的引物集为准确的昆虫学调查提供了宝贵的工具,支持有效的病媒控制策略,以减少传播并防止疟疾在GMS重新引入。
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.