改良 PCR 法鉴定致倦库蚊复合体成员（种 V）的发现所导致的新隐种

Modified PCR-based assay for the differentiation of members of Anopheles fluviatilis complex in consequence of the discovery of a new cryptic species (species V).

机构信息

National Institute of Malaria Research, Sector 8, Dwarka, Delhi, 110077, India.

National Institute of Malaria Research, Field Station, BHEL Complex, Sector-III Dispensary, Ranipur, Hardwar, 249403, India.

出版信息

Malar J. 2020 Feb 27;19(1):96. doi: 10.1186/s12936-020-03172-1.

DOI:10.1186/s12936-020-03172-1

PMID:32103759

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7045375/

Abstract

BACKGROUND

Anopheles fluviatilis is a species-complex comprising of four cryptic species provisionally designated as species S, T, U and V. Earlier, a 28S-rDNA based allele-specific polymerase chain reaction (ASPCR) assay was developed for the differentiation of the then known three members of the An. fluviatilis complex, i.e., species S, T, and U. This assay was modified in consequence of the discovery of a new cryptic member, species V, in the Fluviatilis Complex to include identification of new species.

METHODS

In the modified procedure, the ASPCR assay was performed first, followed by restriction digestion of PCR product with an enzyme BamH I, which cleaves specifically PCR amplicon of species V and the resultant PCR-RFLP products can differentiate all the four cryptic members of the complex. Morphologically identified An. fluviatilis samples were subjected to sibling species identification by modified PCR-based assay and standard cytotaxonomy. The result of PCR-based assay was validated through cytotaxonomy as well as DNA sequencing of some representative samples.

RESULTS

The modified PCR-based assay differentiates all four sibling species. The result of modified PCR-based assay tested on field samples was in agreement with results of cytotaxonomy as well as DNA sequencing of representative samples.

CONCLUSIONS

The modified PCR-based assay unambiguously differentiates all four known members of the An. fluviatilis species complex. This assay will be useful in studies related to bionomics of members of the Fluviatilis Complex in their role in malaria transmission.

摘要

背景

致倦库蚊是一个包含四个隐种的种复合体，暂命名为 S、T、U 和 V 种。先前，基于 28S-rDNA 的等位基因特异性聚合酶链反应（ASPCR）检测方法被开发出来，用于区分已知的致倦库蚊复合体的三个成员，即 S、T 和 U 种。由于在致倦库蚊复合体中发现了一个新的隐种 V，因此对该检测方法进行了修改，以包括对新种的鉴定。

方法

在修改后的程序中，首先进行 ASPCR 检测，然后用 BamH I 酶对 PCR 产物进行酶切，该酶特异性地切割 V 种的 PCR 扩增子，所得的 PCR-RFLP 产物可区分复合体的所有四个隐种成员。对形态学上鉴定为致倦库蚊的样本进行改良的基于 PCR 的检测和标准细胞分类学鉴定。通过细胞分类学以及一些代表性样本的 DNA 测序对基于 PCR 的检测结果进行验证。