用聚合酶链反应区分我国大劣按蚊复合体A和D种  

DIFFERENTIATION OF CRYPTIC SPECIES A AND D OF ANOPHELES DIRUS COMPLEX BY POLYMERASE CHAIN REACTION 

作　　者：徐晓春[1] 徐建农[1] 瞿逢伊[1] 

机构地区：[1]第二军医大学寄生虫学教研室

出　　处：《中国寄生虫学与寄生虫病杂志》1998年第3期172-175,共4页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金

摘　　要：目的：建立一种鉴别大劣按蚊复合体Ａ和Ｄ种的ＰＣＲ检测技术。方法：根据大劣按蚊Ａ和Ｄ种核糖体ＤＮＡ第二内转录间隔区的序列差异，设计种特异引物，通过ＰＣＲ扩增出种特异长度（Ａ种为３７４ｂｐ，Ｄ种为６６３ｂｐ）的片段区分蚊种。结果：该法敏感性达１／１６００单蚊抽提ＤＮＡ或１／５单条蚊腿水匀浆ＤＮＡ。应用该法检测大劣按蚊ＡＦＲＩＭＳ实验室品系１０例，ＨＮ实验室品系２０例，以及采自海南省白沙、和平、罗葵、毛阳等地野外样本１４８例，均显示Ａ种特异带；云南省勐腊地区野外样本３０例，全部为Ｄ种特异带。结论：提供一种简便可靠的蚊种鉴别ＰＣＲ法，确认我国海南和云南的大劣按蚊分别为Ａ种和Ｄ种，为深入研究我国大劣按蚊复合体不同成员种的地理分布、生态习性和传疟作用创造了条件。AIM: To distinguish cryptic species A and D of Anopheles dirus complex using polymerase chain reaction (PCR). METHODS: A diagnostic PCR assay of species was developed by use of three primers, one derived from highly conservative 5.8 S coding sequences and two from different interspecies sequence in the second internal transcribed spacer (ITS2) of ribosomal DNA. RESULTS: Using the PCR method, specific fragments were amplified in both species, the size of fragments is 374 bp for species A and 663 bp for species D. Thirty samples of species A from AFRIMS and HN laboratory colony and seven samples of the species D from Yunnan Province were correctly identified by PCR. Satisfactory results were obtained from the amount of DNA as little as 1/1 600 of extracted DNA of a single mosquito or 1/5 of DNA derived from one leg of a mosquito triturated in water. A total of 148 field collected specimens of Anopheles dirus from Heping(HP), Baisha(BS) , Loukui( LK) , and Maoyang(MY) in Hainan Province revealed fragment characteristic of species A, while 30 specimens from Mengla(ML) in Yunnan Province showed the specific fragment of species D. CONCLUSION: A simple and reliable method was developed to identify cryptic species A and D of Anopheles dirus complex and it was further verified that Anopheles dirus from Hainan and Yunnan Provinces is the species A and the species D, respectively.

关 键 词：聚合酶链反应 大劣按蚊 核糖体 DNA A种 D种 

分 类 号：R384.1[医药卫生—医学寄生虫学]