中国寄生虫学与寄生虫病杂志 ›› 1998, Vol. 16 ›› Issue (3): 172-175.

• 论著 • 上一篇    下一篇

用聚合酶链反应区分我国大劣按蚊复合体A和D种

徐晓春; 徐建农; 瞿逢伊   

  1. 第二军医大学寄生虫学教研室
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:1998-06-30 发布日期:1998-06-30

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

Xu Xiaochun; Xu Jiannong; Qu Fengyi   

  1. Department of Parasitology, Second Military Medical University, Shanghai 200433
  • Received:1900-01-01 Revised:1900-01-01 Online:1998-06-30 Published:1998-06-30

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。

关键词: 聚合酶链反应, 大劣按蚊, 核糖体DNA第二内转录间隔区

Abstract:

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.

Key words: Polymerase chain reaction, Anopheles dirus, second internal transcribed spacer of ribosomal DNA