常见嗜尸性蝇类蛹壳样品在种属分子鉴定中的应用

中国寄生虫学与寄生虫病杂志

常见嗜尸性蝇类蛹壳样品在种属分子鉴定中的应用

李学博1，宁淑华2，张贵芹3，余红丽3，王清山4，李红卫4 *

1 山东省高校证据鉴识重点实验室（山东政法学院），济南 250014；2 重庆市公安局江北分局刑侦支队，重庆 400020；
3 石家庄市公安局刑科所，石家庄 050021；4 重庆市公安局物证鉴定中心，重庆 400021

出版日期:2015-02-28 发布日期:2015-05-04

Species Identification of Puparium Samples of Common Sarcosaphagous Flies Based on Molecular Marker Analysis

LI Xue-bo1，NING Shu-hua2，ZHANG Gui-qin3，YU Hong-li3，WANG Qing-shan4，Li Hong-wei4 *

1 Shandong Provincial Key Laboratory of Evidence Identification in Colleges and Universities， Shandong University of Political Science and Law，Jinan 250014，China；2 Jiangbei Branch of Chongqing Public Security Bureau，Chongqing 400020，China；3 Institute of Forensic Science，Shijiazhuang Public Security Bureau，Shijiazhuang 050021，China；4 Institute of Forensic Science， Chongqing Public Security Bureau，Chongqing 400021，China

Online:2015-02-28 Published:2015-05-04

摘要/Abstract

摘要：

目的探讨常见嗜尸性蝇类蛹壳样品在法医昆虫学种属鉴定中的应用价值。方法收集不同保存时间的嗜尸性蝇类成虫及其羽化后的蛹壳成套样品共55套，根据形态学特征鉴定为2科6属8种。以不同保存时间，将上述样品分为＜2年组（n=23）、2～5年组（n=20）和＞5年组（n=12）。取单个成蝇胸部和空蛹壳，以十六烷基三甲基溴化铵（CTAB）法提取各样品mtDNA，并测定其浓度和纯度。PCR扩增COⅠ基因，获得COⅠ基因序列Ⅰ和Ⅱ，分别截取498 bp和841 bp的序列。在GenBank数据库对所获序列作BLAST搜索，进行同源性比对分析。结果所有样品均成功提取了mtDNA。保存时间≤5年的成虫胸肌和＜2年的空蛹壳DNA浓度多在1.0～3.0 μg/μl之间，A260/A280值多在1.6～1.8之间。保存时间＞5年的成虫胸肌和≥2年的空蛹壳DNA浓度多低于1.0 μg/μl，A260/A280值多低于1.6。随着样品保存时间的延长，各样品DNA浓度均明显下降（P＜0.01）。保存时间＜2年，成虫胸部样品和空蛹壳样品均成功扩增出COⅠ序列Ⅰ和序列Ⅱ。随着保存时间的延长，扩增成功率明显下降，序列Ⅱ表现更为明显（P＜0.01）。8种嗜尸性蝇类COⅠ基因序列Ⅰ和序列Ⅱ分别有6个和7个种属的同源性比对结果完全正确且样品序列比对的Max ident值均＞97%。结论嗜尸性蝇类空蛹壳样品可作为mtDNA提取和种属比对的检材。

关键词: 嗜尸性蝇类, 蛹壳, 种属鉴定

Abstract:

Objective To investigate the application value of empty puparia in species identification of common sarcosaphagous flies. Methods Fifty-five samples of adult flies and their empty puparia were collected. All the samples were identified as 2 families， 6 genera and 8 species by morphological characteristics. The samples were divided into 3 groups according to their time period between eclosion and our analyses： less than 2 years（n=23）， 2-5 years（n=20）， and more than 5 years（n=12）. The mtDNA of each sample was extracted by CTAB method. The purity and concentration of DNA were tested. PCR products were amplified using two sets of primers. Two sequences of COⅠ gene（sequenceⅠ： 498 bp， sequenceⅡ： 841 bp） from each sample were compared to the sequences in GenBank using BLAST for species identification. Results The mtDNA was extracted successfully from all the samples. DNA concentration of adult chest muscle preserved less than or equal to 5 years and empty puparia preserved less than 2 years ranged from 1.0 to 3.0 μg/μl， and the value of A260/A280 ranged from 1.6 to 1.8. The purity and concentration was lower than 1.6 and 1.0 μg/μl， when the adult chest muscle and empty puparia preserved more than 5 years and 2 years， respectively. DNA concentration of the samples significantly decreased with the prolonged preservation time（P＜0.01）. Two sequences of COⅠ gene was amplified in adult chest muscle and empty puparia which preserved less than 2 years. The success rates of amplification decreased with the prolonged preservation time， especially for the sequence Ⅱ（P＜0.01）. The morphological identification of 8 species did not match exactly with the results based on the COI gene， correct species identification occurred in 6 and 7 species out of 8 based on the two sequences， respectively， and their Max ident value exceeded 97%. Conclusion Empty puparium samples can be used to extract mtDNA and identify species.

Key words: Sarcosaphagous fly, Puparium, Species identification

李学博1，宁淑华2，张贵芹3，余红丽3，王清山4，李红卫4 *. 常见嗜尸性蝇类蛹壳样品在种属分子鉴定中的应用[J]. 中国寄生虫学与寄生虫病杂志.

LI Xue-bo1，NING Shu-hua2，ZHANG Gui-qin3，YU Hong-li3，WANG Qing-shan4，Li Hong-wei4 *. Species Identification of Puparium Samples of Common Sarcosaphagous Flies Based on Molecular Marker Analysis[J]. Chinese Journal of Parasitology and Parasitic Diseases.