摘要
【目的】改进和评估无形态损伤提取粉螨DNA并保留完整凭证标本的方法。【方法】用改良Chelex-100法,STE法和碱裂解法提取单只椭圆食粉螨Aleurolyphus ovatus基因组DNA,并进行凭证标本回收和形态观察。超微量分光光度计测定DNA的纯度。通过对COⅠ基因的扩增,验证所提取的DNA是否满足PCR扩增。【结果】3种方法处理后的凭证标本整体保持完整,但碱裂解法处理后,躯体刚毛易脱落,凭证标本透明过度,影响标本的形态鉴定。改良Chelex-100法的A_(260)/A_(280)比值为1.95,其余两种方法A_(260)/A_(280)均低于1.8。3种方法所提单只螨DNA均满足PCR扩增,但STE法的扩增效果较差。【结论】改良Chelex-100法保存凭证标本完整,不影响标本的形态鉴定;提取的DNA纯度高,可以满足PCR扩增和后续相关分子生物学研究,且简单高效,适于单只粉螨DNA的无形态损伤提取。
[Objectives]To evaluate three methods of extracting DNA from acaroid mites without causing morphological damage,thereby preserving complete voucher specimens.[Methods]Genomic DNA of a single Aleurolyphus ovatus was extracted using the modified Chelex-100 method,the STE method,and the Alkaline lysis method,followed by voucher specimen recovery and morphological comparison.The purity of DNA was determined using an ultra microspectrophotometer.The COI gene was amplified to verify whether the extracted DNA met PCR amplification requirements.[Results]Voucher specimens subjected to one of the 3 methods remained intact as a whole,but the alkaline lysis method caused excessive transparency of the cuticle and detachment of body bristles,compromising the morphological identification of specimens.The A_(260)/A_(280)ratio of the modified Chelex-100 method was 1.95,whereas that of the other two methods was<1.8.DNA extracted by either of the three methods met the standard for PCR amplification,although the degree of amplification achieved using the STE method was relatively poor.[Conclusion]The modified Chelex-100 method had the least effect on the morphology of voucher specimens and did not compromise subsequent morphological identification.DNA extracted using this method also has high purity,sufficient to meet the requirements of PCR amplification and subsequent molecular research.The Chelex-100 method is simple and efficient,and allows DNA to be extracted from single acaroid mites without morphological damage.
作者
范海页
朱永航
陈慧林
刘璐瑶
赵薇
曹玉祥
叶长江
孙恩涛
FAN Hai-Ye;ZHU Yong-Hang;CHEN Hui-Lin;LIU Lu-Yao;ZHAO Wei;CAO Yu-Xiang;YE Chang-Jiang;SUN En-Tao(School of Laboratory Medicine,Wannan Medical College,Wuhu 241002,China)
出处
《应用昆虫学报》
CAS
CSCD
北大核心
2023年第6期1886-1891,共6页
Chinese Journal of Applied Entomology
基金
国家自然科学基金项目(31870352)
安徽省级大学生创新创业项目(S202110368044)
皖南医学院2021年度校大学生科研资助金项目(WK2021XS20)
作者简介
第一作者:范海页,E-mail:fhy2743317586@163.com;通讯作者:孙恩涛,E-mail:asdentao@126.com
