提取陈旧阳性血膜疟原虫DNA方法的研究  

Extraction Method of Malaria Parasite DNA from Preserved Positive Blood Smears

作　　者：徐超[1] 魏庆宽[1] 李瑾[1] 肖婷[1] 贾凤菊[1] 王卫艳[1] 尹昆[1] 付婷霞[1] 赵桂华[1] 刘功振[1] 黄炳成 

机构地区：[1]山东省医学科学院,山东省寄生虫病防治研究所,山东省疟疾诊断参比实验室,济宁272033

出　　处：《中国寄生虫学与寄生虫病杂志》2015年第5期372-376,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：全球基金中国疟疾项目(No.201201055);山东省自然科学基金(No.2012ZRC03040,ZR2014YL036)~~

摘　　要：目的建立一种能够提取陈旧阳性血膜疟原虫DNA的方法,为疟原虫基因溯源研究奠定基础。方法应用DNA抽提试剂盒(QIAampDNA Mini Kit)改良后提取41份吉氏染色镜检阳性血膜的疟原虫DNA,并与Chelex-100和Na2HPO4法进行比较。巢式PCR扩增疟原虫SSU r RNA基因片段,鉴定疟原虫虫种。PCR结果进行测序和序列比对,统计分析20世纪80年代与近10年血膜、不同血膜处理方法以及不同质量血膜的PCR阳性检出率差异。结果改良试剂盒法PCR结果总阳性检出率为70.7%(29/41)。80年代和近10年血膜PCR阳性检出率分别为78.6%(11/14)和66.7%(18/27),两组间差异无统计学意义(χ2=0.63,P>0.05)。经去油、脱色处理和未经处理组血膜的PCR阳性检出率分别为62.5%(15/24)和82.4%(14/17),两组间差异无统计学意义(χ2=1.89,P>0.05)。质量较好和较差血膜的PCR阳性检出率分别为93.3%(28/30)和9.1%(1/11),两组间差异有统计学意义(χ2=27.59,P<0.01)。测序结果表明扩增目的片段与预期结果一致。Chelex-100和Na2HPO4法PCR未能扩增出特异性目的条带。结论试剂盒(QIAampDNA Mini Kit)改良法提取80年代血膜疟原虫DNA可获得较高PCR阳性检出率,且染色剂和镜检油渍对血膜疟原虫DNA提取效果无明显影响。Objective To develop a method for DNA extraction from malaria parasites on preserved blood smears, to provide basis for research on malaria genetic traceability. Methods The improved DNA extraction kit （QIAamp DNA Mini Kit） was used to extract plasmodium DNA from 41 giemsa-stained blood smears, and the ex- traction was compared with that using the Chelex-100 and Na2HPO4 methods. Nested PCR was used to amplify small subunit ribosomal RNA to identify Plasmodium parasite. The PCR products underwent sequencing and sequence alignment, to analyze the difference in PCR positive rates between blood smears prepared in the 1980s and in re- cent 10 years, between blood smears with and without deoil/decoloration, and between blood smears with different qualities. Results The total PCR positive rate for the improved kit method was 70.7% （29/41）. The PCR positive rate for blood smears prepared in the 1980s and in recent 10 years was 78.6% （11/14） and 66.7% （18/27） re- spectively, with no significant difference （X2=0.63, P〉0.05）. The PCR positive rate for blood smears with and with- out deoil/decoloration was 62.5% （15/24） and 82.4% （14/17） respectively, also with no significant difference （X2= 1.89, P〉0.05）. However, the PCR positive rate was significantly higher in blood smears with high quality [93.3% （28/30）] than those with low quality [9.1%（1/11）]（x2=27.59, P〈0.01）. Sequence alignment showed that the PCR products were consistent with the target DNA fragments. However, DNA extracted using the Chelex-100 and Na2HPO4 methods showed negative PCR results. Conclusions the improved Kit （QIAamp DNA Mini Kit） shows a of oil for microscopic examination do not affect DNA DNA extracted from blood smears prepared in the 1980s using high PCR positive rate. Besides, blood smear staining and use extraction.

关 键 词：疟原虫 吉氏染色血膜 DNA提取 巢式PCR 

分 类 号：R382.31[医药卫生—医学寄生虫学]