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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 establish a method to extract DNA from old Plasmodium falciparum and lay a foundation for the study of Plasmodium genetic tracing. Methods DNA was extracted from 41 positive samples of Chlamydia pneumoniae by QIAamp DNA Mini Kit and compared with Chelex-100 and Na2HPO4. Nested PCR amplified fragment of SSU r RNA of Plasmodium to identify Plasmodium species. PCR results were sequenced and sequence alignment, statistical analysis of the 20th century, 80 years and nearly 10 years of blood film, different methods of blood treatment and blood quality of different PCR positive detection rate differences. Results The total positive detection rate of the modified PCR method was 70.7% (29/41). The positive rate of PCR in the 1980s and the past 10 years were 78.6% (11/14) and 66.7% (18/27), respectively. There was no significant difference between the two groups (χ2 = 0.63, P> 0.05). The positive rates of PCR in the blood films of untreated and untreated groups were 62.5% (15/24) and 82.4% (14/17), respectively, with no significant difference between the two groups (χ2 = 1.89, P> 0.05). The detection rate of positive PCR was 93.3% (28/30) and 9.1% (1/11) respectively with good quality and poor blood flow, the difference was statistically significant (χ2 = 27.59, P <0.01). Sequencing results showed that the amplified fragments were consistent with the expected results. Chelex-100 and Na2HPO4 PCR failed to amplify specific target bands. Conclusion The higher detection rate of PCR was obtained by using QIAamp DNA Mini Kit modified method to extract P. pastoris DNA in the 1980s. Staining and microscopic examination showed no significant effect on the DNA extraction of Plasmodium.