关键词: 疟原虫, 吉氏染色血膜, DNA提取, 巢式PCR

Abstract:

 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 extraction 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 recent 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） respectively, with no significant difference （χ2=0.63, P>0.05）. The PCR positive rate for blood smears with and without deoil/decoloration was 62.5% （15/24） and 82.4% （14/17） respectively, also with no significant difference （χ2=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）］（χ2=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  DNA extracted from blood smears prepared in the 1980s using the improved Kit  （QIAamp DNA Mini Kit） shows a high PCR positive rate. Besides, blood smear staining and use of oil for microscopic examination do not affect DNA extraction.

Key words: Plasmodium, Giemsa-stained blood smears, DNA extraction, Nested PCR