巢氏PCR检测血片中低密度间日疟原虫的研究
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Study on the detection of lowdensity plasmodium vivax from stained blood smears using nested PCR
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    摘要:

    【摘要】目的 探讨巢式PCR应用于检测低密度疟原虫血片DNA的方法。 方法 以健康人抗凝血为稀释液提取不同浓度梯度间日疟原虫血片DNA,测定其最低检出限,将一份含有高密度间日疟原虫的抗凝血样按照1:10、1:100、1:1000、1:2000、1:4000、1:8000、1:16000、1:32000比例进行稀释,制成9个浓度梯度血样。每个浓度血样按照厚血膜5ul、薄血膜2ul同时制作2张血片,连续稀释(9个稀释度)一份含有高密度间日疟原虫的EDTA抗凝血样本,制作标准血片,同时进行所制得的血片中厚、薄血膜通过显微镜计数后,分别用以提取DNA,进行和巢式PCR检测。 结果 间日疟原虫血片厚血膜的检出限为1:16000(08个虫/μl血),薄血膜检出限为1:100,与镜下计数水平一致。 结论 巢式PCR适用于低密度血片中间日疟原虫DNA的检测,具有较高的检测意义。

    Abstract:

    【Abstract】Objective To explore the detection limits of Plasmodium vivax from stained blood smears using nested PCR. Methods Serial dilutions (1:10, 1:100, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000 and 1:32000) of a peripheral blood sample containing highdensity P. vivax were prepared using a blood sample drawn from a healthy person as diluent solution. Blood smears for each concentration were made with 5 μl sample for the thick blood film and 2ul for the thin blood filmStandard blood smears (at 9 degrees of dilution) were prepared using highdensity P. vivax from a peripheral blood sample. After microscopic examination, DNA was extracted from the thick blood film and thin blood film respectively and nested PCR were conducted in the same time. Results The detection threshold for P. vivax from the thick blood film was 1:16000 (08 parasite/uL). As for the thin blood film, the detection threshold was 1:100. Conclusion Nested PCR can be used as an alternative and useful tool for the detection of lowdensity P. vivax from stained blood smears, and it’s valuable for the molecular diagnosis of malaria.

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  • 在线发布日期: 2018-03-05
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