不同检材微量血间日疟原虫PCR检测模板制备方法的研究

目的 探寻不同检材微量血间日疟原虫PRC检测模板制备的方法.方法 全血标本采用酚/氯仿抽提法、洗涤沉淀法、Chelex煮沸法和洗涤水煮法；滤纸干血滴及厚血膜标本以Chelex煮沸法和洗涤沉淀法制备模板.以间日疟原虫特异性引物作PCR扩增检测模板制备的效果.结果 采用上述4种不同的方法制备的全血间日疟原虫DNA模板与采用Chelex法和洗涤沉淀法制备的滤纸干血滴以及厚血膜疟原虫DNA模板均可被扩增出1条341bp的间日疟原虫特异性DNA条带,与预期的扩增片断大小一致；不同方法制备的10份间日疟患者血样DNA模板用于PCR扩增检测,结果无明显差异.其中,Chelex煮沸法和洗涤沉淀法对不同检材的疟原虫感染血样DNA模板均能有效制备,且可在同一离心管中进行,减少了交叉污染的机会.结论 Chelex法和洗涤沉淀法简便、实用,适用于不同检材微量血疟原虫PRC模板的制备.

A study on the methods for preparing DNA templates of Plasmodium vivax from different trace blood samples for PCR detection

Objective To establish a method for preparing Plasmodium vivax DNA templates from different trace blood samples.Methods The DNA templates of whole blood samples were prepared by the phenol/chloroform extraction,washing precipitation,Chelex-boiling,and water-boiling methods,and the DNA templates of filter paper dried blood drops and thick blood film specimens were prepared by Chelex boiling method and washing precipitation method.Results The DNA templates from the blood samples prepared with the different methods could be used in polymerase chain reaction for detection of Plasmodium vivax.Among the different preparation methods,no significant difference was observed in respect of effect of DNA extraction.Chelex-boiling and washing precipitation methods could be used in the DNA templates preparation of the different trace blood samples in one centrifuge tube,thus the chance of cross contamination reduced.Conclusions The Chelex-boiling method and washing precipitation method were simple and effective in the DNA preparation for PCR detection of Plasmodium vivax in different blood samples.