疟原虫核酸检测技术建立及在献血员筛查应用的研究

目的建立血液中疟原虫检测的核酸技术，探讨应用于献血员筛查的效果。方法根据红细胞内期疟原虫ssurRNA编码基因序列，设计合成三条扩增引物。将5份待检血样混合，采用蛋白酶K消化裂解法制备模板，在同一反应体系中扩增恶性疟原虫和间日疟原虫DNA，诊断鉴别恶性疟和间日疟。与厚血膜镜检法比较评价其灵敏度及特异性。结果将3份恶性疟原虫及10份间日疟原虫感染者的血样随机混入5000份无偿献血者血样中，每5份为一个检测单元，采用PCR方法从感染血样中扩增出分别为436bp和341bp的恶性疟原虫及间日疟原虫特异性的DNA片段，并经序列测定证实扩增片段的正确性；10份间日疟患者血样以及3份恶性疟患者血样均被检出，献血者血样PCR检测结果均为阴性，结果与厚血膜方法完全一致。结论所建立的疟原虫核酸检测技术灵敏度高、特异性好，且可以批量处理，提高了检测的效率，能有效降低疟原虫的输血传播，具有很好的推广使用价值。

Establishment of a PCR- based method for detection of malaria parasites and its application in screening of blood donors

Objective To establish a polymerase chain reaction(PCR)based method for detection of Plasmodim vivax(P.v)and falciparum(P.f)in blood samples and apply it for screening blood donors.Methods Three primers was designed based plasmodium blood-stage SSUrDNA sequence,and DNA templets of blood samples were prepared using protein enzyme K digestion method.Results The DNA products with the sizes of 341 bp for P.v and 431 bp for P.f were amplified from blood samples in a single PCR reaction.10 P.v infected blood samples and 3 P.f infected blood samples randomly mixed with the 5 000 blood samples were successfully detected by the PCR based method,and the 5 000 blood donor samples was negative by PCR.The coincidence rate of PCR was 100% with that of microscopy in detection of p.f and p.v infection in blood donors.Conclusion The PCR based method was specific and sensitive for detection of P.v and p.f in blood samples and it can be used as a complement tool for screening blood donors from malaria en demic areas.