聚合酶链反应扩增谷氨酸富有蛋白基因片段应用于恶性疟原虫基因分型

GENOTYPING OF PLASMODIUM FALCIPARUM ISOLATES BY AMPLIFICATION OF GLUTAMATE RICH PROTEIN GENE USING POLYMERASE CHAIN REACTION

目的：建立恶性疟原虫谷氨酸富有蛋白（ＧＬＵＲＰ）基因分型诊断鉴别系统。方法：设计并合成两对针对恶性疟原虫ＧＬＵＲＰ基因的特异引物，采用套式聚合酶链反应（ＰＣＲ）技术，扩增ＧＬＵＲＰ基因Ｒ２多态区目的基因，并应用于泰国Ｂｏｒａｉ疟区恶性疟患者虫株基因分型。结果：在自然感染的恶性疟原虫株群中，ＧＬＵＲＰ基因存在明显的多态性。在１５４份恶性疟感染血样中，共检查出２９０个基因株，它们属于１２种ＤＮＡ片段长度不同的ＧＬＵＲＰ基因型；其中以７７０ｂｐ片段基因型较为常见，１１００ｂｐ片段基因型较少见。４３％以上病人为不同恶性疟原虫基因株混合感染者。在９个月的流行季节中，１２种不同基因株的频率构成比无明显变化。结论：建立ＧＬＵＲＰ基因分型诊断鉴别系统，将有助于疟原虫虫株分类和致病性研究，对疟疾的流行病学研究与防治具有实用意义。

AIM: To develop a genotyping method based on amplifying glutamate rich protein(GLURP) gene for the diagnosis and identification of Plasmodium falciparum . METHODS:Two pairs of primers specific for GLURP gene of P.falciparum were designed and synthesized.R 2 polymorphic domain of GLURP gene was amplified by nested PCR, which was applied to genotyping of P.falciparum isolates obtained from patients attending the malaria clinic at the village of Borai, Thailand. RESULTS: Conspicuous polymorphism of GLURP alleles in natural populations of P.falciparum was found. 290 GLURP alleles were detected in 154 P. falciparum infections. Among the above mentioned alleles, 12 different GLURP genotypes were distinguished according to different DNA sizes . Of them, the most frequently found allele was a variant of 770 bp, the least allele was that of 1 100 bp. More than 43% of the patients were found to be infected with mixed alleles. No apparent change for frequencies of the 12 different alleles was found in the 9 month longitudinal study. CONCLUSION: A genotyping method is developed for the research of strain taxonomy and pathogenesis of malaria parasites.