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目的了解我国恶性疟原虫分离株Pfcrt基因K76T及Pf mdr1基因N86Y和D1246Y的点突变特征及发生率,并分析上述分子标志与恶性疟原虫对氯喹敏感性的关系。方法从我国恶性疟流行区云南和海南省采集恶性疟现症患者血样,根据恶性疟原虫Pfcrt基因和Pf mdr1基因序列设计巢式PCR引物,以血样中的恶性疟原虫DNA为模板,进行巢式PCR-RFLP分析,并对部分PCR产物进行测序验证。采用世界卫生组织制定的体外微量法测定同一批血样中恶性疟原虫对氯喹的敏感性。结果云南、海南省恶性疟原虫Pfcrt基因K76T的突变发生率分别为86.7%和64.3%;云南、海南省恶性疟原虫Pf mdr1 N86Y突变发生率分别为46.5%和3.4%。未发现云南、海南省恶性疟原虫分离株存在Pf mdr1 D1246Y突变。体外微量测定法显示Pfcrt 76T突变发生率氯喹抗性株与敏感株间差异有统计学意义(χ2=24.70,P<0.01)。Pf mdr1 86Y突变发生率氯喹抗性株与敏感株间差异无统计学意义(χ2=0.20,P=0.65)。结论在云南省和海南省现场未发现恶性疟原虫Pf mdr1 D1246Y点突变,抗氯喹恶性疟原虫的Pf mdr1 N86Y突变发生率与敏感株间无显著差异。Pfcrt K76T作为分子标志在我国恶性疟原虫氯喹抗性监测中具有应用价值。
Objective To understand the point mutation characteristics and incidence of Pfcrt gene K76T and Pf mdr1 gene N86Y and D1246Y of P. falciparum isolates in China and analyze the relationship between the above molecular markers and susceptibility to Plasmodium falciparum. Methods Blood samples of patients with P. falciparum were collected from Yunnan and Hainan Provinces in the endemic area of P. falciparum in our country. According to the Pfcrt gene and Pf mdr1 gene sequence of P. falciparum, nested PCR primers were designed. Plasmodium falciparum DNA in blood sample was used as a template for nested PCR-RFLP analysis and sequencing of some PCR products. The sensitivity of Plasmodium falciparum to chloroquine in the same blood samples was determined by the in vitro microtiter system developed by the World Health Organization. Results The incidences of Pfcrt gene K76T mutation in P. falciparum in Yunnan and Hainan Province were 86.7% and 64.3%, respectively. The incidence of Pf mdr1 N86Y mutation in P. falciparum in Yunnan and Hainan was 46.5% and 3.4% respectively. No Pf mdr1 D1246Y mutation was found in the isolates of P. falciparum from Yunnan and Hainan Provinces. In vitro microassay showed that the incidence of Pfcrt 76T mutation between chloroquine resistant and susceptible strains was statistically significant (χ2 = 24.70, P <0.01). The incidence of Pf mdr1 86Y mutation was not statistically different between chloroquine resistant and susceptible strains (χ2 = 0.20, P = 0.65). Conclusions Pf mdr1 D1246Y point mutation was not found in Yunnan and Hainan Provinces. There was no significant difference between Pf mdr1 N86Y mutation and susceptible strains in Plasmodium falciparum isolates. Pfcrt K76T as a Molecular Marker in Our Country Plasmodium falciparum chloroquine resistance monitoring has application value.