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目的对河南省2015年输入性恶性疟患者血样的恶性疟原虫氯喹抗性转运蛋白(Plasmodium falciparum chloroquine resistant transporter,Pfcrt)基因进行检测,分析其基因多态性。方法采集河南省2015年132例输入性恶性疟患者的血样,并收集患者相关信息。提取患者血样中恶性疟原虫基因组DNA,采用已有的Pfcrt基因序列引物,进行巢式PCR扩增,扩增产物经ApolⅠ酶切和测序,分析输入性恶性疟原虫Pfcrt基因多态性及其分布情况。结果132例输入性恶性疟患者以男性青壮年为主,均为自非洲19个国家的劳务返乡人员,其中以西非居多,占38.6%(51/132),其他依次为中非(占26.5%,35/132)、南非(25.0%,33/132),东非(占8.3%,11/132)和北非(占1.5%,2/132)。患者血样DNA经巢式PCR扩增,均获得约145 bp的特异性片段。扩增产物经ApolⅠ酶切,66.7%(88/132)患者血样的扩增产物被完全酶切,出现114 bp和31 bp两个片段;32.6%(43/132)不能被酶切,仅出现145 bp单个片段;0.8%(1/132)则酶切不完全,产生145 bp、114 bp和31 bp等3个片段。测序结果显示,132份患者血样的扩增产物经测序获得的Pfcrt基因序列,与氯喹敏感株3D7序列比对,其中43份血样(占32.6%)的Pfcrt基因序列编码的74、75和76位氨基酸碱基由ATG、AAT和AAA突变为ATT、GAA和ACA,即M74I、N75E和K76T,为突变型(CVIET);1份血样(占0.8%)的Pfcrt基因序列编码的74~76位氨基酸碱基则突变为ATG/T、A/GAA/T和AA/CA,为混合型(CVM/I、N/E/D/K、T/K);88份血样(占66.7%)的Pfcrt基因序列未发生改变,为野生型(CVMNK)。自西非输入的恶性疟病例突变型所占比例最多,为41.2%(21/51),其他依次为东非(4/11)、南非(30.3%,10/33)和中非(22.9%,8/27),三者比较差异无统计学意义(χ2=4.07,P>0.05)。自北非输入的2例均为野生型;1例基因混合型自西非输入。结论2015年河南省自非洲19个国家输入的恶性疟原虫Pfcrt基因存在3种类型,野生型(CVMNK)、突变型(CVIET)和混合型(CVM/I、N/E/D/K、T/K),野生型(CVMNK)所占比例最高(66.7%);自西非地区输入的突变型(CVIET)所占比例最多(41.2%)。
Objective To detect the genetic polymorphisms of Plasmodium falciparum chloroquine resistant transporter (Pfcrt) in blood samples of imported falciparum malaria patients from 2015 to 2015 in Henan province. Methods A total of 132 blood samples of imported falciparum malaria patients from Henan Province in 2015 were collected and relevant information was collected. Plasmodium falciparum genomic DNA was extracted from the blood samples of patients and the existing Pfcrt gene sequence primers were used for nested PCR amplification. The amplified product was digested with Apol Ⅰ and sequenced to analyze the Pfcrt gene polymorphism and distribution of imported Plasmodium falciparum Happening. Results 132 cases of imported falciparum malaria were mainly male and young, all of whom were workers returning from 19 countries in Africa, most of them were in West Africa, accounting for 38.6% (51/132), followed by Central Africa (26.5% (35.0%, 35/132), South Africa (25.0%, 33/132), East Africa (8.3%, 11/132) and North Africa (1.5%, 2/132). The DNA of the patient’s blood sample was amplified by nested PCR, and a specific fragment of about 145 bp was obtained. The amplified product was digested with ApolⅠ, and the amplified products of the blood samples of 66.7% (88/132) patients were completely digested with two fragments of 114 bp and 31 bp. Only 32.6% (43/132) could not be digested, 145 bp single fragment; 0.8% (1/132) incomplete digestion, resulting in 145 bp, 114 bp and 31 bp and other three fragments. Sequencing results showed that the Pfcrt gene sequence of 132 blood samples was aligned with that of chloroquine sensitive strain 3D7. The Pfcrt gene sequences of 43 blood samples (32.6%) encoded 74, 75 and 76 Amino acid bases were mutated from ATG, AAT and AAA to ATT, GAA and ACA, ie, M74I, N75E and K76T, were mutated (CVIET); one copy of the Pfcrt gene of blood samples (0.8% The bases were mutated to ATG / T, A / GAA / T and AA / CA and were mixed (CVM / I, N / E / D / K, T / K); 88 blood samples (66.7% The gene sequence did not change, the wild type (CVMNK). The highest percentage of mutant cases of falciparum malaria imported from West Africa was 41.2% (21/51), followed by East Africa (4/11), South Africa (30.3%, 10/33) and Central Africa (22.9%, 8% / 27), there was no significant difference between the three (χ2 = 4.07, P> 0.05). Two cases imported from North Africa were wild type; one case of gene mixed type imported from West Africa. Conclusions There are three types of Pfcrt gene of Plasmodium falciparum imported from 19 countries in Henan Province in 2015, including CVMNK, CVIET and CVM / I, N / E / D / K, T / K), wild type (CVMNK) accounted for the highest proportion (66.7%) and CVIET (41.2%) imported from West Africa.