【摘要】 目的 本研究旨在检测恶性疟原虫(Plasmodium falciparum)氯喹抗性转运蛋白(Pfcrt)基因的多态性，分析其结构区域的序列变异，以期为掌握辽宁省恶性疟的耐药性情况进展和提供临床有效治疗方案提供科学的理论依据。方法 收集了从2019-2021年期间的辽宁省输入性恶性疟疟疾病例患者的全血，采集了相关患者的流行病学信息。采用巢氏多重PCR的方法对恶性疟患者的全血样本进行Pfcrt耐药基因的扩增并且对其进行基因序列比对，对其目前输入性恶性疟原虫耐药基因的多态性进行详尽分析以及阐述其流行分布特征。本次共检测到18份病例患者的血样，扩增出的目的产物经测序获得到了Pfcrt基因序列，并在基因库中与恶性疟原虫的标准株3D7(PF3D7-265519)序列进行Blast比对分析，使用Mega5.05软件对其进行了序列多态性的分析，并计算出序列之间的有效变异位点，遗传距离和氨基酸突变位点构成比。结果 本研究收集的辽宁省输入性恶性疟患者样本病例全部来自非洲，包括安哥拉、刚果布、刚果金、几内亚、尼日利亚、乌干达、喀麦隆。经过比对分析目前辽宁省恶性疟原虫Pfcrt基因存在着3种类型，即CVMNK型、CVIET型和CVMNT型。分析得出在突变型基因(CVIET)中的突变位点的发生主要在74、75、76位氨基酸，它的碱基突变分别为ATG→AAT、AAT→GAA和AAA→ACA,导致氨基酸的变化：M74I、N75E和K76T。这些突变类型与氯喹耐药性密切相关，尤其是K76T突变已被广泛认为是氯喹耐药性的重要标志。结论 CVMNK型是最常见的Pfcrt基因类型，且氯喹敏感型仍占主导地位。研究结果提示，目前辽宁省氯喹依然可以作为恶性疟原虫有效的治疗选择。但CVIET型等突变型的存在也提示氯喹耐药性在部分地区可能逐渐发展，因此需要进一步加强对氯喹耐药性的监测和评估。尽管本文研究的样本较少，但结果为氯喹耐药性的分布和演变提供了有效信息，仍需范围内开展进一步的监测工作，以应对潜在的抗疟药物耐药性风险。更多还原

【Abstract】 Objective This study aims to detect the polymorphism of the chloroquine resistance transporter(Pfcrt) gene of Plasmodium falciparum,analyze the sequence variations in its structural regions, and provide a scientific theoretical basis for understanding the progress of drug resistance in falciparum malaria in Liaoning Province and offering effective clinical treatment plans. Methods Whole blood samples from patients with imported falciparum malaria in Liaoning Province from 2019 to 2021 were collected, and the epidemiological information of the relevant patients was gathered. The nested multiplex PCR method was used to amplify the Pfcrt drug resistance gene in the whole blood samples of falciparum malaria patients, and the gene sequences were compared. A detailed analysis of the polymorphism of the current imported falciparum malaria parasite drug resistance gene and an elaboration of its distribution characteristics were conducted. A total of 18 blood samples from patients were detected. The amplified target products were sequenced to obtain the Pfcrt gene sequence, which was then compared with the standard strain 3D7(PF3D7-265519) sequence of P. falciparum in the gene bank by Blast analysis. Mega5.05 software was used to analyze the sequence polymorphism, and the effective variation sites, genetic distance and the composition ratio of amino acid mutation sites between the sequences were calculated. Results All the imported cases of falciparum malaria in Liaoning Province collected in this study originated from Africa, including Angola, Congo-Brazzaville, Congo-Kinshasa, Guinea, Nigeria, Uganda and Cameroon. After comparative analysis, it was found that there are three types of Pfcrt genes in falciparum malaria in Liaoning Province at present, namely CVMNK type, CVIET type and CVMNT type. The analysis shows that the mutation sites in the mutant gene(CVIET) mainly occur at amino acids 74,75 and 76,with base mutations of ATG→AAT,AAT→GAA and AAA→ACA,resulting in amino acid changes of M74I,N75E and K76T. These mutation types are closely related to chloroquine resistance, especially the K76T mutation, which has been widely recognized as an important marker of chloroquine resistance. Conclusion The CVMNK type is the most common type of Pfcrt gene, and the chloroquine-sensitive type still dominates. The research results suggest that chloroquine can still be an effective treatment option for P. falciparum in Liaoning Province at present. However, the existence of mutant types such as CVIET also indicates that chloroquine resistance may gradually develop in some areas. Therefore, further monitoring and assessment of chloroquine resistance are needed. Although the sample size of this study is small, the results provide effective information on the distribution and evolution of chloroquine resistance. Further monitoring work should be carried out on a larger scale to deal with the potential risk of antimalarial drug resistance.更多还原