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目的对云南省及边境地区恶性疟原虫的裂殖子表面蛋白(merozoite surface protein,MSP)1,2基因进行分型研究,确定其等位基因的类型和分布特征,结合当地的恶性疟原虫株流行病学信息,为该地区疟疾的防治提供科学依据。方法从缅甸拉咱、那威和我国云南省西双版纳勐腊、德宏瑞丽、保山腾冲采集恶性疟患者血样。用巢式PCR(nest-PCR)扩增18S rRNA基因,确定感染疟原虫的种类。对检测为恶性疟原虫以及恶性疟原虫/间日疟原虫混合感染的样本,进行恶性疟原虫MSP-1、MSP-2基因的扩增并作测序、验证与序列分析。结果共采集89份恶性疟样本,经18S rRNA基因检测,确定间日疟9例,恶性疟78例和混合感染2例。在检测为恶性疟和混合感染的80份样本中,69例扩增出MSP-1基因片段,77例扩增出MSP-2基因片段。在MSP-1等位基因中,以MAD20型68.75%为主,RO33型23.75%和K1型20.00%次之。来源于勐腊的样本均未检出RO33型和K1型;MSP-2等位基因FC27型和3D7型的感染率均为91.25%,无明显的优势虫株;MSP-1和MSP-2基因多克隆样本所占百分比与多重性感染(multiplicity of infection,MOI)分别为22.50%、1.81和86.25%、3.51。MSP-1和MSP-2等位基因目的片段多样性与其原虫密度之间存在相关性(Spearman’s r=0.496,P<0.05;Spearman’s r=0.240,P<0.05)。MSP-1和MSP-2等位基因测序结果表明,在FC27型基因序列3′端发现1个新的APK序列,在3D7基因型序列中检测到1个新的PAT重复序列和其它19个新的序列。结论云南省及边境地区恶性疟原虫分离株MSP-1等位基因存在MAD20型、K1型和RO33型3种类型,以MAD20型为优势虫株,勐腊样本未发现K1型和RO33型;MSP-2等位基因存在FC27型和3D7型2种类型,其优势均不明显。
Objective To study the typing and distribution of merozoite surface protein (MSP) 1 and 2 gene in Plasmodium falciparum in Yunnan Province and its border areas, and to determine the type and distribution of its alleles. Epidemiological information, to provide a scientific basis for the prevention and treatment of malaria in the area. Methods The blood samples of patients with falciparum malaria were collected from Lhasa, Nawei, Myanmar and Mengla, Dehong Ruili and Baoshan Tengchong in Xishuangbanna, Yunnan Province, China. The 18S rRNA gene was amplified by nested PCR (nest-PCR) to determine the type of Plasmodium infection. Plasmodium falciparum MSP-1 and MSP-2 genes were amplified and sequenced, validated and sequenced for samples tested for mixed infection with Plasmodium falciparum and Plasmodium falciparum / Plasmodium vivax. Results A total of 89 samples of P. falciparum were collected. Nineteen cases of P. vivax, 78 cases of P. falciparum and two cases of mixed infection were identified by 18S rRNA gene test. Of 80 samples tested for falciparum malaria and mixed infections, 69 had MSP-1 gene fragments amplified and 77 had MSP-2 gene fragments amplified. Among the MSP-1 alleles, 68.75% were predominant in MAD20, 23.75% in RO33 and 20.00% in K1. None of the samples from Mengla were detected with type RO33 and type K1. The infection rates of type F27 and type 3D7 of MSP-2 allele were 91.25%, and no obvious predominant strains were found. Many MSP-1 and MSP-2 genes The percentage of clonal samples and multiplicity of infection (MOI) were 22.50%, 1.81 and 86.25%, 3.51, respectively. There was a correlation between the diversity of the MSP-1 and MSP-2 alleles and their protozoa (Spearman’s r = 0.496, P <0.05; Spearman’s r = 0.240, P <0.05). The sequencing results of the MSP-1 and MSP-2 alleles showed that a new APK sequence was found on the 3 ’end of the FC27 gene sequence and one new PAT repeat sequence and 19 new ones were detected in the 3D7 genotype sequence the sequence of. Conclusion MSP-1 alleles of MSP-1 isolates from Yunnan Province and the border areas are of three types: MAD20, K1 and RO33, with MAD20 as the dominant strain and no K1 and RO33 strains found in Mengla samples. MSP- 2 alleles exist in two types of FC27 and 3D7, the advantages are not obvious.