武汉市输入性恶性疟原虫裂殖子表面蛋白1、2基因分型研究

摘要/Abstract

摘要：

目的了解武汉市输入性恶性疟原虫（Plasmodium falciparum）裂殖子表面蛋白1（merozoite surface protein 1,MSP1）和MSP2的等位基因型特征。方法于2010-2015年采集武汉市从非洲疟疾流行区回国人员中恶性疟现症患者血样,收集患者流行病学资料。提取患者血样中的恶性疟原虫DNA,采用巢式PCR分别扩增恶性疟原虫MSP1、MSP2基因型特异性片段,分析各等位基因型的构成比和不同感染来源地患者的基因型频数分布,分析重症患者不同基因型的相关性。不同基因型的重症患者比例差异采用χ²检验。结果共采集输入性恶性疟患者血样175份,其中轻度症状患者血样137份,重症患者血样38份。巢式PCR结果显示,MSP1等位基因K1、RO33、MAD20型分别扩增出260~390、270和150 bp目的条带;MSP2等位基因3D7、FC27型分别扩增出200~330、330~500 bp目的条带。MSP1和MSP2基因均未检出的有9份。检出MSP1等位基因136份,检出率为77.7%,等位基因MAD20、K1、RO33、MAD20 + K1、MAD20 + RO33、K1 + RO33、MAD20 + K1 + RO33型的构成比分别为5.1%（7/136）、37.5%（51/136）、20.6%（28/136）、5.9%（8/136）、4.4%（6/136）、16.9%（23/136）、9.5%（13/136）。检出MSP2等位基因143份,检出率为81.7%,FC27、3D7和FC27 + 3D7型的构成比为20.2%（29/143）、39.9%（57/143）、39.9%（57/143）。MAD20型和K1以南非最多,分别占10.5%（4/38）和34.2%（13/38）;RO33型以东非最多,占2/7;FC27型东非和北非均未检出,南非占比最高,为18.4%（7/38）;3D7型以东非最多,占4/7。MSP1基因MAD20、K1、RO33、MAD20 + K1、MAD20 + RO33、K1 + RO33、MAD20 + K1 + RO33型的重症患者分别占1/7、19.6%(10/51)、32.1%（9/28）、1/8、4/6、13.0%(3/23)、46.2%（6/13）,MAD20 + RO33、MAD20 + K1 + RO33和RO33（P > 0.05）,与其他4种基因型间差异有统计学意义（P < 0.05）。MSP2基因3D7、FC27、FC27 + 3D7型的重症患者比例分别为19.3%(11/57)、20.7%(6/29)、24.6%（14/57）,各基因型间差异无统计学意义（P > 0.05）。结论武汉市输入性恶性疟原虫MSP1存在MAD20、Kl和R033等3种单基因型以及4种多基因型,以K1型和R033型为优势基因型;MSP2存在FC27、3D7和FC27 + 3D7型,3D7型的比例大于FC27型。

关键词: 恶性疟原虫, 裂殖子表面蛋白1, 裂殖子表面蛋白2, 基因分型

Abstract:

Objective To identify the genotypes of merozoite surface protein 1 (MSP1) and merozoite surface protein 2 (MSP2) of imported Plasmodium falciparum in Wuhan. Methods Blood samples were collected from returnees infected with P. falciparum from endemic areas of Africa, from 2010 to 2015 in Wuhan City, and epidemiological data were collected. The Plasmodium falciparum DNA was extracted from the blood samples. Nested PCR was used to amplify gene fragments of MSP1 and MSP2. The constituent ratios of the allelic families, the frequencies of each allelic family in patients from different countries, and the association of different allelic families with disease severity were analyzed. Analysis of proportions of severe cases with different genotypes were analyzed with χ² test. Results A total of 175 blood samples of patients with falciparum malaria were collected, which including 38 severe cases and 137 mild cases. Nested PCR for MSP1 allelic families K1, RO33 and MAD20 produced bands of 260-390, 270 and 150 bp, and MSP2 allelic families 3D7 and FC27 produced bands of 200-330 and 330-500 bp. A total of 175 patients with falciparum malaria were involved in this study, of whom 9 patients showed negative results for both MSP1 and MSP2 amplification. The MSP1 allelic families were detected in 136 patients, with a detection rate of 77.7%. The constituent ratios of MAD20, K1, RO33, MAD20 + K1, MAD20 + RO33, K1 + RO33, and MAD20 + K1 + RO33 were 5.1% (7/136), 37.5% (51/136), 20.6% (28/136), 5.9% (8/136), 4.4% (6/136), 16.9% (23/136), and 9.5% (13/136), respectively. The MSP2 allelic families were detected in 143 patients, with a detection rate of 81.7%. The constituent ratios of FC27, 3D7, and FC27 + 3D7 were 20.2% (29/143), 39.9% (57/143), and 39.9% (57/143), respectively. The MAD20 allelotype was predominantly from South Africa (10.5%, 4/38), the K1 was predominantly from South Africa (34.2%, 13/38), and the RO33 was predominantly from East Africa (2/7). The FC27 was not detected in East and North Africa, but was predominantly from South Africa (18.4%, 7/38). The 3D7 was predominantly from East Africa (4/7). The proportions of severe cases in the MAD20, K1, RO33, MAD20 + K1, MAD20 + RO33, K1 + RO33, and MAD20 + K1 + RO33 allelic families were 1/7, 19.6% (10/51), 32.1% (9/28), 1/8, 4/6, 13.0% (3/23), and 46.2% (6/13), respectively (P > 0.05 among MAD20 + RO33, MAD20 + K1 + RO33 and RO33; P < 0.05 for MAD20 + RO33, MAD20 + K1 + RO33 and RO33 versus the other four). The proportions of severe cases with 3D7, FC27, and FC27 + 3D7 were 19.3% (11/57), 20.7% (6/29), and 24.6% (14/57), respectively (P > 0.05). Conclusion There are three single allelic families of MSP1（MAD20, K1 and RO33） and four mixed allelic families in imported falciparum malaria in Wuhan, of which K1 and R033 are the dominant genotypes. There are three allelic families for MSP2（FC27, 3D7 and FC27 + 3D7）, of which 3D7 has a higher frequency than FC27.

Key words: Plasmodium falciparum, Merozoite surface protein 1, Merozoite surface protein 2, Genotype

中图分类号:

R382.312

贾西帅, 周水茂, 杨燕, 徐明星, 吴凯. 武汉市输入性恶性疟原虫裂殖子表面蛋白1、2基因分型研究[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(5): 434-439.

Xi-shuai JIA, Shui-mao ZHOU, Yan YANG, Ming-xing XU, Kai WU. Genotyping of merozoite surface protein 1 and 2 of imported Plasmodium falciparum in Wuhan City[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(5): 434-439.

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扩增基因 Amplified gene	引物 Primer		序列（5′→3′） Sequence（5′→3′）	片段长度/bp Fragment length/bp	等位基因型 Allelic family
裂殖子表面蛋白1 Merozoite surface protein 1	第1轮 First round	PF	AATAAGGCTATGTAAAATG
	第1轮 First round	PR	GGATCAGTAAATAAACTATA
	第2轮 Second round	M2F	GGTTCAGGTAATTCAAGACGATC	150	MAD20
		K2F	TCTTAAATGAAGAAATTACTACAAA	260~390	K1
		R2F	TAAAGGATGGAGCAAATACTCAAGT	270	RO33
		C3R	ACATATGATTGGTTAAATCAAAGAG
裂殖子表面蛋白2 Merozoite surface protein 2	第1轮 First round	M2P1	ATGAAGGTAATTAAAACATTGTCTATTA
	第1轮 First round	M2P2	TATGAATATGGCAAAAGATAAAACAAGT
	第2轮 Second round	FC27F	TGGCAAATGAAGGCCCTAATAC
		FC27R	GCTTTGGGTCCTTCTTCAGTTG	330~500	FC27
		3D7F	GCAGAAAGTAAGCCTCCTACTGG
		3D7R	GATTTGTTTCGGCATTTTTATGA	200~330	3D7

感染来源地 Infection source country	病例数 No. cases	MSP1等位基因型 Allelic family of MSP1							MSP2等位基因型 Allelic family of MSP2
感染来源地 Infection source country	病例数 No. cases	MAD	K1	RO33	MAD + K1	MAD + RO33	K1 + RO33	MAD + K1 + RO33	FC27	3D7	FC27 + 3D7
北非North Africa 苏丹Sudan	2	0	1	0	0	0	1	0	0	0	0
利比亚Libya	1	0	0	0	0	0	0	0	0	1	0
东非East Africa 埃塞俄比亚Ethiopia	2	0	1	0	0	0	0	0	0	2	0
坦桑尼亚Tanzania	2	0	0	1	0	0	0	1	0	1	1
乌干达Uganda	3	0	1	1	0	0	0	1	0	2	1
南非South Africa 安哥拉Angola	24	2	8	5	1	3	3	1	3	3	13
莫桑比克Mozambique	8	2	2	0	1	0	0	1	3	3	1
赞比亚Zambia	6	0	3	2	0	1	0	0	1	4	1
西非West Africa 几内亚Guinea	8	0	2	0	1	0	3	0	1	1	5
加纳Ghana	6	0	2	1	1	0	1	0	1	1	4
科特迪瓦Cote d'Ivoire	4	1	0	0	0	0	1	0	0	1	2
利比里亚Liberia	23	0	6	5	1	0	2	1	4	11	4
尼日利亚Nigeria	28	0	6	5	0	2	7	1	4	9	9
塞拉利昂Sierra Leone	6	1	2	0	0	0	0	0	3	2	1
贝宁Benin	3	0	1	1	0	0	0	0	1	1	0
马里Mali	2	0	2	0	0	0	0	0	0	1	0
中非Central Africa 赤道几内亚Equatorial Guinea	21	0	7	2	1	0	2	4	2	8	7
刚果Congo-Kinshasa	18	0	6	5	1	0	2	2	4	4	4
喀麦隆Cameroon	3	1	0	0	0	0	1	0	1	0	2
中非Central Africa	1	0	1	0	0	0	0	0	0	0	1
尼日尔Niger	1	0	0	0	0	0	0	1	0	1	0
加蓬Gabon	3	0	0	0	1	0	0	0	1	1	1
合计 Total	175	7	51	28	8	6	23	13	29	57	57