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目的采用分子生物学方法鉴定云南省疟疾参比实验室镜检疟原虫疑难血样。方法选取2012年8月-2015年4月云南省疟疾疫情报告病例中疟原虫形态学鉴定疑难血样,对其18srRNA基因进行巢式PCR扩增和产物测序,测序结果在NCBI和Mega6.06软件与4种疟原虫参比虫株序列(恶性疟原虫P.falciparum:KC906781;间日疟原虫P.vivax:X13926;三日疟原虫P.malariae:M54897;卵形疟原虫P.ovale:L48987)进行相似性和同源性分析。结果38份疑难血样中,采自缅甸感染、云南感染、非洲感染者的血样分别为81.58%(31/38)、15.79%(6/38)和2.63%(1/38)。12份P.malariae及感染红细胞的镜检形态特点为:疟原虫体积占红细胞的体积>1/4,可见不同发育期的疟原虫形态,但同时可见“带状”滋养体、“梅花状”裂殖体典型形态的仅有4例,占33.33%(4/12),“带状”滋养体、“梅花状”裂殖体单独可见的比例分别为83.33%(10/12)和75.0%(9/12),100%(12/12)的疟原虫感染红细胞体积不胀大或略为缩小。5份P.ovale及感染红细胞的镜检形态为:疟原虫体积占红细胞的体积>1/4,可见各期疟原虫,红细胞体积正常或缩小的发生率为100%(5/5),红细胞呈伞矢状、拖尾、红细胞边缘呈刺突变形的比例分别为80%(4/5)、100%(5/5)和80%(4/5)。11份可疑为P.vivax的形态仅剩疟色素样或疟原虫细胞核样物体。10份P.falciparum的形态不同于以往云南省常见的疟原虫胞浆、胞核纤细、致密的恶性疟原虫,胞浆粗大,虫体占红细胞体积的三分之一。上述血样经18S rRNA基因PCR扩增和DNA测序分析,阳性符合率为100%(38/38),4种疟原虫的18SrRNA基因DNA序列与4种参比序列的相似性在81%~100%之间,也与各自的参比序列聚类成彼此分离的进化分枝,即序列分类与形态识别结果吻合。结论云南省的输入性三日疟、卵形疟病例时有发生,对疑难形态疟原虫的虫种鉴定须借助分子生学技术加予验证。
Objective To identify the blood samples of malaria parasite in malaria reference laboratory of Yunnan Province by molecular biology method. Methods The blood samples of malaria parasite morphologically identified in Yunnan Province from August 2012 to April 2015 were collected. The 18s rRNA gene was amplified by nested PCR and the products were sequenced. The sequencing results were compared between NCBI and Mega6.06 software Four Plasmodium reference insect strain sequences (P. falciparum: KC906781; P. vivax: X13926; P. malariae: M54897; P. ovale: L48987) Similarity and homology analysis. Results The blood samples collected from Burma, Yunnan and Africa were 38.5% (31/38), 15.79% (6/38) and 2.63% (1/38) respectively in 38 difficult blood samples. The morphological characteristics of 12 P. malariae and infected erythrocytes were as follows: the volume of erythrocytes in the malaria parasite was> 1/4 and the morphology of the parasite at different developmental stages was observed, but at the same time the “zonal” trophozoites were visible, There were only 4 cases (23.33%, 4/12) of the typical form of plum-shaped schizonts and 83.33% of the bands of trophozoites and plum-shaped schizonts, respectively (10/12), 75.0% (9/12), and 100% (12/12) infected red blood cells did not bulge or slightly shrink. The microscopic examination of 5 P.ovale and infected erythrocytes showed that the volume of the malaria parasite was> 1/4 of that of the erythrocytes, and the incidence of normal or contracted volume of red blood cells was 100% (5/5) The ratio of sagittal, trailing and erythrocyte edge was 80% (4/5), 100% (5/5) and 80% (4/5) respectively. Of the 11 suspicious P.vivax species, only malachite-like or plasmodium-like nuclear-like objects remained. The morphology of 10 P. falciparum is different from that of the common Plasmodium falciparum cytoplasm and nucleus of Plasmodium falciparum in Yunnan Province. The cytoplasm is thick and the worms account for one third of the volume of erythrocytes. According to the 18S rRNA gene PCR amplification and DNA sequencing analysis, the positive coincidence rate of the above blood samples was 100% (38/38). The DNA sequence of 18S rRNA gene of four kinds of Plasmodium had 81% -100% similarity with 4 reference sequences, , Also clustered with their respective reference sequences into separate evolutionary branches, ie, the sequence classification agrees well with the morphological recognition results. Conclusions The incidence of imported malaria and malaria cases in Yunnan Province have occurred from time to time. Identification of the species of malaria parasite with difficult morphology should be validated by molecular biology techniques.