發(fā)布時間：2018-05-22 10:28

  本文選題：云南 + 瘧原蟲��； 參考：《中國病原生物學雜志》2017年03期

【摘要】：目的采用分子生物學方法鑒定云南省瘧疾參比實驗室鏡檢瘧原蟲疑難血樣。方法選取2012年8月-2015年4月云南省瘧疾疫情報告病例中瘧原蟲形態(tài)學鑒定疑難血樣,對其18srRNA基因進行巢式PCR擴增和產(chǎn)物測序,測序結(jié)果在NCBI和Mega6.06軟件與4種瘧原蟲參比蟲株序列(惡性瘧原蟲P.falciparum:KC906781;間日瘧原蟲P.vivax:X13926;三日瘧原蟲P.malariae:M54897;卵形瘧原蟲P.ovale:L48987)進行相似性和同源性分析。結(jié)果38份疑難血樣中,采自緬甸感染、云南感染、非洲感染者的血樣分別為81.58%(31/38)、15.79%(6/38)和2.63%(1/38)。12份P.malariae及感染紅細胞的鏡檢形態(tài)特點為:瘧原蟲體積占紅細胞的體積1/4,可見不同發(fā)育期的瘧原蟲形態(tài),但同時可見"帶狀"滋養(yǎng)體、"梅花狀"裂殖體典型形態(tài)的僅有4例,占33.33%(4/12),"帶狀"滋養(yǎng)體、"梅花狀"裂殖體單獨可見的比例分別為83.33%(10/12)和75.0%(9/12),100%(12/12)的瘧原蟲感染紅細胞體積不脹大或略為縮小。5份P.ovale及感染紅細胞的鏡檢形態(tài)為:瘧原蟲體積占紅細胞的體積1/4,可見各期瘧原蟲,紅細胞體積正�；蚩s小的發(fā)生率為100%(5/5),紅細胞呈傘矢狀、拖尾、紅細胞邊緣呈刺突變形的比例分別為80%(4/5)、100%(5/5)和80%(4/5)。11份可疑為P.vivax的形態(tài)僅剩瘧色素樣或瘧原蟲細胞核樣物體。10份P.falciparum的形態(tài)不同于以往云南省常見的瘧原蟲胞漿、胞核纖細、致密的惡性瘧原蟲,胞漿粗大,蟲體占紅細胞體積的三分之一。上述血樣經(jīng)18S rRNA基因PCR擴增和DNA測序分析,陽性符合率為100%(38/38),4種瘧原蟲的18SrRNA基因DNA序列與4種參比序列的相似性在81%~100%之間,也與各自的參比序列聚類成彼此分離的進化分枝,即序列分類與形態(tài)識別結(jié)果吻合。結(jié)論云南省的輸入性三日瘧、卵形瘧病例時有發(fā)生,對疑難形態(tài)瘧原蟲的蟲種鑒定須借助分子生學技術加予驗證。
[Abstract]:Objective to identify the difficult blood samples of Plasmodium falciparum in malaria reference laboratory of Yunnan Province by molecular biological method. Methods from August 2012 to April 2015, difficult blood samples of malaria parasite were identified in Yunnan Province. The 18srRNA gene was amplified by nested PCR and its products were sequenced. The results were compared with four Plasmodium falciparum: KC906781, P.vivax: X13926, P.malariae: M54897 and P.ovale1: L48987 by NCBI and Mega6.06 software. The results showed that P. falciparum: KC906781; P. vivax: X13926; P. malariae: M54897; P. ovale: L48987; P. falciparum: KC906781; P. vivax: X13926; P. malariae: M54897; P. Results 38 difficult blood samples were collected from Myanmar infection and Yunnan infection. The blood samples of the infected people in Africa were 81.58% and 15.79% (6 / 38) and 2.63% / 38%, respectively, and the microscopic morphological characteristics of 12 P.malariae samples and infected red blood cells were as follows: the volume of Plasmodium parasites accounts for 1 / 4 of the volume of red blood cells, and the morphology of malaria parasites at different stages of development can be seen. But at the same time, "banded" trophozoites were seen, and only 4 cases of "plum blossom" merozoite were typical. Accounting for 33.33% 4 / 12%, "banded" trophozoites, "plum blossom" splits were found separately in 83.33% 10 / 12) and 75.0% of 9 / 12% / 100% 12 / 12).) the erythrocyte volume of Plasmodium parasites infected by Plasmodium falciparum did not swell or decreased slightly, and the microscopic morphology of the infected red blood cells was as follows: Plasmodium parasites accounted for the volume of the infected erythrocytes, and the microscopic morphology of the infected erythrocytes was: Plasmodium plasmodium accounted for the volume of the parasite. The volume of red blood cells is 1 / 4, and you can see the malaria parasites at all stages. The incidence of normal or reduced red blood cell volume is 100%, 5 / 5%, red blood cell is sagittal and tail. The proportion of erythrocyte edge deformed by spines was 80 / 4 / 5 / 100 / 100 / 5) and 80 / 4 / 5 / 5, respectively. Of the 11 suspected forms of P.vivax, only malarial pigment or plasmodium nucleoloid body. 10 P.falciparum were different from the plasmodium cytoplasm and nucleus of Plasmodium that were common in Yunnan Province in the past. Compact Plasmodium falciparum with a large cytoplasm, accounting for 1/3 of erythrocyte volume. By PCR amplification of 18s rRNA gene and DNA sequencing, the positive coincidence rate was 100%. The similarity between the DNA sequence of 18SrRNA gene of four Plasmodium species and the four reference sequences was between 81% and 100%. That is, the result of sequence classification is consistent with that of morphological recognition. Conclusion the cases of imported 3-day malaria and oval malaria occur from time to time in Yunnan Province. The identification of difficult form malaria parasites should be verified by molecular technique.
【作者單位】： 云南省寄生蟲病防治所云南省瘧疾研究中心云南省蟲媒傳染病防控研究重點實驗室;
【基金】：國家自然科學基金地區(qū)科學基金項目(No.81660559) 云南省科技廳面上項目(No.2011FB136)
【分類號】：R382.31
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本文編號：1921694