检测4种人体疟原虫多重PCR体系的建立和应用  

Establishment and Application of Multiplex PCR System for Detecting Four Human Plasmodium Species

作　　者：李美[1] 夏志贵[1] 汤林华[1] 

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所卫生部寄生虫病原与媒介生物学重点实验室世界卫生组织疟疾血吸虫病和丝虫病合作中心,上海200025

出　　处：《中国寄生虫学与寄生虫病杂志》2015年第2期91-95,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家质检总局公益性行业科研专项(No.201210056)~~

摘　　要：目的 应用新建的多重PCR体系检测4种人体疟原虫。 方法 应用DNAman软件比较分析4种人体疟原虫的18S rDNA基因序列，采用Oligo 6.0软件，在其第5和第6保守区间的变异区设计4条下游引物序列，并以含4种疟原虫18S rDNA部分序列的质粒DNA为模板，检测多重PCR体系的特异性和敏感性。此外，将新建的多重PCR体系与NP-1993体系的第1轮属特异引物组合，形成新的巢式PCR扩增体系（M-Nest体系），并用该体系和新建的多重PCR体系对不同稀释倍数的恶性疟和间日疟患者血样DNA进行扩增，检测这两种体系的敏感性。应用M-Nest体系和NP-1993体系检测广西2013年自加纳疟疾高流行区返乡人员的307份血样，比较两者的检测结果是否一致。最后，应用M-Nest和NP-2002体系对广西2014年1～5月份报告的66份镜检以卵形疟原虫感染为主的血样进行复核，比较两者的检测结果是否一致。 结果 应用新建的多重PCR体系得到的恶性疟原虫、三日疟原虫、卵形疟原虫和间日疟原虫的扩增片段大小分别为268、446、394和323 bp，且不同原虫的扩增条带恰好位于50 bp DNA标志物条带之间，更易于区分，但不同疟原虫的Tm值较为接近，不易区分和鉴别虫种。新建体系对4种疟原虫18S rDNA基因的最低检出浓度分别为：恶性疟原虫5.58×102 拷贝/μl，三日疟原虫1.56×103 拷贝/μl，卵形疟原虫1.66×103 拷贝/μl，间日疟原虫1.80×102 拷贝/μl。新建体系对恶性疟原虫血样的最低检测浓度为1.43×102～8.84×103 拷贝/μl或5.10×10～4.92×102个原虫/μl，高于间日疟原虫的17.4～69.1 拷贝/μl和13.5～83.2个原虫/μl。与单独的多重PCR体系相比，应用M-Nest检测体系将对疟原虫的最低检测浓度降低了10～100倍。M-Nest体系和NP-1993体系对从加纳返乡的307份血样的检测结果不一致，并且M-Nest体系还检测到2例卵形疟原虫感染，而NP-1993体系则未能检�Objective To establish a multiplex PCR detection system for identifying 4 human Plasmodium species and evaluate its applicability. Methods The sequences of 18S rDNA gene of the 4 human Plasmodium species were compared using DNAman software， and 4 downstream primers were designed using Oligo 6.0 software， which targeted the region of variability between conserved regions 5 and 6 of the sequences. Using these primers， the specificity and sensitivity of the multiplex PCR system were evaluated， with plasmids containing the 18S rDNA gene sequence as a template. Further， a new nest PCR system（M-Nest） was established by combining the multiplex PCR system with the first-cycle genus-specific primer of the NP-1993 system. The sensitivities of the multiplex PCR system and the M-nest system were evaluated in serial dilutions of blood DNA samples from patients infected by P. falciparum and P. vivax. In addition， the NP-1993 and M-Nest systems were applied to screen the Plasmodium species in 307 blood samples from people returning to Guangxi from Ghana， a malaria epidemic area. And the NP-2002 and M-Nest systems were applied to re-check Plasmodium species in 66 blood samples collected in Guangxi from 2014 January to May， which were identified by microscopy to be infected mainly by P. ovale. Results The sizes of multiplex PCR products for P. falciparum， P. vivax， P. ovale， and P. malariae were 268 bp， 323 bp， 394 bp， and 446 bp， respectively， located in-between 50-bp DNA ladders. However， their melting curves had similar Tm values， thus could not be used to identify the 4 species. The minimum detection limits of P. falciparum， P. malariae， P. ovale， and P. vivax 18S rDNA gene by the multiplex PCR system were 5.58×102， 1.56×103， 1.66×103， and 1.80×102 copies/μl. The minimum detection limit of blood DNA from falciparum malaria patients by the multiplex PCR system was 1.43×102-8.84×103 copies/μl or 5.10×10-4.92×102 parasites/μl， higher than that of P. vivax（17.4-69

关 键 词：多重PCR 巢式PCR 卵形疟原虫wallikeri亚种 

分 类 号：R531.3[医药卫生—内科学]