检测4种人体疟原虫多重PCR体系的建立和应用

发布时间：2018-03-02 02:17

本文关键词： 多重PCR 巢式PCR 卵形疟原虫wallikeri亚种　出处：《中国寄生虫学与寄生虫病杂志》2015年02期 　论文类型：期刊论文

【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 r DNA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 r DNA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 r DNA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体系则未能检测到该感染。此外,M-Nest体系和NP-2002体系对66份镜检以卵形疟原虫感染为主的血样的复核结果一致。结论新建的多重PCR检测体系可同时检测4种人体疟原虫,具有良好的现场适用性。
[Abstract]:Objective to detect the application of multiplex PCR for the new 4 species of human Plasmodium. Comparative analysis of 18S R DNA gene sequences of 4 species of human Plasmodium method using DNAman software, using Oligo 6 software, the design of 4 downstream primer sequences in the fifth and sixth conserved mutation interval, and the plasmid DNA containing 4 18S R DNA of Plasmodium sequence as a template, a multiplex PCR system for detection of specificity and sensitivity. In addition, the multiplex PCR system and NP-1993 system in the new round of first specific primer combinations, the formation of new nested PCR amplification system (M-Nest system), and the different dilution of Plasmodium falciparum and Plasmodium vivax DNA system and the blood samples of patients the new multiplex PCR amplification, detection sensitivity of these two systems. 307 blood samples by M-Nest system and NP-1993 system for the detection of the 2013 Guangxi home Garner malaria epidemic area personnel, comparison of the two The detection results are consistent. Finally, the application of M-Nest and NP-2002 system of Guangxi 1~5 in 2014 reported 66 microscopic examination of blood samples to Plasmodium falciparum infections mainly review, compare the results is consistent with the test results. The multiple application of PCR system of the new three Plasmodium falciparum malaria parasite, Plasmodium falciparum and amplified fragment size Plasmodium vivax were 268446394 and 323 BP, and different protozoa amplified bands located just 50 BP DNA markers between bands, more easy to distinguish, but different Plasmodium Tm value is close, not easy to distinguish and identify species. The new system of 4 species of Plasmodium 18S R DNA gene of the minimum detectable concentration respectively: 5.58 * 102 copies of Plasmodium falciparum / L, three, 1.56 * 103 copies / Plasmodium L, p.ovale 1.66 * 103 copies / L, 1.80 * 102 copies of Plasmodium vivax / L. new system for malignant The parasite samples the lowest detection concentration of 1.43 * 102~8.84 * 103 copies / L or 5.10 * 10~4.92 * 102 / L is higher than that of protozoa, Plasmodium vivax copies of 17.4~69.1 / L and 13.5~83.2 / L. protozoa and multiplex PCR system alone compared to the application of M-Nest detection system will be the lowest detection concentration of Plasmodium reduce the 10~100 times of.M-Nest system and NP-1993 system of Garner from returning 307 blood samples. The detection results are not consistent, and the M-Nest system is detected 2 cases of Plasmodium falciparum infection, while the NP-1993 system failed to detect the infection. In addition, M-Nest system and NP-2002 system to review the blood samples of 66 microscopic examination to Plasmodium falciparum infection the results are consistent. Conclusion the new multiplex PCR detection system for simultaneous detection of 4 species of human Plasmodium, has good applicability.

【作者单位】：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;
【基金】：国家质检总局公益性行业科研专项(No.201210056)~~
【分类号】：R531.3

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