高通量基因芯片在口岸疟原虫检测中的应用
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摘要
目的验证及应用一种同时快速检测4种疟原虫的高通量基因芯片方法。方法收集上海口岸现场经显微镜镜检及荧光PCR确认后的间日疟、恶性疟、三日疟、卵形疟病例血样,采用高通量基因芯片对4份疟原虫阳性血样同时进行验证检测。提取4种疟原虫DNA,采用实时荧光定量PCR进行定量,使用芯片检测经梯度稀释的疟原虫DNA样品,评价芯片的灵敏度。将4种疟原虫DNA和相同数量级浓度的诺氏疟原虫、微小隐孢子虫、结肠小袋纤毛虫DNA进行交叉反应检测,评价芯片的特异性。采集50例云南现场疑似疟疾病例血样,采用芯片进行检测,并与镜检结果进行比较。结果芯片检测上海口岸4份疟原虫阳性血样的结果与镜检和PCR结果相符,检测过程仅需2 h。芯片灵敏度检测结果显示,其对疟原虫DNA的检测下限分别为间日疟原虫78 copies/μl、三日疟原虫87 copies/μl、恶性疟原虫135 copies/μl、卵形疟原虫302 copies/μl。芯片特异性检测结果显示, 4种疟原虫均可检出,诺氏疟原虫、微小隐孢子虫、结肠小袋纤毛虫均未检出,无交叉反应。云南现场采集的50份疑似疟疾病例血样芯片检测结果显示,间日疟原虫阳性18份、恶性疟原虫阳性12份、三日疟原虫阳性1份,与镜检结果相符。结论该高通量基因芯片可以同时检测4种疟原虫,具有快速、准确、灵敏度高的特点,适合现场筛检及疟疾疫情防控的需要。
Objective To validate the application of a sensitive and specific high-throughput DNA microarray chip method for rapid detection of four species of Plasmodium simultaneously. Methods The blood samples with infections of different species of Plasmodium(P. vivax, P. falciparum, P. ovale, P. malariae) identified by microscopy and fluorescent PCR were collected from quarantined passengers at airport, and used for validating the sensitivity and specificity of the high-throughput DNA microarray chip to detect Plasmodium spp.. DNAs were extracted from the blood samples and quantified by the quantitative real-time PCR(RT-PCR). The sensitivity of the DNA microarray was determined by the series dilutions of extracted DNA samples. The specificity of the DNA microarray was determined by cross reacting with the specific Plasmodium sample mixed with the same amount of DNA extracted from P. knowlesi, Cryptosporidium parvum and Balantidium coli. The microarray chip was also used for detecting 50 blood samples collected from patients with suspected malaria from the border area of Yunnan Province, compared with re-sults of microscopy. Results The outcome of the DNA microarray detection of plasmodium spp. completely matched the results detected by microscopy and PCR. The whole procedure only took 2 hours. The chip was able to detect as low as 78 copies/μl of P. vivax, 87 copies/μl of P. malariae, 135 copies/μl of P. falciparum and 302 copies/μl of P. ovale. There was no cross-reaction with P. knowlesi, C. parvum and B. coli. The microarray chip was used to detect 50 blood samples from suspected malaria patients from Yunnan border, 18 of them was identified as infec-tion with P. vivax, 12 for P. falciparum, and 1 for P. malariae. The chip results were totally consistent with the microscopic results. Conclusion The high-throughput DNA microarray is a rapid, sensitive and specific method to detect different species of Plasmodium infections with no cross-reaction to other species of protozoans.
Objective To validate the application of a sensitive and specific high-throughput DNA microarray chip method for rapid detection of four species of Plasmodium simultaneously. Methods The blood samples with infections of different species of Plasmodium(P. vivax, P. falciparum, P. ovale, P. malariae) identified by microscopy and fluorescent PCR were collected from quarantined passengers at airport, and used for validating the sensitivity and specificity of the high-throughput DNA microarray chip to detect Plasmodium spp.. DNAs were extracted from the blood samples and quantified by the quantitative real-time PCR(RT-PCR). The sensitivity of the DNA microarray was determined by the series dilutions of extracted DNA samples. The specificity of the DNA microarray was determined by cross reacting with the specific Plasmodium sample mixed with the same amount of DNA extracted from P. knowlesi, Cryptosporidium parvum and Balantidium coli. The microarray chip was also used for detecting 50 blood samples collected from patients with suspected malaria from the border area of Yunnan Province, compared with re-sults of microscopy. Results The outcome of the DNA microarray detection of plasmodium spp. completely matched the results detected by microscopy and PCR. The whole procedure only took 2 hours. The chip was able to detect as low as 78 copies/μl of P. vivax, 87 copies/μl of P. malariae, 135 copies/μl of P. falciparum and 302 copies/μl of P. ovale. There was no cross-reaction with P. knowlesi, C. parvum and B. coli. The microarray chip was used to detect 50 blood samples from suspected malaria patients from Yunnan border, 18 of them was identified as infec-tion with P. vivax, 12 for P. falciparum, and 1 for P. malariae. The chip results were totally consistent with the microscopic results. Conclusion The high-throughput DNA microarray is a rapid, sensitive and specific method to detect different species of Plasmodium infections with no cross-reaction to other species of protozoans.
引文
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[13]Farcas GA,Zhong KJ,Lovegrove FE,et al.Evaluation of the Binax NOW ICT test versus polymerase chain reaction and microscopy for the detection of malaria in returned travelers[J].Am J Trop Med Hyg,2003,69(6):589-592.
[14]Singh B,Bobogare A,Cox-Singh J,et al.A genus-and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies[J].Am J Trop Med Hyg,1999,60(4):687-692.
[15]Fuehrer HP,Fally MA,Habler VE,et al.Novel nested direct PCR technique for malaria diagnosis using filter paper samples[J].J Clin Microbiol,2011,49(4):1628-1630.
[16]Mangold KA,Manson RU,Koay ES,et al.Real-time PCRfor detection and identification of Plasmodium spp[J].J Clin Microbiol,2005,43(5):2435-2440.
[2]周水森,王漪,李雨.2010年全国疟疾疫情分析[J].中国寄生虫学与寄生虫病杂志,2011,29(6):401-403.
[3]张丽,丰俊,张少森,等.2015年全国疟疾疫情分析[J].中国寄生虫学与寄生虫病杂志,2016,34(6):477-481.
[4]张丽,丰俊,张少森,等.2017年全国消除疟疾进展及疫情特征分析[J].中国寄生虫学与寄生虫病杂志,2018,36(3):201-209.
[5]丰俊,夏志贵.2004-2013年中国疟疾发病情况及趋势分析[J].中国病原生物学杂志,2014,9(5):442-446.
[6]张丽,丰俊,张少森,等.2016年全国疟疾疫情分析[J].中国寄生虫学与寄生虫病杂志,2017,35(3):515-519.
[7]夏志贵,杨曼尼,周水森.2011年全国疟疾疫情分析[J].中国寄生虫学与寄生虫病杂志,2012,30(6):419-422.
[8]Harris I,Sharrock WW,Bain LM,et al.A large proportion of asymptomatic Plasmodium infections with low and submicroscopic parasite densities in the low transmission setting of Temotu Province,Solomon Islands:challenges for malaria diagnostics in an elimination setting[J].Malar J,2010,9:254.
[9]Schneider P,Bousema JT,Gouagna LC,et al.Submicroscopic Plasmodium falciparum gametocyte densities frequently result in mosquito infection[J].Am J Trop Med Hyg,2007,76(3):470-474.
[10]Bednár M.DNA microarray technology and application[J].Med Sci Monit,2000,6(4):796-800.
[11]Kallioniemi OP.Biochip technologies in cancer research[J].Ann Med,2001,33(2):142-147.
[12]江莉,王真瑜,张耀光,等.3种疟疾检测方法的应用分析[J].中国寄生虫学与寄生虫病杂志,2017,35(1):53-58.
[13]Farcas GA,Zhong KJ,Lovegrove FE,et al.Evaluation of the Binax NOW ICT test versus polymerase chain reaction and microscopy for the detection of malaria in returned travelers[J].Am J Trop Med Hyg,2003,69(6):589-592.
[14]Singh B,Bobogare A,Cox-Singh J,et al.A genus-and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies[J].Am J Trop Med Hyg,1999,60(4):687-692.
[15]Fuehrer HP,Fally MA,Habler VE,et al.Novel nested direct PCR technique for malaria diagnosis using filter paper samples[J].J Clin Microbiol,2011,49(4):1628-1630.
[16]Mangold KA,Manson RU,Koay ES,et al.Real-time PCRfor detection and identification of Plasmodium spp[J].J Clin Microbiol,2005,43(5):2435-2440.