高通量基因芯片在口岸疟原虫检测中的应用

doi:10.12140/j.issn.1000-7423.2019.01.011

中国寄生虫学与寄生虫病杂志 ›› 2019, Vol. 37 ›› Issue (1): 61-65.doi: 10.12140/j.issn.1000-7423.2019.01.011

高通量基因芯片在口岸疟原虫检测中的应用

曹继红¹(), 张子龙², 李深伟², 李美³, 张晓航², 田桢干^2,^*()

1 江西省九江市第三人民医院,九江 332000
2 上海国际旅行卫生保健中心,上海 200335
3 中国疾病预防控制中心寄生虫病预防控制所,国家热带病研究中心,世界卫生组织热带病合作中心,科技部国家级热带病国际联合研究中心,卫生部寄生虫病原与媒介生物学重点实验室,上海 200025

收稿日期:2018-08-16 出版日期:2019-02-28 发布日期:2019-03-18
通讯作者: 田桢干
作者简介:
作者简介：曹继红（1975-）,女,本科,副主任医师,从事传染病研究。E-mail: 13970288510@163.com
基金资助:
国家科技重大专项（No. 2018ZX10101003-002-005）;上海科委项目（No. 18495810900）;质检总局课题（No. 2016IK219）

Application of high-throughput DNA microarray for rapid detection of Plasmodium spp. at port

Ji-hong CAO¹(), Zi-long ZHANG², Shen-wei LI², Mei LI³, Xiao-hang ZHANG², Zhen-gan TIAN^2,^*()

1 The Third People’s Hospital of Jiujiang City, Jiujiang 332000, China;
2 Shanghai International Travel Healthcare Center, Shanghai 200335, China
3 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention; Chinese Center for Tropical Diseases Research; WHO Collaborating Centre for Tropical Diseases; National Center for International Research on Tropical Diseases, Ministry of Science and Technology; Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai 200025, China

Received:2018-08-16 Online:2019-02-28 Published:2019-03-18
Contact: Zhen-gan TIAN
Supported by:
Supported by National Science and Technology Major Project(No. 2018ZX10101003-002-005), Shanghai Science and Technology Committee Project(No. 8495810900), and General Administration of Quality Supervision, Inspection and Quarantine Project(No. 2016IK219)

摘要/Abstract

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。

关键词: 高通量芯片, 疟原虫, 快速检测

Abstract:

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 results 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 infection 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.

Key words: High-throughput DNA microarray, Plasmodium, Rapid detection

中图分类号:

R382.31

曹继红, 张子龙, 李深伟, 李美, 张晓航, 田桢干. 高通量基因芯片在口岸疟原虫检测中的应用[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(1): 61-65.

Ji-hong CAO, Zi-long ZHANG, Shen-wei LI, Mei LI, Xiao-hang ZHANG, Zhen-gan TIAN. Application of high-throughput DNA microarray for rapid detection of Plasmodium spp. at port[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2019, 37(1): 61-65.

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参考文献 16

[1]	黄勇. 人的社会行为因素对疟疾防治效果的影响[J]. 中国血吸虫病防治杂志, 2002, 14(6): 470-471.
[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 sub-microscopic 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 PCR for detection and identification of Plasmodium spp[J]. J Clin Microbiol, 2005, 43(5): 2435-2440.

检测方法Detection method		基因芯片检测 Microarray detection					符合率/% Accuracy/%
检测方法Detection method		间日疟原虫阳性 P. vivax	恶性疟原虫阳性 P. falciparum	三日疟原虫阳性 P. malariae	阴性Negative	合计Total	符合率/% Accuracy/%
显微镜镜检 Microscopy	间日疟阳性P. vivax	18	0	0		18	100
	恶性疟阳性P. falciparum	0	12	0		12	100
	三日疟阳性P. malariae	0	0	1		1	100
	阴性Negative				19	19	100
	合计Total	18	12	1	19	50	100

高通量基因芯片在口岸疟原虫检测中的应用

Application of high-throughput DNA microarray for rapid detection of Plasmodium spp. at port

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