甲型H1N1流感病毒FAST-LAMP检测方法的建立

doi:10.13560/j.cnki.biotech.bull.1985.2015.05.013

摘要/Abstract

摘要： 建立一种新型等温扩增检测方法，用于提高等温扩增反应的检测速度，应用于甲型H1N1流感病毒检测。根据HA基因设计一组引物，包括外引物、内引物、环引物及套内引物，套内引物的加入增加了引物与模板的接触位点，提高扩增效率，缩短检测时间。结果显示，FAST-LAMP检测法比普通LAMP检测法时间缩短45 min左右，比加入环引物的LAMP检测方法缩短20 min，大大缩短了反应的检测时间。检测灵敏度可达到1×10² 拷贝。FAST-LAMP是一种高效、更快的检测方法。

关键词: FAST-LAMP, 甲型H1N1流感病毒, 检测

Abstract: It was to establish a new loop-mediated isothermal amplification（FAST-LAMP）for rapidly detecting influenza A（H1N1）virus. A set of primers were designed according to HA gene, including the outer primers, internal primers, loop primers and nest internal primers. The nest internal primers increased the contact sites of primers and nucleic acid template, so that the amplification efficiency could be improved and detection time reduced. The FAST-LAMP method could be 45 less than LAMP method, and 20 minutes less than LAMP method with loop primers, i.e. detection time reduced significantly detecting sensitivity reached 1×10² copies cDNA template/tube. It proved that the established FAST-LAMP method is an efficient and reliable method for detecting influenza A（H1N1）virus.

Key words: FAST-LAMP, H1N1 influenza virus, detection

吕沁风, 罗鹏, 何蕾, 杨永耀, 郑伟, 李莉, 吴忠华,. 甲型H1N1流感病毒FAST-LAMP检测方法的建立[J]. 生物技术通报, 2015, 31(5): 78-83.

Lü Qinfeng, Luo Peng, He Lei, Yang Yongyao, Zheng Wei, Li Li, Wu Zhonghua. The Establishment of FAST-LAMP Method for Detecting Influenza A（H1N1）Virus[J]. Biotechnology Bulletin, 2015, 31(5): 78-83.

参考文献

[1] Notom T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acid Research, 2000, 28（12）：e63.
[2] Nagamine K, Watanabe K, Ohtsuka K, et al. Loopmediated isothermal amplification reaction using a nondenatured template[J]. Clin Chem, 2001, 47：1742-1743.
[3] Goto M, Honda E, Ogura A, et al. Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxyl naphthol blue[J]. Biotechniques, 2009, 46：167-172.
[4] Mori Y, Nagamine K, Tomita N, Notomi T. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation[J]. Biochem Biophys Res Commun, 2001, 289：150-154.
[5] Lyu QF, Che Y, Zhang W, et al. Establishment of reserve transcrip-tion loop-mediated isothermal amplification method for Yellow fever virus[J]. Microbiology China, 2014, 41（1）：184-190.
[6] Zhao Z, Fan B, Wu G, et al, Development of loop-mediated isother-mal amplification assay for specific and rapid detection of differential goat Pox virus and Sheep Pox virus[J]. BMC Microbiol, 2014, 14（1）：10.
[7] Su JY, Liu XC, Cui HM, et al, Rapid and simple detection of methicillinresistance staphylococcus aureus by orfX loop-mediated isothermal amplification assay[J]. BMC Biotechnol, 2014, 24；14（1）：8.
[8] Tomita N, Mori Y, Kanda H, Notomi T. Loop-mediated isothermal amplification（LAMP）of gene sequences and simple visual detection of products[J]. Nature Protoc, 2008, 3：877-882.
[9] Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers[J]. Mol Cell Probes, 2002, 16（3）：223-229.
[10] Ihira M, Yoshikawa T, Enomoto Y, et al. Rapid diagnosis of human herpesvirus 6 infection by a novel DNA amplification method, loop-mediated isothermal amplification[J]. J Clin Microbiol, 2004, 42（1）：140-145.
[11] Valleron AJ, Cori A, Valtat S, et al. Transmissibility and geographic spread of the 1889 influenza pandemic[J]. Proc Natl Acad Sci USA, 2010, 107（19）：8778-8781.
[12] Mills CE, Robins JM, Lipsitch M. Transmissibility of 1918 pandemic influenza[J]. Nature, 2004, 432（7019）：904-906.
[13] LeBlanc JJ, Li Y, Bastien N, et al. Switching gears for an influenza pandemic：validation of a duplex reverse transcriptase PCR assay for simultaneous detection and confirmatory identification of pandemic（H1N1）2009 Influenza Virus[J]. J Clin Microbiol, 2009, 47（12）：3805-3813.
[14] Influenza A（H1N1）-update 103. World Health Organization. 2010-06-04 Retrieved 2010-10-16
[15] WHO（2013）Influenza update Nu182. 182：3-4. Available：http://www.who.int/influenza/surveillance_monitoring/updates/2013-04-02_surveillance_update 182. pdf.
[16] de la Rosa-Zamboni D, Vázquez-Pérez JA, Avila-Ríos S, et al. Molecular characterization of the predominant influenza A（H1N1）pdm09 virus in Mexico, December 2011-February 2012[J]. PLoS One, 2012, 7（11）：e50116.
[17] Wu C, Cheng X, Wang X, et al. Clinical and molecular characteris-tics of the 2009 pandemic influenza H1N1 infection with severe or fatal disease from 2009 to 2011 in Shenzhen, China[J]. J Med Virol, 2013, 85（3）：405-412.
[18] Brooke RJ, VAN Lier A, Donker GA, et al. Comparing the impact of two concurrent infectious disease outbreaks on The Netherlands population, 2009, using disability-adjusted life years[J]. Epidemiol Infect, 2014, 24：1-10.
[19] Gomez-Barroso D, Martinez-Beneito MA, Flores V. Geographical spread of influenza incidence in Spain during the 2009 A（H1N1）pandemic wave and the two succeeding influenza seasons[J]. Epidemiol Infect, 2014, 27：1-13.
[20] Dakhave M, Khirwale A, Patil K, et al. Whole-genome sequence an-alysis of postpandemic influenza A（H1N1）pdm09 virus isol-ates from India[J]. Genome Announc, 2013, 1（5）：e00727-13.
[21] Chudasama RK, Patel UV, Verma PB. Characteristics of hospitali-zed patients with severe and non-severe pandemic influenza A（H1N1）in Saurashtra region, India（two waves analysis）[J]. J Family Med Prim Care, 2013, 2（2）：182-187.
[22] Kiertiburanakul S, Morasert T, Sirinavin S, et al. Comparisons of clinical characteristics between adult patients with 2009 H1N1 inf-luenza and those with seasonal influenza during the 2009 Epidemic in Thailand[J]. Jpn J Infect Dis, 2014, 67（1）：33-39.
[23] Wang XL, Wong CM, Chan KH, et al. Hospitalization risk of the 2009 H1N1 pandemic cases in Hong Kong[J]. BMC Infect Dis, 2014, 14（1）：32.
[24] North Texas confirmed 20 flu deaths［Last accessed on 2014-01-08] . Available from：http://news. xinhuanet. com/english/world/2014-01/08/c_133026240. htm
[25] http://news.sina.com.cn/c/2013-04-03/222126728529. shtml
[26] Ren YY, Yin YY, Li WQ, et al. Risk factors associated with severe manifestations of 2009 pandemic influenza A（H1N1）infection in China：a case-control study[J]. Virol J, 2013, 10：149.
[27] Choi JH, Kim MS, Lee JY, et al. Development and evaluation of multiplex real-time RT-PCR assays for seasonal, pandemic A/H1pdm09 and avian A/H5 influenza viruses detection[J]. J Microbiol, 2013, 51（2）：252-257.
[28] Mori Y, Kitao M, Tomita N, Notomi T. Real-time turbidimetry of LAMP reaction for quantifying template DNA[J]. J Biochem Biophys Methods, 2004, 59：145-157.
[29] 聂凯, 王大燕, 秦萌, 等. 基于颜色判定的环介导逆转录等温扩增技术检测人甲型H1N1 流感病毒基因[J]. 病毒学报, 2010, 26（2）：81-87.
[30] 王翔, 张骞, 张钫, 等. 甲型H1N1流感病毒HA基因RT-LAMP 检测方法的建立与初步应用[J]. 生物技术通讯, 2011, 22（5）：696-708.