基于ipaH基因的志贺菌LAMP检测方法的建立及应用

doi:10.11924/j.issn.1000-6850.casb2020-0084

摘要/Abstract

摘要：

为建立一种快速、特异、敏感的志贺菌环介导等温扩增(LAMP)检测方法,试验针对志贺菌属侵袭性质粒抗原H基因(invasive plasmid, ipaH)设计一套特异性引物,条件优化及特异性和敏感性验证后建立了该菌的LAMP检测方法,并应用于临床样本的检测。结果显示,62℃恒温扩增1 h能得到明显的扩增产物,除志贺菌标准菌株扩增结果为阳性外,沙门菌、大肠埃希菌、金黄色葡萄球菌、枯草芽孢杆菌和阴性对照均为阴性,检测限值达到120 fg/μL。利用建立的方法检测了20株疑似菌株和38份粪样样品,结果显示20株疑似菌和27份粪样样品为阳性,阳性率分别为100%和71.05%,说明建立的LAMP检测法具有良好的特异性和敏感性,在粪便等临床样本中志贺菌的检测与鉴定方面具有优势和应用前景。

关键词: 环介导等温扩增法, 志贺菌, ipaH基因, 检测

Abstract:

To establish a LAMP detection method which is rapid, specific and sensitive for Shigella, the sequence of invasion plasmid antigen H gene (invasion plasmid, ipaH) of Shigella was used as target sequence to design a set of specific primers, the LAMP method was established after condition optimization, specificity and sensitivity verification, and applied to detect clinical samples. The results showed that the amplification products could be obtained within 1 h at 62℃. The standard Shigella strains showed the positive result, while Salmonella, Escherichia coli, Staphylococcus aureus, Bacillus subtilis and the negative control showed the negative result. The detection limit of the LAMP method for Shigella was 120 fg/μL. 20 suspected strains and 38 fecal samples were tested by the established method. The results showed that 20 suspected bacteria and 27 fecal samples were positive, and the positive rate was 100% and 71.05%, respectively, indicating that this LAMP assay was a specific and sensitive method, and it had advantages and application prospects in the detection and identification of Shigella in clinical samples such as feces.

Key words: loop-mediated isothermal amplification assay, Shigella, ipaH, detect

中图分类号:

S858.2

文英, 刘永生. 基于ipaH基因的志贺菌LAMP检测方法的建立及应用[J]. 中国农学通报, 2021, 37(5): 104-110.

Wen Ying, Liu Yongsheng. Shigella LAMP Detection Method Based on ipaH Gene: Establishment and Application[J]. Chinese Agricultural Science Bulletin, 2021, 37(5): 104-110.

图/表 10

基因	引物名称	引物序列 (5’-3’)
ipaH	LF	GAGTTTTTCCAGCCATGCAG
	LB	TGCCACTGAGAGCTGTGAG
	FIP	CTGTCGAAGCTCCGCAGAGGGGATTCCGTGAACAGGTCG
	BIP	AGTCTTTCGCTGCTGCTGCTGATCCGGAGATTGTTCCATGTG
	F3	ATACCGTCTCTGCACGCA
	B3	GCCTTCTGATGCCTGATGG

表1. 引物序列

试剂	用量
2×反应缓冲液(RM)	12.5 μL
F3	1.0 μL
B3	1.0 μL
Loop-F	1.0 μL
Loop-B	1.0 μL
FIP	1.0 μL
BIP	1.0 μL
DNA模板	2.0 μL
BstDNA聚合酶	1.0 μL
去离子水(DW)	3.5 μL
合计	25.0 μL

表2. LAMP反应体系

图1. 反应温度对LAMP扩增效果的影响

图2. 特异性检测结果 A：浊度;B：反应速度;C：扩增效率;1：阴性对照;2：金黄色葡萄球菌;3：大肠埃希菌;4：枯草芽孢杆菌;5：沙门菌;6：CMCC(B)51252

图3. 特异性检测电泳结果 M：DL-2000 Marker;1：阴性对照;2：CMCC(B)51252;3：大肠埃希菌;4：枯草芽孢杆菌;5：沙门菌;6：金黄色葡萄球菌

图4. 灵敏度检测结果

图5. 疑似菌株检测结果 A：浊度;B：反应速度;C：扩增效率;1~8(A1)：疑似样本;1~3(A2)：疑似样本;4(A2)：CMCC(B)51252;5(A2)：阴性对照

图6. 部分疑似菌株电泳检测结果 M：DL-2000 Marker;1：阴性对照;2~10：疑似菌株

图7. 部分粪样样本的检测结果 A：浊度;B：反应速度;C：扩增效率;1~8(A1)：疑似样本;1~6(A2)：疑似样本;7(A2)：CMCC(B)51252;8(A2)：阴性对照

图8. 部分粪样样本的电泳检测结果 M：DL-2000 Marker;1：阴性对照;2：CMCC(B)51252;3~16：疑似菌株

参考文献 27

[1]	Kotloff K L, Winickoff J P, Ivanoff B, et al. Global burden of Shigella infections: implications for vaccine development and implementation of control strategies[J]. Bulletin of the World Health Organization, 1999,77(8):651-666. URL pmid: 10516787
[2]	黄艳萍, 唐光鹏, 蔡星和, 等. 2005—2011年贵州省细菌性痢疾监测分析[J]. 现代预防医学, 2014,41(11):2092-2094.
[3]	Von Seidlein L, Kim D R, Ali M, et al. A multicentre study of Shigella diarrhoea in six Asian countries: disease burden, clinical manifestations, and microbiology[J]. PLoS Medicine, 2006,3(9):353-359.
[4]	张静, 麻文建, 朱天辉, 等. 一种快速检测桉树焦枯病菌(Cylindrocladium scoparium)的LAMP体系的建立及应用[J]. 微生物学通报, 2015,42(4):791-794. doi: 10.13344/j.microbiol.china.140543 URL
[5]	文英. 不同动物源志贺菌的分离鉴定及耐药性检测[J]. 畜牧与兽医, 2018,50(8):59-63.
[6]	Thiem V D, Sethabutr O, Von Seidlein L, et al. Detection of Shigella by a PCR Assay Targeting the ipaH Gene Suggests Increased Prevalence of Shigellosis in Nha Trang, Vietnam[J]. Journal of Clinical Microbiology, 2004,42(5):2031-2035. doi: 10.1128/jcm.42.5.2031-2035.2004 URL pmid: 15131166
[7]	Houng H S H, Sethabutr O, Echeverria P. A simple polymerase chain reaction technique to detect and differentiate Shigella and enteroinvasive Escherichia coli in human feces[J]. Diagnostic Microbiology & Infectious Disease, 1997,28(1):19-25. doi: 10.1016/s0732-8893(97)89154-7 URL pmid: 9218914
[8]	Sethabutr O, Venkatesan M, Yam S, et al. Detection of PCR products of the ipaH gene from Shigella and enteroinvasive Escherichia coli by enzyme linked immunosorbent assay[J]. Diagn Microbiol Infect Dis, 2000,37(1):11-16. doi: 10.1016/s0732-8893(00)00122-x URL pmid: 10794934
[9]	刘光富, 关荣发, 刘明启. 志贺氏菌的LAMP检测方法研究[J]. 中国食品学报, 2013(8):200-206.
[10]	马晓燕, 张会彦, 宋明明, 等. 环介导等温扩增技术快速检测志贺氏菌的研究[J]. 安徽农业科学, 2019,39(14):8191-8193.
[11]	温来欣, 方丽萍 .应用 LAMP 同时快速检测沙门菌和志贺菌的效果评价[J]. 职业与健康, 2014,30(4):649-654.
[12]	陆冰洋, 刘华栋, 李婷婷, 等. 传染性法氏囊病病毒RT-LAMP 检测方法的建立[J]. 黑龙江畜牧兽医, 2018(15):135-139,243.
[13]	张艳艳, 叶倩, 王正荣, 等. 基于cox2基因的细粒棘球绦虫环介导等温扩增检测方法的初步建立[J]. 中国寄生虫学与寄生虫病杂志, 2017,35(2):169-172.
[14]	Cai T, Lou G Q, Yang J, et al. Development and evaluation of real-time loop-mediated isothermal amplification for hepatitis B virus DNA quantification:A new tool for HBV management[J]. J Clin Virol, 2008,41(4):270-276. doi: 10.1016/j.jcv.2007.11.025 URL pmid: 18296109
[15]	宋海霞. 河南省鸡腹泻重要病原菌的分离鉴定与耐药性及PCR检测方法的研究[D]. 郑州:河南农业大学, 2010.
[16]	朱阵. 牛肠道菌群携带耐药基因分析与志贺菌分离、分型及耐药性研究[D]. 北京:中国农业科学院, 2018.
[17]	马明 .方福平, 朱喜玲, 等. 贵州地区紫云花猪腹泻病原菌的分离鉴定及耐药性分析[J]. 畜牧与兽医, 2019,51(07):96-99.
[18]	蒋建军, 下鹏雁, 剡根强, 等. 集约化兔场高致病性痢疾志贺菌的分离与鉴定[J]. 黑龙江畜牧兽医 2006(8):66-67.
[19]	高凤山, 胡桂学. 幼犬志贺菌感染的微生物学诊断[J]. 安徽农学通报, 2007,13(3):118-142.
[20]	Onyango D M, WandilI S, Kakai R, et al. Isolation of Salmonella and Shigella from fish harvested from the Winam Gulf of Lake Victoria, Kenya[J]. J Infect Dev Ctries, 2009,3(2):99-104. doi: 10.3855/jidc.56 URL pmid: 19755738
[21]	王润锦, 张林, 朱秀高, 等. 藏鸡志贺氏菌的分离鉴定及药敏试验[J]. 中国家禽, 2015,37(22):65-67.
[22]	Buysse J M, Stover C K, Oaks E V, et al. Molecular cloning of invasion plasmid antigen (ipa) genes from Shigella flexneri: analysis of ipa gene products and genetic mapping.[J]. Journal of Bacteriology, 1987,169(6):2561-2569. doi: 10.1128/jb.169.6.2561-2569.1987 URL pmid: 3294797
[23]	蔡其刚, 唐一波, 高小龙, 等. 青藏高原放牧家畜小肠结肠炎耶尔森氏菌LAMP检测方法的建立及应用[J]. 安徽农业科学, 2019,47(24):107-110.
[24]	胡元庆, 沈子晨, 李凤霞, 等. 基于blaCARB-17基因建立水产品中副溶血弧菌的LAMP检测方法[J]. 食品与发酵工业, 2020,46(23):198-206.
[25]	焦文强, 殷相平, 柳纪省. 环介导等温扩增技术原理及其在检测诊断病原微生物中的应用[J]. 生物技术通报, 2009,18(9):54-57.
[26]	吴静怡, 董路宁, 任立松, 等. 痢疾杆菌快速检测方法的研究进展[J]. 中国卫生检验杂志, 2010,20(4):257-259.
[27]	邱索平, 李丹丹, 马保华, 等. LAMP方法快速检测实验猴粪便样品中志贺菌[J]. 中国卫生检验杂志, 2010,20(4):257-259.