Application and evaluation of multiple cross displacement amplification in detection of Listeria monocytogenes
LI Hui1, 2, WANG Yi2, WANG Yan2, LI Hua2, CHEN Zheng-hong1, YE Chang-yun2
1. Department of Microbiology, School of Basic Medical Science, Guizhou Medical University/Key Laboratory of Medical Microbiology and Parasitology of Guizhou Province, Guiyang 550004, China; 2. State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Abstract：Listeria monocytogenes is a significant food-borne pathogen that is capable of causing severe listeriosis in both humans and animals. Traditional techniques such as culture-based approaches have good specificity and sensitivity, while they are time-consuming and tend to be tedious. Hence, we presented a simple and rapid molecular technique using multiple cross displacement amplification (MCDA) targeting L. monocytogenes-specific gene lmo0733 for sensitive and specific detection of the pathogen. L. monocytogenes-MCDA assay used a set of ten primers spanning ten distinct regions of target sequence and was carried out at a constant temperature 61 ℃ to evaluate the specificity, sensitivity and feasibility. A total of three methods were used to confirm MCDA products, including color change of Loopamp Fluorescent Detection Reagent, real-time measurement of turbidity and agarose gel electrophoresis. The limit of detection (LoD) of the MCDA was 10 fg DNA, which was 25-fold and 250-fold more sensitive than that of the LAMP and CPA assay for detecting L. monocytogenes DNA, respectively. The detection ability of MCDA method was identical to the culture-biotechnical method for 153 rat intestinal feces samples, and was better than LAMP, CPA and conventional PCR. The results in this study indicated that the MCDA assay could be used as a rapid, sensitive and effective tool for the detection of L. monocytogenes in the food industry, medical institutions and field.
李慧, 王毅, 王艳, 李华, 陈峥宏, 叶长芸. 多交叉置换扩增技术在单增李斯特菌检测中的应用及评价[J]. 中国人兽共患病学报, 2018, 34(2): 99-104.
LI Hui, WANG Yi, WANG Yan, LI Hua, CHEN Zheng-hong, YE Chang-yun. Application and evaluation of multiple cross displacement amplification in detection of Listeria monocytogenes. Chinese Journal of Zoonoses, 2018, 34(2): 99-104.
[1] Ma AJ, Wang Y, Wang Y, et al. Molecular epidemiological characteristics of Listeria monocytogenes isolated from raw meat samples in some regions of Beijing, China[J]. Chin J Zoonoses, 2015, 31(5): 403-407. doi:10.3969/cjz.j.issn.1002-2694.2015.05.003 (in Chinese) 马爱静, 王艳, 王毅, 等. 北京市一些地区生肉标本中单增李斯特菌的分离及其分子流行病学特征分析[J]. 中国人兽共患病学报, 2015, 31(5): 403-407. [2] Goulet V, Hebert M, Hedberg C, et al. Incidence of listeriosis and related mortality among groups at risk of acquiring listeriosis[J]. Clin Infect Dis, 2012, 54(5): 652-660. doi:10.1093/cid/cir902 [3] Zhang W, Hughes A, Wilt G, et al. The BAX PCR assay for screening Listeria monocytogenes targets a partial putative gene lmo2234[J]. J Food Prot, 2004, 67(7): 1507-1511. [4] Somer L, Kashi Y. A PCR method based on 16S rRNA sequence for simultaneous detection of the genus Listeria and the species Listeria monocytogenes in food products[J]. J Food Prot, 2003, 66(9): 1658-1665. doi:10.4315/0362-028X-66.9.1658 [5] O’grady J, Ruttledge M, Sedano-Balbás S, et al. Rapid detection of Listeria monocytogenes in food using culture enrichment combined with real-time PCR[J]. Food Microbiol, 2009, 26(1): 4-7. doi:10.1016/j.fm.2008.08.009 [6] Wang Y, Wang Y, Ma A, et al. Rapid and sensitive detection of Listeria monocytogenes by cross priming amplification of lmo 0733 gene[J]. FEMS Microbiol Lett, 2014, 361(1): 43-51. doi:10.1111/1574-6968.12610 [7] Wang Y, Wang Y, Ma AJ, et al. Rapid and sensitive isothermal detection of nucleic-acid sequence by multiple cross displacement amplification[J]. Sci Rep, 2015, 5: 11902. doi:10.1038/srep11902 [8] Wang Y, Wang Y, Lan R, et al. Multiple endonuclease restriction real-time loop-mediated isothermal amplification: A novel analytically rapid, sensitive, multiplex loop-mediated isothermal amplification detection technique[J]. J Mol Diagn, 2015, 17(4): 392-401. doi:10.1016/j.jmoldx.2015.03.002. [9] Ryu J, Si HP, Yeom YS, et al. Simultaneous detection of Listeria species isolated from meat processed foods using multiplex PCR[J]. Food Control, 2013, 32(2): 659-664. doi:10.1016/j.foodcont.2013.01.048 [10] Wang D, Zhang G, Lu C, et al. Rapid detection of Listeria monocytogenes in raw milk with loop-mediated isothermal amplification and chemosensor[J]. J Food Sci, 2011, 76(9): M611-M615. doi:10.1111/j.1750-3841.2011.02383.x [11] Liu D, Ainsworth AJ, Austin FW, et al. Use of PCR primers derived from a putative transcriptional regulator gene for species-specific determination of Listeria monocytogenes [J]. Int J Food Microbiol, 2004, 91(3): 297-304. doi:10.1016/j.ijfoodmicro.2003.07.004 [12] Gouin E, Mengaud J, Cossart P. The virulence gene cluster of Listeria monocytogenes is also present in Listeria ivanovii , an animal pathogen, and Listeria seeligeri , a nonpathogenic species[J]. Infect Immun, 1994, 62(8): 3550-3553. [13] Bubert A, Köhler S, Goebel W. The homologous and heterologous regions within the iap gene allow genus-and species-specific identification of Listeria spp. by polymerase chain reaction[J]. Appl Environ Microbiol, 1992, 58(8): 2625-2632.
2018, Vol. 34 
