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Real-time recombinase polymerase amplification for detection of Vibrio parahaemolyticus |
ZHAN Li, XU Chang-ping, ZHANG Yun-yi, CHEN Hong-hu, ZHANG Zheng, CHEN Jian-cai, ZHANG Jun-yan, MEI Ling-ling
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Zhejiang Provincial Center for Disease Control and Prevention,Hangzhou,Zhejiang 310051,China |
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Abstract Objective To establish real-time recombinase polymerase amplification(RPA)for the rapid detection of Vibrio parahaemolyticus(VP).Methods An exo probe and primers were designed according to the conserved sequence of thermolabile hemolysin(tlh)gene of VP and then RPA for detection of VP was established. The sensitivity of the assay was evaluated by detecting different concentration of VP;the specificity was evaluated by detecting different bacteria;the stability was evaluated by repeat trials;the application effect was evaluated by detecting food samples which were simultaneously tested with traditional culture method according to GB 4798.7-2013 Detection of VP.Results A real-time RPA was established to complete VP amplification within 20 min at a constant temperature of 39 ℃. The analytical sensitivity of the assay was five pg per reaction and no cross-reactivity with other pathogenic bacteria observed. The RPA detection results with different concentration of VP and E. coli DNA templates at three time points were consistent. The detection results of 51 food samples by RPA were the same as those by traditional culture method.Conclusion The established real-time RPA can qualitatively detect VP,with simple operation and interpretation of results,which is suitable for rapid detection of VP in public health emergencies and food safety supervision.
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Received: 14 February 2019
Revised: 15 March 2019
Published: 02 July 2019
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[1] 陈洪友,陈敏,盛跃颖. 副溶血性弧菌食源性疾病暴发分离株的血清型、核糖型及毒力基因研究[J]. 中国食品卫生杂志,2011,23(2):114-118. [2] 毛雪丹. 2003—2008年我国细菌性食源性疾病流行病学特征及疾病负担研究[D]. 北京:中国疾病预防控制中心,2010. [3] 吕鹏,章荣华,齐小娟,等. 浙江省食源性疾病监测现状分析[J]. 浙江预防医学, 2015,27(8):828-837. [4] 陈江,章荣华,张荷香,等. 2010—2012年浙江省食源性疾病事件流行病学特征分析[J]. 中国食品卫生杂志,2015,27(2):120-123. [5] 朱大方,周标,陈江,等. 2015年浙江省食源性疾病暴发流行病学特征分析[J]. 预防医学,2016,28(10):1005-1009. [6] 中华人民共和国国家卫生和计划生育委员会. 食品微生物学检验副溶血性弧菌检验:GB 4789.7—2013 [S]. 北京:中国标准出版社,2013. [7] MAFFERT P,REVERCHON S,NASSER W,et a1. New nucleic acid testing devices to diagnose infectious diseases in resource-limited settings[J]. Eur J Clin Microbiol Infect Dis,2017,36(10):1717-1731. [8] 景志刚,董浩,狄栋栋,等. 重组酶聚合酶扩增技术研究进展[J]. 生物技术通报,2016,32(6):47-53. [9] 高威芳,朱鹏,黄海龙. 重组酶聚合酶扩增技术:一种新的核酸扩增策略[J]. 中国生物化学与分子生物学报,2016,32(6):627-634. [10] YAN L,ZHOU J,ZHENG Y,et al.Isothermal amplified detection of DNA and RNA[J]. Mol Biosyst,2014,10(5):970-1003. [11] JAMES A,MACDONALD J.Recombinase polymerase amplification:emergence as a critical molecular technology for rapid,low-resource diagnostics[J]. Expert Rev Mol Diagn,2015,15(11):1475-1489. [12] DAHER R K,STEWART G,BOISSINOT M,et al.Recombinase polymerase amplification for diagnostic applications[J]. Clin Chem,2016,62(7):947-958. [13] LILLIS L,LEHMAN D,SINGHAL M C,et al.Non-instrumented incubation of a recombinase polymerase amplification assay for the rapid and sensitive detection of proviral HIV-1 DNA[J]. PLoS One,2014,9(9):e108189. [14] 彭志,陈刚毅,刘雪飞,等.等温核酸扩增技术在病原体检测中的应用[J]. 生物技术进展,2018,8(4):284-292. [15] ZHANG C,XING D.Single-molecule DNA amplification and analysis using microfluidics[J]. Chem Rev,2010,110(8):4910-4947. [16] TANIGUCHI H,HIRANO H,KUBOMURA S,et al.Comparison of the nucleotide sequences of the genes for the thermostable direct hemolysin and the thermolabile hemolysin from Vibrio parahaemolyticus[J]. Microb Pathog,1986,1(5):425-432. [17] RIZVI A V,BEJ A K.Multiplexed real-time PCR amplification of tlh,tdh and trh genes in Vibrio parahaemolyticus and its rapid detection in shellfish and Gulf of Mexico water[J]. Antonie Van Leeuwenhoek,2010,98(3):279-290. [18] LETCHUMANAN V,CHAN K G,LEE L H.Vibrio parahaemolyticus:a review on the pathogenesis,prevalence,and advance molecular identification techniques[J]. Front Microbiol,2014(5):705. [19] 金晓君,龚雅利,杨莉,等. 重组酶聚合酶扩增在铜绿假单胞菌检测中的应用[J]. 中华烧伤杂志,2018,34(4):233-239. [20] LILLIS L,SIVERSON J,LEE A,et al.Factors influencing recombinase polymerase amplification(RPA)assay outcomes at point of care[J]. Mol Cell Probes,2016,30(2):74-78. |
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