7 12 Vol. 7 No. 12 2016 12 Journal of Food Safety and Quality Dec., 2016 PCR 杨纯佳, 张 * 娟, 周臣清, 霍丽斯, 崔海萍, 王力清 (, ( ), 528300) 摘要 : PCR, PCR,, PCR PCR PCR PCR O157:H7 PCR, 关键词 : PCR; PCR; PCR; Research progress on the application of digital PCR in detection of foodborne pathogenic microorganisms YANG Chun-Jia, ZHANG Juan, ZHOU Chen-Qing, HUO Li-Si, CUI Hai-Ping, WANG Li-Qing * (Guangdong Testing Institute of Product Quality Supervision, China National Quality Supervision and Testing Center for Foods (Guangdong), Foshan 528300, China) ABSTRACT: Foodborne diseases are mostly caused by foodborne pathogens, so it is very important to establish effective detection methods of foodborne pathogenic microorganisms for food safety risk monitoring and people's health ensuring. Compared with the traditional PCR, digital PCR has good accuracy and reproducibility, which has provided a new platform for rapid detection of food safety. This article mainly summarized the basic principles, varieties, application and research progress of digital PCR technology including droplet digital PCR and chip digital PCR. The applications of digital PCR technology in detection of Salmonella, Escherichia coli O157:H7, Listeria monocytogenes, Enterobacter cloacae, Staphylococcus aureus and other foodborne pathogenic microorganisms were also discussed in depth. The technology digital PCR has gotten good applications in quantitative detection of genetically modified organisms and animal derived ingredients, but was remained to be better in the application of foodborne pathogenic microorganisms. KEY WORDS: digital PCR; droplet digital PCR; chip digital PCR; foodborne pathogenic microorganisms 基金项目 : (2016A040403071) Fund: Supported by Science and Technology Project of Guangdong Province (2016A040403071) * 通讯作者 :,, E-mail: sdwlq@21cn.com *Corresponding author: WANG Li-Qing, Professor Senior Engineer, Guangdong Testing Institute of Product Quality Supervision, Foshan 528300, China E-mail: sdwlq@21cn.com
12, : PCR 4727 1 引言,,,,, 70% [1] [2],, [3], PCR, [4-6], PCR,,,, DNA,, DNA, [7] PCR PCR PCR PCR PCR PCR 2 数字 PCR 技术概述 PCR(digital PCR, dpcr) 1992, Sykes [8] PCR, PCR PCR, PCR C t ( ),,,, [9], PCR PCR, PCR PCR [10-14] PCR dpcr(droplet dpcr, ddpcr) dpcr(chip dpcr, cdpcr) PCR PCR,,, PCR,, 1, 0, [15,16] ddpcr PCR(qPCR),,, [17-20] cdpcr PCR,, [21] [22] PCR, PCR 3 dpcr 在食源性致病微生物检测中的应用 3.1 dpcr 检测食品中沙门氏菌,,, [23,24] [25] SD-PMA-ddPCR( - - PCR, sodium deoxycholate-propidium monoazide-droplet dpcr), ddpcr 8.0 copy/20 μl, 10 2 CFU/mL SD-PMA-ddPCR, Rothrock [26] ddpcr,, ddpcr dpcr, 3.2 ddpcr 定量检测大肠杆菌 O157: H7 (enterohemorrhagic Escherichia coli, EHEC) O157:H7,,,, [27] O157:H7, 1982 O157:H7 O157:H7 [28-30] 1999 O157:H7,
4728 7 2, 177, 7 [27], [31] O157: H7 rfbe, ddpcr ddpcr 300 nmol/l O157:H7 DNA 4~1.25 10 5 copy/20 μl ddpcr, ddpcr 0.999 DNA 760~88400 copy/20 μl, (RSD<5%) 4 copy/20 μl, 3 copy/20 μl, ddpcr, 13, PCR Bian [32] ddpcr O157:H7, PCR, ddpcr 10 CFU/mL PCR, dpcr 3.3 dpcr 定量检测单核细胞增生李斯特氏菌 (Listeria monocytogenes),,,, 20%~50% [33] 4,,, [34] [35] SD-PMA-ddPCR, (sodium deoxycholate, SD), (propidium monoazide, PMA) DNA, DNA ddpcr SD-PMA-ddPCR 2.0 copy/20 μl, Rothrock [26] ddpcr,, ddpcr Bian [32] ddpcr, 10 CFU/mL SD-PMA-ddPCR, 3.4 dpcr 定量检测阴沟肠杆菌 1980 (Enterobacter sakazakii) ( ), 50% [36] dpcr, qpcr,, 8.9 copy/μl,, 3.5 dpcr 定量检测金黄色葡萄球菌 (Staphylococcus aureus, SA),, [37-39], 33%, 45%, [40-42] Kelley [43] ddpcr, qpcr ddpcr SA0140(SA) (MECA) 397, qpcr ddpcr, ddpcr qpcr,,, dpcr, 4 结语与展望 dpcr,, :(1)dPCR, ; (2) PCR, ;(3)dPCR PCR, PCR PCR,,, ;, 参考文献 [1] Groundwater PW, Todd A, Worsley A J, et al. The technology and techniques used in the detection of pathogenic bacteria [J]. Pharm J, 2009, 283(7569): 281 282. [2]. PCR [J]., 2006, CFI(11): 44 46.
12, : PCR 4729 Fang P. Application of PCR in rapid detection of some food-borne pathogenic microorganisms [J]. Acad Forum, 2006, CFI(11): 44 46. [3],,. [J]., 2015, 6(9): 3405 3413 Chen YM, Cheng N, Xu WT. Novel technologies for foodborne pathogenic microorganism detection [J]. J Food Saf Qual, 2015, 6(9): 3405 3413. [4],,,. [J]., 2013, 34(18): 110 115. Li JY, Zhao Z, Jia XC, et al. Advance on detection of foodborne pathogenic bacteria [J]. Food Res Dev, 2013, 34(18): 110 115. [5],,. PCR [J]., 2010, 24(4): 8 12. Zhang R, Dong HQ, Zhang TW. Development and standardization discussion of PCR detection for foodbornemicro-organis [J]. J Shandong Inst Light Ind, 2010, 24(4): 8 12. [6]. [J]., 2014, 5(1): 173 186. Chen AL. Technology application status and development trend of rapid detection of foodborne pathogenic microorganisms [J]. J Food Saf Qual, 2014, 5(1): 173 186. [7],. [J]., 2007, 28(10): 632 637. Zeng QM, Zhang DD. Bacterial detection methods in foods [J]. Food Sci, 2007, 28(10): 632 637. [8] Sykes PJ, Neoh SH, Brisco MJ, et al. Quantitation of targets for PCR by use of limiting dilution [J]. Biotechniques, 1992, 13(3): 444. [9],. PCR [J]., 2014, 24(12): 2415 2423. Lin CQ, Yao B. Recent advance in digital PCR [J]. Prog Chem, 2014, 24(12): 2415 2423. [10],,,. PCR PCR [J]., 2015, 25(19): 3221 3230. Yang DY, Yang YC, Li H, et al Comparation of digital PC and fluorescent PCR for detecting kitchen-waste vegetable oil [J]. Chin J Health Lab Technol, 2015, 25(19): 3221 3230. [11] Kim TG, Jeong SY, Cho KS Comparison of droplet digital PCR and quantitative real -time PCR for examining population dynamics of bacteria in soil [J]. Appl Microbiol Biotechnol, 2014, 98(13): 6105 6113 [12] Hindon BJ, Ness KD, Masquelier DA, et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number [J]. Anal Chem, 2011, 83(22): 8604 8610. [13] Phillip B, Stephanie CT, John FR, et al. Droplet digital PCR measurement of HER2 copy number alteration in formalin-fixed paraffin-embedded breast carcinoma tissue [J]. Clin Chem, 2013, 59(6): 991 994 [14] Christopher MH, John RC, Hilary AB, et al Absolute quantification by droplet digital PCR versus analog real-time PCR [J]. Nat Methods, 2013, 10(10): 1003 1005 [15],,,. PCR [J]., 2016, 37(08): 187 191. Miao L, Zhang XP, Chen J, et al Quailtitative analysis of bovine aild porcine ingredients in meat products by droplet digital PCR [J]. Food Sci, 2016, 37(08): 187 191. [16] Massanellam, Singhania A, Bellakova BN, et al Differential gene expression in HIV-infected individuals following ART [J]. Antiviral Res, 2013, 100(2): 420 428 [17],,. PCR PCR [J]., 2015, 411(7): 38 41. Wang S, Li ZJ, Miao L. Comparison of microsphere digital PCR and real-time fluorescence PCR for detection mutton-derived and porcine-derived ingredients in mutton products [J]. Meat Ind, 2015, 411(7): 38 41. [18] Hindson CM, Chevillet JR, Briggs HA, et al. Absolute quantification by droplet digital PCR versus analog real-time PCR [J]. Nat Methods, 2013, (10): 1003 1005. [19] Sanders R, Huggett JF, Bushell CA, et al. Evaluation of digital PCR for absolute DNA quantification [J]. Anal Chem, 2011, 83(17): 6474 6484. [20] Pinheiro LB, Coleman VA, Hindson CM, et al. Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification [J]. Anal Chem, 2012, 84(2): 1003 1011. [21],,,. PCR [J]. ( ), 2015, 42(6): 786 789 Zhan C, Yang L, Wang L, et al The development and application of digital PCR [J]. Fudan J (Med Ed), 2015, 42(6): 786 789. [22],,,. PCR [J]., 2013, 34(3): 545 550. Zhu QY, Yang WX, Gao YB, et al Microfluidic digital chip for absolute quantification of nucleic acid amplification [J]. Chem J Chin Univ, 2013, 34(3): 545 550. [23],,,. PCR [J]., 2011, 44(16): 3395 3402. Li XL, Liu B, Dan XL, et al. Development and application of an IAC-PCR kit for the rapid detection of Salmonella spp [J]. Sci Agric Sin, 2011, 44(16): 3395 3402. [24],,,. PCR [J]., 2008, 30(3): 220 224. Zhong WJ, Zhao MQ, Deng ZP, et al. Establishment and preliminary application of real-time PCR method for detection of Salmonella in food [J]. Chin J Prev Vet Med, 2008, 30(3): 220 224. [25],,,. SD-PMA-ddPCR [J]., 2016, 37(10): 67 71. Wang J, Zhang HM, Wei W, et al. Detection of Salmonella cells based on SD-PMA-ddPCR [J]. Sic Technol Food Ind, 2016, 37(10): 67 71. [26] Rothrock MJ, Hiett KL, Kiepper BH, et al. Quantification of zoonotic bacterial pathogens within commercial poultry processing water samples using droplet digital PCR [J]. Sci Res, 2013, 3: 403 411. [27],,,. PCR O157:H7[J]., 2010, 31(08): 212 215. Chao QG, Yang XM, Ge Y, et al. Rapid PCR detection of enterohemorrhagic Escherichia coli O157:H7 in foods [J]. Food Sci, 2010, 31(08): 212 215. [28],,,. O157:H7 PCR [J]., 2011, 32(12): 278 282. Hu H, Chen YJ, Duan ZG, et al Real-time PCR detection of specific gene in Escherichia coli O157:H7 [J]. Food Sci, 2011, 32(12): 278 282.
4730 7 [29] Fach P, Perelle S, Grout J, et al. Comparison of different PCR teats for detecting shigatoxin-producing Escherichia coli O157 and development of an ELISA-PCR assay for specific identification of the bacteria [J]. J Microbiol Methods, 2003, 55(2): 389 392. [30] Scotter S, Aldridge M. Validation of a method for the detection of E.coli O157:H7 in foods [J]. Food Control, 2000, 11(2): 85 95. [31],,,. O157: H7 PCR [J]., 2013, 43(3): 319 324. Dong LH, Zhang L, Jiang J, et al. Development of droplet digital polymerase chain reaction for quantifying Escherichia coli O157: H7 [J]. Chin J Anal Chem, 2013,43(3): 319 324. [32] Bian XJ, Jing FX, Gang L. A microfluidic droplet digital PCR for simultaneous detection of pathogenic Escherichia coli O157 and Listeria monocytogenes[j]. Biosens Bioelectron, 2015, 74(15): 770 777. [33],,. [J]., 2009, 30(15): 281 284. Duan X, Lai WH, Zhang LL. Research progress on detection of Listeria monocytogenes in food [J]. Food Sci, 2009, 30(15): 281 284. [34],,. PCR [J]., 2008, 01(024): 95 99. Xu W, Li SF, Liu J. The development of Listeria monocytogenes assay by PCR [J]. Biotechnol Bull, 2008, 01(024): 95 99. [35],,,. SD-PMA-ddPCR [J]., 2016, 10, DOI: 10.13344/j.microbiol. china.150746. Wang J, Liu YM, Li CX, et al. Detection of Listeria monocytogenes cells in food based on SD-PMA-ddPCR [J]. Microbiol china, 2016, 10, DOI: 10.13344/j.microbiol.china.150746. [36],,,. PCR [J]., 2016, 48(3): 1 4. Yu SY, Tang LF, Chen XQ, et al. Establishment of the digital PCR assay for detection of Enterobacter cloacae [J]. Anim Husb Vet Med, 2016, 48(3): 1 4. [37],,,. DPO-PCR [J]., 2016, 35(4): 419 423. Li DD, Xu YG, Qiu SP, et al. A DPO-PCR Method for the rapid detection of Staphylococcus aureus [J]. J Food Sci Biotechnol, 2016, 35(4): 419 423. [38],,,. [J]., 2008, 8(3): 88 93. Suo YJ, Yu HE, Ling W, et a1. Analysis on the contamination of Staphylococcus aureus in food [J]. J Chin Inst Food Sci Technol, 2008, 8(3): 88 93. [39] Rail VL, Vieira FP, Rail R, et a1. PCR detection of staphylococcal entero toxin genes in Staphylococcus aureus strains isolated from raw and pasteurized milk [J]. Vet Microbiol, 2008, 132(3 4): 408 413. [40],,,. PCR [J]., 2011, 30(1): 84 89. Xu XK, Wu QP, Zhang JM, et a1 Studies on detection of Staphylococcus aureus in foods by multiplex PCR [J]. J Food Sci Biotechnol, 2011, 30(1): 84 89. [41],,,. PCR [J]., 2006, 39(5): 990 996. Yang Y, Zhang W, Yuan YW, et a1. Detection of Staphylococcus aureus in dairy products by polymerase chain reaction assay [J]. Sci Agric Sin, 2006, 39(5): 990 996. [42]. [J]., 2004, 16(1): 3 9 Liu XM. Studies on the techniques for the monitoring and controlling foodbome illness [J]. Chin J Food Hyg, 2004, 16(1): 3 9. [43] Kelley KS, Cosman A, Belgrader P, et al. Detection of methicillin-resistant Staphylococcus aureus by a duplex droplet digital PCR assay [J]. Am Soc Microbiol, 2013, 51(7): 2033 2039. 作者简介 ( 责任编辑 : 姚菲 ) 杨纯佳, 助理工程师, 主要研究方向为食品质量检验 E-mail: 237611106@qq.com 王力清, 教授级工程师, 主要研究方向为食品检验技术 E-mail: sdwlq@21cn.com