Establishment of a Triple Droplet Digital PCR Quantitative Detection Method for Typical Pathogenic Bacteria in Livestock and Poultry Manure

doi:10.13560/j.cnki.biotech.bull.1985.2022-0944

Abstract

Abstract:

In order to accurately and rapidly detect typical pathogenic microorganisms in livestock and poultry manure and reduce the risk of transmission of zoonotic diseases,three common pathogenic bacteria,Staphylococcus aureus,Escherichia coli（O157：H7）and Salmonella enteritidis,were used as the research object. By screening its specific primers and probes as well as optimizing the reaction system,a fast and stable multiplex droplet digital PCR（ddPCR）reaction system was established. The specificity of the system was verified by detecting different strains,the detection limit of pathogenic bacteria in livestock and poultry waste was determined,and a rapid detection method of multiple droplet digital PCR was developed. The results showed that each pair of primers and probes amplified the target strains without cross-reaction in the ddPCR system. The absolute quantitative lower limit of detecting S. enteritidis was 0.68 copies/µL,and that for S. aureus was 0.79 copies/µL,and that for E. coli was 1.02 copies/µL. The high-efficiency and high-precision detection of three typical pathogenic bacteria in livestock and poultry manure may be achieved by the method established in this study.

Key words: livestock waste, Salmonella enteritidis, Escherichia coli, Staphylococcus aureus, triple droplet digital PCR

CHENG Shen-wei, ZHANG Ke-qiang, LIANG Jun-feng, LIU Fu-yuan, GAO Xing-liang, DU Lian-zhu. Establishment of a Triple Droplet Digital PCR Quantitative Detection Method for Typical Pathogenic Bacteria in Livestock and Poultry Manure[J]. Biotechnology Bulletin, 2022, 38(9): 271-280.

Figures/Tables 15

References 23

[1]	杨天学, 刘鸿志, 李东阳, 等. 种养废弃物协同污染控制现状及发展对策建议[J]. 中国环境科学, 2021, 41(1):473-481.
	Yang TX, Liu HZ, Li DY, et al. The Current situation and development suggestions of waste pollution cooperative control from planting and breeding industry[J]. China Environ Sci, 2021, 41(1):473-481.
[2]	吴玉高, 李卓阳, 方菁. 畜禽粪便还田所致环境污染现况及其健康危害[J]. 环境与职业医学, 2021, 38(11):1284-1290.
	Wu YG, Li ZY, Fang J. Environmental pollution and health hazards caused by animal agriculture manure[J]. J Environ Occup Med, 2021, 38(11):1284-1290.
[3]	中华人民共和国卫生部. 食品安全国家标准食品微生物学检验沙门氏菌检验:GB 4789. 4-2010[S]. 北京: 中国标准出版社, 2010.
	Ministry of Health of the People's Republic of China. National food safety standard Food microbiological examination:Salmonella:GB 4789. 4-2010[S]. Beijing: Standards Press of China, 2010.
[4]	Postollec F, Falentin H, Pavan S, et al. Recent advances in quantitative PCR(qPCR)applications in food microbiology[J]. Food Microbiol, 2011, 28(5):848-861. doi: 10.1016/j.fm.2011.02.008 pmid: 21569926
[5]	Sykes PJ, Neoh SH, Brisco MJ, et al. Quantitation of targets for PCR by use of limiting dilution[J]. BioTechniques, 1992, 13(3):444-449. pmid: 1389177
[6]	Corbisier P, Bhat S, Partis L, et al. Absolute quantification of genetically modified MON810 maize(Zea mays L. )by digital polymerase chain reaction[J]. Anal Bioanal Chem, 2010, 396(6):2143-2150. doi: 10.1007/s00216-009-3200-3 pmid: 19816678
[7]	杨冬燕, 杨永存, 李浩, 等. 数字PCR与荧光PCR方法鉴别餐厨废弃植物油的比较[J]. 中国卫生检验杂志, 2015, 25(19):3221-3225, 3230.
	Yang DY, Yang YC, Li H, et al. Comparation of digital PCR and fluorescent PCR for detecting kitchen-waste vegetable oil[J]. Chin J Heal Lab Technol, 2015, 25(19):3221-3225, 3230.
[8]	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. doi: 10.1007/s00253-014-5794-4 pmid: 24831026
[9]	Hindson 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. doi: 10.1021/ac202028g pmid: 22035192
[10]	Belgrader P, Tanner SC, Regan JF, 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. doi: 10.1373/clinchem.2012.197855 pmid: 23358413
[11]	Hindson CM, Chevillet JR, Briggs HA, et al. Absolute quantification by droplet digital PCR versus analog real-time PCR[J]. Nat Methods, 2013, 10(10):1003-1005. doi: 10.1038/nmeth.2633 pmid: 23995387
[12]	魏咏新, 马丹, 李丹, 等. 食品中Escherichia coli O157:H7微滴数字PCR绝对定量检测方法的建立[J]. 食品科学, 2020, 41(16):259-265.
	Wei YX, Ma D, Li D, et al. Establishment of droplet digital PCR system for absolute quantitative detection of Escherichia coli O157:H7 in foods[J]. Food Sci, 2020, 41(16):259-265.
[13]	Kelley K, Cosman A, Belgrader P, et al. Detection of methicillin-resistant Staphylococcus aureus by a duplex droplet digital PCR assay[J]. J Clin Microbiol, 2013, 51(7):2033-2039. doi: 10.1128/JCM.00196-13 URL
[14]	赵新, 兰青阔, 陈锐, 等. 应用微滴数字PCR技术快速检测食用菌中沙门氏菌[J]. 食品与生物技术学报, 2017, 36(3):315-321.
	Zhao X, Lan QK, Chen R, et al. Rapid detection of salmonellla spp. in edible fungi by droplet digital PCR[J]. J Food Sci Biotechnol, 2017, 36(3):315-321.
[15]	刘聪, 蒋克明, 刘聪, 等. 微滴技术的数字PCR研究现状及发展趋势[J]. 化学研究与应用, 2018, 30(7):1041-1047.
	Liu C, Jiang KM, Liu C, et al. Research status and trend of digital PCR based on microdroplet technology[J]. Chem Res Appl, 2018, 30(7):1041-1047.
[16]	Sharma VK. Real-time reverse transcription-multiplex PCR for simultaneous and specific detection of rfbE and eae genes of Escherichia coli O157:H7[J]. Mol Cell Probes, 2006, 20(5):298-306. doi: 10.1016/j.mcp.2006.03.001 URL
[17]	马薇, 孔义军, 吴伟健, 等. 水中大肠菌群微滴数字PCR定量检测方法的建立[J]. 环境保护科学, 2019, 45(1):58-66.
	Ma W, Kong YJ, WU WJ, et al. The establishment of quantitative detection of total coliforms in water based on ddPCR technology[J]. Environ Prot Sci, 2019, 45(1):58-66.
[18]	赵丽青, 王静, 秦燕, 等. PMA结合ddPCR检测食品中金黄色葡萄球菌的研究[J]. 微生物学杂志, 2017, 37(1):105-109.
	Zhao LQ, Wang J, Qin Y, et al. Detection of Staphylococcus aureus in foods by PMA combined with ddPCR[J]. J Microbiol, 2017, 37(1):105-109.
[19]	Wang M, Yang JJ, Gai ZT, et al. Comparison between digital PCR and real-time PCR in detection of Salmonella typhimurium in milk[J]. Int J Food Microbiol, 2018, 266:251-256. doi: 10.1016/j.ijfoodmicro.2017.12.011 URL
[20]	中华人民共和国国家质量监督检验检疫总局. 化妆品微生物检验方法第11部分:金黄色葡萄球菌多重实时荧光PCR法 S N/T 2206. 11-2014[S]. 北京: 中国标准出版社, 2014.
	General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China. Determination of microorganism in cosmetics- Part 11:Multiplex fluorescent real-time PCR method for Staphylococcus aureus SN/T 2206. 11-2014[S]. Beijing: China Standard Press, 2014.
[21]	Malorny B, Bunge C, Helmuth R. A real-time PCR for the detection of Salmonella Enteritidis in poultry meat and consumption eggs[J]. J Microbiol Methods, 2007, 70(2):245-251. doi: 10.1016/j.mimet.2007.04.013 URL
[22]	Kawasaki S, Fratamico PM, Horikoshi N, et al. Multiplex real-time polymerase chain reaction assay for simultaneous detection and quantification of Salmonella Species, Listeria monocytogenes, and Escherichia coli O157:H7 in ground pork samples[J]. Foodborne Pathog Dis, 2010, 7(5):549-554. doi: 10.1089/fpd.2009.0465 pmid: 20132032
[23]	崔元, 陈萍, 李佳暖, 等. 各类PCR技术的优缺点比较研究[C]北京: 北京畜牧兽医学会, 2016.
	Cui Yuan, Chen Ping, Li Jianuan, et al. Comparative study on the advantages and disadvantages of various PCR techniq-ues[C]Beijing: Beijing Animal Husbandry and Veterinary Association, 2016.

菌株名称Species	编号Code	来源Origin
金黄色葡萄球菌	CMCC（B）26073	中国医学细菌保藏管理中心
大肠埃希氏菌（O157：H7）	ATCC 25922	美国菌种保藏中心
肠炎沙门氏菌	CMCC（B）50041	中国医学细菌保藏管理中心

菌株名称Species	编号Code	来源Origin
金黄色葡萄球菌	CMCC（B）26073	中国医学细菌保藏管理中心
大肠埃希氏菌（O157：H7）	ATCC 25922	美国菌种保藏中心
肠炎沙门氏菌	CMCC（B）50041	中国医学细菌保藏管理中心

菌株名称Species	靶基因Target gene	引物探针序列Primer sequence（5'-3'）
金黄色葡萄球菌	nuc	CCTGAAGCAAGTGCATTTACGA
		CTTTAGCCAAGCCTTGACGAACT
		HEX-TGGACGTGGCTTAGCGTATATTTATGCTGATG-BHQ1
大肠埃希氏菌	rfbE	TCAAAAGGAAACTATATTCAGAAGTTTGA
		CGATATACCTAACGCTAACAAAGCTAA
		CY5-AATAAATTTGCGGAACAAAACCATGTGCAA-BHQ2
肠炎沙门氏菌	invA	GCGGCGTTGGAGAGTGATA
		AGCAATGGAAAAAGCAGGATG
		FAM-CATTTCTTAAACGGCGGTGTCTTTCCCT-BHQ1

菌株名称Species	靶基因Target gene	引物探针序列Primer sequence（5'-3'）
金黄色葡萄球菌	nuc	CCTGAAGCAAGTGCATTTACGA
		CTTTAGCCAAGCCTTGACGAACT
		HEX-TGGACGTGGCTTAGCGTATATTTATGCTGATG-BHQ1
大肠埃希氏菌	rfbE	TCAAAAGGAAACTATATTCAGAAGTTTGA
		CGATATACCTAACGCTAACAAAGCTAA
		CY5-AATAAATTTGCGGAACAAAACCATGTGCAA-BHQ2
肠炎沙门氏菌	invA	GCGGCGTTGGAGAGTGATA
		AGCAATGGAAAAAGCAGGATG
		FAM-CATTTCTTAAACGGCGGTGTCTTTCCCT-BHQ1

样品名称 Name of samples	总微滴数 Total number of droplets	肠炎沙门氏菌 S. enteritidis/ （Cp·µL^-1）	阳性微滴 Number of positive droplets	金黄色葡萄球菌 S. aureus/ （Cp·µL^-1）	阳性微滴 Number of positive droplets	大肠埃希氏菌 E. coli/（Cp·µL^-1）	阳性微滴 Number of positive droplets
沙门-阳性	27018	59.9	932	0	0	0	0
金黄-阳性	26349	0	0	75.7	1 143	0	0
大肠-阳性	26983	0	0	0	0	76.8	1 187
三重-阳性	25796	61.6	915	79.2	1 169	81.7	1 206