Triple ERA Rapid Detection Method for Pathogens in Refrigerated Meat Products

doi:10.12141/j.issn.1000-565X.240169

Abstract

Abstract:

Meat products, being rich in nutrients, often provide an ideal environment for microbial growth.Even under low temperature storage, most microorganisms remain active and retain partial virulence. Therefore, screening for pathogenic bacteria in meat products is crucial. This study developed a triple-fluorescence ERA (enzyme-mediated recombinase polymerase amplification) rapid detection method based on enzyme recombinase amplification (ERA) technology. Performance analyses were conducted on the method’s specificity, sensitivity, and detection limits. Additionally, the applicability of the method was validated using commercially available refrigerated meats and compared to the industry standard SN/T 1870—2016 real-time fluorescent polymerase chain reaction (PCR) method. The results show that a triple-fluorescent ERA rapid detection method was successfully constructed for three key pathogens: Staphylococcus aureus, Salmonella and Listeria monocytogenes. After optimization, the method could simultaneously detect the three pathogens within 12 min from a single reaction. The minimum detection limit is 10^-3 ng/μL. In artificially contaminated samples, after 6 hours of pre-enrichment, the method can simultaneously detect contamination levels of 1 CFU/mL for all three pathogens. The sampling results of commercially available refrigerated meat show that the detection rates for Staphylococcus aureus, Salmonella, and Listeria monocytogenes were 19.35%, 12.90%, and 6.45%, respectively, which align with the detection results of the industry standard SN/T 1870―2016. These fully demonstrate the accuracy and applicability of the method. This study not only helps reduce the risk of disease caused by foodborne pathogens but also provides solid technical support for future rapid detection of foodborne pathogens.

Key words: refrigerated meat products, foodborne pathogens, triple-fluorescence ERA, rapid detection

CLC Number:

TS207.4

YANG Yange, WU Zhanwen, LIU Tong, WANG Shuai, WEI Ying, ZHAO Jiansong, LI Hongna, LI Tao, ZHANG Feng. Triple ERA Rapid Detection Method for Pathogens in Refrigerated Meat Products[J]. Journal of South China University of Technology(Natural Science Edition), 2025, 53(4): 135-146.

Figures/Tables 9

Table 1

Information of the strains used in this study"

编号	种类	菌株	拉丁名称	菌株号
1	金黄色葡萄球菌	金黄色葡萄球菌	Staphylococcus aureus	ATCC27664
2		金黄色葡萄球菌	Staphylococcus aureus	ATCC13565
3		金黄色葡萄球菌	Staphylococcus aureus	CMCC26003
4		金黄色葡萄球菌	Staphylococcus aureus	ATCC25923
5		金黄色葡萄球菌	Staphylococcus aureus	ATCC12600
6	沙门氏菌	鼠伤寒沙门氏菌	Salmonella typhimurium	ATCC14028
7		鼠伤寒沙门氏菌	Salmonella typhimurium	CMCC50013
8		肠道沙门氏菌	Salmonella enterica	ATCC10708
9		猪霍乱沙门氏菌	Salmonella choleraesuis	ATCC53885
10		猪霍乱沙门氏菌	Salmonella choleraesuis	CMCC50018
11	单核细胞增生李斯特氏菌	单核细胞增生李斯特氏菌1/2a血清型	Listeria monocytogenes	ATCC15313
12		单核细胞增生李斯特氏菌1/2b血清型	Listeria monocytogenes	IQCC22291
13		单核细胞增生李斯特氏菌1/2b血清型	Listeria monocytogenes	ATCC-BAA-751
14		单核细胞增生李斯特氏菌1/2c血清型	Listeria monocytogenes	ATCC19112
15		单核细胞增生李斯特氏菌4b血清型	Listeria monocytogenes	ATCC19115
16		单核细胞增生李斯特氏菌4e血清型	Listeria monocytogenes	ATCC19118
17		单核细胞增生李斯特氏菌4a血清型	Listeria monocytogenes	ATCC19114
18	其他致病菌	绵羊/伊氏李斯特菌	Listeria ivanovii	ATCC19119
19		格氏李斯特菌	Listeria grayi	ATCC25401
20		斯氏/西尔李李斯特氏菌	Listeria seeligeri	ATCC35967
21		英诺克李斯特氏菌	Listeria innocua Seeliger	ATCC33090
22		威尔斯李斯特氏菌	Listeria welshimeri	ATCC35897
23		马尔蒂氏李斯特氏菌	Listeria marthii	ATCC BAA-1595
24		大肠埃希氏菌O157：H7	Escherichia coli O157：H7	IQCC10193
25		大肠埃希氏菌	Escherichia coli	IQCC10138
26		宋内氏志贺氏菌	Shigella sonnei	ATCC29930
27		福氏志贺氏菌	Shigella flexneri	CMCC51571
28		小肠结肠炎耶尔森氏菌	Yersinia enterorcolitica	ATCC27729
29		副溶血弧菌	Vibrio parahaemolyticus	ATCC 33847
30		河流弧菌	Vibrio fluvialis	ATCC33809
31		溶藻弧菌	Vibrio alginolyticus	ATCC17749
32		铜绿假单胞菌	Pseudomonas aeruginosa	ATCC25619
33		荧光假单胞菌	Pseudomonas fluorescens	ATCC 17397
34		恶臭假单胞菌	Pseudomonas putida	ATCC17485
35		蜡样芽孢杆菌	Bacillus cereus	ATCC10876
36		枯草芽孢杆菌	Bacillus subtilis	ATCC11774
37		乙型溶血性链球菌	Streptococcus hemolytic-β	CMCC32210

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菌	名称	序列（5ʹ-3ʹ）	基因
金黄色葡萄球菌	金F	CTTATAGGGATGGCTATCAGTAATGTTTCG	nuc
	金R	TCTATTTACGCCATTATCTGTTTGTGATGC
	金P^a	ACGCAAAGAGGTTTTTCTATTTCGCTAC［ROX-dT］A［THF］［BHQ1-dT］TGTTTAGTGTTAAC［c3-spacer］
沙门氏菌	沙F	ATATTACCAGATATATATTAGAGCAATGGAAAA	fimY
	沙R	ATAGCCGAGGTAGTTATCAGTTGTAATTATTG
	沙P^a	TTTAAGAAATGCCAAAGACTGCGCCTGCCG［HEX-dT］T［THF］［BHQ1-dT］CACTCTCCAACGCCG［c3-spacer］
单核细胞增生李斯特菌氏菌	单1F	TCGATCACTCTGGAGGATACGTTGCTCAATTC	hlyA
	单1R	GTTACCAGGCAAATAGATGGACGATGTGAAAT
	单1P^a	CATTTCTTGGGATGAAGTAAATTATGATCC［Cy5-dT］GA［THF］GG［BHQ2-dT］AACGAAATTGTTC［c3-spacer］
	单2F	GAAAATAAAGCACCATTTATAGAAAAACAAG	actA
	单2R	CACATTATTAGCTGGTTCGCTTTCTCCTAC
	单2P	CGGCCAATTGATATGCCGAGCCTACCAG［FAM-dT］A［THF］［BHQ1-dT］CCAAAAAGAAGTTACC［c3-spacer］
	单3F	CTAGCTCATTTCACATCGTCCATCTATTTGC	hlyA
	单3R	CATCAATTACCGTTCTCCACCATTCCCAAGC
	单3P	CGTCCATCTATTTGCCWGGTAACGCRAGAAAT［FAM-dT］A［THF］T［BHQ1-dT］AATGTTTACGCYAAAG［c3-spacer］