Escherichia coli, Staphylococcus aureus, Salmonella, Vibrio alginolyticus, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Clostridium perfringens, Listeria monocytogenes and Vibrio vulnificus are common foodborne pathogens in shellfish such as oyster. To provide technical support for rapid screening of foodborne pathogens, a multiplexed tandem polymerase chain reaction (MT-PCR) assay was developed. By designing specific primers and optimizing reaction parameters, the specificity and sensitivity of MT-PCR was further evaluated and applied in oyster samples. The optimized MT-PCR has equal quantitative ability as qPCR and can simultaneously detect nine pathogenic bacteria. The sensitivity of MT-PCR for detecting nine pathogens was less to 102 CFU/mL in mixed culture, especially, the sensitivity of MT-PCR was 10 times higher than qPCR for detecting E. coli, S. aureus, P. aeruginosa, C. perfringens, L. monocytogenes and V. vulnificus. The MT-PCR can detect nine target strains at 102-103 CFU/g in spiked oyster samples. The MT-PCR assay could achieve high-throughput, rapid and accurate detection of these nine pathogens in oyster samples, which is of great significance to ensure food safety and evaluate the risk of foodborne pathogens contamination.
MA Chunyang
,
GAO Shijue
,
JIANG Liting
,
YANG Chuhua
,
TANUSHREE B GUPTA
,
NIMA AZARAKHSH
,
WU Xiyang
. A multiplexed tandem PCR assay for simultaneous detection of nine different pathogens in oyster[J]. Food and Fermentation Industries, 2022
, 48(21)
: 247
-253
.
DOI: 10.13995/j.cnki.11-1802/ts.031373
[1] MA B, LI J L, CHEN K, et al.Multiplex recombinase polymerase amplification assay for the simultaneous detection of three foodborne pathogens in seafood[J].Foods (Basel, Switzerland), 2020, 9(3):278.
[2] DUMEN E, EKICI G, ERGIN S, et al.Presence of foodborne pathogens in seafood and risk ranking for pathogens[J].Foodborne Pathogens and Disease, 2020, 17(9):541-546.
[3] LEHEL J, YAUCAT-GUENDI R, DARNAY L, et al.Possible food safety hazards of ready-to-eat raw fish containing product (sushi, sashimi)[J].Critical Reviews in Food Science and Nutrition, 2021, 61(5):867-888.
[4] 陈嘉茵, 方苓, 吴诗微, 等.一步法微滴数字PCR检测生菜中GII型诺如病毒[J].食品科学, 2019, 40(4):332-337.
CHEN J Y, FANG L, WU S W, et al.Detection of Norovirus genogroup II in lettuce by droplet digital PCR[J].Food Science, 2019, 40(4):332-337.
[5] 赵峰, 佟利惠, 杨敏, 等.牡蛎中诺如病毒的感染及其防控研究进展[J].南方水产科学, 2021, 17(4):133-140.
ZHAO F, TONG L H, YANG M, et al.Progress and prospects of infection, prevention and control of Norovirus in oyster[J].South China Fisheries Science, 2021, 17(4):133-140.
[6] FODDAI A C G, GRANT I R.Methods for detection of viable foodborne pathogens:Current state-of-art and future prospects[J].Applied Microbiology and Biotechnology, 2020, 104(10):4 281-4 288.
[7] MAILLET A, BOUJU-ALBERT A, ROBLIN S, et al.Impact of DNA extraction and sampling methods on bacterial communities monitored by 16S rDNA metabarcoding in cold-smoked salmon and processing plant surfaces[J].Food Microbiology, 2021, 95:103705.
[8] WEI C J, ZHONG J L, HU T, et al.Simultaneous detection of Escherichia coli O157:H7, Staphylococcus aureus and Salmonella by multiplex PCR in milk[J].3 Biotech, 2018, 8(1):1-7.
[9] ATTWOOD L O, FRANCIS M J, HAMBLIN J, et al.Clinical evaluation of AusDiagnostics SARS-CoV-2 multiplex tandem PCR assay[J].Journal of Clinical Virology, 2020, 128:104448.
[10] LAU A, STANLEY K, SORRELL T.Multiplex-tandem PCR for fungal diagnostics[J].Methods in Molecular Biology (Clifton, N.J.), 2013, 968:195-201.
[11] WEI S, WANG C G, ZHU P Y, et al.A high-throughput multiplex tandem PCR assay for the screening of genetically modified maize[J].LWT, 2018, 87:169-176.
[12] 魏霜, 汪天杰, 龙阳, 等.多重富集定量PCR(ME-qPCR)同时检测4种食源性病原弧菌[J].中国农业科学, 2016, 49(23):4 619-4 627.
WEI S, WANG T J, LONG Y, et al.Multiplex enrichment quantitative PCR assays for the detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae[J].Scientia Agricultura Sinica, 2016, 49(23):4 619-4 627.
[13] XIE G Y, YU S, LI W, et al.Simultaneous detection of Salmonella spp., Pseudomonas aeruginosa, Bacillus cereus, and Escherichia coli O157:H7 in environmental water using PMA combined with mPCR[J].Journal of Microbiology (Seoul, Korea), 2020, 58(8):668-674.
[14] NAGPAL R, OGATA K, TSUJI H, et al.Sensitive quantification of Clostridium perfringens in human feces by quantitative real-time PCR targeting alpha-toxin and enterotoxin genes[J].BMC Microbiology, 2015, 15:219.
[15] GRACIAS K S, MCKILLIP J L.A review of conventional detection and enumeration methods for pathogenic bacteria in food[J].Canadian Journal of Microbiology, 2004, 50(11):883-890.
[16] COSTA J, MAFRA I, KUCHTA T, et al.Single-tube nested real-time PCR as a new highly sensitive approach to trace hazelnut[J].Journal of Agricultural and Food Chemistry, 2012, 60(33):8 103-8 110.
[17] TAO J, LIU W W, DING W, et al.A multiplex PCR assay with a common primer for the detection of eleven foodborne pathogens[J].Journal of Food Science, 2020, 85(3):744-754.
[18] D’SOUZA C, KUMAR B K, RAI P, et al.Application of gyrB targeted SYBR green based qPCR assay for the specific and rapid detection of Vibrio vulnificus in seafood[J].Journal of Microbiological Methods, 2019, 166:105747.
[19] ZHANG Z H, XIAO L L, LOU Y, et al.Development of a multiplex real-time PCR method for simultaneous detection of Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella spp.in raw shrimp[J].Food Control, 2015, 51:31-36.
