Milk is a putrescible commodity that is extremely prone to microbial contamination. Primarily, milk and dairy products are believed to be easily contaminated by pathogenic microorganisms, including Listeria monocytogenes, Salmonella spp., and Staphylococcus aureus. The microbiological quality of raw milk and dairy products regarding foodborne pathogens is of paramount importance due to concern of human health. In this study 400 buffalo raw milk samples were screened for assessing the prevalence of L. monocytogenes, Salmonella spp., and S. aureus. This study implemented uniplex-polymerase chain reaction (u-PCR) and multiplex-polymerase chain reaction (m-PCR) assays for the fast simultaneous detection of these pathogens comparing to the conventional culturing methods. Raw milk samples were found contaminated with the prevalence of 2.2%, 4.0%, and 14.2% for L. monocytogenes, Salmonella spp., and S. aureus, respectively. These pathogens were detected with the optimized polymerase chain reaction assays after 6 h of enrichment. u-PCR and m-PCR demonstrated the limit of detection as 104, 102, and 10 cells/mL after 6, 12, 18, and 24 h for each culture of the pathogens. A high sensitivity (10 colony-forming unit [CFU]/mL) of the m-PCR protocol was noted. The developed protocol is a cost-effective and rapid method for the simultaneous detection of pathogens associated with raw milk and dairy industries.
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References
1.
AbulreeshHH.Salmonellae in the Environment. In: Salmonella: Distribution, Adaptation, Control Measures and Molecular Technologies. (Annous BA, Gurtler JB. eds.) IntechOpen: Croatia,, 2012; pp. 19–50; doi: 10.5772/28201
2.
Akrami-MohajeriF, DerakhshanZ, FerranteM, et al.The prevalence and antimicrobial resistance of Listeria spp in raw milk and traditional dairy products delivered in Yazd, central Iran (2016). Food Chem Toxicol, 2018; 114:141–144; doi: 10.1016/j.fct.2018.02.006
3.
AlarcónB, García-CañasV, CifuentesA, et al.Simultaneous and sensitive detection of three foodborne pathogens by multiplex PCR, capillary gel electrophoresis, and laser-induced fluorescence. J Agric Food Chem, 2004; 52:7180–7186; doi: 10.1021/jf049038b.
4.
AmaglianiG, GiammariniC, OmiccioliE, et al.Detection of Listeria monocytogenes using a commercial PCR kit and different DNA extraction methods. Food Control, 2007; 18:1137–1142.
5.
AmaguañaRM, HammackTS, AndrewsWH. Methods for the recovery of Salmonella spp. from carboxymethylcellulose gum, gum ghatti, and gelatin. J AOAC Int, 1998; 81:721–726.
6.
AndrewsW, HammackT.Food Sampling and Preparation of Sample Homogenate. In: Bacteriological Analytical Manual, 8th ed. Center for Food Safety and Applied Nutrition (CFSAN), Food and Drug Administration: College Park, MD, 2003, Chapter 1.
7.
AroraS, AgarwalR, BistB. Comparison of ELISA and PCR vis-à-vis cultural methods for detecting Aeromonas spp. in foods of animal origin. Int J Food Microbiol, 2006; 106:177–183; doi: 10.1016/j.ijfoodmicro.2005.06.019
8.
BaileyJ.Detection of Salmonella cells within 24 to 26hours in poultry samples with the polymerase chain reaction BAX system. J Food Protect, 1998; 61:792–795.
9.
BeaubrunJJG, ChengCM, ChenKS, et al.The evaluation of a PCR-based method for identification of Salmonella enterica serotypes from environmental samples and various food matrices. Food Microbiol, 2012; 31:199–209; doi: 10.1016/j.fm.2012.03.016
10.
BélecL, AuthierJ, Eliezer-VanerotMC, et al.Myoglobin as a polymerase chain reaction (PCR) inhibitor: A limitation for PCR from skeletal muscle tissue avoided by the use of Thermus thermophilus polymerase. Muscle Nerve, 1998; 21:1064–1067; doi: 10.1002/(sici)1097-4598(199808)21:8<1064::aid-mus11>3.0.co;2-u
11.
BennettR, LancetteG. Staphylococcus aureus. In: Bacteriological Analytical Manual, 8th ed. Center for Food Safety and Applied Nutrition (CFSAN), Food and Drug Administration: College Park, MD, 1998, Chapter 12.
12.
BrakstadO, AasbakkK, MaelandJ. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J Clin Microbiol, 1992; 30:1654–1660.
13.
CappuccinoJ, ShermanN.Microbiology: A Laboratory Manual, 3rd ed. Cummings Publishing: New York, NY; 1992.
14.
ChenJ, TangJ, LiuJ, et al.Development and evaluation of a multiplex PCR for simultaneous detection of five foodborne pathogens. J Appl Microbiol, 2012; 112:823–830; doi: 10.1111/j.1365-2672.2012.05240.x
15.
CocolinL, ManzanoM, CantoniC, et al.Use of polymerase chain reaction and restriction enzyme analysis to directly detect and identify Salmonella typhimurium in food. J Appl Microbiol, 1998; 85:673–677; doi: 10.1111/j.1365-2672.1998.00575.x
16.
DhamaK, VermaAK, RajagunalanS, et al.Listeria monocytogenes infection in poultry and its public health importance with special reference to food borne zoonoses. Pak J Biol Sci, 2013; 16:301–308; doi:10.3923/pjbs.2013.301.308
17.
DingT, SuoY, ZhangZ, et al.A multiplex RT-PCR assay for S. aureus, L. monocytogenes, and Salmonella spp. detection in raw milk with pre-enrichment. Front Microbiol, 2017; 8:989; doi: 10.3389/fmicb.2017.00989
18.
El-BazAH, El-SherbiniM, AbdelkhalekA, et al.Prevalence and molecular characterization of Salmonella serovars in milk and cheese in Mansoura city, Egypt. J Adv Vet Animal Res, 2017; 4:45–51.
19.
Food Safety and Standards Authority of India (FSSAI). Food Safety, and Standards (Contaminants, Toxins, and Residues) Regulations. Food Safety and Standards Authority of India, Ministry of Health and Family Welfare: New Delhi; 2011.
20.
FurrerB, CandrianU, HoefeleinC, et al.Detection and identification of Listeria monocytogenes in cooked sausage products and in milk by in vitro amplification of haemolysin gene fragments. J Appl Bacteriol, 1991; 70:372–379; doi: 10.1111/j.1365-2672.1991.tb02951.x
21.
GebretsadikS, KassaT, AlemayehuH, et al.Isolation and characterization of Listeria monocytogenes and other Listeria species in foods of animal origin in Addis Ababa, Ethiopia. J Infect Public Health, 2011; 4:22–29; doi: 10.1016/j.jiph.2010.10.002
22.
GuldimannC, JohlerS. An introduction to current trends in foodborne pathogens and diseases. Curr Clin Microbiol Rep, 2018; 5:83–87; doi: 10.1007/s40588-018-0093-y
23.
HayashiM, NatoriT, Kubota-HayashiS, et al.A new protocol to detect multiple foodborne pathogens with PCR dipstick DNA chromatography after a six-hour enrichment culture in a broad-range food pathogen enrichment broth. Biomed Res Int, 2013; 2013:295050; doi: 10.1155/2013/295050
24.
HitchinsAD, JinnemanK, ChenY.Detection of Listeria monocytogens in Foods and Environmental Samples and Enumeration of Listeria monocytogenes in Foods. In: Bacteriological Analytical Manual, 8th ed. Center for Food Safety and Applied Nutrition (CFSAN), Food and Drug Administration: College Park, MD, 2003, Chapter 10.
25.
KarmakarA, DuaP, GhoshC. Biochemical and molecular analysis of Staphylococcus aureus clinical isolates from hospitalized patients. Can J Infect Dis Med Microbiol, 2016; 2016:9041636; doi: 10.1155/2016/9041636
26.
KaushikP, KumariS, BhartiSK, et al.Isolation and prevalence of Salmonella from chicken meat and cattle milk collected from local markets of Patna, India. Vet World, 2014; 7:62–65.
27.
KhanJ, RathoreR, KhanS, et al.In vitro detection of pathogenic Listeria monocytogenes from food sources by conventional, molecular and cell culture method. Braz J Microbiol, 2014a:44:751–758; doi: 10.1590/s1517-83822013000300013
28.
KhanJ, RathoreR, KhanS, et al.Antibiotic resistance profile of L. monocytogenes isolated from food sources. Indian J Biotechnol Pharmaceut Res, 2014b;2:21–24.
29.
KimCH, KhanM, MorinD, et al.Optimization of the PCR for detection of Staphylococcus aureus nuc gene in bovine milk. J Dairy Sci, 2001; 84:74–83.
30.
KimJH, YooJG, HamJS, et al.Direct detection of Escherichia coli, Staphylococcus aureus, and Salmonella spp. in animal-derived foods using a magnetic bead-based immunoassay. Korean J Food Sci Anim Resour, 2018; 38:727–736; doi: 10.5851/kosfa.2018.e11
31.
LawJWF, Ab MutalibNS, ChanKG, et al.Rapid methods for the detection of foodborne bacterial pathogens: Principles, applications, advantages and limitations. Front Microbiol, 2014; 5:770; doi: 10.3389/fmicb.2014.00770
32.
LeuenbergerA, SartoriC, BossR, et al.Genotypes of Staphylococcus aureus: On-farm epidemiology and the consequences for prevention of intramammary infections. J Dairy Sci, 2019; 102:3295–3309; doi: 10.3168/jds.2018-15181
33.
LiuY, CaoY, WangT, et al.Detection of 12 common food-borne bacterial pathogens by TaqMan real-time PCR using a single set of reaction conditions. Front Microbiol, 2019; 10:222; doi: 10.3389/fmicb.2019.00222
34.
Mansouri-NajandL, KianpourM, SamiM, et al.Prevalence of Listeria monocytogenes in raw milk in Kerman, Iran. Vet Res Forum, 2015; 6:223–226.
35.
MaryM, ShrinithivihahshiniN.Pervasiveness of Listeria monocytogenes in milk and dairy products. J Food Microbiol Safety Hyg, 2017:2:125; doi: 10.4172/2476-2059.1000125
36.
O'GradyJ, RuttledgeM, Sedano-BalbasS, et al.Rapid detection of Listeria monocytogenes in food using culture enrichment combined with real-time PCR. Food Microbiol, 2009; 26:4–7; doi: 10.1016/j.fm.2008.08.009
37.
OmarD, Al-AshmawyM, RamadanH, et al.Occurrence and PCR identification of Salmonella spp. from milk and dairy products in Mansoura, Egypt. Int Food Res J, 2018; 25:446–452.
38.
ParichehrM, MohammadK, AbbasD, et al.Developing a multiplex real-time PCR with a new pre-enrichment to simultaneously detect four foodborne bacteria in milk. Future Microbiol, 2019; 14:885–898; doi: 10.2217/fmb-2019-0044
39.
PercivalSL, ChalmersR, EmbreyM, et al.Microbiology of Waterborne Diseases. San Diego, CA: Elsevier Academic Press, 2004.
40.
PriyankaB, PatilRK, DwarakanathS. A review on detection methods used for foodborne pathogens. Indian J Med Res, 2016; 144:327–338; doi: 10.4103/0971-5916.198677
41.
QamarW, KhanMR, ArafahA. Optimization of conditions to extract high quality DNA for PCR analysis from whole blood using SDS-proteinase K method. Saudi J Biol Sci, 2017; 24:1465–1469; doi: 10.1016/j.sjbs.2016.09.016
42.
RegasaS, MengistuS, AbrahaA. Milk safety assessment, isolation, and antimicrobial susceptibility profile of Staphylococcus aureus in selected dairy farms of Mukaturi and Sululta town, Oromia region, Ethiopia. Vet Med Int, 2019; 2019:3063185; doi: 10.1155/2019/3063185
43.
SambrookJ, RussellDW. Molecular Cloning: A Laboratory Manual, 3rd edition. Vol. 1. NY: Cold Spring Harbor Laboratory Press, 2001.
44.
SeyoumET, WoldetsadikDA, MekonenTK, et al.Prevalence of Listeria monocytogenes in raw bovine milk and milk products from central highlands of Ethiopia. J Infect Dev Ctries, 2015; 9:1204–1209; doi: 10.3855/jidc.6211
45.
SharmaS, SharmaV, DahiyaDK, et al.Prevalence, virulence potential, and antibiotic susceptibility profile of Listeria monocytogenes isolated from bovine raw milk samples obtained from Rajasthan, India. Foodborne Pathog Dis, 2017; 14:132–140; doi: 10.1089/fpd.2016.2118
46.
ShrinithivihahshiniN, MariyaselvamS, DuraisamyM, et al.Occurrence of Listeria monocytogenes in food and ready to eat food products available in Tiruchirappalli, Tamil Nadu, India. World J Life Sci Med Res, 2011; 1:70–75.
47.
SinghP, SinghR, GuptaB, et al.Prevalence study of Salmonella spp. in milk and milk products. Asian J Dairy Food Res, 2018; 37:7–12.
48.
SonnierJL, KarnsJS, LombardJE, et al.Prevalence of Salmonella enterica, Listeria monocytogenes, and pathogenic Escherichia coli in bulk tank milk and milk filters from US dairy operations in the National Animal Health Monitoring System Dairy 2014 study. J Dairy Sci, 2018; 101:1943–1956; doi: 10.3168/jds.2017-13546
49.
SowmyaN, ThakurM, ManonmaniH. Rapid and simple DNA extraction method for the detection of enterotoxigenic Staphylococcus aureus directly from food samples: Comparisons of PCR and LAMP methods. J Appl Microbiol, 2012; 113:106–113; doi: 10.1111/j.1365-2672.2012.05315.x
50.
SreejaS, MoorthyK, UpasaniVN. Prevalence of Listeria monocytogenes in raw and pasteurized milk samples from Tiruchengode (TN), India. Int J Innov Res Sci Eng Technol, 2016; 5:1419–1424; doi: 10.1568/0/IJIRSET.2016.0502034
51.
SudhanthirakodiS, JainP, ChattopadhyayUK, et al.Non-typhoidal Salmonella isolates from livestock and food samples, in and around Kolkata, India. J Microbiol Infect Dis, 2016; 6:113–120; doi: 10.5799/jmid.vi.328974
52.
SuriyapriyaS, SelvanP, PorteenK, et al.Prevalence of Listeria spp. in traditional Indian dairy products from Chennai Metropolis, Tamil Nadu. Proc Food Sci, 2016; 6:230–234; doi: 10.1016/j.profoo.2016.02.025
53.
ToubarSME, ElbialyAA, ZakyMMM, et al.Prevalence of Staphylococcus aureus in raw milk and some dairy products in port said governorate. Am J Zool, 2018; 1:40–46.
54.
UpadhyayBP, UtrarachkijF, ThongshoobJ, et al.Detection of Salmonella invA gene in shrimp enrichment culture by polymerase chain reaction. Southeast Asian J Trop Med Public Health, 2010; 41:426–435.
55.
VidicJ, VizziniP, ManzanoM, et al.Point-of-need DNA testing for detection of foodborne pathogenic bacteria. Sensors, 2019; 19:1100; doi: 10.3390/s19051100
56.
WaiGY, TangJYH, NewCY, et al.A review on Listeria monocytogenes in food: Prevalence, pathogenicity, survivability and antibiotic resistance. Food Res, 2020; 4:20–27.
57.
WangRF, SlavikMF, CaoWW, et al.Development of DNA probes specific for Campylobacter jejuni. J Rapid Methods Auto Microbiol, 1992; 1:83–92.
58.
WidjojoatmodjoM, FluitA, TorensmaR, et al.Evaluation of the magnetic immuno PCR assay for rapid detection of Salmonella. Eur J Clin Microbiol Infect Dis, 1991; 10:935–938.
59.
ZengL, WangL, HuJ. Current and Emerging Technologies for Rapid Detection of Pathogens. In: Biosensing Technologies for the Detection of Pathogens. (Rinken T, Kiviran K. eds.) Intechopen: Croatia,, 2018; pp. 6–19; doi: 10.5772/intechopen.73178
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