Simplex and duplex PCR assays for species specific identification of cattle and buffalo milk and cheese

A polymerase chain reaction, amplifying a fragment of the mitochondrial DNA D loop region was developed for species specific detection of cattle and buffalo milk. The method was simultaneously extended for detection of HTST pasteurized milk samples and cheeses of bovine and buffalo origin. A common forward primer was used with two different species specific reverse primers that resulted amplification of a 126 bp and 226 bp products for cattle and buffalo, respectively, in simplex as well as in multiplex polymerase chain reaction. The primers successfully amplified DNA extracted by conventional protocol from minimal amount of raw milk, heat treated milk and cheese of either bovine or buffalo origin. The primers showed a high degree of specificity. The sensitivity of the assay was excellent with detection level of 0.1 percent adulteration of cow and buffalo milk or cheese (0.15 ng buffalo and 0.04 ng cattle DNA). The assay represents a sensitive and simple method for identification of adulteration in milk and cheese.     

Development and application of molecular markers for poultry meat identification in food chain

Every year, large quantities of poultry and game meat are consumed. Thus, efficient techniques to identify the meat species origin are required which interest traders, consumers and organizations. In this study, two mitochondrial DNA (mtDNA) genes, Cytochrome b (Cyt b) and 12S ribosomal RNA (12S rRNA) were tested as putative discrimination markers in samples of raw and processed poultry meat (chicken, turkey, duck, goose, pheasant, partridge, woodcock, ostrich, quail and song thrush), applying the PCR–RFLP technique with universal primers and ten different restriction enzymes. Digestion of 12S rRNA by AciI successfully distinguished all avian species, producing species-specific patterns. We conclude that the 12S rRNA gene is more informative than Cyt b gene for avian species identification purposes. Moderate process treatment did not prevent the species identification, presenting similar patterns with the raw meat. Finally, this method was considered sufficient to detect mixtures of meat, making it a valuable tool for checking possible adulterations.     

Rapid and sensitive identification of buffalo’s,cattle’s and sheep’s milk using species-specific PCR and PCR–RFLP techniques

For the rapid, specific and sensitive identification of buffalo’s, cattle’s and sheep’s milk, species-specific PCR and PCR–RFLP techniques were developed. DNA from small amount of fresh milk (100 μL) was extracted to amplify the gene encoding species-specific repeat (SSR) region and the mitochondrial DNA segment (cytochrome-b gene). PCR amplification size of the gene encoding SSR region was 603 bp in both buffalo’s and cattle’s milk, while in sheep’s milk was 374 bp. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) technique was used to discriminate between buffalo’s and cattle’s milk. Restriction analysis of PCR–RFLP of the mitochondrial cytochrome-b segment (359 bp) analysis showed difference between buffalo’s and cattle’s milk. Where, the fragment length (bp) generated by TaqI PCR–RFLP were 191 and 168, whereas no fragments were obtained in cattle’s milk for cytochrome-b gene (359 bp). The proposed PCR and PCR–RFLP assays rep resent a rapid and sensitive method applicable to the detection and authentication of milk species-specific.     

Meat species identification based on the loop mediated isothermal amplification and electrochemical DNA sensor

An easy, rapid and sensitive method of detection of the presence of meat species in raw or processed foods is important from cultural, religious, health and commercial perspectives. In this study we have tried to distinguish species-specificity in control and processed pork, chicken and bovine meats using loop mediated isothermal amplicons (LAMP) and disposable electrochemical printed (DEP) chips. LAMP is a nucleic acid amplification method that amplifies target DNA with high specificity, efficiency and rapidity under isothermal condition (63 °C). Electrochemical genosensor with the DEP chips detects the amplicons by Linear Sweep Voltammetry (LSV) observation of DNA–Hoechst33258 interaction on the chip surface. Hoechst33258 interacts with DNA in solution without immobilization of DNA onto the electrode surface eliminating the time consuming probe immobilization step. Our method is more specific and free of unwanted amplifications compared to Multiplexed PCR (M-PCR) method and gave limits of detection of ∼20.33 ng/μl (3 × 10⁴ copies/reaction), ∼78.68 pg/μL (3 × 10² copies/reaction) and ∼23.63 pg/μL (30 copies/reaction) for pork, chicken and bovine species, respectively. Our method of detection is quick, taking only an hour, and it may be useful for food administration laboratories to carry out meat species identification in raw and processed foods.     

Application of an optimized 18-h method involving one step culturing and single primer-based PCR assay for detection of Salmonella spp. in foods

A polymerase chain reaction (PCR) using 20-mer oligonucleotide single primer (named primer 3) randomly designed on the basis of Salmonella Typhimurium gatD gene encoding galactitol-1-phosphate dehydrogenase could produce the specific DNA product of approximate 770-bp in all 38 Salmonella strains used. No 770-bp DNA band was amplified from any DNA samples of 20 non-Salmonella bacteria. The DNA band was detected by 1% agarose gel electrophoresis and ethidium bromide staining. The sensitivity of the RAPD–PCR assay for detection of pure genomic DNA from S. Typhimurium ATCC 13311 and Salmonella Enteritidis DMST 15676 was as few as 0.01 ng. When simple boiling in TE buffer for 5 min was used for extraction both Salmonella spp. DNA, the detection limits were at least 230 and 320 cells, respectively. By using the RAPD–PCR assay following at least 14 h pre-enrichment in nutrient broth (NB), as few as 1 CFU of S. Typhimurium ATCC 13311 or S. Enteritidis DMST 15676 per 25 g of autoclaved chicken meat was detected. When the optimized 18-h method involving pre-enrichment in NB and DNA extraction by boiling protocol followed by RAPD–PCR using primer 3 was evaluated in comparison with the conventional method on 195 possibly naturally-contaminated food samples, 36 samples were found positive by both methods. In addition, the results of the developed RAPD–PCR-based assay proved to be identical to those by the conventional method. The optimized 18-h method was simple, rapid and sensitive, achieved the same detection limit as the conventional method and produced a zero level of false-negative results.     

Beef specific polymerase chain reaction assay for authentication of meat and meat products

The aim of this study was to develop species-specific polymerase chain reaction (PCR) assay for specific detection of beef using self-designed primer pair based on D-loop region of mitochondrial gene for amplification of 513 bp DNA fragments from fresh, processed and autoclaved meat and meat products. The beef-specific primer pair was self-designed based on the available gene sequences on NCBI nucleotide database. The primer pair was individually optimized for amplification of desired 513 bp DNA fragments from isolated DNA of fresh beef. After successful amplification of desired DNA fragments by this primer pair, the PCR assay was evaluated for their efficiency to amplify DNA extracted from cooked and autoclaved meat and meat emulsion. The level of detection of this beef-specific primer pair was found to be less than 1 percent using PCR assay, even in admixed meat products containing meat of beef, buffalo meat, pork, chevon, mutton and chicken. No adverse effect of heat treatment, processing conditions and ingredients was observed on amplification pattern. The experiments were repeated for several time and results was found to be repeatable every-time.     

A multiplex PCR assay for fraud identification of deer products

Attempts were made to established one-step multiplex PCR assay for the fraud identification of the mostly used species in deer products (bovine, ovine, porcine and poultry). Primers were selected from published papers or designed in the well-conserved region of tRNA-Val and 16S rRNA mitochondrial genes after alignment of the available sequences in the GenBank database. The primers generated specific fragments of 124, 183, 225 and 290 bp length for bovine, poultry, ovine and porcine, respectively. The detection limit was 1 ng for porcine and ovine primers, 5 ng for poultry primers and 0.5 ng for bovine primers. The results demonstrated that fraud phenomena are very epidemic in the deer products, especially heart, blood, penis and antler products.The multiplex PCR described in this study, proved to be very sensitive and reliable in species identification, could be considered as a further improvement of traditional methods based assay for the identification of deer products.     

Authentication of meat and commercial meat products from common pigeon (Columba livia) woodpigeon (Columba palumbus) and stock pigeon (Columba oenas) using a TaqMan real-time PCR assay

A species-specific real-time polymerase chain reaction (PCR) assay using TaqMan^® probes has been developed for the identification of meat and meat products from common pigeon (Columba livia), woodpigeon (Columba palumbus) and stock pigeon (Columba oenas). The method combines the use of species-specific primers and TaqMan^® probes that amplify small fragments (amplicons < 200 base pairs) of the mitochondrial 12S rRNA gene, and an endogenous control primer pair that amplifies a 141 bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. Analysis of experimental raw and heat-treated binary mixtures as well as of commercial meat products from the target species, demonstrated the suitability of the assay for the detection of the target DNAs. The PCR assay reported in this work could be useful in inspection programs to verify the correct labelling of raw and heat-treated pigeon meat products.     

Mislabelling of beef and poultry products sold in Malaysia

Meat species specification is important for consumer protection and increases concern in food labelling regulations enforcement. Although regulations exist for processed meat products, information on the prevalence of meat products mislabelling and regulatory compliance in Malaysia is lacking. In this study, 143 prepacked (beef and poultry) meat products (sausages, cold cut meats, cooked whole muscle meats, breaded products, meatballs and ground meats) were purchased from several national and international supermarket chains in Malaysia. These samples were analysed for the presence of common meat species (buffalo, cattle, chicken, goat, sheep, duck and goose) and meats prohibited by Islamic laws (“Haram”) (cat, dog, monkey, pig and rat) using species-specific primers. The results showed that 112 (78.3%) samples were mislabelled, attributed by the false declaration of species and/or presence of undeclared meat species. The mislabelled products consisted of 17/28, 3/4, 6/8, 19/25, 48/56, and 19/22 of sausage, cold cuts, cooked whole muscle meat, breaded product, ground meat, and meatball samples, respectively. Buffalo DNA was detected in 40 out of the 58 samples labelled as beef. The presence of undeclared chicken and buffalo DNA were detected in 33/58 and 62/84 of beef and chicken products, respectively. The five “Haram” meat sources, however, were not detected in all meat products tested. The presence of chicken or buffalo DNA in these products could be attributed to unintentional cross contamination from food processing equipment, especially meat grinder, and lack of proper cleaning or inadequate hygiene. In conclusion, this study shows that majority of the samples are not legally compliant, signifying that substitution and mislabelling of meat products are commonplace in Malaysia. Strict implementation of the Malaysia Food Regulations 1985 alongside with regular surveillance and monitoring programmes are compulsory to alleviate and deter mislabelling issues.     

Detection of Listeria monocytogenes using a commercial PCR kit and different DNA extraction methods

The aim of our work was to evaluate a new commercial test kit for the detection of Listeria monocytogenes by PCR, using different DNA extraction methods. Food samples (pork sausage and “mozzarella” cheese) were spiked with known concentrations of L. monocytogenes and culture-enriched for 24 h. DNA extracted using three commercial kits and two standard methods, was amplified in species-specific PCR employing a L. monocytogenes PCR Detection Kit (Diatheva). The PCR-based method proved to be a reliable means of detecting the pathogen in food samples independently from the extraction procedure used, even for a contamination cell number of 1 cfu/g before culture enrichment. The molecular assay, showing perfect agreement with standard microbiological tests and a considerably shortened analysis time, provides a sensitive and rapid alternative for applications in the testing of foods for microbiological contamination, and highlights the potential of PCR technology in routine food control.     

Species identification of commercial jerky products in food and feed using direct pentaplex PCR assay

The direct pentaplex PCR assay was developed for simultaneous identification using species-specific primer sets and a universal eukaryotic primer set in processed jerky products without DNA extraction. The specific primer sets of target meat species amplified the expected 83-, 133-, 166-, and 204-bp PCR products for pork, chicken, beef, and duck, respectively, and obtained no cross-reactivity against a total of sixteen animal species. A universal eukaryotic primer set amplified a 99-bp conserved fragment in all meat species. To evaluate the sensitivity of this assay, the different percentages of jerky samples were prepared with the meat species having the possibility to be mixed. Adulterated beef jerky samples contaminated with 0.1, 0.5, 1, 5, 10, and 50% pork and adulterated duck jerky samples contaminated with 0.1, 0.5, 1, 5, 10, and 50% chicken were prepared in a laboratory. The detection level of direct pentaplex PCR was below 0.1% pork in adulterated beef jerky and 0.1% chicken in adulterated duck jerky. The optimized assay was also applied to the analysis of commercial food and feed jerky products. The meat species in commercial jerky products were successfully identified without DNA extraction.     

Biogenic amines content,histamine-forming bacteria and adulteration of bonito in tuna candy products

Thirty-four tuna candy products sold in retail markets and supermarkets in Taiwan were purchased and tested to determine the biogenic amine, histamine-forming bacteria, and adulteration of bonito meat. The levels of pH value, water activity (Aw), water content, total volatile basic nitrogen (TVBN), aerobic plate count (APC) and total coliform (TC) in all samples ranged from 5.3 to 6.1, 0.47 to 0.65, 7.37% to 17.32%, 12.1 to 54.6 mg/100 g, <1 to 6.2 log CFU/g and <3 to 1700 MPN/g, respectively. None of these sample contained Escherichia coli. The average content of various biogenic amines in all tested samples was less than 1.0 mg/100 g. Four histamine-producing bacterial strains isolated from tested samples produced 1.02–1.74 ppm of histamine in trypticase soy broth supplemented with 1.0% l-histidine (TSBH) after incubation at 35 °C for 24 h. Assay of multiplex polymerase chain reaction (PCR) revealed that adulteration rate of bonito meat was 26.5% (9/34) in tuna candy samples. Tuna species in tuna candy samples was identified as Thunnus albacares for 29 samples (85.3%), Thunnus alalunga for four samples (11.8%) and Thunnus obesus for one sample (2.9%) by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP).     

Simultaneous detection of Escherichia coli O175:H7, Salmonella spp., and Listeria monocytogenes by multiplex PCR

The wide application of nucleic acid amplification techniques and the increasing industrial interest toward rapid methods has led to the development and application of PCR based methods for the detection of microbial pathogens in food. In the present paper we describe the development of a multiplex PCR method for simultaneous detection of Salmonella enterica serovar Typhimurium, Listeria monocytogenes and Escherichia coli O157:H7 in a complex food matrix (liquid whole egg).Four different DNA extraction procedures were evaluated for their application on food and, among these, Chelex resin combined with a DNA purification step were found to better perform on the food system considered.A multiplex PCR system was developed, based on the evaluation and combination of published primer sets, and applied to the simultaneous detection of the target pathogens plus an internal amplification control, both in culture media and in a model food system.The overall system proposed, based on an overnight enrichment step followed by DNA isolation and multiplex PCR, was satisfactorily tested for its specificity and sensitivity and allowed the detection of the presence of bacterial DNA and the identification of the target pathogens down to 10 cells/25 g liquid whole egg.     

Effects of gamma irradiation and frozen storage on microbial,chemical and sensory quality of chicken meat in Iran

Irradiation is considered one of the most efficient technological processes for the reduction of microorganisms in food. It can be used to improve the safety of food products, and to extend their shelf lives. The aim of this study was to evaluate the effects of gamma irradiation and frozen storage as a combination process for improvement of chicken meat shelf life. Broiler chicken were treated with 0 (non irradiated), 0.75, 3.0, and 5.0 kGy of gamma irradiation and held frozen for 9 months. The control and irradiated samples were stored at −18 °C and underwent microbial analysis, chemical characteristics and sensory evaluation at 3 months intervals. Microbial analysis indicated that irradiation and freezing storage had a significant effect (P < 0.05) on the reduction of microbial loads. There was no significant difference in sensory quality and chemical characteristics during freezing storage in chicken meat. The combination of frozen storage plus irradiation resulted in greater overall reductions on microbial loads, extending shelf-life of chicken meat for commercial application and critical condition.     

Sensory quality and food safety of boneless chicken breast portions thawed rapidly by submersion in hot water

Boneless chicken breast portions were thawed by submersion in hot water (60 °C) and compared to refrigerator thawing. Thawing in hot water was significantly quicker (2–8.5 min) than refrigerator thawing (10–15.5 h). Thawing time in hot water increased with an increase in meat thickness. Sensory panelists could not distinguish a difference between hot water versus refrigerator thawed and subsequently grilled chicken breast portions. A model for Salmonella growth predicts that thawing chicken breast at the slowest rate in this study (0.5 °C/min) would result in a lower increase in the Salmonella concentration than that expected for room temperature storage for 4 h.     

A potential methodology for differentiation of ciguatgoxin-carrying species of moray eel

A food poisoning incident due to ingestion of an unknown moray eel occurred in Taiwan. To identify the species of causative moray eel implicated in food poisoning, a 376-bp fragment sequence of cytochrome b gene was successfully obtained from four species of moray eel by using a pair of primers (L newest-1/H 15149). This fragment could be amplified using fish meat treated with different heating processes (100 °C for 30 min, 100 °C for 60 min, 121 °C for 15 min, and 121 °C for 30 min). After sequencing, it was found that no variation in sequence was detected among individuals within each species. The species of causative moray eel implicated in food poisoning was judged as Gymnothorax javanicus based on sequence analysis. In addition, using restriction enzyme analysis, including Taq I and Sau96 I, could distinguish these four species of moray eel and identify the fish species of poisoning sample as G. javanicus. The toxicity of viscera in 24 specimens of four moray eel species was not detected, but the food poisoning sample was found to be toxic. Overall, this study proved that DNA sequence and restriction enzyme analyses are useful in identifying that the causative moray eel species was G. javanicus.     

The effect of high pressure on microbial population,meat quality and sensory characteristics of chicken breast fillet

High hydrostatic pressure (300, 450 and 600 MPa) was used to investigate its effect on microbial population, meat quality and sensory characteristics of chicken breast fillets. Pressures of 450 and 600 MPa almost completely eliminated all the 3 major pathogens Salmonella typhimurium (KCTC 1925), Escherichia coli (KCTC 1682), and Listeria monocytogenes (KCTC 3569) and therefore improved safety of chicken breast fillets. The 600 MPa treatment reduced bacteria count by 6–8 log (CFU/g) for 7–14 days, and the 450 MPa treatment reduced bacteria count by 4–8 log (CFU/g) for 3–14 days, depending on the microorganism. The increased pressure had an impact on flavour, aroma strength and juiciness. The 300 MPa pressure significantly reduced flavour, aroma strength and juiciness, and 450 MPa produced breast fillets with the weakest aroma. Increasing pressure increased cooking loss and colour by increasing L*, a* and b* values. Moreover, elevated pressure increased hardness, cohesiveness, gumminess and chewiness, as well as improved freshness of meat by reducing VBN. Pressure of 450 MPa and higher induced lipid oxidation. The results demonstrate that high pressure treatment is an effective technology in reducing bacterial spoilage and extending shelf-life of chicken breast fillet, however it may have a negative impact on some quality and sensory characteristics.     

A multiplex PCR assay for species identification of raw and cooked bonito

Attempts are made to establish one-step multiplex PCR assay for distinguishing five species of raw and cooked bonito including Euthynnus pelamis, Euthynnus affinis, Auxis rochei, Auxis thazard, and Sarda orientalis. After constructing the 1141 bp complete mitochondrial cytochrome b genes of five bonito species and other five contrastive Scombridae species, five sets of species-specific primer were designed to amplify different cytochrome b gene fragments in each species individually. The amplified lengths of fragments were respectively 143 bp for A. rochei, 236 bp for E. pelamis, 318 bp for A. thazard, 398 bp for E. affinis and 506 bp for S. orientalis, which could be obviously differentiated from each other on DNA electrophoresis. The five sets of species-specific primer were mixed and applied to simultaneously detect bonito species. All species from 12 commercial raw fish and five species out of eight cooked bonito fillets were successfully identified by the multiplex PCR assay. Experiments carried out demonstrate that the multiplex PCR assay was useful for identifying species of non-overheating fish product.     

Authentication of meats from quail (Coturnix coturnix), pheasant (Phasianus colchicus), partridge (Alectoris spp.), and guinea fowl (Numida meleagris) using polymerase chain reaction targeting specific sequences from the mitochondrial 12S rRNA gene

Polymerase chain reaction (PCR) based on oligonucleotide primers targeting the mitochondrial 12S rRNA gene has been applied to the specific identification of meats from quail (Coturnix coturnix), pheasant (Phasianus colchicus), partridge (Alectoris spp.), and guinea fowl (Numida meleagris). The use of specific primers pairs for quail, pheasant, partridge and guinea fowl allowed the selective amplification of the desired avian sequences. The specificity of each primer pair was verified by PCR analysis of DNA from meats of various game and domestic bird and mammalian species. The assay can be useful for the accurate identification of meats from game bird species, avoiding mislabelling or fraudulent species substitution in meat products.