Application of a real-time PCR assay for the detection of ostrich (Struthio camelus) mislabelling in meat products from the retail market

A rapid and highly species-specific real-time polymerase chain reaction (PCR) assay has been developed for the authentication of ostrich meat (Struthio camelus). The method combines the use of ostrich-specific primers, that amplify a 155 bp fragment of the mitochondrial 12S rRNA gene, and a positive control primer pair that amplifies a 141 bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. SYBR^® Green dye or TaqMan^® fluorogenic probes were used to monitor the amplification of the target genes. Results obtained with the use of TaqMan^® probes as detection platform increased the specificity of the real-time PCR assay in comparison with the results obtained using SYBR^® Green. Analysis of 100 commercial ostrich meat products from the market demonstrated the suitability of the technique for the detection of ostrich DNA. The results obtained suggest that this method may be routinely applied to verify the correct labelling of ostrich meat 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.     

A novel common primer multiplex PCR (CP-M-PCR) method for the simultaneous detection of meat species

A novel common primer multiplex PCR (CP-M-PCR) was applied to detect four kinds of meats (chicken, cattle, pig and horse) as raw materials. A common adapter was designed in the 5′-end of species-specific reverse primers which matched with the species-specific DNA sequences for each species and also used as the common primer (CP). CP-M-PCR primers were designed to uncover different length fragments of 239, 292, 412, and 451 bp from chicken, cattle, pig and horse meats, respectively. The bands of specific DNA fragments amplified by CP-M-PCR method still appeared until the concentration of species-specific primers diluted to 0.015 pmol and primer sensitivity was increased by 100 times compared with conventional multiplex PCR without CP. CP-M-PCR detection limit of the DNA samples was 0.1 ng (36.4 copies) for single kind of meat as well as four kinds of meats. CP-M-PCR method simplified the PCR reaction system and conquered the disparate amplified efficiency from different primers. The CP-M-PCR method could be widely applied in practical detection for simultaneous identification of other meat species and their products.     

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.     

Development of an efficient real-time PCR assay to quantify enterotoxin-producing staphylococci in meat products

A SYBR Green-based real-time PCR (qPCR) procedure for the rapid and specific detection of enterotoxin-producing Staphylococcus spp. in meat products has been developed. For this, a specific primer pair based on conserved regions of enterotoxin genes was designed for detecting most of the described staphylococcal enterotoxins. No cross-reactivity with other microorganisms or non-enterotoxin-producing Staphylococcus spp. was detected. The detection limits of the assay were about 2–40 cfu/g for artificially contaminated meat products after 8 h enrichment period at 30 °C. Total time for assay completion was approximately 12 h. Thus, the qPCR method offers a useful, rapid and efficient tool for screening enterotoxin-producing Staphylococci in meat products. This tool could be also used for monitoring these foodborne pathogens in food safety preventive programs.     

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.     

Identification of meat and poultry species in food products using DNA barcoding

DNA barcoding is a promising method for the sequencing-based identification of meat and poultry species in food products. However, DNA degradation during processing may limit recovery of the full-length DNA barcode from these foods. The objective of this study was to investigate the ability of DNA barcoding to identify species in meat and poultry products and to compare the results of full-length barcoding (658 bp) and mini-barcoding (127 bp). Sixty meat and poultry products were collected for this study, including deli meats, ground meats, dried meats, and canned meats. Each sample underwent full and mini-barcoding of the cytochrome c oxidase subunit I (COI) gene. The resulting sequences were queried against the Barcode of Life Database (BOLD) and GenBank for species identification. Overall, full-barcoding showed a higher sequencing success rate (68.3%) as compared to mini-barcoding (38.3%). Mini-barcoding out-performed full barcoding for the identification of canned products (23.8% vs. 19.0% success), as well as for turkey and duck products; however, the primer set performed poorly when tested against chicken, beef, and bison/buffalo. Overall, full barcoding was found to be a robust method for the detection of species in meat and poultry products, with the exception of canned products. Mini-barcoding shows high potential to be used for species identification in processed products; however, an improved primer set is needed for this application.     

Targeting double genes in multiplex PCR for discriminating bovine,buffalo and porcine materials in food chain

Beef, buffalo and pork are the major meat of economic, religious and health concern. Current methods to authenticate these materials in food chain are based on mainly single gene targets which are susceptible to break down by food processing treatments. We, for the first time, described here a double gene targeting short-amplicon length multiplex polymerase chain reaction assay for discriminating bovine, buffalo and porcine materials in a single assay platform. The advantage of the assay is evidenced in terms of fidelity, cost and time since it is highly unlikely that two different targets would be missing even in a decomposed specimen. Detection of multiple targets in a single assay definitely saves analytical cost and time. Mitochondrial cytochrome b (cytb) and ND5 genes were targeted and six different targets (length: 90–146 bp), two for each of cow (120 and 106bp), buffalo (90 and 138bp) and pig (73 and 146bp), were amplified from raw, boiled, autoclaved and microwaved cooked meat under pure and mixed matrices. The detection limit was 0.02 ng DNA under pure states and 0.1% meat in binary mixtures and meatball products. Screening of Malaysian meatball products revealed all beef products were buffalo positive in which 35% were totally replaced. In contrast, all pork products were found uncontaminated from beef and buffalo.     

Development and validation of fast Real-Time PCR assays for species identification in raw and cooked meat mixtures

Five species-specific Real-Time PCR protocols were developed, standardised and validated for the identification of turkey, chicken, beef, pork and sheep meat in complex food products. Specific primers and probes were designed for each assay. 16S rRNA and cyt-b target genes located in the mitochondrial DNA were used for this purpose. The limit of detection of the Real-Time PCR methods ranged between 0.02 pg and 0.80 pg of template DNA, with an efficiency between 95% and 100%. All methods were able to detect the target species when spiked at 1% in any other species and no relevant difference was observed between the Ct values of raw and cooked samples. An Internal Amplification Control was used to detect possible false negatives due to inhibitory substances eventually present in the sample matrix. The assays were tested on meat mixtures to evaluate the diagnostic sensitivity and specificity.     

Development of a real time PCR assay for detection of allergenic trace amounts of peanut (Arachis hypogaea) in processed foods

Peanut allergic reactions can result from the ingestion of even very small quantities of peanut and represent a severe threat to the health of sensitized individuals. In order to protect the allergic consumer, efficient and reliable methods are required for the detection of allergenic ingredients. For this purpose, we have developed a TaqMan real-time PCR method for specific detection of peanut in commercial food products. The assay uses two genetic makers in the Arah2 (125 bp) and ITS1 (90 bp) gene respectively, and TaqMan probes. The nuclear 18S rRNA gene was employed as a positive amplification control. Results obtained on sensitivity of both Arah2 and ITS primers with mixtures spiked with different concentrations of the target showed detection levels of 10 and 0.1 ppm, respectively. The applicability of the real-time PCR protocol to detect the presence of peanut DNA in commercial food products was determined through analysis of 123 different commercial products with the ITS primers which aimed higher sensitivity than Arah2 primer pair. The performance of the real-time PCR proposed herein allows a highly sensitive detection of peanut DNA in all the different food products, which declared to contain peanut material as well as in others were the presence of peanut or its traces wasn't declared in the labeling.     

Comparison of real-time PCR and ELISA-based methods for the detection of beef and pork in processed meat products

Two commonly used methodologies for species detection within processed meat products are real-time polymerase chain reaction (PCR), a DNA-based method, and enzyme-linked immunosorbent assay (ELISA), a protein-based method. In this study, a real-time PCR assay was compared to a commercial ELISA kit based on sensitivity, specificity, agreement among duplicate samples, cost, time, and ease of use. Fifteen reference samples containing known percentages (0.1–99.9%, w/w) of pork and beef were analyzed in duplicate using both methods. Thirty commercial products, including sausages, pet treats, and canned meats, were also tested in duplicate with each method. Reference sample analysis showed real-time PCR was able to detect pork in duplicate samples at 0.10% and beef at 0.50% in the binary mixtures. ELISA detected pork in duplicate samples at 10.0% and beef at 1.00% in the binary mixtures. When the results of reference and commercial samples were combined, real-time PCR demonstrated the greatest agreement among duplicate samples, at 96.7%, compared to 95.6% agreement for ELISA. The real-time PCR assay used in this study was found to be less expensive, while ELISA was less time-consuming and easier to perform. Both methods were successful at identifying species in ground meats, sausage, and deli meat samples; however, pet treats and canned meats proved more challenging. Overall, it was determined that the real-time PCR assay was optimal for species identification in processed meat products when a low detection limit is required; however, the ELISA kit may be advantageous in other situations due to its ease of use.     

Quantitative detection of goats’ milk in sheep’s milk by real-time PCR

The need to support food-labeling legislation has provided a driving force for development of analytical techniques for the analysis of food ingredients. In this study, the development of a method for quantification of goats’ milk in sheep’s milk mixtures is described. The technique involves the use of a real time PCR technique, based on the amplification of a fragment of the mitochondrial 12S ribosomal RNA gene (rRNA). The method combines the use of goat-specific primers that amplify a 171 bp fragment from goat DNA, and mammalian-specific primers amplifying a 119 bp fragment from mammalian species DNA, which are used as endogenous control. An internal fluorogenic probe (TaqMan) that hybridizes in the “goat-specific” and also in the “mammalian” DNA fragments is used to monitor the amplification of the target gene. A comparison of the cycle number (C_t) at which mammalian and goat-specific PCR products are first detected, in combination with the use of reference standards of known caprine content, allows the determination of the percentage of goats’ milk in a milk mixture. The assay was used to analyze raw and heat-treated milk binary mixtures (goat/sheep), enabling the quantification of goats’ milk in the range 0.6–10%. The reported PCR assay may represent a rapid and straightforward tool applicable to the authentication of milk and other dairy products.     

A validated strip-based lateral flow assay for the confirmation of sheep-specific PCR products for the authentication of meat

Molecular methods, such as PCR and real-time PCR, have been developed to detect species in meat and meat products. Despite good specificity and sensitivity, they are not widely implemented in food control programs due to complex operation or financial reasons. In the present study, a simple, rapid and affordable method, Sheep-PCR-Strip [Sheep specific polymerase chain reaction-Strip], was developed for the authentic identification of raw and heat-treated mutton. The assay is based on PCR amplification of sheep DNA, followed by detection of the PCR product by a strip format; the result can be read within 5 min by the naked eye. There is a real advantage of the strip approach rather in the reduced time (5 min versus electrophoresis) and avoidance of chemicals (e.g. ethidiumbromide). The sensitivity of the Sheep-PCR-Strip test was established to be 0.01% for the detection of adulterated meat; the limit of detection (LOD) was up to 0.01 pg of sheep DNA. The assay was also specific for sheep, and no cross-reactions were observed in other non-target species. It is a promising new tool for sheep identification and can be rapidly modified for other meat detection and widely used for solving problems related to food quality assurance, species authentication and traceability.     

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.     

Investigation on prevalence of Listeria spp. and Listeria monocytogenes in animal-derived foods by multiplex PCR assay targeting novel genes

Chilled and frozen animal-derived food can be contaminated by Listeria spp., emerging foodborne pathogens in food industry. The objective of this study was to mine novel target genes by comparative genomics approach for multiplex PCR detection and differentiation of Listeria monocytogenes and other Listeria spp. in food. Multiplex PCR assay targeting the genetic markers LMOf2365_2721, AX25_00730, lin1814, int, lwe1673, and Oxidoreductase gene, resulted in the amplification of DNA fragments of 583 bp, 703 bp, 421 bp, 994 bp, 345 bp, and 201 bp from L. monocytogenes, Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, and Listeria grayi, respectively. The detection limits of the multiplex assays were as low as 89 fg/μL genomic DNA and 910 CFU/mL of bacterial culture. The prevalence of Listeria spp. was determined using the developed multiplex PCR assay and standard microbiological method in a total of 200 food samples collected from different supermarkets and traditional agri-product markets in Nanjing, China. A total of 28 samples were found to be positive for the presence of Listeria, including 10.9% (6/55) of livestock meat samples, 22% (11/50) of poultry samples, 15% (6/40) of shellfish samples, 13.3% (4/30) of octopus samples and 4% (1/25) of freshwater fish samples. Of these, 13 isolates were classified as L. monocytogenes, 11 were classified as L. innocua, 2 were classified as L. ivanovii and 3 were classified as L. welshimeri. These results demonstrate that the multiplex PCR assay based on novel target genes is able to rapidly detect the Listeria spp. in 12 h with high accuracy and sensitivity, which may be used in the future for detection of Listeria spp. in animal-derived food products.     

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.     

Real-time PCR for quantitative meat species testing

A method for quantitative meat speciation is described which combines the use of real-time PCR with species specific and ‘universal’ primers to measure individual species content and total meat content respectively. A comparison of the cycle number at which universal and species specific PCR products are first detected, in combination with the use of reference standards of known species content, is used as the basis for determining the percentage of a given species in a mixed sample. Importantly, the use of universal primers allows differences in DNA quality between samples and reference standards to be taken into account, while the use of real-time PCR allows measurement at an early stage in the PCR process which is inherently more accurate than the end point analysis associated with gel-based systems. This paper describes the quantification of beef in mixed samples to illustrate the principle of this approach.     

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.     

Developing an SCAR and ITS reliable multiplex PCR-based assay for safflower adulterant detection in saffron samples

Saffron (Crocus sativus L.), one of the most important and expensive medicinal spice products traded internationally, is subject to adulteration by design or default with safflower stamens and corn stigmas, leading to poor quality of saffron samples. The present study aims at the development of specific, sensitive and reproducible PCR-based markers to detect these adulterants in traded saffron. Six putative RAPD markers generated by random primers, OPA-14, MG-11, MG-12 and AJ-05, were identified as saffron specific by comparative RAPD analysis of genuine saffron, safflower and corn. These specific RAPD markers were cloned, sequenced and six pairs of SCAR primers were designed. Specific designed primers were able to amplify reproducible saffron DNA with expected sizes and no amplification in corn and safflower DNA. In this study, a primer pair was also designed based on ITS sequences for specific amplification of safflower DNA. PCR reactions were also specifically amplified 613 bp of ITS region in safflower genome. The multiplex PCR assays were further established for the joint use of some SCAR and ITS markers efficiently. The special feature of this new molecular method was technically rapid and convenient practically and suitable for analyzing large numbers of samples. Thus, the simple rapid PCR-based molecular method could be used as a helpful assistant tool for the identification of adulterant saffron samples. This study described the development of a new SCAR and ITS maker-based multiplex PCR assay for the rapid molecular detection of substitutes in saffron.