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.     

A multiplex-conventional PCR assay for bovine,ovine, caprine and fish species identification in feedstuffs: Highly sensitive and specific

A multiplex PCR assay was developed for rapid and reliable identification of bovine, ovine, caprine and fish species in feedstuffs simultaneously. The method merges the use of bovine, ovine, caprine and fish primers that amplify fragments (ovine; 119 bp, caprine; 142 bp, fish; 224 bp and bovine; 271 bp) of the mitochondrial t.glu gene forward and cyt b reverse, 12S rRNA, 12S rRNA, and ATPase subunit 8 genes respectively, and a universal 18S rRNA primers that amplifies a 99 bp from eukaryotic DNA. To evaluate the effect of heat treatment, a severe sterilization condition (133 °C at 300 kPa for 20 min) was applied. Multiplex analysis of the reference feedstuff samples showed that the detection limit of the assay was 0.01% for each species. Taken together, all data indicated that this multiplex PCR assay was a simple, rapid, sensitive, specific, and cost-effective detection method for bovine, ovine, caprine and fish species in feedstuffs.     

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 novel target-enriched multiplex PCR (Tem-PCR) assay for simultaneous detection of five foodborne pathogens

In the present study, a novel target-enriched multiplex PCR (Tem-PCR) assay was developed for simultaneous detection of Salmonella spp., Listeria (L.) monocytogenes, Staphylococcus (S.) aureus, Escherichia (E.) coli O157:H7 and Shigella spp.. DNA primers including universal primer and composite primer pairs were used in this Tem-PCR assay. Of which, the universal primer was linked to the 5'-end of each specific primer designed targeting Salmonella invA gene, Staphylococcus aureus femA gene, Shigella ipaH gene, Listeria monocytogenes hly gene and Escherichia coli O157:H7 rfbE gene, respectively, generating the composite primers. During PCR amplification, the composite primer pairs were employed to enrich target genes of different pathogens in initial cycles and the universal primer was employed to enrich the amplicons produced with the composite primers priming in later PCR cycles. Significantly, the Tem-PCR assay overcomes the amplification disparity resulting from primers competition observed in conventional multiplex PCR assay. With the evaluation of the Tem-PCR assay for the detection of these five foodborne pathogens, the assay showed high specificity to the target bacteria with an analytical detection limit of <2.0 × 10² CFU/mL for each, and practical detection capability, suggesting a novel multiplex PCR method for detecting foodborne pathogens.     

Rapid species identification of fresh and processed scallops by multiplex PCR

Food control policies regarding to seafood label authenticity have become a global issue due to increased incidence of species substitution or mislabelling. Proper species-level identification in processed scallop products is hindered by the lack of morphological characters such as their valves. In order to identify four commercially important scallop species (Argopecten purpuratus, Argopecten irradians, Mizuhopecten yessoensis, Pecten albicans) a species-specific multiplex PCR reaction is described herein. Novel reverse species-specific primers in combination with one universal forward primer designed to amplify a partial region of the mitochondrial 16S rRNA gene were assayed in fresh as well as in manufactured scallop samples. All PCR reactions showed a high specificity allowing an unambiguous species authentication.     

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.     

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.     

Identification and verification of porcine DNA in commercial gelatin and gelatin containing processed foods

Gelatin, derived from bovine and porcine sources, has been used in many foods and pharmaceutical products. To ensure the compliance of food products with halal regulations, the reliable analytical methods are very much required. In this study, polymerase chain reaction (PCR) assay using species-specific primers was performed to evaluate the halal authenticity of commercial pure gelatin and gelatin-containing processed food products. Based on the specificity and cross-reactivity results of the seven species-specific primers by conventional PCR, the porcine species primer No. 2 was selected and it was able to detect species DNA in 12 out of 36 processed foods. The cloning, sequencing, and blasting at NCBI confirmed the presence of pork DNA in 5 out of 12 porcine DNA positive food samples. The maximum identity (homology) with pork sequence available in NCBI Gene Bank for the five samples ranged from 87% to 97% and the Query Cover ranged from 94% to 100%. The real-time PCR assay detected more positive samples (27 positive amplifications) compared to 12 positive samples with conventional PCR using porcine specific primer No. 2. PCR using species specific primers is a very useful and effective technique for halal authenticity of gelatin and gelatin-containing food 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.     

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.     

Using the PCR–RFLP method to identify the species of different processed products of billfish meats

A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method had been developed for the detection of five billfish species Xiphias gladius, Makaira nigricans, M. indica, Istiophorus platypterus and Tetrapturus audax in raw, frozen and heat-treated meats. The primers L-CYTBF and H-CYTBF were designed in the mitochondrial cytochrome b (cytb) gene and the molecular weight of amplified fragment was 348 bp and amplified the fragment from processed billfish meats. The results obtained from the BsaJI, Cac8I and HpaII enzymes digestion could be used to distinguish the five billfish species in frozen and heat-treated meats. Using the PCR–RFLP method, species of 10 commercial samples including raw fish fillets, frozen fish meats and fried fish meats could be identified. It was determined that two commercial samples of billfish products were not made from billfish. The method is sensitive, rapid and valid to detect fraudulent billfish products substituted from cheaper fish.     

Contamination of Opisthorchis viverrini and Haplorchis taichui metacercariae in fermented fish products in northeastern Thailand markets

Consumption of raw or inadequately cooked cyprinid fish as well as related products (often contaminated with Opisthorchis viverrini and/or Haplorchis taichui) is one of the major causes of fish-borne trematode (FBT) infection, which is still endemic in the Greater Mekong Subregion including northeastern Thailand. This study surveyed FBT metacercariae (FBTM) in fermented fish dishes (pla-ra and pla-som) obtained from 73 local markets in 20 provinces of northeastern Thailand during April to November 2011. Fish were identified and examined for FBTM under a microscope. In addition, the coexistence of H. taichui in O. viverrini-positive samples was confirmed by multiplex PCR. FBTM were detected in fermented fish dishes from markets located in five provinces: Si Sa Ket, Sakon Nakhon, Mukdahan, Khon Kaen and Udon Thani. FBTM contamination was found in 9.58% (7/73) of markets, mainly in pla-ra. FBTM were found in four species of fish: Henicorhynchus siamensis, Puntius bimaculatus, Puntius orphoides and Hampala dispar. Multiplex PCR revealed 186 and 330 bp PCR products in most of FBTM-positive samples, indicating the coexistence of H. taichui and O. viverrini in fermented fish dishes. These results suggest that fermented fish dishes are frequently contaminated with FBTM and may serve as important sources of FBT infection in people who typically eat raw or undercooked fish dishes. This study might provide evidence leading to improved public health awareness for surveillance and control of FBT contamination in fermented fish dishes.     

Simultaneous detection of Listeria species isolated from meat processed foods using multiplex PCR

Listeriosis is a foodborne disease caused by the pathogenic Listeria monocytogenes and is considered as a serious health problem due to the severity of symptoms and its high mortality rate. Listeria genus is divided into six species and especially L. monocytogenes is an important foodborne pathogen in humans and livestock. Recently, other Listeria species are reported as pathogenic strains in decayed foods and environments as well. High mortality rate of listeriosis demands for rapid methods to detect the potential presence of the food pathogens in the food industry. We have developed a multiplex PCR for rapid and simultaneous detection of six Listeria species including Listeria grayi, Listeria innocua, Listeria ivanovii, L. monocytogenes, Listeria seeligeri and Listeria welshimeri to identify specific Listeria species in processed foods. The optimized multiplex PCR in this study utilized one Listeria genus specific and each Listeria species-specific primer pairs. Each primer pair yields the products of 370-bp for Listeria genus-specific, 201-bp for L. grayi-specific, 749-bp for L. innocua-specific, 463-bp for L. ivanovii-specific, 509-bp for L. monocytogenes-specific, 673-bp for L. seeligeri-specific and 281-bp for L. welshimeri-specific. We have successfully applied multiplex PCR strategy to 93 Listeria isolates from processed meat products to determine specific Listeria species and out of which 81 strains of L. monocytogenes, 10 strains of L. innocua and 2 strains of L. welshimeri were identified. This established multiplex PCR provides rapid and reliable results and will be useful for the detection of Listeria species in contaminated food products and clinical samples.     

Development of triplex PCR for simultaneous detection of maize,wheat and soybean

A reliable and fast detection of important food plants, such as maize (Zea mays L.), wheat (Triticum aestivum L.), and soybean (Glycine max L.) is of particular interest for food authenticity and safety assessment. In this study, the novel multiplex polymerase chain reaction (PCR) method was developed for the rapid qualitative detection of soybean, maize and wheat. To this purpose, new soybean-specific and maize-specific PCR primers were designed. Their specificity was assayed by uniplex PCRs with different plant species, namely maize, soybean, wheat, oats (Avena sativa), and barley (Hordeum vulgare L). Gel electrophoresis of the amplification products demonstrated high specificity of both primer pairs for identification of relevant species. Subsequently, based on the developed DNA markers, the species-specific triplex PCR targeting maize invertase gene, soybean lectin gene and wheat low-molecular-weight glutenin subunit was developed and optimized for simultaneous identification of these three plant species. The developed PCR method enables specific, effective and rapid detection of maize, wheat and soybean and may be used for food analysis.     

Occurrence of pathogenic bacteria in raw milk, cultured pasteurised milk and naturally soured milk produced at small-scale dairies in Zimbabwe

The presence of selected pathogenic bacteria in raw milk (RM), cultured pasteurised milk (CPM), and naturally soured raw milk (NSRM) sold at three small-scale dairies in Zimbabwe was determined. Mean levels of Escherichia coli in RM, CPM and NSRM were 4.5, 7.1 and 7.8 log₁₀ CFU ml⁻¹, respectively. Enterotoxigenic E. coli (ETEC), producing heat-stable enterotoxin (ST1) was found in 16 of the samples. Presumptive Staphylococcus aureus was identified in 7 of 12 RM, 15 of 27 CPM and 20 of 21 NSRM samples at mean levels of 5.2, 7.3 and 7.8 log₁₀ CFU ml⁻¹, respectively. Klebsiella pneumonia spp., Enterobacter cloacae, Proteus mirabilis, Hafnia alvei, Citrobacter spp., Serratia marcescens and Aeromonas hydrophilia were found in 27 samples. The high number of S. aureus, E. coli, identification of ETEC-ST1 and the other pathogenic microorganisms found in pasteurised and unpasteurised milk products represent a health hazard to the consumers and emphasises the need for improved hygiene practice at all levels in the dairy.     

Simultaneous detection of Pathogenic Vibrio species using multiplex real-time PCR

We developed a multiplex real-time (RTi) PCR method for the simultaneous detection of Vibrio cholerae, V. parahaemolyticus, and V. vulnificus using zot, vmrA, and vuuA as the respective target genes. A set of primer pairs specific for those target genes was designed and employed in the SYBR Green-based multiplex RTi-PCR assay. Quantitative analyses with ten-fold serially diluted genomic DNA of each target organism resulted in a linear correlation between C_T values and the amount of each target genome per reaction, with a lower detection level of less than ten genome copies per reaction. Similar sensitivities were observed for Vibrio-spiked seafood samples (oyster, crab meat, and raw fish). After 8 h of enrichment culture of the seafood homogenate in alkaline peptone water, our optimized multiplex RTi-PCR was shown to achieve theoretical maximum sensitivity (ca. 10⁰ CFU/gram food homogenate). Our proposed method is simple, robust and readily adaptable in routine laboratories, allowing for high-throughput surveillance of pathogenic Vibrio species in seafood.     

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.     

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.     

Inhibition of food poisoning and pathogenic bacteria by Lactobacillus plantarum strain 2.9 isolated from ben saalga, both in a culture medium and in food

The bacteriocinogenic strain Lactobacillus plantarum 2.9 isolated from the traditional pearl millet-based African fermented food ben saalga was tested for inhibition of food poisoning and pathogenic bacteria in MRS broth and in a malted millet flour slurry. In MRS broth, strain 2.9 completely eliminated Bacillus cereus, Escherichia coli O157:H7 and Salmonella enterica cells within 48 h incubation at 22–30 °C. A much lower inhibition was observed at 15 °C. The inhibitory effect of strain 2.9 on the above-mentioned target bacteria was corroborated in the malted millet flour slurry, reducing viable cell counts below detection levels after 8 h storage for B. cereus or after 24 h for S. enterica and 48 h for E. coli. An Enterobacter aerogenes strain was only moderately inhibited in the slurry. Results from the present study suggest that strain 2.9 could be used as starter culture to improve the microbiological safety of fermented ben saalga.