Double gene targeting multiplex PCR-RFLP detects Crocodylus porosus in chicken meatball and traditional medicine

Crocodiles have been hunted and consumed for centuries for skins, nutrients, and medicines. These indomitable trends have overpowered restrictions from wildlife and conservation agencies, continuing the illegal trades of crocodiles across the world. This paper described the development of a very stable, fast, and secured polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for the confirmed detection of Crocodylus porosus under any matrices and decomposing treatments. Two very short-sites (77 and 127-bp) of atp6 and cytb genes of C. porosus were controlled digested with AciI enzyme; producing distinctive RFLP patterns (83, 54, 44 & 23 bp). The enzyme digested assay was stable following extreme boiling, autoclaving, and microwaving treatments that break down DNA. The sensitivity was tested and validated in model meatballs and it was suitable for detecting 0.01% crocodile meatball matrices. The optimized RFLP assay was used to screen 3 commercial meatballs and 21 traditional medicines (TM). While no crocodile DNA was found in commercial chicken meatballs, 4/21 TM products were found correctly labelled to contain C. porosus DNA. The novel assay demonstrated sufficient merit to be used by regulatory agencies for any forensic and/or archaeological identification of C. porosus even under the state of decomposition.     

Quantitative duplex real-time polymerase chain reaction assay with TaqMan probe detects and quantifies Crocodylus porosus in food chain and traditional medicines

Consumption and exploitation of crocodiles have been rampant for their exotic, nutritive and medicinal attributes. These depredations are alarming and although they have continued to be monitored by wildlife and conservation agencies, unlawful trading of crocodiles shows an increasing trend worldwide. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays for crocodile have been documented but they are only suitable for identification and cannot quantify adulterations. We described here a quantitative duplex real-time PCR assay with probes to quantify contributions from Crocodylus porosus materials simultaneously. A very short amplicon size of 127bp was used because longer targets could have been broken down in samples, bringing considerable uncertainty in molecular analysis. We have validated a TaqMan probe-based duplex real-time PCR (qPCR) assay for the detection of 0.004 ng DNA in pure state and 0.1% target meat in model chicken meatball. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 12 model chicken meatballs adulterated with C. porosus reflected 96.3?120.2% target recovery at 0.1?10% adulterations. A validation test of 21 commercial food and traditional medicine (TM) crocodile-based products showed 100% effectiveness. Short amplicon sizes, alternative complementary target, exceptional stability and superior sensitivity suggested the assay could be used for the identification and quantitative determination of C. porosus in any food or TM samples even under degraded conditions.     

Canine-Specific PCR Assay Targeting Cytochrome b Gene for the Detection of Dog Meat Adulteration in Commercial Frankfurters

This report described a cytochrome b (cytb)-based polymerase chain reaction (PCR) assay for the detection of canine tissues in commercial frankfurters. Discriminating detection of canine derivatives in processed food products has important application in halal authentication as well as in health, religions, and fare trades. The assay based on a pair of canine-specific primers that targeted a 100 bp region of canine mithochondrial-cytb gene which is present in multiple copies and highly conserved within the same species. The specificity of the assay was tested against dog and eight most common animal meat species as well as five plant species commonly found in frankfurter formulation. The stability and specificity of the assay were verified under different thermal processing conditions under pure and complex matrices. Three commercial brands of chicken and beef frankfurters were tested in triplicate, and specific PCR products were obtained only from deliberately contaminated formulations. The detection limit of the assay was 0.1 % (0.02 ng DNA) of canine meat spiked with other meats in a typical frankfurter formulation. Shorter amplicon length, superior stability, and higher sensitivity of the assay suggested its potential application in the screening of canine-origin biomaterials in processed food products.     

不同养殖环境、不同部位鳄鱼肉的营养品质和加工特性

本文初步研究不同养殖环境和采肉部位对鳄鱼肉营养品质和加工特性的影响,以室内和室外两个饲养环境的鳄鱼为原料,对尾部、躯干、腿部三个部位的部分营养成分和加工特性进行了分析。结果表明:营养品质方面,室外养殖鳄鱼的尾部蛋白质最高为20.88%,其躯干有最高灰分含量为0.12%,其腿部有最低脂肪含量为0.88%和水分最高为76.09%(P<0.05),不同养殖环境不同部位肉的氨基酸共检测出17种,其中腿部的总氨基酸含量最高(P<0.05),室外养殖鳄鱼躯干和腿部的总氨基酸含量均低于室内养殖鳄鱼。加工特性方面,鳄鱼尾部的亮度值L*最高,蒸煮损失率最小,持水力最大,pH最低,室外养殖鳄鱼肉形成的凝胶在保水性、质构和流变特性方面优于室内养殖鳄鱼。可见养殖环境和采肉部位对鳄鱼肉的营养品质和加工特性有明显影响,室外养殖鳄鱼的尾部肉具有更高营养品质和加工特性。     

Swine-Specific PCR-RFLP Assay Targeting Mitochondrial Cytochrome B Gene for Semiquantitative Detection of Pork in Commercial Meat Products

Verification of pork adulteration in commercial meat products is increasingly important for the authentication of Halal labels in processed foods. Here, we documented a PCR–restriction fragment length polymorphism (RFLP) assay with high precision and reproducibility for the tracing of porcine DNA in commercial meat products. The assay combined the species-specific primers to selectively amplify a short fragment (109 bp) of porcine cytochrome b gene from a heterogeneous background of genomic DNAs followed by RFLP analysis to authenticate real amplicon. The analysis of PCR products and restriction digests was automated in a chip-based capillary electrophoresis incorporated in Agilent 2100 bioanalyzer. The swine specificity of the assay was checked with 11 different meat-providing animal and fish species. Model experiments, mimicking the processed foods, were performed in binary and ternary mixtures after mechanical grinding and prolonged autoclaving. Finally, four types of the most popular finished meat products (meatball, streaky beacon, frankfurter, and burger) which are prevalent in the Malaysian food market were analyzed in order to verify the assay performance. The assay was sensitive enough to detect 0.0001 ng of swine DNA in pure formats and 0.01% (w/w) spiked pork in extensively processed ternary mixture of pork, beef, and wheat flour.     

A suitable method to detect potential fraud of bringing Malayan box turtle (Cuora amboinensis) meat into the food chain

Malayan box turtle (Cuora amboinensis) has been a wildlife-protected vulnerable turtle species in Malaysia since 2005. However, because of its purported usage in traditional medicine, tonic foods and feeds, clandestine black market trade is rampant. Several polymerase chain reaction (PCR) assays for the taxonomic detection and classification of turtle species have been proposed. These assays are based on long-length target amplicons which are assumed to break down under compromised states and, hence, might not be suitable for the forensic tracing and tracking of turtle trafficking. For the first time this paper develops a very short-amplicon-length PCR assay (120 bp) for the detection of Malayan box turtle meat in raw, processed and mixed matrices, and experimental evidence is produced that such an assay is not only more stable and reliable but also more sensitive than those previously published. We checked the assay specificity against 20 different species and no cross-species detection was observed. The possibility of any false-negative detection was eliminated by a universal endogenous control for eukaryotes. The assay detection limit was 0.0001 ng of box turtle DNA from pure meat and 0.01% turtle meat in binary and ternary admixtures and commercial meatballs. Superior target stability and sensitivity under extreme treatments of boiling, autoclaving and microwave cooking suggested that this newly developed assay would be suitable for any forensic and/or archaeological identification of Malayan box turtle species, even in severely degraded specimens. Further, in silico studies indicated that the assay has the potential to be used as a universal probe for the detection of nine Cuora species, all of which are critically endangered.     

Carcass and meat characteristics of the Nile crocodile (Crocodylus niloticus)

Seven Nile crocodiles (Crocodylus niloticus) of 1300 mm length were slaughtered in order to established baseline values for component yields and expected percentage of lean meat, fat and bone for this species. The skin presents nearly 20% of the live weight of the Nile crocodile, while a dressing percentage of 56.5% was derived. The tail realised 18 and 33% of the live weight and empty carcass weight respectively. Values of 60.8, 12.2 and 26.6% of carcass weight were obtained for total lean meat, fat and bone respectively. A pH value of ± 6.5 at 24 h post‐mortem in both tail and leg muscles and a decreasing pH towards 48 h post‐mortem illustrated that rigor mortis is still not complete when crocodile carcasses are processed. While fat content differed statistically (P < 0.05) from 91.1 g kg⁻¹ in raw torso samples to 29.4 g kg⁻¹ in raw neck samples, protein content was relatively constant around a mean of 220.8 g kg⁻¹ in raw meat. Cooking did not have any influence of practical value on proximate, amino acid or mineral composition. Crocodile meat is characterised by a lower iron, magnesium and sodium content than either beef or chicken. Of the total fatty acids present in the tail samples, 37.7% were saturated, 51.1% monounsaturated and 10.7% polyunsaturated. Oleic acid was predominant (43.1%), whilst palmitic acid (25.4%), stearic acid (9.9%) and linoleic acid (9.1%) were also present in high concentrations. © 2000 Society of Chemical Industry     

TaqMan probe real-time polymerase chain reaction assay for the quantification of canine DNA in chicken nugget

This paper describes a short-amplicon-based TaqMan probe quantitative real-time PCR (qPCR) assay for the quantitative detection of canine meat in chicken nuggets, which are very popular across the world, including Malaysia. The assay targeted a 100-bp fragment of canine cytb gene using a canine-specific primer and TaqMan probe. Specificity against 10 different animals and plants species demonstrated threshold cycles (Ct) of 16.13 ± 0.12 to 16.25 ± 0.23 for canine DNA and negative results for the others in a 40-cycle reaction. The assay was tested for the quantification of up to 0.01% canine meat in deliberately spiked chicken nuggets with 99.7% PCR efficiency and 0.995 correlation coefficient. The analysis of the actual and qPCR predicted values showed a high recovery rate (from 87% ± 28% to 112% ± 19%) with a linear regression close to unity (R² = 0.999). Finally, samples of three halal-branded commercial chicken nuggets collected from different Malaysian outlets were screened for canine meat, but no contamination was demonstrated.     

The detection of commercial duck species in food using a single probe-multiple species-specific primer real-time PCR assay

A real-time PCR assay for the simultaneous detection of Mallard and Muscovy duck is described. Species-specific primers were designed for Mallard or Muscovy duck using the mitochondrial cytochrome b gene sequence. These primer sets were multiplexed with a single duck probe to produce a simple, rapid and robust real-time PCR assay. This assay was shown to be specific for duck compared to a wide range of commercially important meat species and was used for the successful detection of duck meat in complex food matrices. This is the first report of an assay that will detect all species of commercially available duck in commercial products using real-time PCR.     

Duplex real-time PCR assay using SYBR Green to detect and quantify Malayan box turtle (Cuora amboinensis) materials in meatballs,burgers, frankfurters and traditional Chinese herbal jelly powder

The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected species widely used in exotic foods and traditional medicines. Currently available polymerase chain reaction (PCR) assays to identify MBT lack automation and involve long targets which break down in processed or denatured tissue. This SYBR Green duplex real-time PCR assay has addressed this research gap for the first time through the combination of 120- and 141-bp targets from MBT and eukaryotes for the quantitative detection of MBT DNA in food chain and herbal medicinal preparations. This authentication ensures better security through automation, internal control and short targets that were stable under the processing treatments of foods and medicines. A melting curve clearly demonstrated two peaks at 74.63 ± 0.22 and 78.40 ± 0.31°C for the MBT and eukaryotic products, respectively, under pure, admixed and commercial food matrices. Analysis of 125 reference samples reflected a target recovery of 93.25–153.00%, PCR efficiency of 99–100% and limit of detection of 0.001% under various matrices. The quantification limits were 0.00001, 0.00170 ± 0.00012, 0.00228 ± 0.00029, 0.00198 ± 0.00036 and 0.00191 ± 0.00043 ng DNA for the pure meat, binary mixtures, meatball, burger and frankfurter products, respectively. The assay was used to screen 100 commercial samples of traditional Chinese herbal jelly powder from eight different brands; 22% of them were found to be MBT-positive (5.37 ± 0.50–7.00 ± 0.34% w/w), which was reflected through the Ct values (26.37 ± 0.32–28.90 ± 0.42) and melting curves (74.63–78.65 ± 0.22°C) of the amplified MBT target (120 bp), confirming the speculation that MBT materials are widely used in Chinese herbal desserts, exotic dishes consumed with the hope of prolonging life and youth.     

Identification of barramundi (Lates calcarifer) and tilapia (Oreochromis spp.) fillets by DNA- and protein-analytical methods

Fish and fillet of barramundi (Lates calcarifer) and tilapia (Oreochromis species) obtained from wholesale and retail trade were assigned to species by sequencing of PCR products. Two segments (358 and 464 bp) of the cytochrome b gene (cytb) were amplified using universal primers. The amplicons gave characteristic patterns in SSCP-analysis (single strand conformation polymorphism) suitable for differentiation of Lates calcarifer from Lates niloticus and Lateolabrax japonicus. Intra-specific variation of sequences and SSCP patterns were observed for barramundi. In case of tilapia species, it was found to be difficult to identify samples by BLAST due to the high similarity of cytb sequences of O. niloticus, O. mossambicus, O. aureus and Sarotherodon galileus. Four different patterns of single strand DNA (ssDNA) were obtained by SSCP analysis of the 464 bp amplicon of tilapia. Different patterns of ssDNA matched to variations in sequences. Protein profiles obtained by IEF (isoelectric focusing) of water-soluble proteins from raw fillet were found to be suitable for rapid differentiation of Lates calcarifer from Lateolabrax japonicus, but the three different Oreochromis species expressed only minor differences in protein patterns. The patterns of the tilapia and barramundi species showed a number of acidic, heat-stable proteins, presumably representing parvalbumin.     

Lab-on-a-chip-based PCR-RFLP assay for the confirmed detection of short-length feline DNA in food

Wider availability but lack of legal market trades has given feline meat a high potential for use as an adulterant in common meat and meat products. However, mixing of feline meat or its derivatives in food is a sensitive issue, since it is a taboo in most countries and prohibited in certain religions such as Islam and Judaism. Cat meat also has potential for contamination with of severe acute respiratory syndrome, anthrax and hepatitis, and its consumption might lead to an allergic reaction. We developed a very short-amplicon-length (69 bp) PCR assay, authenticated the amplified PCR products by AluI-restriction digestion followed by its separation and detection on a lab-on-a-chip-based automated electrophoretic system, and proved its superiority over the existing long-amplicon-based assays. Although it has been assumed that longer DNA targets are susceptible to breakdown under compromised states, scientific evidence for this hypothesis has been rarely documented. Strong evidence showed that shorter targets are more stable than the longer ones. We confirmed feline-specificity by cross-challenging the primers against 10 different species of terrestrial, aquatic and plant origins in the presence of a 141-bp site of an 18S rRNA gene as a universal eukaryotic control. RFLP analysis separated 43- and 26-bp fragments of AluI-digest in both the gel-image and electropherograms, confirming the original products. The tested detection limit was 0.01% (w/w) feline meat in binary and ternary admixed as well as meatball matrices. Shorter target, better stability and higher sensitivity mean such an assay would be valid for feline identification even in degraded specimens.     

The yield and nutritional value of meat from African ungulates, camelidae, rodents, ratites and reptiles

The current knowledge of the yield and nutritional (proximate and fatty acid) composition of meat derived from African ungulates, camelidae, rodents, ratites and reptiles is reviewed. Although most of the species discussed give low cholesterol levels consistent with their low meat lipid contents, the tegu lizard gives a very low level (18.2mg/100g tissue). The fatty acid profiles of the various species all have low saturated fatty acids and high polyunsaturated fatty acids resulting in favourable saturated to polyunsaturated fatty acid ratios. Although the springbok, camel, ostrich and crocodile are marketed and exported to sophisticated markets, the rodents are the species that show most promise in becoming large commercial commodities. Not only is their meat desirable and nutritional, but they are also highly adaptable to extensive and intensive production systems.     

Development and application of highly specific PCR for detection of chicken (Gallus gallus) meat adulteration

In order to prevent fraud in the sale and strengthen quality assurance, authentic identification of chicken meat is essential. In the present investigation, a chicken (Gallus gallus)-specific polymerase chain reaction (PCR) was developed for the unambiguous identification of chicken meat. The PCR assay employs pair of primers designed against chicken nuclear 5-aminolevulinate (ALA) synthase gene. Highly chicken-specific diagnostic amplicon of 288 bp was established upon PCR and was evident in all the nine breeds/strains of chicken species. Sensitivity of PCR in detecting chicken meat adulteration was established to be at 0.1 % in the foreign meat matrix, while limit of detection (LOD) of chicken DNA was 10 pg. Suitability of the developed chicken-specific PCR was validated and confirmed in raw, cooked/heat treated (60, 80, 100, and 121 °C), and micro-oven cooked meat samples. Possibility of cross-amplification of adulterating DNA was excluded by cross-checking the developed PCR assay with several animal and avian species. The PCR assay developed in this study is highly promising for applications involving circumstances that require authentic identification of chicken meat.     

A lab-on-a-chip-based multiplex platform to detect potential fraud of introducing pig,dog, cat,rat and monkey meat into the food chain

Food forgery has posed considerable risk to public health, religious rituals, personal budget and wildlife. Pig, dog, cat, rat and monkey meat are restricted in most religions, but their sporadic adulteration are rampant. Market controllers need a low-cost but reliable technique to track and trace suspected species in the food chain. Considering the need, here we documented a lab-on-a-chip-based multiplex polymerase chain reaction (PCR) assay for the authentication of five non-halal meat species in foods. Using species-specific primers, 172, 163, 141, 129 and 108-bp sites of mitochondrial ND5, ATPase 6 and cytochrome b genes were amplified to detect cat, dog, pig, monkey and rat species under complex matrices. Species-specificity was authenticated against 20 different species with the potential to be used in food. The targets were stable under extreme sterilisation (121°C at 45 psi for 2.5 h) which severely degrades DNA. The assay was optimised under the backgrounds of various commercial meat products and validated for the analysis of meatballs, burgers and frankfurters, which are popular fast food items across the globe. The assay was tested to detect 0.1% suspected meats under commercial backgrounds of marketed foods. Instead of simplex PCR which detects only one species at a time, such a multiplex platform can reduce cost by at least fivefolds by detecting five different species in a single assay platform.     

鳄鱼肉与其他肉类营养成分的比较分析

为了进一步挖掘鳄鱼肉的营养价值,以鳄鱼肉、猪肉、牛肉、鸡肉、羊肉、河虾肉和鲫鱼肉为研究对象,采用国家标准检测方法,分析和测定其含有的主要营养成分并进行营养评价.结果表明:与其他肉类相比,鳄鱼肉中磷含量最高;鳄鱼肉中钙含量显著高于其他畜禽肉(P<0.05),是一种优质补钙食品,鳄鱼肉中水溶性维生素含量最高,鲜味氨基酸含量...     

A Method for the Detection of Potential Fraud of Bringing Feline Meat in Food Chain

Adulteration of meat in processed food is a sensitive issue since certain meat species are prohibited in some religions such as Islam and Judaism. Some meat types are also potential carrier of some deadly diseases such as severe acute respiratory syndrome, anthrax, and hepatitis. Furthermore, unconscious consumption of certain meat might lead to an allergic reaction. Feline meat is not only taboo in most cultures but also under religious prohibition. However, cat meat has been consumed in certain countries including Cambodia, South Korea, China, and Vietnam. Several polymerase chain reaction assays are proposed for the detection of feline meat. However, those assays are not tested under processed food matrices. They are also based on longer targets (672, 331, 274, 180, and 108 bp) which breakdown under compromised states. Here we documented a very short-amplicon-length (69 bp) polymerase chain reaction assay and produced strong evidence that short targets are more stable than the longer ones. Feline specificity was confirmed by cross-challenging against 17 non-target species and target stability was tested after boiling, microwaving, and autoclaving treatments under complex matrices. The tested detection limit was 0.01% (w/w) of feline meat in ternary mixtures and 0.1% (w/w) in cooked meatballs.     

多重位点特异性PCR快速检测牛肉中常见掺假动物源性成分

目的:基于动物线粒体cytb基因的多态性位点,建立一种特异性多重PCR体系检测牛肉、猪肉和鸡肉的方法。方法:提取肉类的基因组DNA,利用不同物种mtDNA cytb基因序列的SNP位点的差异,设计特异性引物。进行多重PCR扩增,利用扩增产物片段大小不同,检测牛肉中常见的掺假动物源性成分。通过灵敏性试验,确定最低检测量。结果:实验设计的引物特异性良好,在同一反应体系中,在同一退火温度52℃条件下,牛肉DNA扩增后产生149 bp的特异性条带,猪肉DNA扩增片段为261 bp,鸡肉DNA扩增片段为554 bp,未发生非特异性扩增。且检测的最低浓度达到100 pg/μL,具有高度的灵敏性和适用性。结论:根据动物线粒体cytb基因的差异性位点,开发的多重PCR体系,可一次性地同时检测牛肉、猪肉和鸡肉,可快速、灵敏、高通量地分析食品中掺假动物成分的来源。     

鳄鱼肉主要营养成分及与其他畜禽肉的比较

为了充分了解鳄鱼肉的营养特性,更好地利用鳄鱼资源,本实验分析鳄鱼肉的主要组分、脂肪酸及氨基酸的构成,并与羊肉、猪肉、牛肉和鸡肉进行比较.结果表明:与其他畜禽肉相比,鳄鱼肉是一种高蛋白、低脂肪的优质肉品,多不饱和脂肪酸含量丰富,尤其是花生四烯酸、二十碳五烯酸(EPA)和二十二碳六烯酸(DHA).鳄鱼肉富含必需氨基酸,尤其是赖氨酸.且根据必需氨基酸指数,鳄鱼肉的营养价值高于其他畜禽肉.综合看来,鳄鱼肉是较理想的动物蛋白及脂肪来源.     

Polymerase chain reaction coupled with peptide nucleic acid high-performance liquid chromatography for the sensitive detection of traces of potentially allergenic hazelnut in foodstuffs

A new DNA extraction method suitable for a wide variety of complex food matrices has been devised and applied in combination with a new polymerase chain reaction (PCR) method for the sensitive detection of a specific DNA sequence univocally identifying the presence of potentially allergenic hazelnut (Corylus avellana). A 156 base pair amplicon corresponding to an internal region of the complementary DNA of the major hazelnut allergen (Cor a 1) was designed, and was found to be highly specific; the method was tested on both pure and complex food matrices and was found to be able to confirm the presence of hazelnut down to 5 pg of its DNA. The sequence of the amplicon was further confirmed by high-performance liquid chromatography (HPLC) analysis with a specific peptide nucleic acid (PNA) probe. A 15-mer PNA probe was expressly designed and synthesized to hybridize an internal sequence of the previously described amplicon. Given its reported sequence specificity and high hybridization efficiency, the PNA probe was used to develop an anion-exchange HPLC method allowing for a fast and reliable confirmation of the identity of the amplified products. The PCR–HPLC method was successfully tested on commercial samples, allowing for the detection of the presence of potentially hidden allergens even in products where the presence of hazelnut as an ingredient or possible contaminant was not reported.