Meat Species Research Articles

A digital PCR method for identifying and quantifying adulteration of meat species in raw and processed food.

Meat adulteration is a worldwide concern. In this paper, a new droplet digital PCR (ddPCR) method was developed for the quantitative determination of the presence of chicken in sheep and goat meat products. Meanwhile, a constant (multiplication factor) was introduced to transform the ratio of copy numbers to the proportion of meats. The presented ddPCR method was also proved to be more accurate (showing bias of less than 9% in the range from 5% to 80%) than real-time PCR, which has been widely used in this determination. The method exhibited good repeatability and stability in different thermal treatments and at ultra-high pressure. The relative standard deviation (RSD) values of 5% chicken content was less than 5.4% for ultra-high pressure or heat treatment. Moreover, we confirmed that different parts of meat had no effect on quantification accuracy of the ddPCR method. In contrast to real-time PCR, we examined the performance of ddPCR as a more precise, sensitive and stable analytical strategy to overcome potential problems of discrepancies in amplification efficiency discrepancy and to obtain the copy numbers directly without standard curves. The method and strategy developed in this study can be applied to quantify the presence and to confirm the absence of adulterants not only to sheep but also to other kinds of meat and meat products.

Chemiluminescent optical fibre genosensor for porcine meat detection

An easy, rapid and sensitive method for the detection of meat species is important to identify adulteration or fraudulent substitution in raw meats with undeclared species. We report herein the development of an optical fibre genosensor able to detect low level of pork (Sus Scrofa) meat in mixtures of minced meat obtained by an optimized and faster DNA extraction protocol. Our approach uses highly specific and sensitive DNA capture and secondary probes designed out of the mitochondrion genome of Sus Scrofa. Once DNA capture probes were immobilized on optical fibres, we demonstrate their putative application as genosensors in detecting Sus Scrofa DNA in purified samples obtained first using a commercial DNA extraction kit and thereafter using our improved extraction method. Furthermore, analysis of beef (Bos taurus) minced meat samples also show that the assay can determine 1% (w/w) of Sus Scrofa DNA. The sensitivity obtained by the optical fibre and the short time required for the results (about 2.5h) indicate the usefulness of the system. We envision that this genosensor may serve as a diagnostic tool for food sample screening using a technology that is accurate, simple to perform and has the potential to be used in portable format.

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.

Practices and exploration on competition of molecular biological detection technology among students in food quality and safety major.

With the increasing importance in the application of the molecular biological detection technology in the field of food safety, strengthening education in molecular biology experimental techniques is more necessary for the culture of the students in food quality and safety major. However, molecular biology experiments are not always in curricula of Food quality and safety Majors. This paper introduced a project "competition of molecular biological detection technology for food safety among undergraduate sophomore students in food quality and safety major", students participating in this project needed to learn the fundamental molecular biology experimental techniques such as the principles of molecular biology experiments and genome extraction, PCR and agarose gel electrophoresis analysis, and then design the experiments in groups to identify the meat species in pork and beef products using molecular biological methods. The students should complete the experimental report after basic experiments, write essays and make a presentation after the end of the designed experiments. This project aims to provide another way for food quality and safety majors to improve their knowledge of molecular biology, especially experimental technology, and enhances them to understand the scientific research activities as well as give them a chance to learn how to write a professional thesis. In addition, in line with the principle of an open laboratory, the project is also open to students in other majors in East China University of Science and Technology, in order to enhance students in other majors to understand the fields of molecular biology and food safety. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(4):343-350, 2017.

A rapid, semi-quantitative test for detection of raw and cooked horse meat residues

Intentional mislabeling and adulteration of meat products with undeclared horse meat is a concern for religious, ethnic, and health reasons and is illegal under regulations mandated and enforced by food regulatory agencies and the Federal Meat Inspection Act. Nonetheless, recent analysis of the meat industry has revealed an apparent increase in the frequency of meat adulteration including intentional horse meat contamination, necessitating a broader use of meat authentication testing. As existing methods for meat speciation are cumbersome and require specialized equipment and/or training, we developed a highly specific lateral flow immunoassay that can rapidly identify raw and cooked horse meat down to 0.01% and 1.0% contamination, respectively in xenogeneic meat sources in about 35 min with no false positive signals observed. Specificity analysis revealed no cross-reactivity with serum albumins or meat derived from chicken, turkey, pig, cow, lamb, and goat. The results of method comparison showed that the assay had similar if not better sensitivity than the commercial ELISA kit and PCR, and required considerably less time to perform than either method. The development of a highly robust and rapid test method capable of detecting trace amounts of horse meat residues should aid food control authorities in their continued efforts to monitor for horse meat adulteration.

Loop-Mediated Isothermal Amplification Assays for the Rapid Identification of Duck-Derived Ingredients in Adulterated Meat

Meat adulteration is a growing concern in the marketplace today. To protect consumer rights and prevent unfair competition, it is essential to use an efficient assay to identify rapidly the species of meat being sold. In this context, a loop-mediated isothermal amplification (LAMP) assay coupled with a lateral flow dipstick (LFD) or hydroxynaphthol blue dye (HNB) was developed for the detection of duck genes in meat products. The LAMP-LFD and LAMP-HNB assays were performed at 65 °C for 30 min, with no cross-reactivity against four other species of meat. Sensitivity evaluation showed that the two assays can detect 3 pg of duck DNA per reaction, which is 10 times higher than that of the real-time quantitative polymerase chain reaction (qPCR). Through testing the experimental adulteration models, which were prepared by mixing duck meat with beef at different concentrations (0.01 to 10%), the detection limits of the two assays were confirmed as 0.1% duck meat. Combining the simple DNA extraction assay and the LAMP-LFD or LAMP-HNB assay, adulteration with only 1 mg duck meat can be identified within 40 min. The LAMP-LFD and LAMP-HNB assays are simple and highly sensitive, which provide valuable tools for the identification of duck gene from adulterated meat. The simple DNA extraction assay further enables our LAMP assays to be applicable in the field.

Quality Assurance of Meat and Poultry products using Advanced Biotechnological Methods

Nowadays, meat adulteration constitutes an important problem in Egypt. Adulteration causes many problems that need to be considered as economic issues arising from replacement of high commercial value meat species with cheaper or undesired meats, religious issues as pork meat prohibited for Muslims and allergic reactions arising from consumption of some special meats as equine meat species and others. The current study includes a surveillance study aimed to detect the adulteration of beef and poultry meat products with equine and or pork meat species in Damanhur city, El Behera Governorate, Egypt. Over a limited period of one year from February, 2015 to February, 2016, A total of 156 samples of meat and chicken products were collected and divided into two groups; Group (A) includes 96 packaged high brands of three meat processing plants including beef luncheon, burger and kofta (18 samples each), chicken luncheon (18), chicken burger (12) and chicken kofta (12) and Group (B) includes 60 non-packaged low brand samples of beef luncheon, burger, kofta, minced meat and sausage(6 samples each) and chicken luncheon, burger, kofta, minced meat and sausage (6 samples each) from meat shops. All samples were analyzed for detection of adulteration with equine and/or pork meat by Polymerase Chain Reaction (PCR) technique using specific primers for equine and pork species. The obtained result revealed that adulteration rate of processed meat products with equine meat species was found to be 3.2 % (5 positive samples out of 156 examined samples) so it was recommended that rapid movement of responsible authorities with cooperation with scientific academies to protect health of consumers and prevent illegal substitution of meat.

Identification of meat species in different types of meat products by PCR

Et urunlerinde tur tayini gida guvenligi acisindan onemli bir konudur. Etikette bildirilmeyen cesitli hayvan turlerine ait etlerin kullanimi, tuketici sagligi, ulke ekonomisi, dini ve yasal duzenlemeler ile iliskilendirilmektedir. Bu calismada, sucuk, salam, sosis gibi urunlerde farkli turlere ait etlerin varliginin tespiti amaclanmistir. Arastirmada Ankara’da cesitli marketlerde satisa sunulan toplam 102 adet islenmis et urunu tur tayini amaciyla PZR yontemi kullanilarak incelenmistir. Yapilan analizler sonucunda, incelenen 37 adet sucuk orneginden etikette belirtilmeyen 5 (%13.5) kanatli, 1 (%2.7) kanatli ve tek tirnakli eti tespit edilmistir. Benzer seklide, 32 salam orneginin yedisinde (%21.8), 33 adet sosis orneginin de ikisinde (%6.1) kanatli eti bulunmustur. Urunlerin etiket bilgileri karsilastirildiginda 15 adet (% 14.7) ornegin etiketinde belirtilmeyen farkli hayvan turlerine ait etlerden uretildigi tespit edilmistir. Sonuc olarak, Ankara’da farkli marketlerden alinan islenmis et urunleri orneklerinde, etiketlerinde belirtilmeyen hayvan turlerine ait etlerin kullanildigi ve bu durumun potansiyel halk sagligi sorunlari yaninda tuketicinin ekonomik olarak da kayba ugramasina sebep olabilecegi ortaya konmustur

Beef and horse meat discrimination and storage time classification using a portable device based on DSP and PCA method

Food authenticity is an issue of major concern. The adulteration of meat products with horse meat drew attention to the development of robust techniques for meat species classification. This work presents an instrument and a method to discriminate among horse and beef meat and to classify their degree of spoilage based on meat colour. The proposed device employs charge-coupled device (CCD) imaging techniques, digital image processing, digital signal processor (DSP), processing techniques and liquid crystal display (LCD) screen. Samples were placed under cold storage at 4°C for two weeks. Two colour models are used to define beef and horse meat: red, green, and blue (RGB) and hue, saturation and intensity (HSI). Principal component analysis (PCA) was employed to optimise the data matrix. Results show that the device was able to distinguish between beef and horse meat and to classify them according to the number of days spent in cold storage.

Isolation and identification of Pseudomonas species in meat and meat product and cold storage depotsIsolation and identification of Pseudomonas species in meat and meat product and cold storage depots

Isolation and identification of Pseudomonas species in meat and meat product and cold storage depotsIsolation and identification of Pseudomonas species in meat and meat product and cold storage depots

Treatment Optimisation and Sample Preparation for the Evaluation of Lipid Oxidation in Various Meats Through TBARs Assays before Analysis

The aim of this work was to evaluate the critical points while using a TBARs test to measure the lipid oxidation in meat samples; this method includes a distillation method, extracting acid solutions (7% trichloroacetic acid—TCA, 4% perchloric acid—HClO4 and 4 N hydrochloric acid—HCl), butylated hydroxytoluene (BHT) concentrations and their interactions. The TBARs test method has been evaluated in different animal meat species and in different kinds of meat: fresh, stored, frozen and at different times of defrosting. Moreover, the influence of sample management was evaluated. The best results were obtained after using a distillation and a cold extraction with 7% TCA. The presence of the antioxidant agent (BHT) was essential and was more important in frozen samples than in fresh meat and especially when it was added at negligible concentrations immediately before defrosting, within 2 min of sample withdrawal from the freezer. The general management of the sample requires careful attention, avoiding storage and/or freezing, and lean meat appears to be more susceptible than fatty meat to the oxidative process. Practical Applications: Optimized TBARs test, including sample management, may represent a line guide to its application such that the results of future studies can be comparable and successfully employed.

Effect of Meat Processing and Cooking on DNA Extraction and Detection of Meat Adulteration in Mutton Rista (Kashmiri Meat Product)

The processing and cooking of meat, during meat product preparation, affects the DNA quality and its concentration during DNA isolation. In this study, the effect of processing and cooking, during Rista preparation, on meat speciation of beef and buffalo meat in mutton Rista was studied. The study material involved three types of meat i.e. unprocessed meat, Rista emulsion and the final cooked Rista product. In each type of meat, pure meat samples of mutton, beef and buffalo meat were studied along with the adulterated mutton sample having 10% beef and 10% buffalo meat adulteration level. The meat samples were subjected to mtDNA isolation and multiplex PCR analysis. The results of this study showed that processing and cooking decreases the concentration of extracted DNAs but does not affect the detection of beef and buffalo meat in adulterated mutton Rista (unprocessed, processed and cooked) at 10% level of adulteration.

Molecular assay using PCR based technology to identify fraud and adulteration of some meat products

Meat species specification is a vital field to ensure the food safety to the consumers which corresponds to the laws relatedto meat and meat products. The adulteration of inferior quality meat into superior quality one is a common practice allover the world. In this study, 30 beef product samples including minced beef, luncheon and sausage (10 samples each)were collected from different markets in Cairo and Giza Governorate, transferred to the laboratory for detection ofadulteration of beef with pig, equine and dog meat using Simple and reliable multiplex-polymerase chain reaction(multiplex-PCR) for the partial-length of cytochrome b (cyt b) gene of mitochondrial DNA (mtDNA) which indicated thesuccessful detection of little as 0.05 ng % adulteration of meat products. Multiplex PCR can also be applied to detectauthentication with equal efficiency to fresh, cooked or putrefied mixed meat samples. The obtained results revealed thatone examined sample (10%) of minced beef was intermixed with equine meat only, while two samples (20%) of beefsausage were adulterated with addition of equine meat only. Moreover, the examined luncheon samples were free fromadulteration with either pork or equine meat. All examined samples were found to be free from adulteration with dogmeat. The bad effects of fraud and adulteration of meat products was discussed

Comparison of DNA extraction methods for meat analysis.

Preventing adulteration of meat and meat products with less desirable or objectionable meat species is important not only for economical, religious and health reasons, but also, it is important for fair trade practices, therefore, several methods for identification of meat and meat products have been developed. In the present study, ten different DNA extraction methods, including Tris-EDTA Method, a modified Cetyltrimethylammonium Bromide (CTAB) Method, Alkaline Method, Urea Method, Salt Method, Guanidinium Isothiocyanate (GuSCN) Method, Wizard Method, Qiagen Method, Zymogen Method and Genespin Method were examined to determine their relative effectiveness for extracting DNA from meat samples. The results show that the salt method is easy to perform, inexpensive and environmentally friendly. Additionally, it has the highest yield among all the isolation methods tested. We suggest this method as an alternative method for DNA isolation from meat and meat products.

Authentication of sheep (Ovis aries) and goat (Capra hircus) meat species using species-specific polymerase chain reaction

Small ruminants (sheep and goat) immensely contribute to the Indian meat food basket; yet regional preference for either sheep or goat meat varies. Owing to the differences in the market prices, these two closely resembling species meats are often misrepresented interchangeably. Prevention of such fraudulent practices requires authentic and sensitive speciation tools. A novel mitochondrial D-loop based species-specific polymerase chain reaction (PCR) technique was developed for the precise detection of origin of sheep and goat meats. Species-specific primers (SSP-G and SSP-S) developed for PCR in this study amplify 229 and 425 base pairs products specific to goat and sheep species. An additional herteroplasmic PCR product of size 350 bp was evident only in sheep. Primers designed in this study were checked for the absence of cross-species amplification in closely related animal species and was successfully applied for the authentication of a forensic case. Our results indicated prerogative applications of the novel species-specific PCR for the authentic identification of origin of sheep and goat species thereby aiding in the prevention of the menace of misrepresentation of meats.

Nitarsone, Inorganic Arsenic, and Other Arsenic Species in Turkey Meat: Exposure and Risk Assessment Based on a 2014 U.S. Market Basket Sample.

Background:Use of nitarsone, an arsenic-based poultry drug, may result in dietary exposures to inorganic arsenic (iAs) and other arsenic species. Nitarsone was withdrawn from the U.S. market in 2015, but its use in other countries may continue.Objectives:We characterized the impact of nitarsone use on arsenic species in turkey meat and arsenic exposures among turkey consumers, and we estimated cancer risk increases from consuming turkey treated with nitarsone before its 2015 U.S. withdrawal.Methods:Turkey from three cities was analyzed for total arsenic, iAs, methylarsonate (MA), dimethylarsinate, and nitarsone, which were compared across label type and month of purchase. Turkey consumption was estimated from NHANES data to estimate daily arsenic exposures for adults and children 4–30 months of age and cancer risks among adult consumers.Results:Turkey meat from conventional producers not prohibiting nitarsone use showed increased mean levels of iAs (0.64 μg/kg) and MA (5.27 μg/kg) compared with antibiotic-free and organic meat (0.39 μg/kg and 1.54 μg/kg, respectively) and meat from conventional producers prohibiting nitarsone use (0.33 μg/kg and 0.28 μg/kg, respectively). Samples with measurable nitarsone had the highest mean iAs and MA (0.92 μg/kg and 10.96 μg/kg, respectively). Nitarsone was higher in October samples than in March samples, possibly resulting from increased summer use. Based on mean iAs concentrations in samples from conventional producers with no known policy versus policies prohibiting nitarsone, estimated lifetime daily consumption by an 80-kg adult, and a recently proposed cancer slope factor, we estimated that use of nitarsone by all turkey producers would result in 3.1 additional cases of bladder or lung cancer per 1,000,000 consumers.Conclusions:Nitarsone use can expose turkey consumers to iAs and MA. The results of our study support the U.S. Food and Drug Administration’s removal of nitarsone from the U.S. market and further support its removal from the global marketplace.Citation:Nachman KE, Love DC, Baron PA, Nigra AE, Murko M, Raber G, Francesconi KA, Navas-Acien A. 2017. Nitarsone, inorganic arsenic, and other arsenic species in turkey meat: exposure and risk assessment based on a 2014 U.S. market basket sample. Environ Health Perspect 125:363–369; http://dx.doi.org/10.1289/EHP225

Molecular detection of goat and sheep meat origin using mitochondrial cytochrome b gene

The present study was conducted to discriminate between sheep and goat species meat origin utilizing mitochondrial cytochrome b (Cyt b) gene fragment. A total of 46 ear tissue and meat samples were collected from different slaughterhouses and farms of Mymensingh and Rangpur districts. Genomic DNA was extracted using GeNet Bio DNA isolation kit and DNA concentration and purity was quantified by NanoDrop spectrophotometer. Two pairs species specific primer were used to amplify Cyt b gene fragments. Selected primers were highly conserved across the breed within a species and worked well with the species of indigenous goat and sheep resulting similar size of the amplicons 330 and 585 bp respectively. The duplex PCR condition would enable to detect adulteration from goat and sheep mixed samples which revealed by two precise bands (330 and 585 bp) in a single reaction. This study suggests an accurate molecular technique for identification of sheep and goat meat species origin and differentiates species present in adulterate meat samples. In conclusion, this DNA based marker could be used for prevention of fraudulent practice in slaughterhouse and chain shops in Bangladesh.Bang. J. Anim. Sci. 2016. 45 (2): 41-45

Development and validation of short-amplicon length PCR assay for macaques meat detection under complex matrices

ABSTRACTThe macaque (Macaca fascicularis) monkeys are the third largest primate population in tropical forests. Being the potential carrier of Simian Immunodeficiency and Ebola and Corona viruses, as well as being a religiously-protected and wildlife-protected species could not save the macaques from being over-hunted and consumed. Despite having needs, methods to detect monkey species in foods are rarely documented. To fill this gap, this article describes a monkey-specific polymerase chain reaction assay targeting a short-site (120 bp) of mitochondrial d-loop gene because the short-length targets are thermodynamically more stable than the longer ones under degrading states. Specificity was tested against 17 terrestrial and aquatic meat and fish species and no cross-species amplification was detected under raw, processed, and admixed states. The sensitivity of the assay was 0.0001 ng DNA under pure states and 0.1% monkey meats in binary meat mixtures. Finally, the assay was validated by digesting the polymerase chain reaction products with AluI and CViKI-1 and distinctive restriction fingerprints for macaque species were demonstrated.

Protein markers for discrimination of meat species in raw beef, pork and poultry and their mixtures

The purpose of this study was to find discrimination markers for four major meat species such as beef, pork, chicken and duck. Myofibrillar and sarcoplasmic proteins isolated from each meat type were analyzed by one-dimensional gel electrophoresis and some proteins were identified through LC-MS/MS analysis. We confirmed that troponin I (TnI), enolase 3, l-lactate dehydrogenase (LDH) and triose-phosphate isomerase (TPI) could be useful markers for discrimination of mammals from poultry due to their different electrophoretic mobility. Tropomyosin 1 and carbonic anhydrase 3 were observed as muscle fiber type-related proteins and these could also be markers to distinguish mammals from poultry. Species-specific peptides identified by LC-MS/MS spectra allow the identification of each species regardless of the same protein. Therefore, it is easy to discriminate between mammals and poultry by comparing the electrophoretic mobility of TnI, enolase 3, LDH, TPI and CA3, and each species could be identified through LC-MS/MS analysis.

Rapid analysis and identification of meat species by laser-ablation electrospray mass spectrometry (LAESI-MS).

Laser-ablation electrospray ionization mass spectrometry (LAESI-MS) was applied to analyze fresh meat species without sample pretreatment. The study demonstrates that the LAESI-MS technique is a promising, rapid and accurate method for meat identification using a protocol combining principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). A focused IR-laser was used for meat sample ablation at a wavelength of 2940nm. The ablated particulates were carried through a transfer PTFE tube using air as carrier gas, delivered to the electrospray plume and ionized. A TOF-MS was used to detect the ion signal. The raw mass spectra were analyzed using the PCA and PLS-DA protocol. Five fresh meat samples, chicken, duck, pork, beef and mutton, were identified by the developed LAESI-MS technique using the protocol combining PCA and PLS-DA. The discrimination accuracy of all meat species is 100%, and the score plot also shows good identifying ability. Five fresh meat samples were analyzed using the LAESI-MS technique. Each set of raw mass data was collected within 30s and analyzed by the PCA and PLS-DA protocol. Eighteen, 19, 18, 17, and 15 markers for chicken, duck, pork, beef, and mutton, respectively, have been selected successfully for meat identification. The results demonstrate that LAESI-MS is a new reliable and rapid method for meat identification. Copyright © 2016 John Wiley & Sons, Ltd.