IDENTIFICATION AND DIFFERENTIATION OF POULTRY MEAT AND PRODUCTS USING PCR-RFLP TECHNIQUE

The mitochondrial cyt b gene plays a serious role in investigating untruthful meat species. This study aimed to authenticate the species of poultry products (Escallop, Nugget, Steak, and Sausage) depending on cyt b gene by using universal cyt b primer. DNA was isolated, and then a band of 359 bp of a mitochondrial cytochrome b gene was produced during the PCR amplification. The PCR products were exposed to Hinf1 and Rsa I restriction enzymes. The restricted fragments produced by restriction fragment length polymorphism technique (RFLP), were run by agarose gel electrophoresis. Results showed that all products had a similar band except sausage product does not follow the rule and showed mislabeling product by the REs, Two bands were yielded by HinfI RE for all products (114 and 245) bp with the differentiated sausage among other products based on the fake product (63 and 296) bp, while digestion by Rsa I produced three bands for escallop, nugget, and steak, (63, 100, 196), but only two bands for sausage was generated (148 and 211). As result, the study offered that analyzing meat products to detect the origin species via a PCR-RFLP technique by using these restriction enzymes can give reliable results. In short sausage is considered as fraud products because the results showed different bands as compared with poultry meat.


INTRODUCTION
In most regions of the world, meat and their products intake carry on to increase and particularly in developing nations (cawthorn et al., 2013 and keyvan et al., 2017). Meat has always been eaten by people in different types, both after thermal processing or prepared in a way that requires long-term packing, such as dry sausages ….etc. These based products have become rare and expensive throughout the year, they are sold on the market at parentally excessive expenses, and for this purpose they are desirable goals for the meat commercial fraud. Furthermore, it is a critical part of the administrative problem of sustenance as tainted and substitution of meat has been a reliable concern for various reasons, for example, community health, religious aspects, future health and unwanted task in meat markets (rashid et al., 2014; hou et al., 2015 and farag et al., 2015).
In order to protect clients and avoid unfair opposition, the detection and prevention of deceptive practices requires easy and rapid research methods that are appropriate for routine applications of these products. Different approaches based on the study of species-specific components consisting of protein and DNA have been established to distinguish meat and their products originating from different species of animal and to prevent fraudulent practices. It is possible to classify organisms by analyzing proteins via various methods using immunological, chromatographic and electrophoretic methods (Kang'ethe et al., 1986;Berger et al., 1988;Zerifi et al., 1992;Amstrong andLeach, 1992 andGallardo et al., 1995). Nevertheless, even in the identical species, protein denaturation in meat during warmth treatment or other technological process and variation in protein composition decreases these methods. In addition, these techniques may be insufficient to discriminate between species that are closely related and not suitable for routine use, as it is difficult and time consuming to isolate speciesspecific protein. Moreover, these techniques can be insufficient to differentiate between species that are closely related and are not appropriate for using routinely, as the isolation of species-precise proteins is hard and time-consuming as it is tough and labor to isolate of species-specific proteins is (Hofmann, 1987;Jemmi and Schlosser, 1992and Koh et al., 1998and Kesmen et al., 2010.
Various molecular approaches have been developed to recognize origin meat species. These methods can reduce the insufficiencies of common techniques (Girish et al., 2005). PCR, AFLP, RAPD, DNA hybridization and RFLP are included in molecular markers (Rodriguez et al., 1991;Arslan et al., 2005 andAlves et al., 2002). Polymerase chain response-based methods are outstandingly fast and reliable, and presently they have proved to be a standard for meat distinguishable confirmation in the industry (Kesmen et al., 2010). Particularly mtDNA has been the foremost studied eukaryotic genomes district which has expected an essential work being created of population and developmental hereditary qualities (Abou-Hadeed et al., 2011and De Masi et al., 2015. The gene cytochrome b (cyt b) expresses one of the best-known proteins that make up the mitochondrial phosphorylation matrix comlpex III and is the only one expressed by the mitochondrial genome. The cyt b gene is used in valid drug and molecular evolution research as a critical utility tool (Prusak et al., 2004;Al-Sanjary, 2009;Abou-Hadeed et al., 2011and Farag et al., 2015. The aim of the study was to authenticate and detect commercial fraude in certain meat products such as escallop, nugget, steak and sausage obtained from different markets in Erbil-Iraq and the results will be used to compare labels written on products resulting from chicken meat, whether true or not, using the PCR-RFLP molecular technique for mitochondrial cytochrome b gene analysis, using 2 restriction enzymes (HinfI and Rsa I). These restriction enzymes have not been used for identification before in Kurdistan region-Iraq.

MATERIALS AND METHODS Sample Preparation and DNA Isolation
This study was done on meat of four types of poultry products (Escallop, Nugget, Steak and Sausage), in laboratory of molecular genetics in Salahaddin University-Erbil, college of agricultural engineering sciences and a laboratory in genome company in Erbil. Ten samples in each type were collected from different markets in Erbil governorate (mixed together to make 4 main samples as a polled sample from same origin), and chicken meat in different parts (breast, wing, leg) was taken as used as a positive control for comparisons. Meat samples of the concerned products were stored in aluminum foil at -20 °C (until the all samples were collected for about 2 weeks) for DNA extraction. DNA was extracted by using Blood-Animal-Plant DNA Preparation Kit (Spin column based genomic DNA purification, Jena Bioscience GmbH, Germany) according to manufacturer's instructions. The isolated DNA was labeled and stored at -20 °C for the next stage. The purity of DNA was checked by Nanodrop spectrophotometer (Thermo scientific UK) and gel electrophoresis.

PCR Primers
Polymerase chain Reaction (PCR) occurred utilizing a modification of the forced restriction fragment length polymorphism (RFLP) strategy. The primer sequences used for this research were showed in Table 1.

PCR Amplification
The target DNA (mtcyt b gene, forward and reverse primers) for each species was amplified by PCR (Applied Biosystems® Veriti® 96-Well Fast Thermal Cycler, USA). The final reaction volume for each one was of 25µl. The PCR component for amplification of cyt b gene is shown in Table 2. The cycling conditions consisted of an initial denaturation at 95°C for 5 min, followed by 35 cycles consisting of denaturation at 95°C for 0.30 min, annealing 50°C 1 min, and extension at 72°C for 45 sec, with final extension 72°C for 7 min then holded at 4 °C for infinity. The PCR products were screened in a 2% agarose gel containing ethidium bromide (LOT:110802BB197, Bio Basic Inc.) in Tris-borate EDTA buffer and visualized under UV transillumination (Biostep-UST-20M-8K).

RESULTS AND DISCUSSION
Mitochondrial DNA was isolated successfully and Purity of DNA ratio ranged from 1.7 to 1.9. The universal cytochrome b gene was clearly produced 359bp as shown in figure 1, with no differences for each sample when run in 2% agarose gel. Then amplicons were exposed to restriction enzymes HinfI and Rsa I, different positions of amplified DNA were cut by these enzymes. Formerly, digested fragments were separated by 3% agarose gel. The samples displayed different fragment sizes, as shown in Table 3 and Figure 2, 3, when they were imaged via UV Transilluminator and linked with the standard size ladder for comparisons.    In comparison, identification the source of meat and the products by molecular techniques offer satisfied and optimistic result over traditional morphological or protein identification and so on, for that reason DNA based methods are the best method for identifying species in both commercial foods and animal products (Ali et al., 2015;Andrea et al., 2015). Currently, meat adulteration has become a common in many poor countries, therefore it is vital to identify and confirm the commercial fraud meat and products for the public, which cannot be observed with touching or sensing organism, and discover the origin species for those products. Because it is critical for food safety, consumer demands, and law. For these using PCR based method is very trustworthy for the quality of food generally and the meat products specifically (Al-Sanjary, 2009;Farag et al., 2015, Khan et al., 2018and Dilger et al., 2020. As a result of both restriction enzymes that were done all samples separately at the same time, we can notice that the sausage was displayed mislabeling and marked as fraudulent because was not parallel to the chicken meat (positive control) and chicken meat was not used during the process of sausages. Although it was labeled as a chicken meat and there was possibly other meat species that may be replaced with the original meat. exact bands for all samples and there was no need to use statistical analysis to identify significant or nonsignificant among the samples. It is more probable that meat of two or more species is mixed for fraudulent purpose during meat processing such as grinders, cutters, knives, choppers. In order to detect traces of meat mixtures samples have to be kept for PCR-RFLP method due to is a very powerful method (Khan et al., 2018). The results were achieved in utilizing the HinfI, RsaI restriction enzymes absolutely recommended that these results were suitable to authenticity and demonstrate unreal meat source and proposed to utilize other types to detect the real sausages species.
In general, meat identifications previous Study was done based on tissues and mixed processing (Ong et al., 2007, Ghovvati et al., 2009and Ciupa et al., 2012, while in this paper focused on products in general which were commonly used for human daily nutrients, that labeled as a chicken meat, so that the obtained results were a vital and established a very effective determination in all used products by cyt b mt DNA. Moreover, this research approved that there was a satisfactory level of the aimed DNA to amplify in a PCR. Then this is a confirmatory point to settle the benefits of utilizing the mitochondrial DNA more than nuDNA (Pakendorf and Stoneking, 2005). Ayaz et al. (2006) stated that 11 of 28 (39.2%) sausage testers that were affirmed as beef were established mixing beef and poultry meat together. Also, the results were agreement with the research done by (Keyvan and coworker, 2017) that detected the adulteration with sausage products. Kesmen and collegues (2007) used species-specific primers for identification of horse, donkey, pig, beef and sheep DNA in sausage for amplification of various parts of mt DNA, and then they observed one in a hundred ng DNA in every sample.

CONCLUSION
The current study revealed that the RFLP-PCR undeniably is a reliable and prevailing method for detection species meat from Fraud Products beside that using the mitochondrial cytochrome b gene, with the help of two restriction endonucleases (HinfI and RsaI) can be very effective and powerful. Moreover, it is a rapid technique and does not need hardworking and inexpensive method. This research indicated that, the meat source that used to make sausage is not produced with chicken meat, and does not obey the public health, low as well, which offered to a public as a chicken meat product. A fraudulent product in any country is a risk and protection are a must. To keep public health and avoid fake products meat products must be frequently analyzed by quality control in governmental organizations.

ACKNOWLEGEMENTS
The authors are very grateful to the Salahaddin University-Erbil for supporting this research.

DISCLOSURE STATEMENT
The authors declare that they have no conflicts of interest.