Species – specific PCR test for the quick recognition of equine tissue in raw and processed beef meat mixtures

Regardless of performing more strict food labeling regulations locally and all around, the adulteration or misrepresentation of food products to get highlights on the severity keeps to be a typical element of society (Singh & Neelam, 2011; Rahmati et al., 2016). Traditionally, meat had not been affected with corruption since meat was sold as fresh, effortlessly unmistakable (Nakyinsige et al., 2012). On many cases, with the rising costs of commercial meat, the globalization of food business and the intense processing of meat into esteem included items, the incidence of meat adulteration and fraud has become more obvious (Vandendriessche, 2008; Meira et al., 2017). Typical cases of intentional meat corruption incorporate the substitution or expansion of animal proteins (typically less expensive ones) such as donkey or horse meat not expressed all things considered in the fixing list) (Flores-Munguia et al., 2000; Wielogorska et al., 2018).


Introduction
Regardless of performing more strict food labeling regulations locally and all around, the adulteration or misrepresentation of food products to get highlights on the severity keeps to be a typical element of society (Singh & Neelam, 2011;Rahmati et al., 2016).Traditionally, meat had not been affected with corruption since meat was sold as fresh, effortlessly unmistakable (Nakyinsige et al., 2012).On many cases, with the rising costs of commercial meat, the globalization of food business and the intense processing of meat into esteem included items, the incidence of meat adulteration and fraud has become more obvious (Vandendriessche, 2008;Meira et al., 2017).Typical cases of intentional meat corruption incorporate the substitution or expansion of animal proteins (typically less expensive ones) such as donkey or horse meat not expressed all things considered in the fixing list) (Flores-Munguia et al., 2000;Wielogorska et al., 2018).
The capability to recognize questionable species in meat items is critical not only for economic, health, moral and religious reasons, but also to guarantee fair trade and commitment with related laws (Nakyinsige et al., 2012;Abbas et al., 2018).Most indicative strategies used to identify meat have relied upon the revealing of species-particular proteins or DNA (Ballin et al., 2009;Hossain et al., 2017).Today, DNA is considered to be the most appropriate particle for species identifying and distinguishing evidence in foods (Singh & Neelam, 2011).Dn contrast to proteins, is DNA mostly steady at high temperatures; so it can be analyzed in processed, degraded and mixed supplies (Lenstra, 2003;Meira et al., 2017;Abbas et al., 2018).On addition, while the discovery and identification of proteins rely on the type of the tissue examined, DNA exists and is typical in nearly all cells also the variety given by the hereditary code allows the isolation of even entirely-related species (Lockley & Bardsley, 2000;Ballin, 2010;Xiang et al., 2017).DNA-based strategies that have been genuine for species recognizable pieces of proof incorporate DNA hybridization, polymerase chain reaction (PCR), multiplex-PCR, species-specific PCR, restriction-fragment-length-polymorphism (PCR-RFLP) test, real-time PCR and PCR sequencing (Lopez-Andreo et al., 2005;Man et al., 2007;Karlsson & Holmlund, 2007;Chen et al., 2010;Xiang et al., 2017).
This study assesses a technique for equine tissue identification in meat and meat items in the Egyptian street vendors and retail markets utilizing species-specific PCR.The purpose is to build up a system with outstanding sensitivity and specificity that could be utilized as a part of routine control measures to identify undesirable meat species in beef meat products.

Meat samples
Muscle tissue samples from beef, donkey and horse were used as positive control samples in addition to meat and meat product samples which had been collected randomly from street vendors and popular retail markets of Giza governorate .Meat samples were stored at -20 °C until analyzed.

DNA extraction from meats and meat mixtures
DNA was extracted from meat samples by using GF-1 Tissue DNA Extraction Kit (Cat.no.GF-TD-050, Vivantis Technologies, Malaysia) and following the manual instruction with some modifications, where 50 mg of tissue samples were used and DNA was eluted in 50μl of preheated Elution Buffer provided in the kit.

Use of PCR for detection of fraud meat in the Egyptian market
PCR was used to test 96 meat and meat product samples which had been collected randomly from street vendors and popular retail markets of Giza governorate (24 of burger, 16 of minced meat, 24 of kofta, 16 of sausage, 7 of raw meat and 9 of luncheon).The samples stored at 4 ٥ C then transported to the laboratory and processed immediately.

Primer design
Species specific PCR primers for the amplification of bovine, horse and donkey meat were designed (Table 1).All primers were obtained from Vivantis Technologies, Malaysia.

Species-specific Polymerase Chain Reaction (PCR)
The 50-μl reaction mixture was prepared in an Eppendorf tube containing 25 μl of 2X ViRed Taq Master Mix (Cat.no.CLMM01, Vivantis Technologies, Malaysia), 20 pmol of each primer, and 5 μl of target DNA with amplification conditions as described in (Table 2).Electrophoresis was run on 1.5% agarose gel at 100 V for 2 h on a 15-μl portion of the amplified DNA products.The resulting gel was stained visualized.

Specificity of the species-specific primers
Specificity of the species-specific primers and ideal PCR conditions were intended for the identification of beef, donkey, and horse tissues in the meat samples.The primers created species particular products of 145, 153 and 271 bp for beef, donkey and   horse tissue, respectively.The specificity of each species-specific primer was ensured by PCR amplification of beef, donkey and horse genomic DNA with each primer set.These yielded PCR products just from the DNA extracted species that they were planned for, and demonstrated no cross-reactivity with the DNA from alternative species (Figure 1, 2, 3).PCR products were not gotten from negative controls with any of the species-specific primer sets.

Application of species-specific PCR assay to meat products
Testing 96 meat and meat product samples which had been gathered arbitrarily from street vendors and prominent retail markets of Giza governorate (24 of burger, 16 of minced meat, 24 of kofta, 16 of sausage, 7 of raw meat and 9 of launcheon) revealed that 6 positive for donkey tissue (3 from sausage, 2 from minced meat and 1 from kofta) (Figure 8) and 2 positive for horse tissue (from sausage) (Figure 9) as appeared in (Table 3).No cross-reaction, again inhibition or unexpected PCR products was detected in the blended tissues.samples were contaminated with donkey, goat and sheep meat, respectively, Moreover, 6%, 4% and 4% of sausage samples were contaminated with donkey, goat and sheep meat, respectively, in reverse to what was marked., Dn contrary, this was lower than that of Mousa et al. (2017) who revealed that the incidence of adulteration in luncheon, hot dog, sausage and minced meat with horse meat were 24%, 8%, 64% and 56%, respectively in Alexandria city of Egypt.
Adulteration with donkey and horse meat, in the past, had been reported by few specialists in Egypt (El-Shewy, 2007;Abd El-Nasser et al., 2010;Jaayid, 2013;Mousa et al., 2017).However contamination rate with donkey meat in our investigation is higher than that detailed in Assuit governorate in Egypt by Abd El-Nasser et al. (2010) in minced meat (7%) and sausage (8%); this might be ascribed to our stress on the street vendors where there is a powerless control on food.On Egypt, equines are not a legitimately utilized for human feeding.Ots presence affirms adulteration for profit drive point and thus gives a thought that meat has been handled under unhygienic environments constituting conceivable hazard to human health.

Conclusions
The systems applied in this study provide delicate recognition of donkey and horse tissue in beef meat and meat products without any need to add RFLP or sequencing.The streamlined strategy speaks to a legitimate PCR based technique to distinguish equine meat contaminations in fresh and processed beef meat products for quick, precise outcomes.Moreover, use of species-specific PCR does not need expensive devices such as real-time PCR analyses.Finally, this work also highlights on aggravation of the meat adulteration problem in Egypt.

Discussion
Species identification of animal's tissues in meat items is a noteworthy subject to protect consumers against any fraud or corruption for financial, religious and health reasons.The adulteration or misrepresentation of food products for more profit is a common matter all around (Shears, 2010;Doosti et al., 2014;Meira et al., 2017).Substitution with illegally utilized species is so difficult to be identified in such items by visual inspection after grinding and/or heat processing (Abd El-Nasser et al., 2010).
So there are numerous demonstrative techniques which are prepared by depending on protein and DNA investigation (Kesmen et al., 2007;Callao & Ruisanchez, 2018).Advance in DNA innovation has prompted quick development of alternative approaches for species identification (Mousa et al., 2017).
On this study, the species-particular PCR was created for the identification of donkey and horse species in common beef meat and meat products which had been gathered from street vendors and basic retails markets of Giza governorate with a single PCR reaction step.The primers created particular products of 271,145 and 153bp for beef, donkey and horse tissues, respectively as appeared in Figures 1, 2, 3.
The current extraction technique is less complicated and needs less technical requirements in comparison with one beforehand depicted by Di Pinto et al. (2005) and Hossain et al. (2017).The PCR products showed a high specificity of the PCR approach in connection to the outcomes acquired by Di Pinto et al. (2005).
On this work, target DNA was effectively distinguished for each species where intensification was not influenced by spice addition or the cooking procedure as appeared in the sausage samples results (Figures 8, 9) where comparative outcomes were found in fermented sausages (Kesmen et al., 2006).
We tried to distinguish donkey from horse meat since they are nearly associated species and have a high review of sequence homology.However, in this work, no cross-reaction was observed and species-specific PCR products of donkey and horse separated by agarose gel electrophoresis permit perfect species identification (Figures 2, 3).
However this study has exceptionally dedication and identification reach to 1I:10000 (0.01%) of donkey or horse tissue in beef meat.This was superior to that of Kesmen et al. (2007) who detailed a recognition limit of 0.1% foreign meat in the sausage samples for porcine, horse and donkey DNA.This was parallel to that of Matsunaga et al. (1999) who confirmed a recognition limit of 0.25 ng of DNA utilizing a multiplex PCR procedure of the mitochondrial cytochrome b gene from six species and 5 ng of DNA using PCR-RFLP-DHPLC analysis on the mitochondrial CDO gene as reported by Song et al. (2016).
Testing 96 meat and meat product samples which had been gathered arbitrarily from road sellers and mainstream retail markets of Giza governorate (24 of burger, 16 of minced meat, 24 of kofta, 16 of sausage, 7 of raw meat and 9 of launcheon) uncovered18.75%from sausage, 12.5% from minced meat and 4.16% from kofta for donkey and 12.5% for sausage from horse.This is higher than that of Zahran & Hagag (2015) where they reported that 5%, 3% and 4% of minced meat

Table 1 .
The primer pairs used in specific PCR identification of bovine, horse, and donkey meats.

Table 2 .
Primer cycling conditions during PCR.

Table 3 .
Number and percentage of adulterated meat samples with donkey and horse meat (total 96 samples).