Identification of species-specific molecular markers in different farm animals by PCR-RFLP analysis

Meat verification is relevant for public health and economic concerns. The present study identified species origin of raw meat samples of buffalo, cow, sheep, goat and chicken using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) of a mitochondrial cytochrome b gene (359 bp). The amplified PCR products were digested using restriction enzymes Tas1 and Hinf1. DNA fragments of different lengths cleaved by two different enzymes were obtained. Each animal species had a distinctive combination of restriction fragments. PCR-RFLP analysis was performed by resolving digested products through agarose gel electrophoresis. Lengths of most of DNA fragments obtained for different farm animals were same as expected with few exceptions. PCR fragment obtained for buffalo remained uncut by enzyme HinfI. In short, PCR-RFLP method was successfully applied to detect the origin of meat species. This method is simple, quick and reliable for differentiation of meat species. The restriction patterns of cytochrome b are helpful in the discrimination of meat identification in a blind fashion.


Introduction
Meat adulteration is a common practice during preparation of meat products in many countries and causes serious health, economic and religious concerns [1].The identification of meat adulteration in meat products is very important for the implementation of labeling legislation and unfair competition prevention [2].Different identification methods being currently used for species origin detection in raw meat include hair histology, sensory analysis, immune sera diffusion and electrophoresis in ager gel, fat tissues properties, glycogen level in muscle tissue, DNA hybridization and anatomical differences [3].However, these methods have some limitations in their use such as complexity, difficulty, specificity, high cost and inadequacy to differentiate closely related species A ratio between 1.7 and 1.9 indicates a very high purity of DNA.

AGAATGATATTTGTCCTCA-3') as presented earlier by Carr and Marshall [13].
These primers amplified a 359-bp DNA fragment.These primers were designed from the previously developed data of Mitochondrial Cytochrome b genes of Cow, Buffalo, Sheep, Goat and Chicken [14].PCR optimization A precise region of the DNA fiber (target DNA) is amplified by PCR.Amplification of the target DNA (Mitochondrial Cytochrome B gene) was carried out for each species with the help of forward and reverse primers.Each tube contained 2 µL of target DNA, 2.5 µL of 10× PCR buffer, 2.5 µL of MgCl2, 2 µL of deoxynucleotide triphosphate (dNTPs), 0.25 µL of Taq DNA polymerase and 0.5 µL of each primer (forward and reverse primer).Initial denaturation was carried out at 95 °C for 5 minutes followed by 35 cycles of denaturation 94 °C for 30 seconds, annealing of primers at 45 °C for 30 seconds and extension at 72 °C 45 seconds.A final extension step was given at 72 °C for 5 minutes followed by hold at 4 °C for infinity.

RFLP analysis
Products of amplification obtained from PCR were digested with the help of restriction enzymes.Restriction enzymes used were Tas1 and Hinf1; 30 µL of reaction mixture was prepared by adding 10 ul amplified PCR product, 1 ul of each restriction enzyme, 2 µL buffer and adjustable amount of sterile distilled water.The mixture was allowed to incubate at 37°C for 5 hours.After incubation the digested products were analyzed on agarose gel electrophoresis.

Analysis of PCR products
Different bands of DNA were seen under the UV light.DNA bands for five different species digested by two different enzymes (TasI and HinfI) were analyzed.A 50 base pair DNA ladder was used to compare the size of DNA fragments.

RFLP analysis
Cytochrome b gene amplicons were digested by two restriction enzymes TasI and HinfI.Both enzymes cut the gene at different points.
Thus, DNA fragments of various sizes were produced by the two enzymes.The detail of RFLP of five species is given below and can also be seen in (Table 1 & Figure 1).

Gallus gallus domesticus (Chicken)
TasI: The larger fragment produced was of 335 bp while small one was only of 24 bp.HinfI: Two fragments of moderate length 188 bp and 161 bp were generated and a very small one having only 10 bp was obtained.

Conclusion
The present study reveals the authenticity of species identification by the amplification of mitochondrial Cytochrome b gene followed by restriction fragment length polymorphism.359 bp Cytochrome b gene region of each species was amplified by PCR.Then it was cut with two restriction enzymes and agarose gel electrophoresis was performed.Each animal species had a distinctive combination of restriction fragments.Inferred and expected values for fragment lengths obtained by the digestion with TasI restriction enzyme were same for all of the five species under investigation except for one species Bubalus bubalis (Buffalo) where expected fragments were of 161 and 198 bp while three fragments of 161, 155 and 65 bp were obtained.It was noticeable that the buffalo gene remained un-cleaved by the restriction enzyme HinfI.Another controversy in the inferred and expected values of fragment lengths was observed in case of Ovis aries (Sheep) DNA fragment treated by the restriction enzyme HinfI.Expected fragment lengths were 63, 296 bp whereas two fragments of 161 and 198 bp were obtained.The results show that PCR-RLFP is a simple, quick and reliable method for verification of species.This method is equally applicable for animal breeding and protection of biodiversity.