Nested Polymerase Chain Reaction for Identification of Mitochondrial Cytochrome b Gene of Gazella Dorcas

In this study nested PCR, was developed for rapid detection of mitochondrial cytochrome b gene. Whole blood in EDTA were collected from 17 Gazella dorcas from Hilat Kuku Zoo, Elkadaru’s farm and Mozamel Elkurdi’s farm in East of the Nile, Khartoum State. The origins of these animals are Dongola, River Nile- Northern Sudan and Butana- Central Sudan. The product was sent for sequencing to Macrogen Company- World Meridian 10F, Nested PCR used DNA extracted from blood samples from Gazella dorcas by using GZ3 and GZ4 primers. The objective using this technique that nested- PCR is sensitive to detect the part of mitochondrial cytochrome- b gene of Gazella dorcas only. The primers were serially diluted (10 to 10- 6 ) for sensitivity reaction. In all dilutions, the line was clear. The size of the product was 197 bp.


Introduction
One of the most desert-adapted gazelles, dorcas gazelles may go their entire lives without drinking any water, obtaining all needed moisture from the plants which they eat [1]. They can withstand very high temperatures, although during hot weather they are primarily active at dawn, dusk, and throughout the night. Herds wander over large areas searching for food and tend to congregate in areas where recent rainfall has stimulated plant growth [1][2][3][4].
According to [5] dorcas gazelle still ranges in Algeria, Burkina Faso, Chad, Djibouti, Egypt, Eritrea, Ethiopia, Libya, Mali, Mauritania, Morocco, Niger, Somalia, Sudan, Tunisia and Western Sahara; its occurrence in Nigeria is very doubtful, while it is considered extinct in Senegal. Its geographical distribution was acquired from [6].
The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a DNA sequence. Developed [7,8]. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers containing sequences complementary to the target region along with a DNA polymerase are key components to enable selective and repeated amplification [8]. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations [8]. Nested polymerase chain reaction (Nested PCR) is a modification of polymerase chain reaction intended to reduce the contamination in products due to the amplification of unexpected primer binding sites. Polymerase Chain Reaction itself is the process used to amplify DNA samples, via a temperature-mediated DNA polymerase.
Anon (2010) [9] Nested PCR involves two sets of primers, used in two successive runs of (PCR). The second set intended to amplify a secondary target within the first run product [9]. A nested PCR has been developed by [10] to detect a 320 bp DNA segment of the gene encoding the p 57 protein. The sensitivity of the method was increased a hundred fold compared to a conventional PCR method.
[10]. The rapidity, sensitivity and specificity of the nested PCR assay would greatly facilitate detection of mitochondrial cytochrome-b gene [11]. The nested PCR yielded a DNA band of expected 101 bp size. The NS1 gene primer sequences for partial length and nested PCR used in the study were the same as used earlier [8]. The nested PCR assay provides greater specificity because it involves

American Journal of Biomedical Science & Research Am J Biomed Sci & Res
Copy@ Reem Rabie Mohammed Salih primers used to get a larger fragment, which acts as a template for second round of amplification with second set of internal primers [11]. This process provides an additional specificity to the reaction and greatly enhances the efficiency of amplification. The rapidity, sensitivity and specificity of the nested PCR assay would greatly facilitate detection of mitochondrial cytochrome-b gene [11].

Materials and Methods
Blood samples were collected from different species of animals (Gazelles, sheep, goats, cows, camels, donkeys, horses and pigs) in blood container with Ethylene diamine tetra acetic acid (EDTA), these samples were used for DNA extraction. The extraction by used QiAgen commercial kit.

Extraction of DNA from Blood Samples:
For extraction of the DNA from blood samples we used  Taq   DNA polymerase and 5 µl PCR product from first run (GZ1 and GZ2) for dorcas gazelle put in thermal-cycler tubes. All PCR amplification reaction was carried out in a final volume 50 µl.

Nested Polymerase Chain Reaction Sensitivity
To test the sensitivity of species-specific primer, serial dilution of the product from 10 µl to 10 -6 µl were subjected to the reaction

Discussion
Lack of detailed information about threatened groups of animals can hamper conservation efforts [12,13]. Animal or meat species identification has been developed to address different concerns. Authentication of food ingredients is important for consumers because of food fraud was spread. The traceability of meat component in food improves consumer's confidence in food products. The substitution of expensive meat with cheaper one is a major concern. For some consumer groups, such as vegetarians, the contamination of food with meat residue is strictly prohibited.
Another good example of meat identification is the Halal food for the Muslim consumers, who are prohibited from consuming pork.
In this study we used the tissues and cooked meat from Gazella dorcas to detect (mtcy-b gene) using PCR and this in accord with [14][15][16][17][18] findings.
In this study we developed nested-PCR method for very low DNA content products like blood for detection of mitochondrial cytochrome-b gene in Gazella dorcas in Sudan to identify authentic blood, especially from expensive dorcas gazelle's products because the technique was more sensitive, easier, and faster than the conventional PCR. The result of nested PCR in this study is in agreement with [11,19,20] findings.

Results
The results of this study identified mtcyt-b gene in blood from