Validation of Bovine DNA Using Spesific Targeting on Cytocrome-B 
By Real-Time Polymerase Chain Reaction Method

 Nina Salamah1,4, Yuny Erwanto2,3, Sudibyo Martono1 and Abdul Rohman1,3
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia.
2Division of Animal Products Technology, Faculty of Animal Science, Universitas Gadjah Mada, Jl. Fauna No. 3, Bulaksumur, Yogyakarta 55281, Indonesia.
3Research Centre of Halal Products, Universitas Gadjah Mada, Jl. Kaliurang Km 4, Sekip, Yogyakarta 55281, Indonesia.
4Department of Analytical Chemistry, Faculty of Pharmacy, University of Ahmad Dahlan, Jl.Prof Soepomo, Janturan Yogyakarta, Indonesia

Submitted :........................	Reviewed :..........................	Accepted:.....................

ABSTRACT 

Specific primer design is an interesting research topic for the analysis of identification using real-time PCR selectively, specifically, and effectively. This research is very important about the method of analyzing bovine DNA identification using real-time PCR with specific primers in various products to issue halal status. The bovine DNA primer is designed using the NCBI and Primer-BLAST software. Validation parameters performed are specifications,  repeatability test and linearity by real-time PCR. Analysis for the primer specificity performed on fresh tissue (pork and negative control). Repeatability test is done with six replications, and the coefficient of variation (CV) value is calculated. Linearity is carried out at six different DNA concentrations (50000, 10000, 5000, 500, 100, and 50 pg/µL)  to get Efficiency value. The method is expected to specific, sensitive and reliable for the analysis of bovine DNA. The results show that the real-time PCR with primer Cytochrome B specifically able to identify the presence of porcine DNA at optimum annealing temperature 58,70C. The coefficient of variation (CV) on repeatability analysis was 0,57 % and the efficiency value on linearity analysis was 206 %. Real-time PCR using Cytochrome B bovine primer (forward: CTACTGACACTCACATGAATTGG; reverse CACTAGGATGAGGAGAAAGTATAGG) can be used for identification of bovine DNA. 

Keywords: primer, Cytochrome-B, bovine DNA, specific, real-time PCR

.
Corresponding author:
Abdul Rohman, 
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada,
Research Centre of Halal Products, Universitas Gadjah Mada,
Jl. Kaliurang Km 4, Sekip, Yogyakarta 55281, Indonesia,
Email: abdulkimfar@gmail.com
No Hp: 087838445216

INTRODUCTION 
For Muslims, the safety of a product is not enough without being accompanied by its halal status. All Muslims believe that halal is the highest quality of a food product, including safety. For Muslims, a product is not safe if it is not halal. The researchers are always trying to obtain and or develop analytical methods that can detect and even quantify the presence of non-halal components in food products. More importantly the necessity of certainty that the food products are derived from halal materials, one of which is from the raw material of cattle.
Identification of meat in food products lately using DNA-based analysis methods. The method provides advantages because it is fast, specific, and DNA is more stable even though it has undergone processing (Iwobi et al., 2012). The target of this research is to obtain a valid and reliable analysis method for the analysis of the presence of beef gelatin through specific primary analysis in food products to ensure the halalness of pharmaceutical products.
Real-time PCR when compared to conventional PCR, provides a more sensitive, simpler and faster analysis. This method is the development of the PCR method with the results of amplification can be directly observed and analyzed quantitatively by using DNA dyes and fluorescent trackers (Sudjadi, 2008). Another use of real-time PCR is for the detection of single nucleotide polymorphisms and accurate determination of melting temperature based on the results of real-time PCR curves. The use of real-time PCR for analysis based on molecular biology has been developed, especially for halal analysis. Examples of using real-time PCR for halal analysis are pork meat analysis (Himawati, 2013; Martin et al., 2009; Soares et al., 2013; Taufiq, 2014). In general, PCR real-time analysis is divided into three parts, namely the exponential phase, the linear phase, and the flat phase. In the exponential phase, it is possible to obtain PCR amplification products with efficiencies close to 100%, which means DNA duplication occurs in that phase. The magnitude of the efficiency value depends on the PCR condition, the primary characteristics, the purity of the DNA print and the length of the amplification product. PCR results can be analyzed by the correlation method between the values ​​of Cq / Ct with concentration. The correlation obtained will be better when using the Cq value with the logarithm of concentration because a linear and significant correlation is obtained at each concentration. PCR data quality is said to be good if the efficiency value is close to 100% (Yuan et al., 2006).
At this time, a lot of software can be used to facilitate researchers in designing primers. The existence of this software makes it easy for researchers to design primers by modifying the length of the primer, the length of the product of DNA amplification, and the GC content according to the research needs. The use of the program is also integrated with the DNA sequence database so that after the primer is designed, it can be seen the specificity of the target DNA and any mismatches that may arise. Besides the advantage of the program is that researchers can also estimate whether primers that have been arranged are attached to the DNA sequences of other species or are specific to the target DNA. Primary specificity is an important thing that needs to be considered in the PCR technique, so the specific primary design is crucial to the success of the analysis by the PCR method (Sudjadi, 2008).
One of the goals of this research is to obtain a specific primer on bovine mitochondrial DNA for DNA testing in real-time PCR.
 
MATERIALS AND METHOD 

Materials
The primer was designed with software from the NCBI website and subsequently ordered from PT Genetika. The solvents used are chloroform, ethanol, phenol, isoamyl alcohol, and other solvents purchased from E.Merck (Darmstadt, Germany). Sterile aqua from Ikapharmindo (Indonesia). TBE 5x buffer is made using tris base, boric acid, and EDTA (Merck). Loading buffers were made using 0.25% bromophenol blue, glycerol 30% (Merck), and TE buffer. Manufacture of TE buffer using 10 mM Tris HCl pH 8 and 1 mM EDTA (Merck).
The research instruments used were blender (Maspion), micropipette (pipetman, Gilson, France), centrifuge sorvall legend micro 17R (Thermo Scientific, USA), mini transluminator (Biorad), sensitivity analytical balance 0.1 mg (Mettler toledo AB204-5 , USA), real-time PCR CFX 96 (Biorad, USA), EFM 60 shaker (OSK, Seiwariko Co., Ltd), water bath (Haake L Fisons, Karlsruhe, Germany), Thermocycler (BioRad, USA), and tools glassware which is commonly used in analyzes found at LPPT-UGM.

Methods
The stages of the research to be carried out are as follows:
1. Primary design
The primary design for wild boar is done using NCBI-Primer BLAST software by entering the desired criteria data. Primers obtained from the NCBI software are then confirmed their specificity for domesticated pigs (Sus scrofa domestica, taxid: 9825)
2. DNA isolation and purity test results of the isolation
DNA isolation was carried out by grinding 200 mg of each meat sample with a mortar and then inserted into a 2 mL microtube. A warm lysis buffer was added as much as 700 μL to it and 10 μL proteinase-K was added. Then the mixture is homogenized with a vortex device. The mixture was incubated in a water bath at 650C for 2 hours. After incubation, the mixture is vortexed until it is destroyed. 1: 1 volume of phenol-KIAA solution was added to the microtube. The mixture is shaken with a shaker for 30 minutes. Samples were centrifuged at a speed of 12,000 rpm for 5 minutes. In the sample two layers will be obtained. The supernatant is taken and put in a new microtube and then added 2-propanol with a volume of 1: 1. The mixture was incubated in the freezer for 15 hours then centrifuged again at a speed of 12,000 rpm for 5 minutes. DNA will precipitate and is seen at the bottom of the microtube. The supernatant from centrifugation results is then discarded. 500 µl of 70% ethanol solution was added in a microtube and centrifuged at 12,000 rpm for 5 minutes. After centrifugation, the supernatant is completely removed and dried to dry so that DNA pellets form at the bottom of the tube. The DNA pellets were dissolved in a TE 100 µL buffer, homogenized, and incubated at 50 ° C. The isolated sample is then stored in the freezer.
The results of DNA isolation were analyzed for purity by measuring the absorbance of a 20 µL DNA solution in 980 µL sterile aqua at λ 260 nm and 280 nm. DNA concentration was calculated from the absorbance value at λ 260 nm multiplied by the dilution factor and the absorption constant (50 µg / mL).
3. Real-Time PCR Analysis
The real-time PCR analysis was performed using a reaction mixture (20 µL) consisting of 10 µL SYBR Green® universal PCR master mix, 1 µL forward and reverse primer, 1 µL DNA template (50 ng) and 7 µL free nuclease water. The temperature program used is 95 ° C for 30 seconds followed by 30 cycles with a temperature of 95 ° C for 5 seconds for the denaturation stage. The next step is primary attachment at the optimized primary attachment temperature for 10 seconds and 72 ° C for 10 seconds for the advanced stage (extension). The melting curve is carried out at a temperature of 60-90 ° C with a slope of 0.5 ° C / 2 seconds (Himawati, 2013).
4. Optimization of Primary Attachment Temperature
      Optimization of the primary attachment temperature that has been designed for rat DNA was carried out in the temperature range of 52 to 62 ° C using real-time PCR (Bio-Rad, 2006).
5. Primary Specificity Testing
      Primary specificity was confirmed by amplifying 50ng / µL DNA of cattle, pigs and negative controls (without DNA) called NTC (No Template Control)
6. Repeatability Method
      The repeatability of the method was assessed based on the coefficient of variation (CV) of 6 times the replication of DNA amplification from the results of DNA isolation of fresh beef with a concentration of 50 ng / µL.

Data Analysis
	Data analysis for real-time PCR was carried out with a standard curve created by comparing the value of Cq (Quantification circle) against the log value of DNA concentration. Efficiency (E) real-time PCR calculated according to the formula: E = (10 (-1 / slope) - 1) x 100 (Bio-Rad, 2006).

Result and Discussion 
The primary design is the initial stages of the PCR real-time analysis method. In this study, the primary design aims to obtain a specific primary arrangement of bovine mitochondrial DNA (Bos taurus). Sudjadi (2008) states that the determining factor for the success of PCR is the use of primers that are specific to the target so that specific primers are needed to obtain optimal results. Mitochondrial DNA can be targeted for genes or molds in the PCR process because it has many advantages including mitochondrial DNA which is more stable under various conditions during the production process, mitochondrial DNA has a very large number of copies in the cell, and the number of samples needed is small. With these advantages, PCR analysis using mitochondrial DNA as a target is a relatively cheaper, simpler and more sensitive method (Nuraini et al., 2012).
The use of mitochondrial DNA as a target has been widely used in research with both conventional PCR and real-time PCR. Aida (2005), Rahman (2014), and Camma (2012) have developed the PCR method with mitochondrial DNA as a target sequence. Mohamad et al. (2013) stated that the template used in real time-PCR whether mitochondrial DNA or core DNA will affect the sensitivity and specificity of the analysis method.
The method used in the detection of PCR amplification results is SYBR Green. SYBR Green is a fluorosens binding agent with double-stranded DNA that can be detected at λexitation = 498 nm and λemission = 522 nm and the intensity of fluorescence that arises, read by the detector tool. The advantage of SYBR Green compared to other detectors is its universal use in various prints because of its non-specific nature so that it can bind to any double-stranded DNA; cheaper; simpler to use and stable to temperature and does not interfere with DNA Polymerase (McPherson and Simon, 2006)
The primary design is carried out with the NCBI-Primary BLAST software available on the NCBI-Primary BLAST website. The target organism used in the primary design for this study was Bos taurus in the cytochrome b gene region with the GenBank code AF492351. Primary candidates obtained from the primary design with the software carried out by BLAST towards other organisms. Based on preliminary testing of the primary design in silico obtained a pair of primers that will be used in this study as shown in Table I. 

Table 1. The results of the primary design of the mitochondrial DNA Cytochrome B Bos Taurus
Primer	Length	Tm	%GC	Product Length	
Forward	CTACTGACACTCACATGAATTGG	23	57,21	43.48	99 bp	
Reverse	CACTAGGATGAGGAGAAAGTATAGG	25	57,44	44.00		

		The results of the quantitative analysis of DNA isolates with a spectrophotometer (Table II) showed the results of the A260 / A260yang ratio. Clark (2005) states that pure DNA has a ratio of 1.8. Ratios less than 1.8 indicate protein contamination, while ratios of more than 1.8 indicate RNA contamination. The results of the quantitative analysis showed that the results of isolation included imperfect washing so that the DNA obtained was not pure. Codex Alimentarius Commission (2010) states that high DNA purity allows optimal amplification that affects the validity of a PCR method.
Table II. The results of quantitative analysis of meat isolates by spectrophotometer

No	Types of Meat	consentration (µg/mL)	Rasio A260/A280	
1	Porcine	1952,68	2,00	
2	Bovine	1897,80	1,50	

A primer that has been designed as a theoretical Tm based on the GC base content of the primer. Muladno (2010) states that the primary attachment temperature usually ranges from 50 - 60 ° C or approximately 5 ° C below Tm. However, temperature optimization is needed to obtain optimal PCR results that are specific to bovine DNA. Temperature optimization is carried out with bovine and pig DNA targets in the primary attachment temperature range of 53.6 - 59.0 ° C based on the estimated Tm of the primers that have been designed (Table III).

Table III. The results of optimizing the temperature of DNA primer bovine annealing
Optimation Temperatur	Cq	Melt Temperature	Peak Height	
58.7	26,03	77,00	666,82	
59.0	26,01	77,00	603,13	
53.6	26,03	77,00	635,55	
55.0	26,11	77,00	608,09	
The results of this annealing temperature optimization can be seen in Figure 1. Based on these data, it is known that the exact annealing temperature used in the PCR real-time test protocol is a temperature of 58.70C. Because at that temperature, the maximum amplification results and the highest melting temperature among other optimization temperatures.
  
(A)	                                                                       (B)
Figure 1. Results of amplification bovine DNA optimization of primary anealing temperature 
(A)	Cycles (B) Melting temperature

Primary specificity is an important thing that needs to be considered in the PCR technique, so the specific primary design is crucial to the success of the analysis by the PCR method (Sudjadi, 2008). Specific primers are primers which only amplify the target DNA and do not amplify the DNA of other species. In this study, primary DNA specificity testing was only carried out on pig DNA because this method was an initiation method for testing gelatin samples where the available gelatin on the market mostly came from pigs and cows only. This specification test is also carried out on NTC, which is a negative control, so it has no DNA content. Based on the results of the specifications in Figure 2 it appears that only bovine DNA is amplified whereas NTC and pig DNA is not amplified. This result can be said that the primer used is specific.

(A)	                                                                        (B)
Figure 2. The results of the primary amplification (A) values of Cq: 28.22 (B) Tm: 77.00

The method repeatability test was carried out by looking at the results of the amplification of rat meatball samples with a concentration of 100% and the amplification of rat meat that was replicated six times to see the consistency of real-time PCR results (Figure 3 and Table IV)). The Relative Standard Deviation (RSD) or CV of Cq is the basis for making decisions to determine the consistency of the method used. Codex Alimentarius Commission (2010) states that the acceptability of the variation coefficient (CV) of repeatability for PCR is 25%. The CV value of Cq from beef amplification with a concentration of 50 ng / µL was 1.91% while the CV value of Cq from rat meat amplification was 1.01%. These results indicate that the method meets the repeatability requirements.

(A)	                                                                               (B)
Figure 3. DNA repeatability amplification results from beef isolation DNA concentration of 50 ng / µL
(A)	Cycles (B) Melting Curve)

Table IV. The results of Bovine DNA repeatability 
Sample	Cq	Tm	Peak Height	average Cq	SD	CV (%)	

Bovine DNA concentration 50 (ng/ µL)	28.59
28.22
28.19
28.26
28.17
28.17	77.00
77.00
77.00
77.00
77.00
77.00	647.04
778.70
780.05
721.29
696.51
653.12	

28.27	

0.1621	

0.57	

Linear regression equations are also obtained from the standard curve of amplification reaction to DNA isolates from beef (Figures 4 and 5), with R2 values 0.966, slope -2.059, and y-intercept 28.830, so that the curves meet the linearity criteria quite well.

Figure 4. The results of Bovine DNA amplification at various concentrations

Figure 5. The Result of Standard curves of bovine DNA amplification at various concentrations.

The standard curve between log beef concentration and Cq (Figure 5) gives an efficiency result (E) of 206.0%. These values ​​indicate imperfect results due to high-efficiency values. Bio-Rad (2006) states that the results of PCR amplification are stated to be good if the efficiency value is close to 100% (90-105%) and the value of R2> 0.980. Amplification efficiency values ​​that are more than 100% can be caused by less than optimal thermocycling conditions so that the reactions that occur in PCR components are less than optimal (Life Technologies, 2012). Efficiency values ​​can be influenced by several factors, among others, the presence of inhibitors in the master mix PCR, the purity of the reagents used, inconsistencies in the small volume piping process which results in low accuracy, and are also influenced by the testing ability which depends on the primary specificity and target sequence length of the Printed DNA (Svec et al., 2015; Muhammed et al., 2015). Efficiency values ​​that are too large indicate that the target DNA duplicated each cycle is disturbed by the presence of an inhibitor during the reaction, while too small efficiency values ​​indicate imperfect primary attachment, both of which result in template amplification in each cycle being less exponential.

CONCLUSION 
	Primary pairs 5 '- CTACTGACACACACACGAGAATTGG - 3' (forward) and 5 '- CACTAGGATGAGGAGAAAGTATAGG - 3' (reverse) from the cytochrome B area of bovine mitochondria (Bos taurus) are used specifically in the analysis of bovine DNA in real-time PCR with an optimum attachment temperature of 58.7 ° C between pig DNA, so it can be used for halal gelatin analysis methods.

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