Kit for Molecular Detection of Pork Adulteration

Technical Field

[0001 ] Food safety

Background Art

[0002] At present, modern methods for the identification of pork adulteration are

spectroscopic, immunoassay, electromagnetic wave method which these methods available on the market offers the disadvantage of expensive equipment, high production cost, sophisticated working method, low accuracy, low detection speed[1 ]. The most important type of immunoassay is ELISA, but the important thing about this method is that heating changes the protein structure in some of these methods and causes protein denaturation and change the electrophoretic properties of the compound while cross-reaction between the samples may occur.

Summary of Invention

Technical Problem

[0003] Food quality and safety are directly related to human survival and health.

Meat types such as sheep, beef, chicken are mainly used in the community and therefore the use of cheap meats such as pork instead of these meats has increased dramatically. With the rapid expansion of the meat market and frequent fraud in meat content, it is not only economically and religiously but also more likely to harm consumers and other serious consequence. Effective identification of meat adulteration technology is one of the major issues in meat safety that society and organizations need. It therefore requires a high-quality, precise method to produce kits for meat health inspectors to detect pork content in other meat at the site in question. Solution to Problem

[0004] In the present invention, PCR products from pork, beef, sheep are

electrophoresed on agarose gel and characterized by UV irradiation. This method is highly specific for identifying meat products containing pork and is useful for cooked meat products that proteins are denatured during heat, while the ELISA method is not useful for this purpose. In addition, the PCR method is more sensitive than other methods.

Advantageous Effects of Invention

[0005] In the present invention, the DNA extraction kit is capable of isolating

mitochondrial DNA with genomic DNA. Primer design was performed to detect mitochondrial DNA, which makes it possible to identify pork from other meats in processed foods that are destroyed by heat of genomic DNA. In this invention, the process of identification non-halal meat from the halal is carried out using conventional PCR with high sensitivity and specificity and we can specifically identify three gene regions at the pig genome level using a primer pair compared to other meats species. The present method is much lower cost and easier to use than real time PCR.

[0006]

Brief Description of Drawings

[0007] [Figure 1 ] Spectrogram of purified DNA from Gorgan pork (G) and Zirab (Z) at 1 :10 and 1 : 100 concentrations

[0008] [Figure 2] Selecting annealing temperature of pork myoglobin primers in the Primer-BLAST program for the PCR process

[0009] [Figure 3] Image of gel electrophoresis of mixture of beef and pork DNA with different percentages

[0010] [Figure 4] Image of gel electrophoresis of mixture of sheep and pork DNA with different percentage

[0011 ] [Figure 5] Image of gel electrophoresis obtained from a mixture of cooked sheep and pork samples with different percentages [0012] [Figure 6] Image of gel electrophoresis obtained from a mixture of cooked beef and pork samples with different percentages

Description of Embodiments

[0013] DNA Purification

[0014] 20 mg of animal tissue was added to 1.5 ml microtube . 150ul TE buffer and 80ul (1 % SDS) were added to two microtubes and two microtubes were vertexed. Protein kinase k added to the microscope and vortexed and then incubated at 55- 60 °C for 60 min(lncrease the time to 3 hours for better incubation). During incubation the tubes were inverted three or four times every 10 minutes. 100 pl _¬ ot NaCI 5N solution added and mixed well. 80 pL of CTAB / NaCI buffer was added and incubated at 65 °C for 10 minutes .700 pL of phenol was added and immediately vortexed for 30 seconds. Then The microtubes were centrifuged at 14,000 g for 8 minutes and the supernatant were transferred to a 1.5 ml_ tubes and repeated again (14000g centrifuge at 8 minutes). Then three phases were observed the highest phase Separated and was transfered to the new microtube and 420 pL of cold absolute ethanol was added and it was slowly inverted few times and so that a white cloud is forming. The tubes were transferred to -20 ° C for 15 minutes and then centrifuged at 16,000 g for 15 minutes and the

supernatant was discarded carefully. Then the supernatant was discarded and The precipitate formed was separated at the end of the microtube and 1 ml of 85% ethanol was added to the precipitate and gently inverted three times and then centrifuged at 16,000 g for 5 minutes. The microtube was exited from the apparatus and the supernatant was carefully discarded and the pellet was placed in open air for 10 min to evaporate the alcohol.

[0015] UV-VISIBIE Spectroscopy

[0016] 225 pi of distilled water was dropped to 1.5 ml microtube containing 2 pi of each previously purified DNA and vortexed to dilute to 1 /10. Again, 25 ul of the 1 / 10th microtubes were poured into other microtubes, and 225ml of distilled water was added to dilute 1/ 100th. After calibration of distilled water, 200 ul of the sample was poured into the cuvette and read absorbance, and this was done for other samples as well. According to the obtained adsorption, the concentration of each sample was calculated by formula.

[0017] Nanodrop apparatus

[0018] Four microtubes were named. G3-.01 , G3-.1 , Z3-.01 , Z3-.1. First, 100 mI of distilled water was poured into four microtubes, then 10 mI of distilled water was extracted from Z3-.1 and G3-.1 microtubes and Instead of, 10 microliters of the corresponding DNA was added. Then 1 mI_ of distilled water was removed from Z3-0.01 and G3-0.01 and 1 mI_ of the corresponding DNA was added. DNA and cap 10 were selected in the nano-drop device. A drop of distilled water was poured on Flelmacell and then 10 caps were placed on it and the distilled water was calibrated. A drop of Z3-.01 was poured on Flelmacell, then 10 caps were placed on it and the numbers were recorded on the device then removed and the above steps performed on G3-.01 , G3-.1 , Z3-.1 and the numbers were recorded. Subsequently, Cop50 was replaced on the Flelmacell instead of Cop10 and calibration was performed. Therefore, a drop of G3-.01 , Z3-.1 , Z3-.01 , G3-.1 was again poured onto the helma cell, and 50 caps were placed on it and the device showed absorption of each samples.

[0019] PCR Master Mix

[0020] The kit consists of an optimized buffer, PCR enzyme, dNTP compound, gel loading dye, and when run on agarose gel, it is separated into blue and yellow permix simplifies the assembly of the PCR by only adding primers, templates and water and the resulting product can be directly applied to a gel.

[0021] Preparation of TAE 10X gel electrophoresis

[0022] The TAE components include Trise base =12.1 gr ,EDTA=1.86 gr, Acetic acid=2.85 ml, d H20,. The electrophoresis buffer is made up to 250 mI. First, Tris and EDTA powders were weighed in balloons and poured into balloons. Then the acetic acid solution was added to the balloon and the remaining volume of the balloon was filled to 250 ml with distilled water and the lid was parafilm and and shaken well on the table to allow the powders to dissolve well. The solution was then poured into a 250 ml_ glass jar and placed in the refrigerator.

[0023] Preparation of agarose gel [0024] .5 g of agarose powder was filled to 50 ml with TAE buffer and placed over the heater to make it transparent. After cooling, 5 microliters of electrophoresis dye was added and the contents of Erlenmeyer were completely poured into the cassette and waited for the gel to form.

[0025] Power Supply device

[0026] A power supply is an electrical device that used as the source of electrical energy required for one or more times electrical load. After amplification of PCR, amplified samples should be inserted into the gel. First, the gel was put into the tanker with the cassette, and the TAE 1 X buffer was poured into the tanker to completely cover the gel. From left to right, the gen ruller, Blank and then DNA samples were added to the gel wells in the same manner as PCR. Then, positive and negative electrodes were attached to the tanker. Before turning on the Power Supply device, first set both current and voltage screws to zero and then switch on. The current was maximized and then the voltage was raised to 140 volts and took 30 minutes.

Examples

[0027] PCR and Gel Electrophoresis Process of cooked Beef and sheep Samples and Pork of Gorgan and Zirab

[0028] Premix was prepared for six microtubes with different DNA Blank, Gorgan, Zirab, Beef, Sheep, G '(non-cocked) and a volume of 2 mI_ of DNA.

G' DNA belongs to unprocessed Gorgan meat purified with phenol as a positive control kit. Six microtubes named Blank, G, Z, B, Sh, G' were prepared and prepared in Blank Permix microtube. First, 65 mI_ of master mix and then 46.2 mI_ of dFI20 followed by 3.3 mI_ of each of the rivers and forward primers were poured into the Blank microtube and then, 18 mI of permix was poured into each of the other five microtubes and 2ul of distilled water was poured into the blanks and the lid was closed. From left to right six microtubes of Blank, G, Z, B, Sh, G', respectively were placed in two rows, at PCR device and the per process was performed at a single annealing temperature of 54.9 °C and with 30 cycles. On this occasion, gel electrophoresis was prepared and placed in a tanker and then the buffer was added. After PCR, from the left to the right, 10 mI of genruler, G, Z, B, Sh, G' were poured into gel wells, respectively. Then turn on the Powersupply device and set the voltage to 140 and wait for 1 hours and 30 minutes for the DNA until the DNA reaches the middle of the gel. Finally, the gel was transferred to the gel duck divice and the photograph was examined.

[0029] PCR and gel electrophoresis of cocked pork mixed with beef

1»>B 100%+P 0% »> 2 ul+0 ul

2»>B 80%+P 20% »> 1.5 ul+.5 ul

3»>B 60%+P 40% »> 1.2 ul+.8 ul

4»>B 40%+P 60% »> .8 ul+1.2 ul

5»>B 20%+P 80% »> .5 ul+1.5 ul

6»>B 0%+P 100% »> 0 ul+2 ul

Permix was prepared for 7 microtubes Blank, BP1 ,BP2,BP3,BP4,BP5,BP6 and a volume of 2 mI_ of DNA.

seven microtubes named BLANK, BP1 ,BP2,BP3,BP4,BP5,BP6 were prepared and prepared in Blank Permix microyubes. First, 77 pL of master mix and then 53.9 pL of dH20 followed by 3.85 pL of each of the rivers and forward primers were poured into the Blank microtube and then, 18 pi of permix was poured into each of the other sex microtubes and 2ul of distilled water was poured into the blanks and the lid was closed then the DNA of cooked beef and pork were added according to their percentage per microtube to reach a total volume of 20 pi. The microtubes were incubated in the PCR device at a single annealing temperature of 54.9 °C for 30 cycles and the PCR process was performed. On this occasion, gel electrophoresis was prepared and placed in a tanker and then the buffer was added. After PCR, from the left to the right, 10 pi of Gene ruler, blank,

BP1 ,BP2,BP3,BP4,BP5,BP6 were poured into gel wells, respectively. Then turn on the power supply device and set the voltage to 140 and wait for 1 hours and 30 minutes for the DNA until the DNA reaches the middle of the gel. Finally, the gel was transferred to the gel duck device and the photograph was examined.

Industrial Applicability

This kit could be used and commercialized for identification of meat adulteration with pig in Muslim and Buddhism countries.

Citation List

[1 ] L. Flongjiang et al.,“Rapid detection kit for pork, beef and mutton and

preparation method of kit,” CN106596955A, 2016.

[2] S. Rahmati, N. M. Julkapli, W. A. Yehye, and W. J. Basirun,“Identification of meat origin in food products-A review,” Food Control, vol. 68, pp. 379-390, 2016.

[3] J. Qinqin,“Rapid detection method for lead content of halal beef and mutton,” CN106338510A, 2016.

[4] T. Rezazadeh, K. Aghaiypour, and Z. Heidari,“Significance of authenticity in meat and meat products in Iran,” vol. 1 , no. 2, pp. 83-88, 2014.