WO2016028138A1 - A method of detecting porcine markers for identifying pork content in a sample - Google Patents

Abstract

The present invention relates to a method of detecting porcine markers for identifying a pork content in a test sample, comprising the steps of: extracting deoxyribonucleic acid (DNA) from the sample; performing polymerase chain reaction (PCR) on the extracted DNA using two pairs of primers, including a first forward primer and a first reverse primer for amplifying a target porcine DNA sequence, and a second forward primer and a second reverse primer (as an internal control) for amplifying a target gene sequence; then subjecting the amplified sequence to electrophoresis to detect the presence of porcine markers in the sample; characterized in that: the first forward primer comprises SEQ ID NO:1, the first reverse primer comprises SEQ ID NO:2, the second forward primer comprises SEQ ID NO:3, and the second reverse primer comprises SEQ ID NO:4.

Description

A METHOD OF DETECTING PORCINE MARKERS FOR IDENTIFYING PORK

CONTENT IN A SAMPLE

Background of the Invention Field of the Invention

This invention relates to a method for food authentication; and more particularly to a method of detecting porcine markers for identifying pork content in food or the like sample. Description of Related Arts

Halal foods are foods that Muslims are allowed to consume under Islamic Shari'ah. Pork is one of the meats that is categorised as non-halal, and thus it cannot be consumed by Muslims. However, food adulteration, which refers to replacement of high cost ingredients with lower grade and cheaper substitutes, often occurs due to substitution of pork and lard for more expensive meats such as beef and lamb.

Many cases of food adulteration have been reported worldwide. For example, in Malaysia, one brand of butter recently had its halal status withdrawn after being confirmed to contain porcine DNA. In another case, porcine DNA was detected in a famous brand chocolate, although the Malaysia Islamic Development Department (Jakim) announce that it was a false result due to possible contamination of the test sample. Nevertheless, these examples illustrate the need for food authentication and sensitive testing to ensure quality control. Reliable state-of-the-art scientific methods for the detection of porcine components are needed in order to monitor adulteration in halal certified food products. Current analytical approaches are broadly based on the detection of porcine protein or DNA. For protein detection, enzyme-linked immunosorbent assays (ELISAs) are frequently used. However, protein denaturation due to the heat and pressure applied during food processing may generate false negative results. Hence, detecting traces of porcine materials in processed samples remains challenging.

Polymerase chain reaction (PCR) may prove to be the answer to this problem. PCR is a standard molecular biology test that has been widely used to amplify a single or a few copies of a particular sequence of DNA. PCR has been widely adopted for analysing food samples due to its simplicity, specificity, and sensitivity.

For example, WO 2009/157750 A1 is a method for identifying traces of pork content in food. The method relies on primer design for the identification of pork in processed food. Samples are analysed for the presence of the pork NADH dehydrogenase (ND5) mitochondrial gene by conducting a PCR test using the forward and reverse primers designed to detect this target.

Another PCR-based method for detecting and quantifying the presence of particular species in food is disclosed in US 2005/01 12592 A1 . This assay is based on detecting a high copy number of short interspersed elements (SINEs) unique to different species, which thereby enables identification of species sources in food samples. US 2007/0009910 A1 is a method that uses a reference plasmid for identifying the species of meat present in an adulterated sample.

The efficiency of a PCR amplification test largely depends on simple and efficient DNA extraction methods. Amplification results can be affected by the presence of PCR amplification inhibitors in the sample, which give rise to false negative results. Therefore, a simple and robust genomic DNA extraction method that provide efficient PCR that could rule out false positive amplification from the sample. is essential. In this newly developed method, a pair of oligonucleotide primers was designed to detect pork content in a test sample, and a second pair of oligonucleotide primers was designed as an internal control to monitor the efficiency and integrity of DNA extraction and for detection of possible false negative results during the amplification process. Summary of Invention

It is the objective of the present invention to provide a method of detecting porcine markers for food authentication. It is also an objective of the present invention to provide a pair of oligonucleotide primers, which target the porcine genome specifically to allow the detection of porcine markers.

Another objective of the present invention is to provide a second pair of oligonucleotide primers as internal control to detect possible false negativity.

Accordingly, these objectives are achieved by the present invention. The present invention relates to a method of porcine marker detection in order to identify even minute amounts of pork content in test samples. Said method comprisesthe following steps: extracting deoxyribonucleic acid (DNA) from samples; performing a polymerase chain reaction (PCR) on the extracted DNA using two pairs of specific primers, including a first forward primer (SEQ ID NO:1 ) and a first reverse primer (SEQ ID NO:2) for amplifying a target porcine DNA sequence, and a second forward primer (SEQ ID NO:3) and a second reverse primer (SEQ ID NO:4) as the internal control; and subjecting the amplicons to electrophoresis to detect the presence of porcine markers in the food sample.

Brief Description of the Drawings

The detailed description of the features of the invention will be more readily understood when read in conjunction with the accompanying drawings:

Figure 1 is an electrophoresis profile of amplified DNA sequences of a) a blank sample as the control, b) pig, c) chicken, d) cattle, e) goat, and f) rabbit, using a 100 bp DNA ladder, generated using the detection method of the present invention. Figure 2 is an electrophoresis profile of amplified DNA sequences of processed food including a) a blank sample as the control, b) chicken sausage brand A, c) chicken sausage brand B, d) chicken nugget, e) chicken meatball, f) pig sausage brand A, and g) pig sausage brand B, using a 100 bp DNA ladder, generated using the detection method of the present invention.

Figure 3 is an electrophoresis profile of amplified DNA sequences of pork samples in different concentrations, using a 100 bp DNA ladder, generated using the detection method of the present invention: a) a blank sample as the control, b) 1 ng porcine DNA, c) 100 pg porcine DNA, d) 10 pg porcine DNA, e) 1 pg porcine DNA, f) 100 fg porcine DNA, g) 10 fg porcine DNA, and h) 1 fg porcine DNA.

Figure 4 is an electrophoresis profile of amplified DNA sequences of bovine meat containing pork in different percentages, using a 100 bp DNA ladder, generated using the detection method of the present invention: a) 100% pork, b) 10% pork, c) 5% pork, d) 1 % pork, e) 0.5% pork, f) 0.1 % pork, g) 0% pork, h) 10 ng porcine DNA, and i) a blank sample as the control.

Detailed Description of the Invention

As required, detailed embodiments of the present invention are disclosed herein; however, note that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting but merely as a basis for claims. The drawings and detailed description are not intended to limit the invention to the particular form disclosed, but rather the invention is to cover all modifications, equivalents, and alternatives falling within the scope of the present invention as defined by the appended claims. Throughout this application, the word "may" is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). Similarly, the words "include," "including," and "includes" mean "including, but not limited to." Furthermore, the words "a" or "an" mean "at least one" and the word "plurality" means one or more, unless otherwise noted. Abbreviations or technical terms are used following the commonly accepted meanings as known in the technical field. For ease of reference, common reference numerals will be used throughout the figures when referring to the same or similar features common to the figures. The present invention will now be described with reference to Figures 1 -4.

The present invention relates to food authentication; more particularly to a method of detecting porcine markers for identifying pork contents in a test sample. In the method, a repetitive element (L1 Non-LTR Retrotransposon from pigs) sequence is used as the marker and is targeted using specific primers for detection of porcine material in a sample. The method of detecting porcine markers for identifying pork contents in a test sample comprising the steps of:

extracting deoxyribonucleic acid (DNA) from the sample; performing polymerase chain reactions (PCR) on the extracted DNA using two pairs of primers, including a first forward primer and a first reverse primer for amplifying a target porcine DNA sequence and a second forward primer and a second reverse primer as an internal control for amplifying a target sequence;

subjecting the amplified sequence to electrophoresis to detect the presence of porcine markers in the test sample; and

characterized in that:

the first forward primer comprises SEQ ID NO:1 , and the first reverse primer comprises SEQ ID NO:2;

the second forward primer comprises SEQ ID NO:3, and the second reverse primer comprises SEQ ID NO:4.

In a preferred embodiment of the method of detecting porcine markers, the test sample is a food sample. The food sample may be raw meat, processed food such as sausage, cooked food, or non-meat based food. In a preferred embodiment of the method of detecting porcine markers, the target sequence is LINE-1 (L1 ) retrotransposons.

In a preferred embodiment of the method of detecting porcine markers, the step of extracting deoxyribonucleic acid (DNA) comprises:

homogenizing the test sample in digestion buffer;

incubating the homogenate with Proteinase K for 16 hours at 55°C with shaking;

heating sample to 90°C for 15 minutes to obtain lysate; and

diluting the lysate with water.

In a preferred embodiment of the method of detecting porcine markers, the PCR comprises the steps of:

initial denaturation at 95°C for 5 minutes;

repeating 35 cycles of denaturation at 95°C for 30 seconds, annealing at

60°C for 30 seconds, extension at 72°C for 30 seconds; and

subsequent extension at 72°C for 5 minutes.

The present invention also provides a kit for detecting porcine markers for identifying pork content in a test sample, characterized by:

a PCR master mix comprising Taq polymerase, deoxynucleotide triphosphate (dNTPs) solution, and polymerase chain reaction (PCR) buffer; a first pair of primers comprising a first forward primer (SEQ ID NO:1 ) and a first reverse primer (SEQ ID NO:2) for amplifying a target porcine DNA sequence; and

a second pair of primers comprising a second forward primer (SEQ ID NO:3) and a second reverse primer (SEQ ID NO:4) as internal control.

The first forward primer and the first reverse primer are targeting a repetitive sequence in the porcine genome, thereby increasing the sensitivity and specificity of the detection. In a preferred embodiment, the pair of primers comprises SEQ ID NO:1 and SEQ ID NO:2 target the LINE-1 (L1 ) retrotransposons contained in the chromosome X of the porcine genome. The targeted L1 retrotransposons has high copy number in the porcine genome and is found specifically only in pigs, therefore giving a high selectivity and sensitivity in the detection of porcine DNA. In a preferred embodiment, the first pair of primers targets the genome of a pig, preferably Sus scrofa. During the PCR process, the first forward primer and the first reverse primer amplify a repetitive region of LINE-1 (L1 ) retrotransposons of the Sus scrofa, L1_SS, thereby enabling the detection of the porcine marker by electrophoresis. The detection limit may be as low as 1 pg of porcine DNA in food derivatives, so even a trace amount of porcine contamination can be detected in case of food adulteration.

The second pair of primers comprises the second forward primer (SEQ ID NO:3) and the second reverse primer (SEQ ID NO:4) as internal control. The second pair of primers is used to detect false negative results that can result from inhibitors present in the PCR test, thereby increasing the sensitivity of the detection result. The second pair of primers also provides an indication of DNA integrity, as they target a region that is conserved in most eukaryotes. Absence of the internal control bands in the electrophoresis results indicates a false negative result.

The PCR products are subjected to gel electrophoresis to visualize the porcine markers. Agarose gel electrophoresis is preferred. Other types of electrophoresis are also applicable for the detection of porcine markers and alternative techniques for separation of nucleic acid mixtures may be applied.

In a further embodiment of the method of detecting porcine markers for identifying pork contaminants in samples, a porcine DNA detection kit can be produced by incorporating said method. The porcine DNA detection kit includes tubes containing buffers for DNA extraction from a test sample, a porcine detection PCR master mix for PCR assay, and an internal control. The test sample may be raw or processed meat. This porcine DNA detection kit is cheap, rapid, sensitive, and specific for the detection of porcine ingredients. The porcine DNA detection kit will help to detect food adulterations and can be used for authentication services.

The present method of detecting porcine markers for identifying pork content in a test sample is simple and environmentally friendly. It does not use hazardous chemicals such as phenol and chloroform for the DNA extraction step. In addition, the robust genomic DNA extraction method may eliminate carry over contamination, thereby reducing sample processing time and cost.

Below is an example of a method of detecting porcine markers for identifying pork in test samples from which the advantages of the present invention may be more readily understood. It is to be understood that the following example is for illustrative purpose only and should not be construed to limit the present invention in any way. Example

A 20 μΙ reaction mixture containing 200 μΜ nucleotides (dNTP) mix, 1 x PCR reaction buffer, 1 .5 imM MgC^, 10 pmol of each forward primer (SEQ ID NO:1 and SEQ ID NO:3) and reverse primer (SEQ ID NO:2 and SEQ ID NO:4), 1 unit Taq DNA polymerase, and 1 μΙ of diluted lysate is prepared. A PCR assay is then performed by initial denaturation at 95 °C for 5 minutes followed by 35 cycles of denaturation at 95 °C for 30 seconds, annealing at 60 °C for 30 seconds, extension at 72 °C for 30 seconds, and a final step of extension at 72 °C for 5 minutes using a BioRad personal cycler. The amplified DNA fragments are separated by electrophoresis on a 2% agarose gel with Tris-Acetate-EDTA buffer (TAE, pH 8.3) and visualized under a UV transilluminator.

Analysis

Various DNAs from different animal species in raw meat and processed meat were tested using the method described above. The SEQ ID NO:3 and SEQ ID NO:4 oligonucleotide primers were used as the internal control.

The results of this experiment are shown in Figures 1-3 in the form of electrophoresis profiles. The internal control bands are present for every sample subjected to PCR amplification, which confirms the absence of false negative results. Figures 1-2 show the accuracy of the present method, wherein the porcine markers were detected as 451 bp amplicon in both raw and processed food sample.

Figure 3 illustrates the sensitivity of the present method. The lowest concentration of DNA that showed a visible band under the UV transilluminator was 1 pg porcine DNA. Figure 4 shows the result of porcine marker detection in adulterated meat samples (porcine and bovine meat). The detection limit was as low as 0.5% porcine meat in 5 g of total meat.

Although the present invention was described above with reference to specific embodiments (also shown in the figures), it will be apparent to those skilled in the art that many variations and modifications of the method can be made within the scope of the invention, as described in the specification and defined in the following claims.

Claims

Claims I/We claim:

1 . A method of detecting porcine markers for identifying pork content in a test sample, comprising the steps of:

extracting deoxyribonucleic acid (DNA) from the samples;

performing polymerase chain reaction (PCR) on the extracted DNA using two pairs of primers, including a first forward primer and a first reverse primer for amplifying a target porcine DNA sequence, and a second forward primer and a second reverse primer as an internal control for amplifying a target gene sequence;

subjecting the amplified sequence to electrophoresis to detect the presence of porcine DNA in the sample; and

characterized in that:

the first forward primer comprises SEQ ID NO:1 and the first reverse primer comprises SEQ ID NO:2; and

the second forward primer comprises SEQ ID NO:3 and the second reverse primer comprises SEQ ID NO:4.

2. The method of detecting porcine markers according to claim 1 , wherein the test sample is a food sample.

3. The method of detecting porcine markers according to claim 1 , wherein the target sequence is LINE-1 (L1 ) retrotransposons.

4. The method of detecting porcine markers according to claim 1 , wherein the step of extracting DNA comprises: homogenizing the test sample in digestion buffer; incubating the homogenate with Proteinase K for 16 hours at 55 °C with shaking; heating the sample to 90 °C for 15 minutes to obtain lysate; and diluting the lysate with water. The method of detecting porcine markers according to claim 1 , wherein the PCR comprises the steps of: initial denaturation at 95 °C for 5 minutes; 35 cycles of denaturation at 95 °C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds; and subsequent extension at 72°C for 5 minutes.

A kit for detecting porcine markers for identifying pork content in a test sample, characterized by:

a PCR master mix comprising Taq polymerase, deoxynucleotide triphosphate (dNTPs) solution, and polymerase chain reaction (PCR) buffer;

a first pair of primers comprising a first forward primer (SEQ ID NO:1 ) and a first reverse primer (SEQ ID NO:2) for amplifying a target porcine DNA sequence; and

a second pair of primers comprising a second forward primer (SEQ ID NO:3) and a second reverse primer (SEQ ID NO:4) as internal control.