By: Joko Hermaniantoa, b, c, Raafqi Ranasasmitac, d, Rina Maulidiyahd
Analysis of laboratory testing for detection of pig contaminants is one of the supporting processes for halal certification. Test results can be used as one of the supporting elements in making decisions on the halal certification process. Various methods for testing pig contaminants have been developed, such as chromatography, isoelectric focusing, FTIR, electronic nose, LC-MS/MS, ELISA, and Polymerization Chain Reaction (PCR). This paper focuses on the detection method of pig contaminants using immunochromatographic test strips and real-time PCR.
Immunochromatographic Test Strip
The immunochromatographic test strip is a fast method of detections pigs, the target is proteins (pig antigens). The principle of the test is the antigen-antibody interaction. The main components of the immunochromatographic test strip include sample location, conjugate membranes, nitrocellulose membranes, and membrane adsorption. The sample location serves as the starting point for the migration (transfer) of the sample solution. Afterward, the sample solution migrates along the membrane to the conjugate membrane. The conjugate membrane contains conjugate antibodies that have been labeled. The marker will be used as an indicator of the presence of pig protein, with the appearance of a red line. In general, the type of label that is often used for detection of pig protein is the Gold Nano Particle (GNP).
A nitrocellulose membrane is a place for sample solutions migration. There are primary and secondary antibodies which are immobilized (placed) in the test line and control line sections. The test line serves as a place to detect pig protein, while the control line functions to show that the migration of the sample solution has been completed.
The examples of test strip schemes and their working mechanisms
The picture above illustrates the immunochromatographic test strip scheme (A) Protein (pig antigen) attached to the sample site, (B1) Conjugate antibodies form complexes with antigens and migrate along the nitrocellulose membrane (B2) The pig antigen conjugate antibody complex is captured by primary antibodies in the test line and the excess is captured by secondary antibodies in the control line (B3).
The test results can be seen visually on the nitrocellulose membrane with the appearance of a red line on the test line and control line.
Real-Time Polymerization Chain Reaction (Real-Time PCR)
Real-time PCR is a technique of detecting gene chains or segments of DNA (Deoxyribo Nucleic Acid). DNA is the smallest unit that composes a highly specific organism gene. Specific pig DNA segments, for example, are cytochrome b. To carry out testing for swine DNA detection, DNA extraction must first be carried out in the sample. The principle of working real-time PCR is denaturation, attachment, and processing of DNA chains. Denaturation aims to separate the structure of double screw DNA into a single thread by heating. Each of these threads becomes a mold for primary attachment. Furthermore, the enzyme in the primer carries out elongation by arranging dNTP (the constituent material of DNA in the form of nucleotides) to become a new thread with anti-sense properties, so that two double threads are mutually bound together.
The process of denaturation, attachment, and elongation is repeated over a certain cycle, in general, 35 cycles are carried out in one test. Amplification/doubling increases the amount of DNA exponentially every cycle. If the amplification of 1 strand DNA is carried out in 35 cycles, then the copy of DNA to be obtained is 235 = 3.43 x 1010copy DNA. This makes real-time PCR a high sensitivity pig detection method.
Real-time PCR has a probe that is bound to a quencher and dye reporter. When hybridization occurs due to doubling of DNA, the probe undergoes separation by the enzyme. The separation of quenchers and dye reporters causes an increase in fluorescence signals. The amount of DNA is known from the intensity of fluorescence emitted. The signal is then received by the real-time PCR detector and translated into a graph that can be read directly.
The visual reading of the PCR real-time test results is done through two graphs, VIC and FAM. The VIC graph is an indicator that there is no damage to the reagent and the real-time PCR device, and there is no inhibition of the sample. If all samples show a positive reaction on this channel, then the test is considered valid. The FAM graph shows the results of the control test and sample test results. The positive control of the FAM chart gives a red line response that cuts the baseline (positive) and the negative control responds to a blue line that does not cut the baseline (negative).
Strengths and Weaknesses of Immunochromatographic Test Strips
The advantages of the immunochromatographic test strip are, among others, easy to use without the need for special skills, fast testing times ranging from 10-15 minutes, and relatively inexpensive. Testing using immunochromatographic test strips for detection of pig contaminants is similar to testing pregnancy test packs, very easy to do even without special expertise.
This detection method has the disadvantage of being only able to detect meat and processed meat, unable to detect pig contaminants in derivative products such as gelatin, capsules, etc. In addition, immunochromatographic test strips have the opportunity to cause positive errors when used to detect pig contaminants in spices, namely spices containing certain ingredients can be detected as if they contain pork, even though the spices are clearly free of pigs. Therefore, detection of pig contaminants using this method should be used only for initial identification, not as a final detection method. The hypochromatographic test strip has a detection limit that varies between 0.1%-1.0%, depending on the design of the manufacturer. This causes the immunochromatographic test strip to be unable to detect pig contamination which is only a contaminant. This method has different sensitivity and specificity capabilities between immunochromatographic test strip manufacturers.
Strengths and Weaknesses Real-time PCR
The advantage of real-time PCR is sensitive and accurate. It can detect the smallest element of an organism, namely DNA, so there is a small chance of errors. This method is able to detect pig contaminants in derivative products, such as gelatin, capsules, etc. Testing time is relatively fast. Testing takes 70 minutes and the test results can be seen visually through a graph formed on a computer that has been connected to a real-time PCR machine.
However, in testing using real-time PCR requires special expertise and more expensive costs.
Joko Hermaniantoa, b, c, Raafqi Ranasasmitac, d, Rina Maulidiyahd
a. A staff of the Department of Food Science and Technology of IPB
b. Expert Staff for the Assessment Institute for Foods, Drugs, and Cosmetics, the Indonesian Council of Ulama (LPPOM MUI)
c. Halal Laboratory Staff of the Assessment Institute for Foods, Drugs, and Cosmetics, the Indonesian Council of Ulama (LPPOM MUI)
d. A staff of the Assessment Institute for Foods, Drugs, and Cosmetics, the Indonesian Council of Ulama (LPPOM MUI)