A membrane chip method that can identify 11 animal-derived components in high flux.

Abstract: Using membrane chip technology, the detection of 11 target species of cattle, sheep, donkeys, yaks, chickens, ducks, rabbits, yaks, foxes, rats and pigs was used to detect the doping of cattle, sheep, donkeys and yak species. The
results show that the method has good specificity and applicability, detection sensitivity and doping sensitivity can reach 0.1%, can quickly and accurately identify cattle, sheep, donkeys, yak, chicken, duck, rabbit, yak, fox, mouse, pig 11 animal-derived ingredients, can meet the meat food samples for cattle, sheep, yak, donkey and other pseudo-identification testing needs.
in recent years, with China's rapid economic development, China's food industry has also entered the golden period of rapid development, the public demand for a variety of meat is also gradually rising.
however, food safety problems are emerging, to the entire food industry and the vast number of consumers have a great impact.
donkey meat because of good taste, low fat content, high nutritional value and so on, by more and more consumers of all ages, but its low yield, high price, sometimes adulteration occurred.
yak is a kind of special animal from the western region, yak amino acid proportion is balanced, fat content is very low, can be used as a source of high-quality protein meat, but its market price is much higher than other meat products, adulteration phenomenon sometimes occurs.
common commercially marketed processed beef, lamb, such as lamb kebabs, dried meat, meatballs, etc., there is often the possibility of adulteration, and even by illegal traders mixed into low-cost pork, chicken, duck and even fox meat.
literature reports indicate that in the pre-packaged and bulk beef products, there are 2 batches and 5 batches of non-cow-derived ingredients, respectively.
screening of beef and mutton in the Beijing area, it was found that the proportion of duck, chicken and pork mixed with it was as high as 34.9%.
donkey meat, beef, beef, mutton doping is reported from time to time, therefore, the above-mentioned meat products for doping identification has become the general public and food testing needs.
but when screening animal-derived ingredients for meat products, the same sample often needs to carry out a variety of suspicious adulterated animal-derived ingredients at the same time testing, the workload is large, for a large number of meat products screening will take a longer time and more human and material resources.
, for some meat products processed from a variety of meat products, the single-re-identification method appears to be inefficient.
therefore, it is urgent to establish a high-volume detection method for a variety of animal-derived components.
, there are many animal-derived component detection techniques, including metabolomics-based detection methods, protein-based detection methods and nucleic acid-based detection methods.
polymerase chain reaction (PCR) technology, because of its high sensitivity, specificity, and simple primer probe design, has become the most widely used animal source component detection technology.
PCR methods include common PCR technology, real-time fluorescent PCR technology and digital PCR technology.
currently in PCR technology, fluorescent PCR method is the most widely used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Although the traditional molecular biology detection technology of animal-derived components has many advantages, but under the requirements of large sample size, high efficiency and rapid inspection, can not meet the testing needs well.
and existing molecular biology testing technology on laboratory instruments, environmental conditions, personnel technology and other requirements are high, so only applicable to laboratory testing, not applicable to large-scale, high-throughput screening work needs, can not meet the current food safety supervision, supervision requirements.
therefore, new methods are needed for high-throughput, multi-indicator detection of animal-derived components.
membrane chip detection method is the product of the development of nucleic acid hybridization technology, which uses the principle of reverse speckle hybridization to sequence a group of oligonucleotide probes on the supporting membrane surface, forming a series of low-density membrane chip probe array.
using multi-PCR technology to amplify the target gene to be tested, the amplification material is cross-breeding with the probe on the membrane chip after the transmutation, and the substrate is color-coded to form a visual test result.
1992, membrane chip technology was used by Fiss and other syphilis to detect mycobacteria, and then applied to food microbiological testing , genetically modified screening , animal medicine , animal medicine , and biological sciences , 29-30 , and many other fields .
because the visual chip typically fixes multiple target sites on the chip at the same time, multiple target genes can be detected at the same time.
Compared with the traditional PCR method, the membrane chip detection method has the technical advantages of high detection flux, short detection cycle, low cost and reliable results, and can be used as a high-throughput animal source component detection method.
this study designed a membrane chip method, can quickly identify donkey meat, yak beef, beef, mutton whether there is chicken, duck, rabbit, slug, fox, rat, pig and other meat source composition of adulteration, for meat source component detection provides a good idea.
1 Materials and Methods 1.1 Materials and Reagents purchase a variety of meat samples from Jinan Market and shopping websites, including fresh samples of cattle, sheep, donkeys, chickens, ducks, pigs and a variety of processed meat products (beef jerky 2, beef jerky 1, sauce 2, kebabs 1).
yak, rabbit, herb, fox, mouse, mule, horse, buffalo, dog, fish and other materials stored in this laboratory.
meat and meat products nucleic acid extraction kit for the German company Kaijie products;
1.2 Instruments and Equipment AB ProFlex PCR Instrument Seroth Usa; 5424R Small Refrigeration Centrifuge Eppendorf, Germany; NanoDrop 2000 ULTRAVIOLET-Visible Spectre Spectre Thermo Fisher Scientific Co., Ltd.; QuantStudio 7 Flex Real-time Fluorescent PCR American ABI; 2000 Purple Diplomacy U.S. Sebemin Scientific Equipment.
1.3 Method 1.3.1 Prime R. 5.0 software to design 11 animal-derived components and internal parameter18S rRNA primers, as well as specific probes that bind dna products through base complementary pairing.
the 5' end of the reverse primer is modified with biotin Biotin, the primer can amplify the biotin-labeled PCR product with a length of 100 to 200 bp.
probe 5' end connected to an amino modification with six carbon atoms as the connecting arm, used to secure the probe to the membrane chip.
also designed a control probe PC and NC to monitor whether the hybridization process is normal.
design 5' end biotin modification, can complement the PC probe of the positive oligonucleotide single chain (CPC), for the quality control membrane chip hybridization process.
the sequence of primer probes for 11 animal-derived components and internal parameters as shown in Table 1.
1.3.2 sample preparation takes 100 g meat source sample, after the four-point reduction, is frozen and ground, at -20 degrees C to save the spare.
1.3.3 DNA extraction and concentration determination take samples of 50 mg of grinding, add 1.5 mL centrifuge tubes, and refer to the kit method for DNA extraction.
1.3.4 Multi-PCR Amplification and Denatured Reference to The Common PCR Reaction System, 11 primers are added to carry out multiple PCR amplification.
multiple PCR reaction system, the total volume of the system is 50 ?L, the final concentration of each substance is: MgCl2 1.5 mmol/L, dATP, dCTP, dGTP, dTTP and dUTP are 0.2 mmol/L, UNG enzyme 1 U/50 ?L, Taq enzyme 2 U/50 ?L, each lead concentration of 20.2.L/L.
was tested with genomic DNA from 11 animals, DNA without these 11 animal components as negative controls, water as blank control, as quality control of PCR amplification system and membrane chip hybrid reaction.
PCR reaction parameters: 37 degrees C, 10 min, 1 cycle; 95 degrees C, 10 min, 95 degrees C, 30 s, 55 degrees C, 30 s, 72 degrees C, 30 s, 35 cycles; 72 degrees C, 10 min, 1 cycle.
product degeneration: the multiple PCR products are annealed to maintain a single chain state. the
degeneration process is 95 degrees C, 5 min, 4 degrees C, 10 min.
single-stranded DNA after annealing needs to be immediately placed at low temperatures.
1.3.5 membrane chip detection 1.3.5.1 11 animal-derived components of the membrane chip design to the square nylon film into 16 small areas, the probe according to the surface design in Figure 1 fixed to different areas.
can determine whether the species has been detected by the color rendering of different dot arrays.
1.3.5.2 Membrane chip manual hybridization process manual hybridization mainly consists of several main steps of deactivation, hybridization, enzyme association, color rendering and reading results: deactivation is to close the active group of the probe that is not fixed to the membrane, thereby increasing the specificity of the membrane chip; The target gene fragment in the PCR product is labeled with biotin, if the PCR product contains the target fragment, the alkaline phosphatase can be connected to the membrane by the action of biotin and streptomycin affinity;
place the membrane chip in the hybrid box and manually cross-breed according to Table 2.
the hybridization process is carried out in the purple diplomatic instrument, and the same experimental effect can be used.
Note: deactivated fluid: 100 mmol/L NaOH; deactivated cleaning fluid, hybrid fluid: 2 x SSPE with 0.1% SDS; hybrid cleaning fluid, enzyme incubation system fluid, incubation cleaning fluid 1: 2 x SSPE with 0.5% SDS; incubation cleaning fluid 2:2 x SSPE; incubating cleaning fluid 2:2 x SSPE SSPE.Saline sodium phosphate EDTA buffer (saline sodium phosphate EDTA), containing 3 mmol/L NaCl, 200 mmol/L NaH2PO4 and 20 mmol/L EDTA, pH 7.4; SDS.decalsulfonate).
2 results and analysis 2.1 meat source component detection specific experiments will be cattle, sheep, donkeys, yaks, chickens, ducks, rabbits, pigs, yaks, foxes, rats, quail, horses, buffaloes, dogs, fish and other animal fresh samples in accordance with 1.3.2 and 1.3.3 sections of DNA extraction, followed by multiple PCR enlargement and product transmutation experiments, PCR reaction pattern 20 to 20 ng0 ng.
can be seen from Figure 2, cattle, sheep, donkeys, yaks, chickens, ducks, rabbits, pigs, yaks, foxes, mice in the probe location has specific hybrid spots, internal ginseng and positive control location hybrid spots color normal, negative control and other locations no hybrid color ingelet spots.
can be seen from Figure 3, the chip on the quay, horse, buffalo, dog, fish, cat species no abnormal hybrid point display, and all kinds of control positions are normal color rendering, no other species of cross-breeding color spots. The results of the
show that the membrane chip has good specificity to 11 species of cattle, sheep, donkeys, yaks, chickens, ducks, rabbits, pigs, slugs, foxes and rats, and no specific hybrid spots are produced on the membrane chip.
2.2 11 kinds of meat source component detection sensitivity experiment This study to cattle, sheep, yak, donkey as an example, to verify the sensitivity of the membrane chip method.
the DNA extracted from cattle, sheep, yaks and donkeys was fully mixed with chicken and duck DNA, and a mixture of 5.0%, 1.0% and 0.1% were prepared, respectively.
perform multiple PCR amplification and hybridization experiments according to the operation of the method.
can be seen from Figure 4, in the 5.0%, 1.0% and 0.1% mixed ratio, cattle, sheep, yak and donkey and chicken, duck mixed DNA on the membrane chip have a color-showing reaction, each control position is normal color rendering, in the chicken, duck's color point has hybrid color spots, indicating that in the mass score of 5.0%, 1.0% and 0.1% of the conditions, the method can detect the composition of the cow, yak, donkey, donkey.
experimental results show that the method has good detection sensitivity to cattle, sheep, yak and donkey composition, and its detection limit can reach 0.1%.
in addition, the sensitivity of adulterated species is detected.
mixed rabbit, pig, slug, fox, mouse DNA with chicken and duck DNA for hybridization experiments.
can be seen from Figure 5, in 0.1% doping ratio, rabbit, pig, slug, fox, mouse components on the membrane chip have color-rendering reaction, each control position is normal color rendering, in chicken, duck color point has hybrid color spots, indicating that in the mass score of 5.0%, 1.0% and 0.1%, the method can detect rabbit, pig, slug, fox, mouse composition, show that the method can have good detection.
2.3 cattle, sheep, yak, donkey ingredient testing applicability verification experiment for the test film chip method of practical application, from the market to buy 6 samples of processed meat products, including beef jerky 2, yak dried 1, donkey meat 2 and lamb kebab 1.
each sample to be tested in accordance with the experimental method seissays PCR amplification and chip hybridization experiments, each sample is repeated 2 times, while the fluorescence method is verified.
the detection of pigs, cattle and sheep targets using SN/T 2051-2008 "Real-time PCR method of animal fluorescence testing method sgoilinofing animals of cattle and sheep in food, cosmetics and feed"; /T/3730.4-2013 "Identification methods for common animal breeds in food and feed Part 4: Donkey ingredient detection real-time fluorescent PCR method" for verification, horse target detection using N/T 3730.