Multiple PCR-capillary electrophoresis detection system for identifying 13 species and special primer combination thereof

Abstract

The invention discloses a multiple PCR-capillary electrophoresis detection system for identifying 13 species and a special primer combination thereof. The primer combination is composed of 26 kinds of DNA (deoxyribonucleic acid) molecules as shown in SEQ ID No. 1 to SEQ ID No. 26. The primer combination is adopted to construct a multiple PCR-capillary electrophoresis detection system, DNA (such as DNA of blood or meat) of 13 species such as chicken, duck, sheep, pig, cattle, mouse, rat, cat, dog, donkey, horse, human and Chinese hamster can be simultaneously identified in a single-tube reaction, the sensitivity to different target DNA is 0.001-0.05 ng, and compared with other researches, the primer combination has obvious advantages in sensitivity and species identification quantity. The method is low in cost and high in efficiency, and has important application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological quarantine, and particularly relates to a multiplex PCR-capillary electrophoresis detection system for identifying 13 species and a special primer combination thereof, and the detection object can be blood or meat tissue. BACKGROUND

[0002] Species identification of unknown biological samples is crucial for forensic evidence analysis. Human and non-human biological samples obtained from crime scenes always contain a large amount of valuable information, which can provide important clues for criminal investigation (Cui W, Jin X, Guo Y, et al. Development and Validation of a Novel Five-Dye Short Tandem Repeat Panel for Forensic Identification of 11 Species [J]. Frontiers in Genetics, 2020, 11: 1005.). In addition, species identification of various animals can also be applied to the field of food safety. In recent years, meat and its processed products have been plagued by adulteration problems (Cui W, Jin X, Guo Y, et al. Development and Validation of a Novel Five-Dye Short Tandem Repeat Panel for Forensic Identification of 11 Species [J]. Frontiers in Genetics, 2020, 11: 1005.). K, Calo-Mata P, Barros-Velázquez J, et al. Review of Recent DNA-Based Methods for Main Food-Authentication Topics[J]. Journal of Agricultural and Food Chemistry, 2019, 67(14): 3854-3864. Li X, Zang M, Li D, et al. Meat food fraud risk in Chinese markets 2012–2021[J]. npj Science of Food, 2023, 7(1): 12.) Unscrupulous businessmen often use inferior meat as good meat, even mix in inedible meat such as rat meat, which poses a significant threat to personal health and public health. Therefore, establishing a rapid, efficient, sensitive, and reliable species identification method has always been a hot research direction in the field of forensic evidence and food safety. The main identification methods for species identification currently include conventional PCR, multiplex PCR, real-time fluorescent quantitative PCR, microdroplet digital PCR, enzyme-linked immunosorbent assay (ELISA), mass spectrometry (MS), electronic nose technology (E-nose), near-infrared spectroscopy (NIR), and surface-enhanced Raman spectroscopy (SERS), etc. However, ELISA technology cannot detect deeply processed biological samples, mass spectrometry technology has the problem of complex biological sample pretreatment, and electronic nose and spectroscopy technology are limited by the lack of standard databases and models, which need further research (Du J, Gan M, Xie Z, et al. Current progress on meat food authenticity detection methods[J]. Food Control, 2023, 152: 109842. Li Y, Liu S, Meng F, et al. Comparative review and the recent progress in detection technologies of meat product adulteration[J]. Comprehensive Reviews in Food Science and Food Safety, 2020, 19(4): 2256-2296.).Therefore, the most widely used species identification method at present is PCR and its derivative techniques which take DNA as the detection object. Among them, the multiplex PCR technique can accurately identify multiple species in a single reaction system by using species-specific primers, which saves time and cost and is the preferred method for specific identification of different species.

[0003] For multiplex PCR, agarose gel electrophoresis (AGE) is the most commonly used method for detecting amplification products, but it has the disadvantages of low resolution and insufficient sensitivity (Li J, Li J, Xu S, et al. A rapid and reliable multiplex PCR assay for simultaneous detection of fourteen animal species in two tubes [J]. Food Chemistry, 2019, 295: 395-402.

[0004] ), which is difficult to meet the supervision and case handling needs of public security organs. Capillary electrophoresis (CE) is widely used in the detection of amplification products due to its faster, more accurate and higher throughput. Although a variety of multiplex PCR detection methods for species identification have been reported, the number of species that can be detected simultaneously cannot meet the increasingly urgent practical application needs. SUMMARY

[0005] The purpose of the present application is to identify 13 species of chicken, duck, sheep, pig, cow, mouse, rat, cat, dog, donkey, horse, human and Chinese hamster, and the detection object is the blood, meat tissue, blood products and / or meat products of the species.

[0006] The present application first protects a primer combination. The primer combination can include primer F1, primer R1, primer F2, primer R2, primer F3, primer R3, primer F4, primer R4, primer F5, primer R5, primer F6, primer R6, primer F7, primer R7, primer F8, primer R8, primer F9, primer R9, primer F10, primer R10, primer F11, primer R11, primer F12, primer R12, primer F13 and primer R13.

[0007] The primer F1 can be a single-stranded DNA molecule as shown in SEQ ID No. 1;

[0008] The primer R1 can be a single-stranded DNA molecule as shown in SEQ ID No. 2;

[0009] The primer F2 can be a single-stranded DNA molecule as set forth in SEQ ID No. 3;

[0010] The primer R2 can be a single-stranded DNA molecule as set forth in SEQ ID No. 4;

[0011] The primer F3 can be a single-stranded DNA molecule as set forth in SEQ ID No. 5;

[0012] The primer R3 can be a single-stranded DNA molecule as set forth in SEQ ID No. 6;

[0013] The primer F4 can be a single-stranded DNA molecule as set forth in SEQ ID No. 7;

[0014] The primer R4 can be a single-stranded DNA molecule as set forth in SEQ ID No. 8;

[0015] The primer F5 can be a single-stranded DNA molecule as set forth in SEQ ID No. 9;

[0016] The primer R5 can be a single-stranded DNA molecule as set forth in SEQ ID No. 10;

[0017] The primer F6 can be a single-stranded DNA molecule as set forth in SEQ ID No. 11;

[0018] The primer R6 can be a single-stranded DNA molecule as set forth in SEQ ID No. 12;

[0019] The primer F7 can be a single-stranded DNA molecule as set forth in SEQ ID No. 13;

[0020] The primer R7 can be a single-stranded DNA molecule as set forth in SEQ ID No. 14;

[0021] The primer F8 can be a single-stranded DNA molecule as set forth in SEQ ID No. 15;

[0022] The primer R8 can be a single-stranded DNA molecule as set forth in SEQ ID No. 16;

[0023] The primer F9 can be a single-stranded DNA molecule as set forth in SEQ ID No. 17;

[0024] The primer R9 can be a single-stranded DNA molecule as set forth in SEQ ID No. 18;

[0025] The primer F10 can be a single-stranded DNA molecule as set forth in SEQ ID No. 19;

[0026] The primer R10 can be a single-stranded DNA molecule as set forth in SEQ ID No. 20;

[0027] The primer F11 can be a single-stranded DNA molecule as shown in SEQ ID No. 21.

[0028] The primer R11 can be a single-stranded DNA molecule as shown in SEQ ID No. 22.

[0029] The primer F12 can be a single-stranded DNA molecule as shown in SEQ ID No. 23.

[0030] The primer R12 can be a single-stranded DNA molecule as shown in SEQ ID No. 24.

[0031] The primer F13 can be a single-stranded DNA molecule as shown in SEQ ID No. 25.

[0032] The primer R13 can be a single-stranded DNA molecule as shown in SEQ ID No. 26.

[0033] The primer combination can specifically consist of the primer F1, the primer R1, the primer F2, the primer R2, the primer F3, the primer R3, the primer F4, the primer R4, the primer F5, the primer R5, the primer F6, the primer R6, the primer F7, the primer R7, the primer F8, the primer R8, the primer F9, the primer R9, the primer F10, the primer R10, the primer F11, the primer R11, the primer F12, the primer R12, the primer F13 and the primer R13.

[0034] In any of the above-mentioned primer combinations, the molar ratio of the primer F1, the primer R1, the primer F2, the primer R2, the primer F3, the primer R3, the primer F4, the primer R4, the primer F5, the primer R5, the primer F6, the primer R6, the primer F7, the primer R7, the primer F8, the primer R8, the primer F9, the primer R9, the primer F10, the primer R10, the primer F11, the primer R11, the primer F12, the primer R12, the primer F13 and the primer R13 can be 1:1:1:1:1:1:1:1:20:20:4:4:2:2:2:2:2:2:2:2:1:1:1:1:1:1.

[0035] In any of the above-mentioned primer combinations, the primer R1, the primer R2, the primer R3, the primer R4, the primer R5, the primer R6, the primer R7 and the primer R8 are labeled with fluorescent FAM; the primer R9, the primer R10, the primer R11, the primer R12 and the primer R13 are labeled with fluorescent HEX. The labeling position can be the 5' end of the primer.

[0036] In any of the above-mentioned primer combinations, the 5' end of the primer R1, the primer R2, the primer R3, the primer R4, the primer R5, the primer R6, the primer R7 and the primer R8 is labeled with 6-FAM; the 5' end of the primer R9, the primer R10, the primer R11, the primer R12 and the primer R13 is labeled with HEX.

[0037] The present application also protects a detection system for identifying 13 species, which can comprise any of the above-mentioned primer combinations; the 13 species can be chicken, duck, sheep, pig, cow, mouse, rat, cat, dog, donkey, horse, human and Chinese hamster.

[0038] In the above-mentioned detection system, the concentration of primer F5 and primer R5 in the detection system is 0.1 μM / L.

[0039] In the above-mentioned detection system, the concentration of primer F1, primer R1, primer F2, primer R2, primer F3, primer R3, primer F4, primer R4, primer F11, primer R11, primer F12, primer R12, primer F13 and primer R13 in the detection system is 0.005 μM / L.

[0040] In the above-mentioned detection system, the concentration of primer F7, primer R7, primer F8, primer R8, primer F9, primer R9, primer F10 and primer R10 in the detection system is 0.01 μM / L.

[0041] In the above-mentioned detection system, the concentration of primer F6 and primer R6 in the detection system is 0.02 μM / L.

[0042] Any of the above-mentioned detection systems can further comprise reagents required for performing PCR amplification reaction; the "reagents required for performing PCR amplification reaction" do not include primers required for PCR amplification reaction.

[0043] The "reagents required for performing PCR amplification reaction" can comprise 2x Multiplex PCR Buffer (Huzhou Shenko, China).

[0044] A kit comprising any of the above-mentioned primer combinations also falls within the protection scope of the present application. The kit is used for identifying 13 species; the 13 species are chicken, duck, sheep, pig, cow, mouse, rat, cat, dog, donkey, horse, human and Chinese hamster.

[0045] The preparation method of any of the above-mentioned detection systems or any of the above-mentioned kits also falls within the protection scope of the present application.

[0046] The preparation method of any of the above-mentioned detection systems or any of the above-mentioned kits can comprise the step of individually packaging each primer in any of the above-mentioned primer combinations.

[0047] The following X1) or X2) also falls within the protection scope of the present application:

[0048] X1) the use of any of the above-mentioned primer combinations or any of the above-mentioned detection systems in the preparation of a kit for identifying 13 species;

[0049] X2) use of any of the above primer combinations or any of the above detection systems in the identification of 13 species;

[0050] The 13 species are chicken, duck, sheep, pig, cow, mouse, rat, cat, dog, donkey, horse, human and Chinese hamster.

[0051] Any of the above uses are for non-disease diagnosis and treatment.

[0052] With the improvement of people's living standards and the continuous change of food supply chain, meat adulteration problems will undoubtedly exist for a long time and change constantly. This application is in line with the actual situation. In the screening process of target species, not only common meat-derived species such as cow, pig, chicken, duck, donkey, sheep, etc., and common forensic identification species such as human, dog, cat, etc. are selected, but also potential adulterated species such as horse, mouse, rat, Chinese hamster, etc. are supplemented based on the detailed investigation of the hot food safety cases in recent years, in order to enhance the practicality of the research and better serve the food safety case identification. The success of the multiplex PCR system depends on the efficient binding ability of species-specific primers to target sequences and the non-interference of each primer when multiple primers exist. In this application, multiple pairs of primers are designed for target species, and the efficiency of primer binding to target sequences is verified by melting curve analysis. In addition, the non-interference of each primer when multiple primers exist is verified by the amplification of a single primer in the presence of other primers.

[0053] The BLAST program strictly evaluates each pair of primers, aligns them with another 12 non-target species DNA sequences, ensures that each pair of primers has no significant mismatch with non-target species, and verifies through a large number of experiments to obtain specific primers without primer dimers and non-specific amplification. In practical application, by detecting the three types of mixtures of sheep, duck, donkey, horse and cow, it is proved that the system has certain mixed sample detection ability; 27 commercial meat samples are detected, and it is found that 5 samples have the problem of false label information, including horse meat pretending to be donkey meat, beef pretending to be donkey meat, and donkey meat mixed with pork or beef, and the detection rate is 18.52%. It can be seen that there are still problems of low-priced meat pretending to be high-priced meat and serious donkey meat adulteration in the meat market at present, and the supervision needs to be strengthened, which also verifies the reliability and necessity of the detection system in practical application. In addition, the application can also identify the adulterated components in the meat products processed by different degrees (including marinating, roasting, drying and frying), which shows that the method also has good applicability to processed foods. At the same time, through the detection of real case samples, the multiple detection ability of the detection system is further verified, and more animal behavior characteristics are revealed, providing multi-dimensional evidence chain for case reconstruction, which shows its application value in forensic material evidence. In addition to the scenarios presented in the application, the application of the multiplex PCR-capillary electrophoresis detection system can also be extended to the screening of human blood product contamination, the identification of unknown biological material species, and the control of the transmission risk of zoonosis. The application selects capillary electrophoresis as the detection platform, which has higher sensitivity and can accurately distinguish multiple DNA fragments of similar length compared with traditional agarose gel electrophoresis. Through a multi-fluorescent labeling system, the resolution capacity can be further improved. The application can detect up to 13 species (chicken, duck, sheep, pig, cow, mouse, rat, cat, dog, donkey, horse, human and Chinese hamster), and the sensitivity of detecting all 13 species is 0.05 ng. At 0.001 ng template amount, 10 species can still be detected, and the false label information, blood, blood products, meat and meat products can be successfully identified. The detection system established in the application has strong specificity and high sensitivity. Through the detection and verification of simulated mixed samples, commercial meat samples and real case samples, it is proved that the system has good application value in forensic material evidence analysis, food safety monitoring and other judicial issues. Compared with other studies, the sensitivity and the number of identified species are obviously superior. The application has important application value. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 The optimized multiplex PCR detection system can accurately distinguish 13 species.

[0055] Figure 2 The species-specific detection results.

[0056] Figure 3 For species-specific detection results.

[0057] Figure 4 For species-specific detection results.

[0058] Figure 5 For sensitivity detection results.

[0059] Figure 6 For sensitivity detection results.

[0060] Figure 7 For detection results of sheep-duck mixed samples in simulated mixed samples.

[0061] Figure 8 For detection results of donkey-horse mixed samples in simulated mixed samples.

[0062] Figure 9 For detection results of cow-pig mixed samples in simulated mixed samples.

[0063] Figure 10 For detection results of case materials. DETAILED DESCRIPTION

[0064] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the application.

[0065] The experimental methods in the following examples are all routine methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0066] In the quantitative test in the following examples, three repeated experiments were set up, and the results were averaged. EXAMPLE

[0067] I. Experimental materials and methods

[0068] 1. Sample collection

[0069] (1) Collect 3 samples for each of the following species.

[0070] Chicken, duck, sheep, pig and cattle muscle tissue samples were purchased from a slaughterhouse in Beijing, and their authenticity was verified by morphology and used. Cat and dog muscle tissue samples were obtained from a pet hospital. Donkey and horse blood samples were collected from breeding farms. Mouse, rat, goose, rabbit, turkey and carp muscle tissue samples were purchased from HuiZhi and Yuan Biotech Co., Ltd. (Suzhou), China. Chinese hamster DNA standard was purchased from BeiNa Biological Company (Beijing). Human blood samples were obtained by elbow venipuncture, and the collection process followed the relevant procedures and protocols. All volunteers signed the informed consent form before sampling.

[0071] (2) Each meat product sample was 3 replicates. Meat product samples were purchased from Beijing market; meat product samples included three kinds of sausages (the label ingredients of the three kinds of sausages were chicken; chicken and pork; chicken, pork and beef;), beef jerky, marinated donkey meat, roast beef, roast mutton, stir-fried beef, stir-fried mutton, raw donkey meat and mutton rolls.

[0072] All samples were transported under ice-cold conditions (4℃), rinsed with 70% ethanol solution and double distilled water for 2-3 times, collected into clean sealed bags, and stored at -20℃ until used for DNA extraction.

[0073] The above sample collection work has been reviewed by the Scientific Ethics Committee of the Ministry of Public Security Identification Center (Approval No.: 2023-002).

[0074] 2、DNA extraction

[0075] DNA of samples was extracted using PureLink TM Genomic DNA Mini Kit (Thermo Fisher, USA), and the specific operation was according to the kit instructions.

[0076] DNA of samples was quantified using Qubit TM dsDNA HS Assay Kit (Thermo Fisher, USA) and 4.0 fluorometer (Thermo Fisher, USA). The DNA of samples was diluted to 1 ng / μL and stored at -20℃ for standby.

[0077] 3、Primer design and synthesis

[0078] (1) Download the mitochondrial genome sequences of 13 species of cattle (NC_006853.1), mouse (NC_005089.1), dog (NC_002008.4), rat (NC_001665.2), pig (NC_000845.1), Chinese hamster (NC_007936.1), cat (NC_001700.1), horse (NC_091244.1), human (NC_012920.1), chicken (NC_053523.1), duck (NC_009684.1), donkey (NC_001788.1), and sheep (NC_001941.1) from the GenBank database (www.ncbi.nlm.nih.gov).

[0079] (2) After step (1) is completed, use Oligo7 software to screen sequences with species specificity, and submit the screened nucleotide sequences to Primer3Plus (https: / / www.primer3plus.com) for primer design; then, use the Basic Local Alignment Search Tool (BLAST) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to check the inter-species specificity of all primers.

[0080] (3) After step (2) is completed, the 5' end of the reverse primer is labeled with a fluorescent dye 6-FAM or HEX, and the primers are synthesized by Shengong Bioengineering (Shanghai) Co., Ltd.

[0081] After multiple experiments, the final screened primer information and the length of the amplification product are shown in Table 1, columns 1-3.

[0082] Table 1

[0083]

[0084]

[0085] Note: The primers containing "F" in the name are upstream primers, and the primers containing "R" in the name are downstream primers.

[0086] 4. Single PCR and multiplex PCR detection

[0087] (1) Single PCR detection

[0088] Single PCR amplification was performed for each target species DNA using the primers synthesized in step 3. The amplification was performed in a 96-well thermal cycler (Bio-Rad, USA). The amplification products were analyzed by a 3500XL capillary electrophoresis instrument (Applied Biosystems, USA).

[0089] The single PCR system was 10 μL, including 2x Multiplex PCR Buffer (Huzhou Shenko, China) 5 μL, upstream primer 1 μL, downstream primer 1 μL, DNA 0.1 ng and nuclease-free water.

[0090] Amplification program: 95℃ 15min; 94℃ 30s, 58℃ 60s, 72℃ 60s, 30 cycles; 72℃ 10min.

[0091] (2) Multiplex PCR detection

[0092] For multiplex PCR, 26 primers in Table 1 with a concentration of 10 μM / L were mixed uniformly, diluted with nuclease-free water to obtain a mixed primer solution with a concentration of 0.02 μM / L. Thirteen samples with a DNA concentration of 5 ng / μL were mixed and diluted with nuclease-free water to obtain a mixed DNA solution with a DNA concentration of 0.1 ng / μL. Then, multiplex PCR amplification was performed in a 96-well thermal cycler. On the basis of the single PCR system, the primer concentration, annealing temperature and other PCR reaction conditions of the multiplex PCR detection system were optimized to ensure that the reaction results did not show non-specific amplification phenomenon, and the optimal primer concentration and reaction conditions were determined; that is, the optimized multiplex PCR detection system and reaction conditions were obtained.

[0093] 5. Species specificity study

[0094] The optimized multiplex PCR detection system was used to detect the DNA of 17 species (including 13 target DNAs and 4 non-target DNAs) extracted, and a negative control with nuclease-free water as a template was set up to evaluate the species specificity of the method.

[0095] 6. Sensitivity study

[0096] The mixture of DNAs of 13 species was added to the corresponding reaction system in an amount of 0.1 ng, 0.05 ng, 0.01 ng, 0.005 ng and 0.001 ng, respectively, for detection to determine the sensitivity of the method. Each template amount was detected in triplicate.

[0097] 7. Detection of simulated mixed samples

[0098] To investigate the mixed sample detection ability of the system, sheep and duck mixed samples, donkey and horse mixed samples, and pig and beef mixed samples were prepared. Specifically, duck meat was mixed into sheep meat at a mass ratio of 10%, 5%, 1%, 0.5%, and 0.1%, respectively, to obtain sheep and duck mixed samples. Horse blood was mixed into donkey blood at a mass ratio of 10%, 5%, 1%, 0.5%, and 0.1%, respectively, to obtain donkey and horse mixed samples. Pig meat was mixed into beef at a mass ratio of 10%, 5%, 1%, 0.5%, and 0.1%, respectively, to obtain pig and beef mixed samples.

[0099] After the sheep and duck mixed samples, donkey and horse mixed samples, and pig and beef mixed samples were fully mixed, DNA was extracted, and amplification detection was performed using the multiplex PCR detection system.

[0100] 8. Commercial meat product samples and case evidence detection

[0101] To investigate the applicability of the system to actual sample detection, 27 commercial meat product samples were purchased from local markets in Beijing, including 3 samples of raw donkey meat, 3 samples of mutton rolls, 3 samples of beef jerky, 3 samples of stewed donkey meat, 3 samples of roast beef, 3 samples of roast mutton, 3 samples of stir-fried beef, and 3 samples of stir-fried mutton; 3 samples of ham sausages, with the label ingredients being chicken, chicken and pork, and chicken, pork, and beef, respectively. DNA extraction was performed on the collected samples, and amplification detection was performed using the multiplex PCR detection system.

[0102] To investigate the applicability of the system in forensic evidence inspection, the on-site materials of an actual case were collected, DNA was extracted from the collected materials, and amplification detection was performed using the multiplex PCR detection system.

[0103] II. Experimental results

[0104] 1. Establishment of multiplex PCR-capillary electrophoresis detection system

[0105] First, 13 target sample DNAs were subjected to single PCR amplification using the 13 primer pairs shown in Table 1, and the reaction system was optimized. The detection results showed that the lengths of the amplification products of the 13 target samples were consistent with the theoretical sizes. On this basis, the primers of the 13 primer pairs shown in Table 1 were mixed, and the multiplex PCR system was optimized, and finally the optimized multiplex PCR detection system and the optimized multiplex PCR reaction conditions were obtained.

[0106] The optimized multiplex PCR detection system was 10 μL, which consisted of 5 μL 2×Multiplex PCR Buffer (Huzhou Shenko, China), 2 μL primer mixture (26 primers mixed in Table 1), 0.1 ng DNA template, and nuclease-free water. In the optimized multiplex PCR detection system, the concentration of each primer in the detection system is shown in column 4 of Table 1.

[0107] Optimized multiplex PCR reaction conditions: 95℃ 15min; 94℃ 30s, 62℃ 60s, 72℃ 60s, 30 cycles; 72℃ 10min.

[0108] The detection results are shown in Figure 1 (Cattle for cattle, Mice for mice, Dog for dog, Rat for rat, Pig for pig, Hamster for hamster, Cat for cat, Horse for horse, Human for human, Chicken for chicken, Duck for duck, Ass for ass, Sheep for sheep) The results showed that the optimized multiplex PCR detection system could accurately distinguish 13 species.

[0109] 2. Species specificity study

[0110] The optimized multiplex PCR system was used to amplify 13 target DNAs and 4 non-target DNAs separately to verify the species specificity of the system.

[0111] The detection results are shown in Figure 2 (a-f are beef samples, mouse meat samples, dog meat samples, rat meat samples, pig meat samples and hamster DNA standard, respectively), Figure 3 (g-l are cat meat samples, horse blood samples, human blood samples, chicken meat samples, duck meat samples and donkey blood samples, respectively), and Figure 4 (m-q are sheep meat samples, goose meat samples, rabbit meat samples, turkey meat samples and carp meat samples, respectively, and r is a negative control). The results showed that the optimized multiplex PCR detection system could accurately distinguish 13 species, and there was no non-specific amplification phenomenon for 4 non-target DNAs.

[0112] 3. Sensitivity study

[0113] The sensitivity detection results are shown in Figure 5 (a is 0.5 ng, b is 0.1 ng, c is 0.05 ng), and Figure 6 (d is 0.01 ng, e is 0.005 ng, f is 0.001 ng). When the amount of mixed DNA template was 0.05 ng, the amplification products of 13 species could be detected; when the amount of mixed DNA template was 0.001 ng, the DNA of 10 species of dog, rat, hamster, cat, horse, human, chicken, duck, donkey and sheep could be detected, the detection limit of mouse DNA was 0.05 ng, the detection limit of pig DNA was 0.01 ng, and the detection limit of cattle DNA was 0.005 ng.

[0114] 4. Detection results of simulated mixed samples

[0115] The results of detecting the simulated mixed samples using the optimized multiplex PCR system are shown in Table 1. Figure 7 (a-e are sheep-duck mixed samples obtained by mixing duck meat into sheep meat at a mass ratio of 10%, 5%, 1%, 0.5%, and 0.1%, respectively), Figure 8 (f-j are donkey-horse mixed samples obtained by mixing horse blood into donkey blood at a mass ratio of 10%, 5%, 1%, 0.5%, and 0.1%, respectively), and Figure 9 (k-o are cow-pig mixed samples obtained by mixing pig meat into cow meat at a mass ratio of 10%, 5%, 1%, 0.5%, and 0.1%, respectively). The duck-derived components in the sheep-duck mixed samples can be detected when the duck meat content is 10%, 5%, 1%, or 0.5%, but not when the duck meat content is 0.1%. The horse-derived components in the donkey-horse mixed samples can be detected when the horse blood content is 10%, 5%, 1%, 0.5%, or 0.1%. The pig-derived components in the cow-pig mixed samples can be detected when the pig meat content is 10%, 5%, 1%, or 0.5%, but not when the pig meat content is 0.1%.

[0116] 5. Results of detecting commercially available meat product samples and case materials

[0117] (1) The 27 collected commercially available meat product samples were detected by amplification using the multiplex PCR system, and the detection results of the 27 commercially available meat product samples are shown in Table 2. Samples 1-4, 7-18, 21-25, and 27 were consistent with the meat-derived components declared in the labels; however, sample 5 detected donkey and pig-derived components, suggesting that the merchant had mixed pig meat into donkey meat; sample 6 only detected horse-derived components, suggesting that the product was horse meat disguised as donkey meat; sample 19 detected donkey and cow-derived components, suggesting that the merchant had mixed cow meat into donkey meat; sample 20 only detected cow-derived components, suggesting that the product was cow meat disguised as donkey meat; and sample 26 detected cow and pig-derived components, suggesting that the merchant had mixed pig meat into cow meat. Five of the 27 samples were detected to have the possibility of adulteration, and the total adulteration detection rate was 18.52%.

[0118] Table 2

[0119]

[0120]

[0121] (2) The case introduction of the actual case is as follows: On March 2025, a case of wild dog attack resulting in death occurred in a certain place in Shaanxi Province, and the clothes fragments of the suspected bite sites of the victim were extracted as materials, named TF1, TF2, or TF3, respectively. The DNA of the materials (TF1, TF2, or TF3) was extracted, and then detected using the optimized multiplex PCR system.

[0122] The detection results are shown in Table 1 Figure 10 (a is TF1, b is TF2, and c is TF3). The test results show that the DNA of human and dog is detected in the three samples, which proves that the stray dog indeed contacted with the victim in this case. In addition, a small amount of pig DNA is detected in sample 2, which is presumably due to the stray dog eating pork and its related meat products before contacting with the victim, resulting in the presence of pig DNA in the clothes fragments at the suspected bite site.

[0123] The above has been described in detail. For those skilled in the art, the present application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the present application and without unnecessary experiments. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, this application intends to include any changes, uses or improvements of the present application, including changes made by conventional techniques known in the art, which are out of the scope disclosed in this application.

Claims

1. A primer combination comprising primer Fl, primer Rl, primer F2, primer R2, primer F3, primer R3, primer F4, primer R4, primer F5, primer R5, primer F6, primer R6, primer F7, primer R7, primer F8, primer R8, primer F9, primer R9, primer F10, primer R10, primer Fl l, primer Rl l, primer F12, primer R12, primer F13 and primer R13; the primer Fl is a single-stranded DNA molecule as shown in SEQ ID No. 1; the primer Rl is a single-stranded DNA molecule as shown in SEQ ID No. 2; the primer F2 is a single-stranded DNA molecule as shown in SEQ ID No. 3; the primer R2 is a single-stranded DNA molecule as shown in SEQ ID No. 4; the primer F3 is a single-stranded DNA molecule as shown in SEQ ID No. 5; the primer R3 is a single-stranded DNA molecule as shown in SEQ ID No. 6; the primer F4 is a single-stranded DNA molecule as shown in SEQ ID No. 7; the primer R4 is a single-stranded DNA molecule as shown in SEQ ID No. 8; the primer F5 is a single-stranded DNA molecule as shown in SEQ ID No. 9; the primer R5 is a single-stranded DNA molecule as shown in SEQ ID No. 10; the primer F6 is a single-stranded DNA molecule as shown in SEQ ID No. 11; the primer R6 is a single-stranded DNA molecule as shown in SEQ ID No. 12; the primer F7 is a single-stranded DNA molecule as shown in SEQ ID No. 13; the primer R7 is a single-stranded DNA molecule as shown in SEQ ID No. 14; the primer F8 is a single-stranded DNA molecule as shown in SEQ ID No. 15; the primer R8 is a single-stranded DNA molecule as shown in SEQ ID No. 16; the primer F9 is a single-stranded DNA molecule as shown in SEQ ID No. 17; the primer R9 is a single-stranded DNA molecule as shown in SEQ ID No. 18; the primer F10 is a single-stranded DNA molecule as shown in SEQ ID No. 19; the primer R10 is a single-stranded DNA molecule as shown in SEQ ID No. 20; the primer Fl l is a single-stranded DNA molecule as shown in SEQ ID No. 21; the primer Rl l is a single-stranded DNA molecule as shown in SEQ ID No. 22; the primer F12 is a single-stranded DNA molecule as shown in SEQ ID No. 23; the primer R12 is a single-stranded DNA molecule as shown in SEQ ID No. 24; the primer F13 is a single-stranded DNA molecule as shown in SEQ ID No. 25; the primer R13 is a single-stranded DNA molecule as shown in SEQ ID No.

26.

2. The primer combination according to claim 1, characterized in that: The primer combination is composed of primer F1, primer R1, primer F2, primer R2, primer F3, primer R3, primer F4, primer R4, primer F5, primer R5, primer F6, primer R6, primer F7, primer R7, primer F8, primer R8, primer F9, primer R9, primer F10, primer R10, primer F11, primer R11, primer F12, primer R12, primer F13 and primer R13.

3. The primer combination according to claim 1, characterized in that: The molar ratio of primer F1, primer R1, primer F2, primer R2, primer F3, primer R3, primer F4, primer R4, primer F5, primer R5, primer F6, primer R6, primer F7, primer R7, primer F8, primer R8, primer F9, primer R9, primer F10, primer R10, primer F11, primer R11, primer F12, primer R12, primer F13 and primer R13 is 1:1:1:1:1:1:1:1:20:20:4:4:2:2:2:2:2:2:2:2:1:1:1:1:1:

1.

4. The primer combination according to any one of claims 1 to 3, wherein: primer R1, primer R2, primer R3, primer R4, primer R5, primer R6, primer R7 and primer R8 are labeled with fluorescent methyl; primer R9, primer R10, primer R11 and primer R12 and primer R13 are labeled with fluorescent ethyl.

5. The primer combination according to claim 4, wherein: the 5' end of primer R1, primer R2, primer R3, primer R4, primer R5, primer R6, primer R7 and primer R8 is labeled with 6-FAM; the 5' end of primer R9, primer R10, primer R11, primer R12 and primer R13 is labeled with HEX.

6. A detection system for identifying 13 species, comprising the primer combination according to any one of claims 1 to 5; the 13 species are chicken, duck, sheep, pig, cow, mouse, rat, cat, dog, donkey, horse, human and Chinese hamster.

7. The detection system according to claim 6, wherein: the concentration of primer F5 and primer R5 in the detection system is 0.1 μM / L; the concentration of primer F1, primer R1, primer F2, primer R2, primer F3, primer R3, primer F4, primer R4, primer F11, primer R11, primer F12, primer R12, primer F13 and primer R13 in the detection system is 0.005 μM / L; the concentration of primer F7, primer R7, primer F8, primer R8, primer F9, primer R9, primer F10 and primer R10 in the detection system is 0.01 μM / L; the concentration of primer F6 and primer R6 in the detection system is 0.02 μM / L.

8. A kit containing the primer combination according to any one of claims 1 to 5; the kit is used for identifying 13 species; the 13 species are chicken, duck, sheep, pig, cow, mouse, rat, cat, dog, donkey, horse, human and Chinese hamster.

9. A method for preparing the detection system of claim 6 or 7 or the kit of claim 8, comprising the step of individually packaging each primer in the primer combination of any one of claims 1 to 5.

10. X1) or X2): X1) use of the primer combination of any one of claims 1 to 5, or the detection system of claim 6 or 7, in the preparation of a kit for identifying 13 species; X2) use of the primer combination of any one of claims 1 to 5, or the detection system of claim 6 or 7, in identifying 13 species; the 13 species are chicken, duck, sheep, pig, cow, mouse, rat, cat, dog, donkey, horse, human and Chinese hamster; the use is for non-disease diagnosis and treatment. ​ ​

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