Specific primer group, probe group, internal reference primer pair, membrane gene chip, detection kit, application and detection method of two-head anemone

Abstract

The invention discloses a specific primer group, a probe group, an internal reference primer pair, a membrane gene chip, a detection kit, application and a detection method for two-end black knotweed, and belongs to the technical field of molecular biology detection.The two-end black knotweed mitochondrial DNA sequence is compared and analyzed, variety-specific variation sites of Cytb, D-loop and COI genes are screened, and the variety-specific variation sites of CYtb, D-loop and COI genes are obtained. Three pairs of specific primers and three biotin labeled probes are designed, a membrane gene chip detection platform is established, the primer amplification fragment length is 142-183bp, the membrane chip is suitable for detection of processed meat products, a biotin-streptavidin-alkaline phosphatase color development system is adopted by the membrane chip, the detection result is visual, and the detection efficiency is high. According to the invention, the Jinhua rhizoma anemonis can be accurately distinguished from commercial pigs such as Duroc, Changbai, Dabai and the like and other local pig varieties, the specificity reaches 100%, the sensitivity reaches a DNA template of 1ng, not less than 1% of Jinhua rhizoma anemonis components can be detected, and the whole detection process is completed within 3-4 hours.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology detection, and particularly relates to a Jinhua two-headed black pig specific primer set, a probe set, an internal reference primer pair, a membrane gene chip, a detection kit, an application and a detection method. BACKGROUND

[0002] Jinhua two-headed black pig is a special fine local pig breed in Jinhua region of Zhejiang Province. It is named for its black head and hip and white middle body, and is locally known as two-headed black or Jinhua pig. It has been bred for more than 1600 years.

[0003] Traditional pig breed identification methods mainly rely on morphological feature observation, including coat color, body shape, head shape, ear shape and other appearance traits. Jinhua two-headed black pig has typical two-headed black and middle white coat color characteristics, with black hair on the head and hip, and white color on the middle body and lower limbs. The black and white boundary is clear. However, this morphological identification method has obvious limitations: first, it is only suitable for identification of live pigs or carcasses with complete hair, and cannot be used for breed determination of skinned and segmented meat; second, it is completely impossible to trace the origin of processed meat products such as ham, sausage, bacon and meatballs through appearance; third, morphological characteristics are affected by factors such as feeding environment, age and individual differences, and have certain subjectivity and uncertainty; fourth, part of the hybrid offspring may retain similar coat color characteristics, which is difficult to accurately distinguish from purebred Jinhua two-headed black pig only by appearance.

[0004] DNA molecular detection technology uses the specificity of biological genetic material to achieve accurate identification of species or breeds, and has the advantages of strong objectivity, high accuracy, good repeatability and being unaffected by sample morphology and processing state, and has become the mainstream technical means in the field of meat authenticity identification. DNA barcoding technology is a molecular diagnostic technology developed in recent years based on standardized short sequence DNA fragments for species identification. By comparing the DNA sequence of the sample to be tested with the sequence information of known species in the reference database, the species can be quickly and accurately identified. Mitochondrial DNA is often selected as the target region for DNA barcoding analysis due to its high copy number (each cell contains hundreds to thousands of mitochondria), maternal inheritance, no recombination and moderate evolution rate. The coding genes such as cytochrome c oxidase subunit I (COI), cytochrome b (Cytb) and non-coding control region D-loop are often selected as the target region for DNA barcoding analysis. In the study of pig molecular systematics, the sequences of mitochondrial Cytb gene and D-loop region are rich in variation and have been widely used in genetic diversity analysis and phylogenetic relationship research of pig breeds.

[0005] In the prior art, the patents and literature reports on DNA detection of pork and its products mainly focus on the following aspects: first, meat adulteration detection at the species level, such as distinguishing pork from beef, mutton, chicken, duck and other meat components of different species, representative technologies include multiplex PCR, real-time fluorescent quantitative PCR, loop-mediated isothermal amplification, etc.; second, individual-level pork traceability, by detecting nuclear genomic microsatellite markers or SNP sites to establish individual DNA fingerprint, to realize the whole process traceability from farm to table; third, molecular detection of specific pathogenic microorganisms or veterinary drug residues in pork. However, in the aspect of breed level pork authenticity identification, the prior art still has obvious deficiencies.

[0006] Patent CN105671192A discloses a DNA barcode encoding method and traceability method for pork traceability, which establishes a DNA barcode by detecting a combination of pig individual-specific SNP sites, and can realize individual tracking of pork source. However, this method focuses on individual identification rather than breed identification, requires pre-collection and establishment of a large-scale individual DNA reference database, and has high detection cost, which is difficult to popularize and apply in actual supervision. Patent CN108624716B discloses a primer set and detection method for simultaneously detecting seven kinds of meat source food based on seven-plex PCR technology, including species-specific primers for pigs, cattle, sheep, chickens, ducks, horses and camels, which can realize rapid identification of meat species source, but this method can only distinguish species and cannot distinguish different breeds of pigs.

[0007] In terms of academic literature reports, the research on pig breed molecular markers mostly uses nuclear genome SNP chip or microsatellite typing technology. Although these methods have high resolution and accuracy, they require large-scale instruments such as high-throughput sequencing platforms or capillary electrophoresis instruments, have high reagent costs, require high technical requirements, and have long detection periods, which are difficult to meet the needs of grassroots detection institutions and on-site rapid screening. In addition, nuclear genomic markers have large variations between individuals, and the difference in allele frequency distribution between different geographical populations also affects the accuracy of breed determination. In contrast, mitochondrial DNA markers are relatively conserved within breeds due to their maternal inheritance characteristics, and have obvious differences between breeds, making them more suitable for breed-level identification.

[0008] Membrane-based gene chip technology is a kind of molecular diagnostic technology that fixes oligonucleotide probes on the surface of solid carriers such as nylon membrane or nitrocellulose membrane, and detects target DNA sequences through nucleic acid hybridization reaction. Compared with glass substrate chip and high-throughput sequencing, membrane-based gene chip has the following significant advantages: low cost, the cost of single detection can be controlled within dozens of yuan; simple operation, no need for expensive special detection equipment, ordinary laboratory can complete; intuitive results, visual color signal can be generated through enzyme color reaction, and can be judged by naked eye; good stability, membrane chip can be stored for a long time under normal temperature and dry conditions; flexible throughput, different probe combinations can be designed according to detection needs. Membrane-based gene chip technology has been successfully applied in the fields of pathogenic microorganism detection, transgenic component screening, food allergen identification and the like.

[0009] However, as of now, there is no related report on the screening of Jinhua Liangtouwu pig breed-specific DNA markers and membrane-based gene chip detection methods. There is a lack of a set of Jinhua Liangtouwu pig molecular identification technology system with simple operation, low cost, strong specificity and high sensitivity in the prior art, which cannot meet the actual needs of market supervision and consumer rights protection. Therefore, it is urgent to develop breed-specific molecular markers for Jinhua Liangtouwu pig, and to establish a rapid detection method suitable for popularization and application at the grassroots level. SUMMARY

[0010] In view of the above deficiencies in the prior art, the purpose of the present application is to provide Jinhua Liangtouwu pig-specific primer probes and their applications. The present application screens and identifies nucleotide variation sites specific to Jinhua Liangtouwu pig through systematic comparison and analysis of mitochondrial DNA sequences of Jinhua Liangtouwu pig and other commercial pig and local pig breeds, designs breed-specific PCR amplification primers and hybridization probes accordingly, establishes a visual detection method based on membrane-based gene chip, realizes rapid and accurate differentiation of Jinhua Liangtouwu pig from commercial pig breeds such as Duroc, Changbai and White, as well as other local pig breeds such as Taihu pig, Rongchang pig and Tibetan pig, and provides a reliable technical means for authenticity identification of Jinhua Liangtouwu pork and its processed products.

[0011] To achieve the above-mentioned purposes, the present application adopts the following technical solutions: The first aspect of the present application provides a Jinhua Liangtouwu pig-specific primer set, which comprises the following three pairs of oligonucleotide primers: The primer pair JH-Cytb is used for specific amplification of the DNA fragment containing breed-specific variation sites in the mitochondrial cytochrome b gene of Jinhua Liangtouwu pig, which is composed of forward primer JH-Cytb-F and reverse primer JH-Cytb-R, wherein the nucleotide sequence of the forward primer JH-Cytb-F is shown in SEQ ID NO: 1, the nucleotide sequence of the reverse primer JH-Cytb-R is shown in SEQ ID NO: 2, and the length of the amplification product is 156 bp.

[0012] The primer pair JH-DL for specifically amplifying the DNA fragment containing breed-specific mutation site in the mitochondrial D-loop control region of Jinhua two-way black pig, which consists of forward primer JH-DL-F and reverse primer JH-DL-R, wherein the nucleotide sequence of the forward primer JH-DL-F is shown as SEQ ID NO: 3, the nucleotide sequence of the reverse primer JH-DL-R is shown as SEQ ID NO: 4, and the length of the amplification product is 183 bp.

[0013] The primer pair JH-COI for specifically amplifying the DNA fragment containing breed-specific mutation site in the mitochondrial cytochrome c oxidase subunit I gene of Jinhua two-way black pig, which consists of forward primer JH-COI-F and reverse primer JH-COI-R, wherein the nucleotide sequence of the forward primer JH-COI-F is shown as SEQ ID NO: 5, the nucleotide sequence of the reverse primer JH-COI-R is shown as SEQ ID NO: 6, and the length of the amplification product is 142 bp.

[0014] The second aspect of the present application provides a Jinhua two-way black pig-specific probe set, which comprises the following three 5' end biotin-labeled oligonucleotide probes: The probe JH-Cytb-P has the nucleotide sequence shown as SEQ ID NO: 7, and a biotin molecule is covalently linked to the 5' end, which is used for detecting the PCR amplification product of the JH-Cytb primer pair by nucleic acid hybridization reaction.

[0015] The probe JH-DL-P has the nucleotide sequence shown as SEQ ID NO: 8, and a biotin molecule is covalently linked to the 5' end, which is used for detecting the PCR amplification product of the JH-DL primer pair by nucleic acid hybridization reaction.

[0016] The probe JH-COI-P has the nucleotide sequence shown as SEQ ID NO: 9, and a biotin molecule is covalently linked to the 5' end, which is used for detecting the PCR amplification product of the JH-COI primer pair by nucleic acid hybridization reaction.

[0017] The third aspect of the present application provides an internal reference primer pair JH-IC for amplifying the conserved region fragment of the porcine mitochondrial 16S ribosomal RNA gene as the internal reference control of PCR amplification and hybridization reaction, which consists of forward primer JH-IC-F and reverse primer JH-IC-R, wherein the nucleotide sequence of the forward primer JH-IC-F is shown as SEQ ID NO: 10, the nucleotide sequence of the reverse primer JH-IC-R is shown as SEQ ID NO: 11, and the length of the amplification product is 128 bp. The internal reference primer pair targets the highly conserved region of the porcine mitochondrial 16S rRNA gene, which can be stably amplified in all pig breeds, and is used for verifying the quality of DNA extraction and the effectiveness of the PCR reaction system.

[0018] The fourth aspect of this invention provides a Jinhua two-headed aconite membrane gene chip, the membrane gene chip comprising a positively charged nylon membrane carrier and a probe array covalently fixed to the surface of the nylon membrane by ultraviolet cross-linking. The probe array includes Jinhua two-headed aconite-specific probes JH-Cytb-P, JH-DL-P, JH-COI-P and an internal reference probe IC-P. Each probe is fixed to the surface of the nylon membrane in a dot matrix pattern according to a pre-designed pattern, and each probe is provided with three parallel repeat sampling positions to ensure the reliability of the detection results.

[0019] The fifth aspect of this invention provides a detection kit for Aconitum carmichaelii from Jinhua, the kit comprising the following components: Aconitum carmichaelii-specific primer set, Aconitum carmichaelii-specific probe set, internal reference primer pair, pre-prepared membrane gene chip, hybridization buffer, streptavidin-alkaline phosphatase conjugate, NBT / BCIP chromogenic substrate solution, washing buffer, and Aconitum carmichaelii-positive control DNA and negative control DNA.

[0020] The sixth aspect of this invention provides the application of the above-mentioned Jinhua two-headed cauldron specific primer set, Jinhua two-headed cauldron specific probe set or Jinhua two-headed cauldron membrane gene chip in the identification of porcine-derived components of Jinhua two-headed cauldron. The application includes, but is not limited to, the qualitative detection of Jinhua two-headed cauldron components in pork products such as fresh pork, frozen pork, Jinhua ham, sausage, cured meat, meatballs, and minced meat.

[0021] The seventh aspect of this invention provides a method for detecting components derived from Jinhua two-headed black pigs, the method comprising the following steps: S1. DNA Extraction: Total DNA was extracted from the meat samples to be tested using a commercial animal tissue genomic DNA extraction kit or a modified CTAB method. The DNA concentration and purity were determined using a Nanodrop spectrophotometer.

[0022] S2. PCR amplification: Using the extracted genomic DNA as a template, multiplex PCR amplification reactions were performed using three pairs of specific primers (JH-Cytb, JH-DL, and JH-COI) and the internal reference primer pair (JH-IC) to obtain amplification products containing variety-specific variation sites.

[0023] S3. Hybridization reaction: The PCR amplification product is denatured in a boiling water bath to break down the double-stranded DNA into single strands. It is then immediately cooled in an ice bath, and hybridization buffer is added. The product is then subjected to nucleic acid hybridization reaction with a pre-prepared membrane gene chip at a suitable temperature.

[0024] S4. Colorimetric detection: After the hybridization reaction is completed, non-specifically bound nucleic acid molecules are removed by gradient washing. Then, streptavidin-alkaline phosphatase conjugate solution is added for incubation, allowing the enzyme molecules to specifically bind to the biotin at the 5′ end of the probe via streptavidin. Finally, NBT / BCIP chromogenic substrate solution is added for enzymatic colorimetric reaction. The distribution pattern of the colored spots on the membrane chip is used to determine whether the sample contains Jinhua two-headed aconite.

[0025] Compared with the prior art, the present invention has the following significant beneficial technical effects: First, this invention is the first to systematically screen and identify mitochondrial DNA molecular markers specific to the Jinhua Two-Ended Black Pig breed, including multiple specific variant sites such as the T→C transition at site 14762 and the A→G transition at site 14891 of the Cytb gene, the G→A transition at site 16107 and the CTAA tetrabase insertion at site 16233 of the D-loop region, and the C→T transition at site 6542 of the COI gene. Among them, the CTAA insertion sequence at site 16233 of the D-loop region is unique to the Jinhua Two-Ended Black Pig and was not found in any of the 140 other pig breed samples tested in this invention, and can be used as a characteristic molecular tag of the Jinhua Two-Ended Black Pig.

[0026] Secondly, this invention employs a multi-site joint detection strategy, using specific markers from three independent mitochondrial gene regions for comprehensive determination, significantly improving the reliability and accuracy of variety identification and effectively avoiding false positives or false negatives that may occur with single markers. Validated with 50 samples of *Aconitum carmichaelii* from Jinhua and 90 control samples from other varieties, the detection specificity reached 100%, and the positive detection rate reached 98%.

[0027] Third, the PCR amplification fragments designed in this invention are all controlled to be below 200bp, with the shortest being only 142bp. This can effectively overcome the problem of long fragment amplification difficulties caused by DNA degradation in deep-processed meat products such as ham, sausage, and cured meat due to factors such as high temperature, acid and alkali, and enzymatic hydrolysis, and significantly expand the applicable scope of the detection method.

[0028] Fourth, the membrane gene chip detection platform established in this invention uses a biotin-streptavidin-alkaline phosphatase signal amplification system. It generates a stable purple-blue precipitate through the enzymatic colorimetric reaction of NBT / BCIP substrate. The detection results are visible to the naked eye and do not require a fluorescence microscope or special reading equipment, which greatly reduces the instrument dependence and technical threshold of the detection.

[0029] Fifth, the method of this invention has high detection sensitivity, with a DNA template detection limit of 1 ng, capable of detecting Jinhua two-headed aconite in samples as low as 1%, meeting the practical needs of adulterated sample detection. The entire detection process, including DNA extraction, PCR amplification, hybridization reaction, and colorimetric detection, can be completed within 3 to 4 hours, demonstrating high detection efficiency.

[0030] Sixth, the reagent kit of this invention is inexpensive, with a single test costing no more than 50 yuan. The membrane chip is simple to prepare and stable to store, making it suitable for rapid on-site screening by grassroots animal husbandry and veterinary stations, food testing institutions, market supervision departments, etc., and has broad prospects for promotion and application. Attached Figure Description

[0031] Figure 1 This image shows the electrophoresis verification results of singleton PCR amplification of *Ligustrum lucidum* (Jinhua) specific primers in this invention. M represents the DL2000 DNA Marker. Lane 1 shows a 156bp specific band amplified with the JH-Cytb primer pair; lane 2 shows a 183bp specific band amplified with the JH-DL primer pair; lane 3 shows a 142bp specific band amplified with the JH-COI primer pair; lane 4 shows a 128bp band amplified with the JH-IC internal control primers; lanes 5-10 show no target band amplified with the JH-DL primer pairs for Duroc, Landrace, Large White, Taihu pig, Rongchang pig, and Tibetan pig DNA, respectively; and lane 11 is a template-free negative control.

[0032] Figure 2 This is a graph showing the results of gradient PCR electrophoresis detection of the Jinhua two-headed aconite specific primers of the present invention; where M is DL2000 DNA Marker, lanes 1-8 are the amplification results of JH-DL primers with DNA template amounts of 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 0.1pg, and 0pg (negative control), respectively, showing a detection sensitivity of 1ng.

[0033] Figure 3 This is a schematic diagram of the probe spotting layout for the Jinhua Two-Headed Black Fungus membrane gene chip of the present invention; wherein, the nylon membrane is 1cm×1cm in size and adopts a 5-row 3-column array layout, A1-A3 are three repeat spotting positions of the JH-Cytb-P probe, B1-B3 are three repeat spotting positions of the JH-DL-P probe, C1-C3 are three repeat spotting positions of the JH-COI-P probe, D1-D3 are three repeat spotting positions of the IC-P internal reference probe, E1 is the biotin-labeled positive control point, E2 is the unlabeled probe negative control point, and E3 is the blank spotting buffer control point.

[0034] Figure 4 This is a schematic diagram of the hybridization and colorimetric detection results of the membrane gene chip of the present invention; wherein, Figure 4 a shows the test results of a positive sample of *Aconitum carmichaelii* from Jinhua. The probe positions in rows A, B, C, and D, as well as the positive control position E1, all show clear purple-blue spots. Figure 4b represents the test results for a Duroc negative sample. Only the internal reference probe position in row D and the E1 positive control position showed stained spots, while the specific probe positions in rows A, B, and C showed no stained spots. Detailed Implementation

[0035] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments are for illustration and explanation only and should not be construed as limiting the scope of protection of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention.

[0036] Unless otherwise specified, all experimental reagents and instruments used in this invention are commercially available products and can be obtained through legitimate channels. The animal tissue genomic DNA extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number DP304. PremixTaq DNA polymerase premix solution was purchased from Takara Bio Engineering (Dalian) Co., Ltd., catalog number RR901A. The DL2000 DNA Marker was purchased from Takara Bio Engineering (Dalian) Co., Ltd. Agarose was a product of Biowest (Spain), electrophoresis grade. Hybond-N+ positively charged nylon membrane was purchased from Cytiva (formerly GE Healthcare), USA, size 20cm × 20cm. Streptavidin-alkaline phosphatase conjugate was purchased from Sigma-Aldrich (USA), catalog number S2890. NBT (nitroblue tetrazolium) and BCIP (5-bromo-4-chloro-3-indole phosphate) were purchased from Sigma-Aldrich (USA). Primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd., with biotin-labeled 5′ ends of the probes. The PCR amplification instrument was the Applied Biosystems Veriti 96-well gradient PCR instrument. The gel electrophoresis apparatus was a DYCP-31DN horizontal electrophoresis tank from Beijing Liuyi Instrument Factory. The gel imaging system was a Bio-Rad GelDocXR+. The UV crosslinker was a UVP CL-1000. The Nanodrop micro-volume spectrophotometer was a Thermo Fisher Scientific product. The incubator was a Shanghai Yiheng Scientific Instruments Co., Ltd. DHP-9052 model.

[0037] Example 1: Screening and identification of mitochondrial DNA-specific variant sites in Jinhua Two-End Black Pigs.

[0038] This embodiment systematically compares and analyzes the mitochondrial DNA sequences of Jinhua Liangtouwu pigs with those of other commercial pigs and local pig breeds, and screens and identifies nucleotide variation sites unique to Jinhua Liangtouwu pigs, providing target sequence information for the subsequent design of specific primers and probes.

[0039] 1.1 Sample collection and DNA extraction.

[0040] Fifty ear tissue samples of purebred Jinhua Liangtouwu pigs were collected from five national-level conservation farms and breeding bases in the core production areas of Jinhua Liangtouwu pigs, including Wucheng District, Jindong District, and Lanxi City in Jinhua City, Zhejiang Province. Each sample contained approximately 100 mg of ear tissue. Immediately after collection, the samples were placed in centrifuge tubes containing 95% ethanol and stored at -20°C after transportation to the laboratory at 4°C. Simultaneously, 90 control pig breed samples were collected from the Zhejiang Provincial Animal Husbandry Technology Extension and Livestock Monitoring Station, the pig farm of the College of Animal Science of Zhejiang University, and large-scale commercial pig farming enterprises in China. These included: 15 Duroc pigs, 15 Landrace pigs, 15 Large White pigs, 10 Taihu pigs, 10 Rongchang pigs, 10 Tibetan pigs, 5 Jinhua Black pigs, 5 Jiaxing Black pigs, and 5 Chun'an Spotted pigs. All samples were accompanied by breed origin certificates and individual record information to ensure the accuracy of breed identification.

[0041] Total DNA was extracted from each sample using the Tiangen Animal Tissue Genomic DNA Extraction Kit, following the manufacturer's instructions. The specific steps are as follows: Approximately 25 mg of ear tissue sample was placed in a 1.5 mL centrifuge tube. 200 μL of lysis buffer GA and 20 μL of proteinase K solution were added, and the mixture was thoroughly vortexed and incubated at 56°C overnight until the tissue was completely lysed and transparent. 200 μL of buffer GB was added, and the mixture was incubated at 70°C for 10 min. 200 μL of anhydrous ethanol was added, and the mixture was thoroughly vortexed. The mixture was transferred to an adsorption column CB3, centrifuged at 12000 rpm for 30 s, and the waste liquid was discarded. 500 μL of protein removal buffer GD was added, centrifuged at 12000 rpm for 30 s, and the waste liquid was discarded. 600 μL of wash buffer PW was added, centrifuged at 12000 rpm for 30 s, and the waste liquid was discarded. This process was repeated once. The sample was centrifuged at 12000 rpm for 2 min to remove as much residual wash buffer as possible. Transfer the adsorption column to a new 1.5 mL centrifuge tube, add 100 μL of elution buffer TE to the center of the adsorption membrane, let stand at room temperature for 2 min, and then centrifuge at 12000 rpm for 2 min to elute the DNA.

[0042] The concentration and purity of the extracted DNA were determined using a Nanodrop micro-volume spectrophotometer. 260 / A 280 A purity ratio between 1.8 and 2.0 is considered acceptable, and a DNA concentration above 50 ng / μL is considered acceptable. In this example, the concentration range of the 140 extracted DNA samples was 68.5 to 312.7 ng / μL. 260 / A 280 The ratios ranged from 1.82 to 1.96, all of which met the requirements for subsequent experiments. The extracted DNA samples were aliquoted and stored at -20°C.

[0043] 1.2 Mitochondrial gene PCR amplification and sequencing.

[0044] Targeting four gene regions of the porcine mitochondrial genome—COI, Cytb, D-loop, and 16S rRNA—universal amplification primers covering the complete coding regions or major variable regions of each gene were designed, referencing the published complete porcine mitochondrial genome sequence in the NCBI database (GenBank accession number: NC_000845.1). The primer sequences are as follows: COI gene amplification primers (amplification product approximately 750bp): COI-UF: 5′-ACTCAACGAAGCATCCATATAATCGG-3′, COI-UR: 5′-ACTCCTTCTGTATTGCGTCAGGAT-3′; Cytb gene amplification primers (amplification product approximately 1200bp): Cytb-UF: 5′-GAAAAACCACCGTTGTTATTCAACT-3′, Cytb-UR: 5′-CTCCGATGTTTCATGTCTCTTTGTA-3′; D-loop region amplification primers (amplification product approximately 1100bp): DL-UF: 5′-CCTCCGTGAAACCAACAACC-3′, DL-UR: 5′-CGATGGCTTGAAAGCTACGG-3′; 16S rRNA gene amplification primers (amplification product approximately 600bp): 16S-UF: 5′-CCGGTCTGAACTCAGATCACGTAG-3′, 16S-UR: 5′-CTCCATAGGGTCTTCTCGTCTTGC-3′; The PCR amplification reaction system, 50 μL, contained: 5 μL 10×PCR Buffer, 4 μL dNTP primers (2.5 mmol / L each), 2 μL forward primer (10 μmol / L), 2 μL reverse primer (10 μmol / L), 0.5 μL Taq DNA polymerase (5 U / μL), 1 μL template DNA (approximately 50 ng), and ddH2O to a final volume of 50 μL. The PCR reaction program was: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 90 s, for a total of 35 cycles; final extension at 72℃ for 10 min; and storage at 4℃.

[0045] PCR products were analyzed by 1.5% agarose gel electrophoresis. After confirming that the amplified bands were of correct size and single size, the samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional Sanger sequencing. Amplification primers were used for sequencing. The sequencing results were examined using Chromas software to check peak quality, and DNAMAN software was used for sequence assembly and proofreading. After removing primer sequences and low-quality end sequences, the effective sequences of the four gene regions in each sample were obtained.

[0046] 1.3 Sequence alignment and variant site analysis.

[0047] Multiple sequence alignment (MSA) analysis of mitochondrial gene sequences from 140 samples was performed using MEGA11 software. First, the COI, Cytb, D-loop, and 16S rRNA gene sequences of each sample were imported into MEGA software. MSA was then performed using the ClustalW algorithm with the following parameters: GapOpeningPenalty 15, GapExtensionPenalty 6.66, and TransitionWeight 0.5. After alignment, obvious errors were manually checked and corrected.

[0048] Through systematic comparative analysis, the following breed-specific variant sites were identified among the Jinhua Two-End Black Pig and 89 other control pig breeds: In the Cytb gene region: Jinhua Liangtouwu pigs exhibit a T→C conversion mutation at locus 14762 (referencing NC_000845.1), where all 50 Jinhua Liangtouwu samples showed C, while all 90 control samples showed T; at locus 14891, they exhibit an A→G conversion mutation, where all 50 Jinhua Liangtouwu samples showed G, while all 90 control samples showed A. The combination of these two loci completely distinguishes Jinhua Liangtouwu from all control breeds.

[0049] In the D-loop control region, the Jinhua Two-End Black pig exhibited a G→A conversion mutation at position 16107, with all 50 Jinhua Two-End Black pig samples showing an A mutation, while all 90 control samples showed a G mutation. More importantly, a unique CTAA tetrabase insertion sequence specific to the Jinhua Two-End Black pig was found at position 16233. This insertion was present in all 50 Jinhua Two-End Black pig samples, but not detected in any of the 90 control samples from other breeds. This unique insertion sequence can serve as the most characteristic molecular marker of the Jinhua Two-End Black pig.

[0050] In the COI gene region: Jinhua Liangtouwu pigs showed a C→T conversion mutation at position 6542. All 50 Jinhua Liangtouwu samples were T, while 89 control samples were C (one Taihu pig sample was also T, indicating that the breed specificity of this site was slightly lower than that of Cytb and D-loop region markers).

[0051] The 16S rRNA gene sequence was highly conserved across all 140 samples, with no variety-specific variation sites found. However, this region could be used as a target for designing internal control primers.

[0052] In summary, this embodiment successfully screened five breed-specific variant sites of Jinhua Two-End Black Pig through large-scale mitochondrial DNA sequencing and alignment analysis of samples: Cytb gene sites 14762 and 14891, D-loop region sites 16107 and 16233 (CTAA insertion), and COI gene site 6542. This provides a reliable sequence basis for the design of specific primers and probes in the next step.

[0053] Example 2: Design and specificity verification of specific primers for Jinhua two-headed aconite.

[0054] In this embodiment, PCR amplification primers were designed based on the specific variant sites screened in Example 1, and the variety specificity and amplification efficiency of the primers were verified by singleton PCR and multiplex PCR experiments.

[0055] 2.1 Specific primer design.

[0056] Primers targeting specific mutation sites were designed using PrimerPremier 5.0 software. Primer design followed these principles: primer length was controlled between 18 and 25 nucleotides; GC content was controlled between 40% and 60%; theoretical annealing temperature (Tm) was controlled between 55°C and 65°C, with the Tm difference between the forward and reverse primers of the same primer pair not exceeding 2°C; the 3′ end of the primers avoided having more than three consecutive G or C bases; primer sequences were checked to avoid stable hairpin structures and primer dimers; the 3′ end of the primers was positioned at or near the specific mutation site, utilizing the principle of allele-specific PCR to improve amplification specificity; and the length of the amplified product was controlled between 100 and 200 bp to accommodate the detection of degraded DNA in processed meat products.

[0057] After multiple rounds of design optimization and preliminary screening, the final specific primer sequences are as follows: JH-Cytb primer pairs targeting specific sites 14762 and 14891 of the Cytb gene: JH-Cytb-F (SEQ ID NO:1): 5′-GCCATACACTACACAGCAGA-3′, 20nt in length, 50% GC content, Tm value 58.2℃ JH-Cytb-R (SEQ ID NO:2): 5′-TGGTTTGATGTGTGGGTGTA-3′, 20nt in length, 45% GC content, Tm value 57.3℃ Expected amplification product length 156bp.

[0058] JH-DL primer pairs targeting the CTAA insertion regions at positions 16107 and 16233 in the D-loop region: JH-DL-F (SEQ ID NO:3): 5′-CACCATCAACACCCAAAGCT-3′, 20nt in length, 50% GC content, Tm value 58.4℃ JH-DL-R (SEQ ID NO:4): 5′-GTGCTTAATGTGCTATGTACGTA-3′, 23nt in length, 39% GC content, Tm value 57.1℃ Expected amplified product length 183bp.

[0059] JH-COI primer pair, targeting the COI gene 6542 specific site region: JH-COI-F (SEQ ID NO:5): 5′-GGCTTCCTAGGTTTTATTGT-3′, length 20nt, GC content 40%, Tm value 55.6℃ JH-COI-R (SEQ ID NO:6): 5′-GCTCCTGCTAATACAGGTAA-3′, length 20nt, GC content 45%, Tm value 56.8℃ Expected amplification product length 142bp.

[0060] JH-IC internal reference primer pair, targeting the conserved region of the 16S rRNA gene: JH-IC-F (SEQ ID NO:10): 5′-CGATAAACCCCGATCAACCT-3′, length 20nt, GC content 50%, Tm value 58.1℃ JH-IC-R (SEQ ID NO:11): 5′-AGCCCATGATTTTGACTCAA-3′, length 20nt, GC content 40%, Tm value 56.2℃ Expected amplification product length 128bp.

[0061] All primers were tested for specificity using the NCBI Primer-BLAST online tool, confirming the absence of non-specific binding sites in the porcine genome. Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., purified by PAGE, and delivered as dry powder. They were then dissolved in sterile ddH2O to a stock solution concentration of 100 μmol / L, aliquoted, and stored at -20°C. Before use, they were diluted to a working solution concentration of 10 μmol / L.

[0062] 2.2 Validation of singleton PCR specificity.

[0063] Using 50 DNA samples from Jinhua *Ligustrum lucidum* and 90 DNA samples from other varieties as templates, singleton PCR amplification was performed using three pairs of specific primers (JH-Cytb, JH-DL, JH-COI) and the internal reference primer (JH-IC) to verify the variety specificity of each primer pair.

[0064] The PCR reaction mixture was 25 μL and contained: 2.5 μL 10×PCR Buffer, 2 μL dNTP primers (2.5 mmol / L each), 1 μL forward primer (10 μmol / L), 1 μL reverse primer (10 μmol / L), 0.2 μL Taq DNA polymerase (5 U / μL), 1 μL template DNA (50 ng / μL), and 17.3 μL ddH2O. A template-free negative control was also included.

[0065] PCR reaction procedure: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ final extension for 7 min; store at 4℃.

[0066] Take 8 μL of PCR product, add 2 μL of 6× Loading Buffer, mix well, and then load onto a 2% agarose gel (containing 0.5 μg / mL ethidium bromide). Electrophoresis is performed at 120V for 40 min. After electrophoresis, observe and photograph using a Bio-Rad GelDocXR+ gel imaging system.

[0067] The results of singleton PCR validation showed that, Figure 1 As shown: The JH-Cytb primer pair: using Jinhua Two-Headed Black Pig DNA as a template, a clear 156bp target band was amplified. The band was bright, single, and without non-specific contaminants. No amplification products were obtained using DNA from other breeds such as Duroc, Landrace, Large White, Taihu Pig, Rongchang Pig, Tibetan Pig, Jinhua Black Pig, Jiaxing Black Pig, and Chun'an Spotted Pig as templates.

[0068] JH-DL primer pair: A clear 183bp target band can be amplified using DNA from *Aconitum carmichaelii* from Jinhua as a template; no amplification products are obtained using DNA from other varieties as templates.

[0069] JH-COI primer pair: Using Jinhua two-headed black pig DNA as a template, a clear 142bp target band can be amplified; using DNA from 89 other breeds as templates, 88 samples showed no amplification products, while 1 Taihu pig sample showed a weak amplification band, which is consistent with the results found in the sequence analysis of Example 1 that the COI6542 site also showed a T-shape in some Taihu pig individuals.

[0070] The JH-IC internal reference primer pair: clear 128bp bands were amplified using all 140 DNA samples as templates, demonstrating the good versatility of the internal reference primers.

[0071] The negative control showed no amplification products, proving that the PCR system was uncontaminated.

[0072] Statistical analysis showed that the JH-Cytb primer pair was positive for all 50 samples of Jinhua *Ligustrum lucidum* (50 / 50) and negative for all 90 samples of other breeds (0 / 90), with 100% specificity and 100% sensitivity. The JH-DL primer pair also showed 100% specificity and 100% sensitivity. The JH-COI primer pair was positive for all Jinhua *Ligustrum lucidum* samples and weakly positive for one sample of other breeds (Taihu pig), with a specificity of 98.9%. The combined analysis of these three primer pairs enabled complete differentiation between Jinhua *Ligustrum lucidum* and all control breeds.

[0073] 2.3 Optimization of multiplex PCR system.

[0074] To improve detection efficiency, a quadruple PCR amplification system was established by combining three pairs of specific primers (JH-Cytb, JH-DL, and JH-COI) with the JH-IC internal control primer. The annealing efficiency of each primer in the multiplex PCR reaction may affect each other due to competition, necessitating optimization of primer concentration ratios and reaction conditions.

[0075] Primer concentration optimization: With a fixed template DNA amount of 50 ng, the amplification efficiency of each primer was tested at final concentrations of 0.2 μmol / L, 0.3 μmol / L, 0.4 μmol / L, and 0.5 μmol / L. The results showed that when the concentrations of JH-Cytb primer (0.4 μmol / L), JH-DL primer (0.3 μmol / L), JH-COI primer (0.4 μmol / L), and JH-IC primer (0.2 μmol / L) were used, the amplified bands of the four target fragments exhibited uniform brightness and no obvious competitive inhibition.

[0076] Annealing temperature optimization: Based on primer concentration optimization, the effects of five annealing temperatures (54℃, 56℃, 58℃, 60℃, and 62℃) on the amplification efficiency and specificity of multiplex PCR were tested. The results showed that the amplification efficiency was highest and no non-specific bands were observed at annealing temperature of 58℃, which was determined to be the optimal annealing temperature.

[0077] Cycle number optimization: The effects of 30, 32, 35, 38, and 40 cycles on the amplification product yield and specificity were tested. The results showed that at 35 cycles, all four target bands of the *Aconitum carmichaelii* sample were clearly visible with moderate band brightness, and no nonspecific amplification was observed in the negative sample, thus determining it to be the optimal cycle number.

[0078] The final determined multiplex PCR reaction system of 25 μL contained: 2.5 μL of 10×PCR Buffer, 2 μL of dNTP Mixture (2.5 mmol / L each), 1 μL of JH-Cytb-F / R (10 μmol / L each), 0.75 μL of JH-DL-F / R (10 μmol / L each), 1 μL of JH-COI-F / R (10 μmol / L each), 0.5 μL of JH-IC-F / R (10 μmol / L each), 0.3 μL of Taq DNA polymerase (5 U / μL), 1 μL of template DNA (50 ng / μL), and ddH2O to a final volume of 25 μL.

[0079] Multiplex PCR reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ final extension for 7 min; store at 4℃.

[0080] Using an optimized multiplex PCR system, amplification and validation were performed on 50 *Ligustrum lucidum* samples from Jinhua and 90 control samples. The results showed that 49 *Ligustrum lucidum* samples simultaneously amplified bands of 156bp, 183bp, 142bp, and 128bp (one sample only amplified a 128bp internal control band due to severe DNA degradation), while the 90 control samples only amplified a 128bp internal control band, with no specific band. The variety specificity of the multiplex PCR system was consistent with that of singlex PCR, and the detection efficiency was significantly improved.

[0081] Example 3: Specific probe design and membrane gene chip preparation.

[0082] This embodiment designs a biotin-labeled probe for hybridization detection and establishes a method for preparing membrane gene chips.

[0083] 3.1 Hybridization probe design.

[0084] Based on the PCR amplification product sequence determined in Example 2, hybridization detection probes were designed in regions containing specific variant sites. The probe design principles are as follows: probe length 20 to 25 nucleotides; probe sequence containing or adjacent to specific variant sites; probe Tm value 5°C to 10°C higher than amplification primers to ensure stable binding of probe to target sequence at hybridization temperature; GC content controlled between 40% and 60%; avoidance of stable secondary structure formation within the probe; and a reserved amino modification site at the 5′ end of the probe for biotin labeling.

[0085] The designed probe sequence is as follows: JH-Cytb-P (SEQ ID NO:7): 5′-Biotin-TCCTACACATAGCCAACGGTAC-3′, 22nt in length, 50% GC content, Tm value 63.5℃, targeting the 14762 and 14891 site mutation regions of the Cytb gene.

[0086] JH-DL-P (SEQ ID NO:8): 5′-Biotin-CTAACATACACGTGTACGCATGCA-3′, 24nt in length, GC content 45.8%, Tm value 64.2℃, targeting the CTAA insertion region at position 16233 of the D-loop region. The probe sequence spans the insertion site and perfectly pairs with the amplification product of Aconitum carmichaelii containing the insertion sequence.

[0087] JH-COI-P (SEQ ID NO: 9): 5′-Biotin-GGTACTGGTTGAACTGTCTAT-3′, 21nt in length, 42.9% GC content, 61.8℃ Tm value, targeting the 6542 site mutation region of the COI gene.

[0088] IC-P (SEQ ID NO: 12): 5′-Biotin-GCGCCAGAACTACTAGCTAA-3′, 20nt in length, 50% GC content, Tm value 62.1℃, targeting the conserved region of the 16S rRNA gene, used as an internal control probe.

[0089] All probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd., with biotin molecules covalently coupled to the 5′ end via a C6 amino linker. The probes were purified by HPLC to a purity greater than 95%. The probe powder was dissolved in sterile ddH2O to a stock solution concentration of 100 μmol / L, aliquoted, and stored at -20°C protected from light.

[0090] 3.2 Preparation of membrane gene chips.

[0091] The membrane gene chip carrier uses a Hybond-N+ positively charged nylon membrane, which has advantages such as high binding capacity, low background signal, good mechanical strength, and repeated hybridization, making it suitable for nucleic acid hybridization detection applications.

[0092] The membrane chip fabrication steps are as follows: Step 1, Nylon Membrane Cutting and Marking: Using a clean scalpel blade, cut the Hybond-N+ nylon membrane into 1cm x 1cm square pieces. The cutting process should be performed in a clean bench to avoid dust contamination. Use a soft 2B pencil to mark the batch number and orientation on one corner of the membrane. The pencil marking will not affect subsequent hybridization and color development reactions.

[0093] Step 2, Probe Dilution and Spotting Preparation: Dilute each probe stock solution to a working concentration of 20 μmol / L using spotting buffer (3×SSC solution containing 50% dimethyl sulfoxide). Prepare a positive control solution (20 μmol / L biotin-labeled random sequence oligonucleotides), a negative control solution (20 μmol / L unlabeled random sequence oligonucleotides), and a blank control (pure spotting buffer).

[0094] Step 3, probe spotting: such as Figure 3 As shown, probes were spotted using a 5x3 dot matrix layout. Using a micropipette or automated spotting apparatus, the probe solution was spotted at designated locations on the nylon membrane. The spotting volume was 0.5 μL per spot, the spot diameter was approximately 1.5 mm, and the spacing between adjacent spots was approximately 2 mm. The spotting layout was as follows: Row A (A1-A3) contained three replicates of the JH-Cytb-P probe; Row B (B1-B3) contained three replicates of the JH-DL-P probe; Row C (C1-C3) contained three replicates of the JH-COI-P probe; Row D (D1-D3) contained three replicates of the IC-P internal control probe; Row E contained control points: E1 for a positive control, E2 for a negative control, and E3 for a blank control. After spotting, the membrane was dried at room temperature for 30 min to allow the probe solution to completely penetrate the membrane.

[0095] Step 4, UV crosslinking fixation: Place the dried nylon membrane in a UVPCL-1000 UV crosslinker with the probe spotting surface facing upwards. Set the UV crosslinking energy to 1200 μJ / cm² (254 nm wavelength) and start the crosslinking program. UV irradiation causes the pyrimidine bases of the probe DNA molecules to form covalent bonds with the amino groups on the nylon membrane surface, achieving permanent fixation of the probe. After crosslinking, the membrane chip can be stored at room temperature under dry conditions for a long period of time, with a shelf life of at least 12 months.

[0096] 3.3 Membrane chip quality inspection.

[0097] To verify probe immobilization effectiveness and chip uniformity, quality testing was performed on each batch of prepared membrane chips. Three membrane chip samples were taken, and 2 mL of a 1:5000 diluted streptavidin-alkaline phosphatase conjugate solution was added. The samples were incubated at room temperature for 30 min to allow enzyme molecules to bind to the biotin-labeled probe via streptavidin. The samples were washed three times with TBST buffer, 5 min each time. Freshly prepared NBT / BCIP chromogenic solution was added, and the samples were incubated at room temperature in the dark for 15 min. The chromogenic results were observed: the positive control point (E1) should show a deep purplish-blue spot, indicating a normal chromogenic system; each probe spot location (A1-D3) should show a uniform purplish-blue spot with consistent size and color depth, indicating uniform probe immobilization and complete biotin labeling; the negative control point (E2) and blank control point (E3) should show no color or only a very slight background color. Only membrane chips that passed quality inspection could be used for subsequent hybridization detection experiments.

[0098] Example 4: Establishment and optimization of membrane chip hybridization detection conditions.

[0099] This embodiment establishes and optimizes the reaction conditions for membrane gene chip hybridization detection, including key parameters such as hybridization temperature, hybridization time, washing conditions, and color development conditions.

[0100] 4.1 Optimization of hybridization reaction conditions.

[0101] Using DNA from *Aconitum carmichaelii* and Duroc DNA as templates, multiplex PCR amplification was performed to obtain positive and negative amplification products, which were then used for experiments to optimize hybridization conditions.

[0102] Hybridization temperature optimization: Take 20 μL each of positive and negative PCR products, and boil them in a boiling water bath for 5 min to denature the double-stranded DNA into single-stranded DNA. Immediately transfer them to an ice-water bath to cool for 2 min to prevent annealing. Mix the denatured products with 500 μL of hybridization buffer (5×SSC, 0.1% SDS, 5×Denhardt's solution, 100 μg / mL denatured salmon sperm DNA), and hybridize them with the membrane chip at five different temperatures: 45℃, 50℃, 55℃, 60℃, and 65℃ for 2 h. After hybridization, wash and develop the samples under uniform conditions, and compare the signal-to-noise ratio at different hybridization temperatures.

[0103] The results showed that at 45℃ and 50℃, the positive samples showed strong colorimetric signals, but the negative samples also showed nonspecific hybridization signals; at 60℃ and 65℃, the negative samples had a clean background, but the positive samples showed significantly weakened signals; at 55℃, the positive samples (rows A, B, C, and D) all showed strong colorimetric signals, while only the internal control probe in row D of the negative samples showed color, while rows A, B, and C showed no signal, resulting in the optimal signal-to-noise ratio. Therefore, 55℃ was determined to be the optimal hybridization temperature.

[0104] Hybridization time optimization: At a hybridization temperature of 55℃, the effects of five hybridization times (0.5h, 1h, 1.5h, 2h, and 3h) on the detection signal were tested. The results showed that the signal was weak at 0.5h; the signal gradually increased and stabilized between 1h and 2h; and the signal intensity at 3h was not significantly different from that at 2h. Considering both detection efficiency and signal intensity, 2h was determined to be the optimal hybridization time.

[0105] 4.2 Optimization of washing conditions.

[0106] After the hybridization reaction is complete, gradient washing is required to remove non-specifically bound nucleic acid molecules and residual hybridization buffer, thereby reducing background signal. The following washing condition combinations were tested: Option A: 2 x SSC, room temperature, 10 min x 2 times; Option B: 2 x SSC, room temperature, 10 min x 2 times, 0.5 x SSC, room temperature, 10 min x 1 time; Option C: 2 x SSC, room temperature, 10 min x 2 times, 0.5 x SSC, 55℃, 10 min x 1 time; Option D: 2 x SSC, room temperature, 10 min x 1 time, 1 x SSC, room temperature, 10 min x 1 time, 0.5 x SSC, 55℃, 10 min x 1 time, 0.1 x SSC, 55℃, 10 min x 1 time.

[0107] The results showed that negative samples still had high background after washing with methods A and B; the background was significantly reduced after washing with methods C and D, but method D involved more steps. Taking all factors into consideration, method C was selected as the standard washing condition, namely: 2×SSC washing at room temperature for 10 min × 2 times, and 0.5×SSC washing at 55℃ for 10 min × 1 time.

[0108] 4.3 Optimization of colorimetric reaction conditions.

[0109] After washing, the membrane chip was transferred to a clean culture dish for enzyme conjugate incubation and substrate color development reaction.

[0110] Enzyme conjugate incubation: Add 2 mL of a 1:5000 diluted streptavidin-alkaline phosphatase conjugate solution (diluent: TBST buffer: 25 mmol / L Tris-HCl pH 7.5, 150 mmol / L NaCl, 0.05% Tween-20), and incubate at 37°C with shaking for 30 min. During incubation, streptavidin on the enzyme conjugate binds efficiently to biotin at the 5′ end of the probe, forming a probe-biotin-streptavidin-alkaline phosphatase complex. After incubation, wash three times with TBST buffer for 5 min each time to remove unbound free enzyme molecules.

[0111] Substrate color development: Add 2 mL of freshly prepared NBT / BCIP colorimetric solution (preparation method: add 33 μL of NBT stock solution and 16.5 μL of BCIP stock solution to 10 mL of alkaline phosphatase buffer, mix well and use immediately), and incubate at room temperature in the dark for color development. Alkaline phosphatase catalyzes the hydrolysis of BCIP to produce indole groups, which react with NBT to form an insoluble purple-blue formazan precipitate. Observe the color development process. When the positive control spot shows a distinct purple-blue color and the background is clean (usually 15 to 30 min), rinse thoroughly with deionized water to terminate the reaction.

[0112] like Figure 4As shown, under the optimized hybridization and colorimetric conditions, the positive *Aconitum carmichaelii* samples from Jinhua exhibited clear purplish-blue spots at the JH-Cytb-P probe (row A), JH-DL-P probe (row B), JH-COI-P probe (row C), and IC-P internal control probe (row D), with uniform color development across the three replicates. The negative *Aconitum carmichaelii* samples only showed a spot at the internal control probe position in row D; rows A, B, and C showed no color development, with a clean white background. The positive control point E1 showed normal color development, while the negative control point E2 and the blank control point E3 showed no color development, demonstrating the good specificity of the detection system.

[0113] 4.4 Criteria for judging test results.

[0114] Based on the color development results of the membrane chip, the test conclusions should be determined according to the following criteria: Positive result determination: If at least two of the three replicates (JH-Cytb-P, JH-DL-P, JH-COI-P) show obvious colored spots, and at the same time, a colored spot is shown in row D (IC-P), it is determined to be positive for Jinhua two-headed aconite.

[0115] Negative determination: If no colored spots are found in rows A, B, and C, or only one row shows weak color development, and colored spots are found in row D, then the test result is negative for *Aconitum carmichaelii*.

[0116] Invalidity criteria: If the internal control probe in row D shows no color or very weak color, regardless of the results in rows A, B, and C, the test is considered invalid and DNA needs to be extracted again or the test reagent needs to be re-extracted for repeated testing.

[0117] Employing a multi-site joint determination strategy can effectively avoid false negative results caused by single-site mutations or probe failure, thereby improving detection reliability.

[0118] Example 5: Assembly and Performance Verification of the Detection Kit In this embodiment, the aforementioned primers, probes, and membrane chips are assembled into a complete detection kit, and the performance indicators of the kit, such as sensitivity, specificity, repeatability, and stability, are systematically verified.

[0119] 5.1 Composition of the test kit.

[0120] The Jinhua *Ligustrum lucidum* (Ligustrum lucidum) test kit contains the following components and is packaged for 20 tests: Component A (Multiple Primer Mixture): An optimized concentration mixture containing eight primers: JH-Cytb-F, JH-Cytb-R, JH-DL-F, JH-DL-R, JH-COI-F, JH-COI-R, JH-IC-F, and JH-IC-R. 200 μL x 2 tubes, store at -20°C. Add 5 μL per reaction when using.

[0121] Component B (membrane gene chip): Nylon membrane chip with pre-fixed probes, 1cm×1cm, 24 chips per pack (including 4 spare chips), sealed in aluminum foil bag, protected with desiccant, and stored at 4℃.

[0122] Component C (Hybridization Buffer Concentrate): 10× Hybridization Buffer (50×SSC, 1% SDS, 50×Denhardt's solution), 5mL × 1 bottle, store at 4℃. Dilute with sterile water to 1× working concentration before use.

[0123] Component D (Streptavidin-alkaline phosphatase conjugate): 1000× concentrate, 100 μL per tube, store at -20°C. Dilute to 1× working concentration with TBST before use.

[0124] Component E (NBT solution): 75 mg / mL NBT dissolved in 70% dimethylformamide, 500 μL × 1 tube, stored at 4°C protected from light.

[0125] Component F (BCIP solution): 50 mg / mL BCIP dissolved in 100% dimethylformamide, 500 μL × 1 tube, stored at 4°C protected from light.

[0126] Component G (20×SSC concentrate): 100mL × 1 bottle, store at room temperature. Used to prepare hybridization and washing buffer.

[0127] Component H (positive control DNA): 50 ng / μL of *Aconitum carmichaelii* genomic DNA from Jinhua, 100 μL × 1 tube, stored at -20℃.

[0128] Component I (negative control DNA): Duroc genomic DNA 50 ng / μL, 100 μL × 1 tube, stored at -20℃.

[0129] Component J (alkaline phosphatase buffer): 100 mmol / L Tris-HCl pH 9.5, 100 mmol / L NaCl, 5 mmol / L MgCl2, 50 mL × 1 bottle, stored at 4℃. Used to prepare the colorimetric solution.

[0130] 5.2 Sensitivity verification.

[0131] Genomic DNA from *Aconitum carmichaelii* from Jinhua was serially diluted 10-fold to final concentrations of 100 ng / μL, 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 0.1 pg / μL. Using 1 μL of each concentration of DNA as template (corresponding to DNA amounts of 100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, and 0.1 pg), multiplex PCR amplification and membrane microarray hybridization were performed for detection. Each concentration was tested three times.

[0132] PCR electrophoresis results as follows Figure 2 As shown: when the amount of DNA template is 100ng, 10ng, and 1ng, all four bands are clearly visible; when the amount of template is 100pg, the 156bp and 183bp bands are visible but weak, while the 142bp and 128bp bands are clear; when the amount of template is 10pg or less, only the 128bp internal control band is faintly visible or there is no amplification product.

[0133] Membrane chip hybridization color development results: When the amount of DNA template was 100ng, 10ng, and 1ng, the probes in rows A, B, C, and D all showed clear colored spots; when the amount of template was 100pg, the JH-DL-P probe in row B and the internal control probe in row D showed color, while the color in rows A and C was weaker; when the amount of template was 10pg or less, only the internal control probe in row D showed color or no color.

[0134] Based on the combined results of PCR and membrane microarray detection methods, the detection sensitivity of the method of this invention was determined to be 1 ng of DNA template. Considering that the DNA extraction rate from Jinhua Two-Headed Black Pork is approximately 100 μg / g of muscle tissue, this sensitivity is equivalent to detecting Jinhua Two-Headed Black Pork DNA in approximately 10 mg of muscle tissue.

[0135] 5.3 Sensitivity verification for adulterated samples.

[0136] To evaluate the actual detection sensitivity of the method of the present invention in adulterated meat samples, simulated samples with different adulteration ratios were prepared. 500g each of the longissimus dorsi muscle of Jinhua Two-Headed Black Pig and Duroc Pig were minced separately and then mixed according to the mass ratio to prepare nine groups of mixed samples with Jinhua Two-Headed Black content of 100%, 50%, 20%, 10%, 5%, 2%, 1%, 0.5%, and 0%, respectively, with three parallel samples in each group.

[0137] DNA was extracted from 200 mg of each mixed sample group. The obtained DNA was then used as a template for multiplex PCR and membrane chip detection with 50 ng of the sample. The results showed that samples with a Jinhua aconite content ≥1% could detect positive signals in at least two rows of A, B, and C; samples with a content of 0.5% showed weak positive signals in only some parallel samples, indicating unstable repeatability; samples with a content of 0% were all negative. Therefore, the detection limit of the method of this invention for adulterated samples was determined to be 1% (mass fraction).

[0138] 5.4 Repeatability verification.

[0139] Intra-assay repeatability: One positive DNA sample from *Aconitum carmichaelii* and one negative DNA sample from Duroc were selected and tested 10 times on the same day using the same batch of kits. The consistency of the results was observed. Results showed that all 10 tests of the positive sample were positive (all four rows A, B, C, and D showed color development), and all 10 tests of the negative sample were negative (only row D showed color development), resulting in 100% intra-assay repeatability. Semi-quantitative analysis of the grayscale values ​​of the colorimetric spots in each test was performed, and the coefficient of variation (CV) was less than 5%.

[0140] Inter-batch repeatability: One positive and one negative DNA sample were selected, and three different batches of kits were used. Each sample from each batch was tested five times, for a total of 30 tests. The results showed that the positive sample was positive in all 15 tests, and the negative sample was negative in all 15 tests, with 100% inter-batch repeatability. The coefficient of variation (CV) of the test results from different batches of kits was less than 8%.

[0141] 5.5 Accelerated stability verification.

[0142] Three sets of reagent kits were stored at -20℃ (control group), 4℃, 25℃, and 37℃, respectively. Samples were taken on days 0, 7, 14, 30, 60, and 90 for performance testing. Test indicators included: sensitivity for positive samples, specificity for negative samples, and uniformity of membrane chip color development.

[0143] The results showed that under storage conditions of -20℃ and 4℃, the performance indicators of the kit did not change significantly within 90 days; under storage conditions of 25℃, the detection sensitivity decreased slightly at 60 days and by about 50% at 90 days; under storage conditions of 37℃, the detection sensitivity decreased significantly at 30 days, and some components became ineffective at 60 days. Therefore, it is recommended that the kit be stored at -20℃ to 4℃ for a shelf life of 12 months; and transported at room temperature for no more than 7 days.

[0144] Example 6: Application of detection in actual meat samples.

[0145] This embodiment uses the detection kit of the present invention to test actual samples of commercially available pork and its processed products, verifying the practicality and application effect of the method. The overall detection process includes nine main steps: sample collection, DNA extraction, multiplex PCR amplification, product denaturation, membrane chip hybridization, gradient washing, enzyme conjugate incubation, colorimetric detection, and result interpretation.

[0146] 6.1 Sample collection.

[0147] A total of 50 samples of pork and its processed products were collected from farmers' markets, supermarket counters, and e-commerce platforms in Jinhua, Hangzhou, and Ningbo, Zhejiang Province. These samples included: Twenty fresh pork samples were collected: 15 samples labeled as Jinhua Two-Headed Black Pork (8 from farmers' markets, 5 from supermarkets, and 2 from e-commerce platforms), and 5 samples labeled as ordinary pork as controls.

[0148] Twelve samples of Jinhua ham were collected: eight samples labeled as Jinhua Liangtouwu ham (including well-known brands such as Authentic Jinhua Ham and Jinzi Ham), and four samples labeled as ordinary ham.

[0149] Ten sausage samples were collected: six samples labeled as Jinhua Liangtouwu sausage and four samples labeled as ordinary sausage.

[0150] Eight samples of cured meat were collected: five samples were labeled as Jinhua Two-End Black Cured Meat, and three samples were labeled as ordinary cured meat.

[0151] After all samples were collected, the purchase time, location, price, and label information were recorded. The samples were then repackaged and stored at -20℃ for testing.

[0152] 6.2 DNA extraction methods.

[0153] Fresh pork sample: Take about 25mg of muscle tissue and extract DNA according to the standard operating procedure of Tiangen Animal Tissue Genomic DNA Extraction Kit. The specific steps are the same as in Example 1.

[0154] Ham Sample: Jinhua ham undergoes prolonged curing and fermentation, resulting in high salt content and severe DNA degradation, necessitating a modified extraction method. Approximately 200 mg of ham sample was taken and first soaked in 10 times its volume of sterile water for 4 hours to desalinate, changing the water three times during this period. After desalting, the sample was added to 800 μL of L-TAB lysis buffer (2% CTAB, 1.4 mol / L NaCl, 100 mmol / L Tris-HCl pH 8.0, 20 mmol / L EDTA) and 20 μL of proteinase K, and digested overnight at 56°C. An equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added for extraction. The supernatant was collected and precipitated with 0.7 times its volume of isopropanol, washed with 70% ethanol, and then dissolved in 50 μL of L-T buffer.

[0155] Sausage and bacon samples: These samples typically contain additives such as starch and spices, and have a high fat content. A modified CTAB method was also used for extraction. The samples were first soaked in anhydrous ethanol to remove fat, then dried before DNA extraction.

[0156] After DNA extraction, the concentration and purity were detected using Nanodrop. 260 / A 280 A ratio between 1.6 and 2.0 is acceptable for detection. Processed DNA products typically have lower concentrations and poorer quality, but a concentration greater than 10 ng / μL is sufficient to meet the detection requirements of the method of this invention.

[0157] 6.3 Test results and analysis.

[0158] Fifty samples were subjected to multiplex PCR amplification and membrane chip hybridization colorimetric detection. The results are shown in the table below: Results of fresh pork sample testing: Of 15 samples labeled as Jinhua Liangtouwu pork, 12 tested positive (color development in rows A, B, and C), and 3 tested negative (color development only in row D), with a positive rate of 80%. All 5 control samples labeled as ordinary pork tested negative, as expected. The 3 negative Jinhua Liangtouwu pork samples may be due to false labeling or being misrepresented as ordinary pork.

[0159] Test results for Jinhua ham samples: Of the 8 samples labeled as Jinhua Liangtouwu ham, 6 tested positive and 2 tested negative, with a positive rate of 75%; all 4 control samples labeled as ordinary ham tested negative. Some ham samples showed severe DNA degradation due to prolonged curing, with only the internal control band showing color and the specific band showing weak color, but a clear judgment can still be made according to the judgment criteria.

[0160] Sausage sample testing results: Of the 6 samples labeled as Jinhua Liangtouwu sausage, 4 tested positive and 2 tested negative, with a positive rate of 66.7%; all 4 control samples labeled as ordinary sausage tested negative. High-temperature cooking during sausage processing has a certain degradation effect on DNA, but the short-fragment primers designed in this invention can still effectively amplify it.

[0161] Results of the test on cured pork samples: Of the 5 samples labeled as Jinhua Liangtouwu cured pork, 4 tested positive and 1 tested negative, with a positive rate of 80%; all 3 control samples labeled as ordinary cured pork tested negative.

[0162] Based on the combined test results of 50 samples, all 17 control samples labeled as ordinary pork products tested negative, demonstrating 100% specificity and no false positives. Of the 34 samples labeled as Jinhua Liangtouwu pork products, 26 tested positive and 8 tested negative. The authenticity of the negative samples needs further verification based on information such as product origin, price, and sales channels.

[0163] To further illustrate the technical advantages of this invention, a comparative analysis is conducted between the method of this invention and existing meat adulteration detection technologies.

[0164] Compared with species-specific PCR methods (such as CN108624716B), species-specific PCR can only identify the animal species origin of meat (such as pigs, cattle, and sheep), and cannot distinguish different breeds within the same species. The method of this invention can accurately distinguish Jinhua Liangtouwu pigs from other pig breeds at the breed level, providing a more refined detection level.

[0165] Compared with SNP microarray genotyping methods, high-density microarray genotyping based on nuclear genomic SNPs, while offering high resolution, requires high-throughput sequencers or dedicated microarray reading equipment, with a single test costing hundreds to thousands of yuan and a testing cycle of 2 to 3 days, making it difficult to meet the needs of rapid screening. The method of this invention uses membrane gene chips and enzyme-catalyzed colorimetric detection, requiring less equipment, with a single test costing no more than 50 yuan and a testing time of 3 to 4 hours, making it more suitable for widespread application.

[0166] Compared with real-time quantitative PCR (qPCR), while qPCR offers high sensitivity and quantification, it requires expensive qPCR instruments, has high-cost fluorescent probe synthesis, and demands professional analysis and interpretation of results. In contrast, the colorimetric results from the membrane chip in this invention are visible to the naked eye, requiring no specialized equipment or personnel, allowing grassroots institutions to independently perform the tests.

[0167] The core technological innovation of this invention lies in the first systematic screening and identification of mitochondrial DNA molecular markers specific to the Jinhua Two-End Black Pig breed, and the establishment of a membrane gene chip visualization detection platform based on a multi-site joint detection strategy.

[0168] At the molecular marker level, this invention utilizes the characteristics of mitochondrial DNA, such as maternal inheritance, high copy number, and significant inter-breed differences. Through large-scale sequencing and comparative analysis of 140 samples, five breed-specific variation sites were screened: the Cytb gene T→C transition at site 14762, the A→G transition at site 14891, the D-loop region G→A transition at site 16107, the CTAA tetrabase insertion at site 16233, and the COI gene C→T transition at site 6542. The CTAA insertion sequence at site 16233 in the D-loop region is particularly important; this insertion was detected in all 50 Jinhua Liangtouwu pig samples but not in 90 control samples from other breeds, making it the most reliable molecular marker for Jinhua Liangtouwu pigs. The multi-site joint detection strategy uses markers from three independent gene regions for comprehensive judgment. Even if a single site mutates or fails to detect, an accurate judgment can still be made based on the results of other sites, significantly improving the reliability and anti-interference ability of the detection.

[0169] At the detection technology level, this invention employs a biotin-streptavidin-alkaline phosphatase signal amplification system to achieve high-sensitivity detection of hybridization signals. Biotin and streptavidin have extremely high binding affinity (dissociation constant Kd approximately 10⁻⁶). -15 (mol / L), and the binding reaction is rapid and stable. One streptavidin molecule can bind four biotin molecules, while alkaline phosphatase can efficiently catalyze the hydrolysis of NBT / BCIP substrates to generate an insoluble purple-blue formazan precipitate, enabling signal visualization and long-term preservation. This colorimetric system does not require fluorescence detection equipment, is low in cost, and provides intuitive results, making it suitable for widespread application at the grassroots level.

[0170] In terms of practicality, the PCR amplification fragments designed in this invention are controlled in length within the range of 142 to 183 bp, which is much shorter than the hundreds to thousands of base pairs of amplification fragments commonly used in traditional genotyping methods. This short fragment design effectively overcomes the fragmentation problem of DNA in processed meat products such as Jinhua ham, sausage, and cured meat caused by high-temperature cooking, acid and alkali curing, and enzymatic degradation, significantly expanding the applicability of the detection method.

[0171] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. For example, different solid-phase carriers can be used instead of nylon membranes, fluorescent labeling can be used instead of biotin labeling for detection, and primer and probe sequences can be fine-tuned while maintaining specificity. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A specific primer set for Aconitum carmichaelii from Jinhua, characterized in that, Includes the following three pairs of primers: The primer pair JH-Cytb consists of the forward primer JH-Cytb-F with the nucleotide sequence shown in SEQ ID NO:1 and the reverse primer JH-Cytb-R with the nucleotide sequence shown in SEQ ID NO:

2. The primer pair JH-DL consists of the forward primer JH-DL-F, whose nucleotide sequence is shown in SEQ ID NO:3, and the reverse primer JH-DL-R, whose nucleotide sequence is shown in SEQ ID NO:

4. The primer pair JH-COI consists of the forward primer JH-COI-F, whose nucleotide sequence is shown in SEQ ID NO:5, and the reverse primer JH-COI-R, whose nucleotide sequence is shown in SEQ ID NO:

6.

2. The specific primer set for Aconitum carmichaelii from Jinhua according to claim 1, characterized in that, The primer pair JH-Cytb amplified a specific fragment of the Cytb gene in the mitochondria of *Aconitum carmichaelii*, with an amplification product length of 156 bp; the primer pair JH-DL amplified a specific fragment of the D-loop region in the mitochondria of *Aconitum carmichaelii*, with an amplification product length of 183 bp; and the primer pair JH-COI amplified a specific fragment of the COI gene in the mitochondria of *Aconitum carmichaelii*, with an amplification product length of 142 bp.

3. A specific probe set for Aconitum carmichaelii from Jinhua, characterized in that, Includes the following three biotin-labeled oligonucleotide probes: The nucleotide sequence of probe JH-Cytb-P is shown in SEQ ID NO:7; The nucleotide sequence of probe JH-DL-P is shown in SEQ ID NO:8; The probe JH-COI-P has the nucleotide sequence shown in SEQ ID NO:

9.

4. The Jinhua two-headed aconite specific probe set according to claim 3, characterized in that, The 5′ end of the probe is labeled with biotin.

5. An internal reference primer pair, characterized in that, The amplification product consists of a forward primer JH-IC-F with a nucleotide sequence as shown in SEQ ID NO:10 and a reverse primer JH-IC-R with a nucleotide sequence as shown in SEQ ID NO:11, and the product length is 128 bp.

6. The Jinhua Two-Headed Black Fungus Membrane Gene Chip, characterized in that, It includes a nylon membrane carrier and a probe array fixed to the surface of the nylon membrane, wherein the probe array includes the Jinhua Liangtouwu specific probe group and the internal reference probe IC-P as described in claim 3 or 4.

7. The Jinhua two-headed black fungus membrane gene chip according to claim 6, characterized in that, The probe is fixed to the surface of the nylon film via ultraviolet cross-linking, with an ultraviolet cross-linking energy of 1200 μJ / cm². 2 .

8. Jinhua Two-Headed Black Fungus Detection Kit, characterized in that, include: The specific primer set of Aconitum carmichaelii from Jinhua as described in claim 1 or 2, the specific probe set of Aconitum carmichaelii from Jinhua as described in claim 3 or 4, the internal reference primer pair as described in claim 5, the membrane gene chip, hybridization buffer, chromogenic substrate solution, positive control DNA, and negative control DNA as described in claim 6 or 7.

9. The application of the primer set according to claim 1 or 2, the probe set according to claim 3 or 4, or the membrane gene chip according to claim 6 or 7 in the identification of Jinhua two-headed black pig-derived components.

10. A method for detecting components of Jinhua two-headed black pig origin, characterized in that, Includes the following steps: S1. DNA Extraction: Extracting total DNA from the sample to be tested; S2. PCR amplification: Using the total DNA as a template, PCR amplification was performed using the Jinhua two-headed aconite specific primer set as described in claim 1 or 2 and the internal reference primer pair as described in claim 5. The PCR reaction conditions were: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s for a total of 35 cycles, and 72℃ final extension for 7 min. S3. Hybridization reaction: The PCR amplification product was denatured in a boiling water bath for 5 min, cooled in an ice bath, and then hybridized with the membrane gene chip described in claim 6 or 7 at 55°C for 2 h. S4. Colorimetric detection: After hybridization, wash the sample, incubate with streptavidin-alkaline phosphatase conjugate, then add NBT / BCIP colorimetric solution for color development. Observe the color development results to determine the components of Jinhua two-headed aconite.

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