Primer probe combination and kit for on-site detection of pig, horse, duck, cattle and sheep derived components

Abstract

The invention discloses a primer probe combination and a kit for on-site detection of pig, horse, duck, cattle and sheep derived components, and relates to the technical field of gene detection. The invention provides a primer probe combination for on-site detection of pig, horse, duck, cattle and sheep-derived components. The primer probe combination comprises a primer pair and a probe for detecting the pig-derived components, the primer pair and the probe are used for detecting horse source components; a primer pair and a probe for detecting the duck-derived component; the primer pair and the probe are used for detecting bovine-derived components; the invention relates to a primer pair and a probe for detecting sheep-derived components. The method for detecting the pig, horse, duck, cattle and sheep derived components by adopting the primer probe combination and the kit integrates sample pretreatment, nucleic acid extraction, purification, amplification and detection, has better sensitivity and accuracy, is simple and convenient to operate, is quick and time-saving, and has a good application prospect in detection of pig, horse, duck, cattle and sheep derived components. The method plays an important role in on-site rapid detection of pig, horse, duck, cattle and sheep source component research and safety management.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of gene detection, in particular to a primer probe combination and kit for on-site detection of pig, horse, duck, cattle and sheep components. BACKGROUND

[0002] With the improvement of living standards, people's demand for meat food types and quality is also increasing, but in recent years, unscrupulous businessmen often add low-value or inferior meat to high-value meat to make profits, which is particularly serious in common meat foods such as beef and mutton, not only damaging the interests of consumers, but also leading to food safety hazards, so it is particularly important to invent a method for rapidly detecting meat components in food.

[0003] At present, the existing animal-derived component identification methods include enzyme-linked immunoassay, nucleic acid detection method, etc. Among them, PCR detection technology has become the most widely used method due to its high detection accuracy and complete quality control means. For example, patent CN107460248B discloses a method for simultaneously detecting pig, horse, cattle and sheep components; patent CN111690753B discloses a method for detecting fifteen kinds of animal-derived components based on high-resolution melting curve detection; and patent CN110144346B discloses a method for simultaneously identifying cattle and sheep components. However, these methods either require complicated sample processing steps, or require expensive detection equipment, or require extremely long detection processes, and cannot meet the needs of on-site detection by markets, customs, etc.

[0004] Based on the above problems, the present inventors have developed a new on-site rapid nucleic acid detection device in prior application CN202110057507.0, which is a "handheld real-time fluorescence PCR all-in-one machine P1000F". After optimization and upgrading of temperature control, the device can complete rapid nucleic acid detection within 35 minutes and achieve on-site automatic detection of "sample in, result out". In order to solve the problem of sample pretreatment, the present inventors have developed a simple and rapid sample processing tube (ZL202320319535.X). Based on the sample processing and detection device, the present application further develops an invention that can better cooperate with the device, i.e., a primer probe combination and integrated closed microfluidic chip kit for joint detection of pig, horse, duck, cattle and sheep components, and application thereof. The kit performs nucleic acid extraction, purification, amplification and detection on the integrated chip, and is applied to on-site rapid nucleic acid detection. SUMMARY

[0005] The application aims to provide a primer probe combination and a kit for detecting pig, horse, duck, cow and sheep components on site.

[0006] To achieve the above-mentioned application purposes, the technical solutions of the application are as follows.

[0007] In one aspect, the application provides a primer probe combination for detecting pig, horse, duck, cow and sheep components on site, wherein the primer probe combination comprises: a primer pair and a probe for detecting pig components, the sequences of which are shown in SEQ ID NO: 1-3; a primer pair and a probe for detecting horse components, the sequences of which are shown in SEQ ID NO: 4-6; a primer pair and a probe for detecting duck components, the sequences of which are shown in SEQ ID NO: 7-9; a primer pair and a probe for detecting cow components, the sequences of which are shown in SEQ ID NO: 10-12; and a primer pair and a probe for detecting sheep components, the sequences of which are shown in SEQ ID NO: 13-18.

[0008] Specifically, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a quencher group.

[0009] Further specifically, the fluorescent reporter group is selected from one or more of FAM, ROX, HEX, CY5, VIC, TET, JOE, CY3, Cy7, RED610, Texas Red, RED670, NED, AMCA, Pacific Blue, ATTO 425, BODIPY FL, AlexaFluor488, YakimaYellow, Quasar 570, Aqua Phluor593, ATTO 590, CY5.5.

[0010] Still further specifically, the fluorescent reporter group is selected from one or more of ATTO425, FAM, HEX, CY3, CY5, CY5.5, VIC, JOE, ROX.

[0011] Further specifically, the quencher group is a fluorescent quencher group.

[0012] Still further specifically, the fluorescent quencher group is selected from one or more of 6-TAMRA, BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse, MGB, QYS-7, SQ1.

[0013] Still further specifically, the fluorescent quencher group is selected from one or more of BHQ2, BHQ3, SQ1, MGB.

[0014] In another aspect, the present application provides the use of the primer-probe combination as described above in the preparation of a product for detecting porcine, equine, duck, bovine, ovine-derived ingredients.

[0015] Specifically, the product includes a separate detection reagent, a chip and a kit.

[0016] In another aspect, the present application provides a kit for detecting porcine, equine, duck, bovine, ovine-derived ingredients on site, which comprises the primer-probe combination as described above.

[0017] Specifically, the kit is an integrated closed microfluidic chip kit.

[0018] Further specifically, the integrated closed microfluidic chip kit comprises a purification reagent and a dry qPCR reagent.

[0019] Specifically, the purification reagent and the dry qPCR reagent can be transported and stored at room temperature.

[0020] Further specifically, the purification reagent comprises a lysis solution, a washing solution, an elution solution and magnetic beads, the lysis solution and the washing solution comprise guanidine hydrochloride, sodium acetate, Triton X-100 and 1,3-butanediol, the elution solution comprises Tris-HCl, and the magnetic beads are dry magnetic beads.

[0021] Further specifically, the lysis solution and the washing solution comprise 4.2M guanidine hydrochloride, 0.28M pH 4.7 sodium acetate, 1.4% Triton X-100 and 30% 1,3-butanediol, and the elution solution comprises 10mM pH8.5 Tris-HCl.

[0022] Specifically, the kit completes the nucleic acid extraction, purification, amplification and detection of the sample on the integrated chip.

[0023] Specifically, the microfluidic chip kit can be used for P1000F rapid nucleic acid detection equipment.

[0024] In another aspect, the present application provides a method for detecting porcine, equine, duck, bovine, ovine-derived ingredients on site, which comprises the preparation of an integrated closed microfluidic chip kit, sample pretreatment and rapid equipment detection.

[0025] Specifically, the method comprises the following steps:

[0026] (1) preparing the primer-probe combination as described above into an integrated closed microfluidic reagent kit;

[0027] (2) adding the sample containing pig, horse, duck, cattle, sheep components into the sample processing tube for processing to obtain the liquid to be detected;

[0028] (3) dropping the liquid to be detected into the integrated closed microfluidic kit;

[0029] (4) placing the kit added with the liquid to be detected on the detection equipment for detection analysis.

[0030] Specifically, the step (2) is specifically adding sample pre-treatment liquid and grinding particles into the sample processing tube; the sample pre-treatment liquid comprises Tris-HCl (pH 8.0), NaCl, EDTA and SDS.

[0031] Further specifically, the concentration of Tris-HCl is 10 mM-100 mM, the concentration of NaCl is 30 mM-1.4 M, the concentration of EDTA is 1.5 mM-20 mM, and the concentration of SDS is 0.05 %-2 %; the diameter of the grinding particles is 0.1 microns-1 millimeter.

[0032] Still further specifically, the concentration of Tris-HCl (pH 8.0) in the sample pre-treatment liquid is 30 mM-55 mM, the concentration of NaCl is 350 mM-700 mM, the concentration of EDTA is 5 mM-10 mM, and the concentration of SDS is 0.5 %-1 %.

[0033] Specifically, the sample in step (2) is pig, horse, duck, cattle, sheep raw meat or products thereof.

[0034] Specifically, the step (4) specifically comprises:

[0035] 1) opening the rapid detection equipment;

[0036] 2) scanning the two-dimensional code;

[0037] 3) inserting the integrated microfluidic chip kit;

[0038] 4) running detection;

[0039] 5) ending, checking the detection results and amplification curve.

[0040] According to some embodiments of the present application, the method comprises the following steps:

[0041] (1) preparing an integrated closed microfluidic kit by combining the primer probe set;

[0042] (2) using a sample processing tube to rapidly pre-treat the sample;

[0043] (3) Using purification reagents on the chip to lyse the sample and extract and purify nucleic acids;

[0044] (4) using the primer-probe combination to rapidly amplify the sample nucleic acid extracted and purified in step (3);

[0045] (5) Analyze the amplification results.

[0046] In another aspect, the present invention provides the use of the above primer-probe combination or the above kit or the above method in detecting source components of raw meat from pigs, horses, ducks, cattle, sheep and their products.

[0047] The beneficial effects of the present invention are:

[0048] The present invention provides a primer-probe combination, an integrated microfluidic chip kit, and a method for detecting porcine, equine, duck, bovine, and ovine ingredients. The method employing the primer-probe combination and kit integrates sample pretreatment, nucleic acid extraction, purification, amplification, and detection, offering high sensitivity and accuracy. The method is simple, rapid, and time-saving. The method can simultaneously detect five ingredients derived from porcine, equine, duck, bovine, and ovine sources, playing an important role in the on-site rapid detection of these ingredients for safety management. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is the sensitivity test result of porcine-derived ingredients.

[0050] Figure 2 This is the sensitivity test result of horse-derived components.

[0051] Figure 3 This is the sensitivity test result of duck-derived ingredients.

[0052] Figure 4 This is the sensitivity test result of the standard sample of bovine origin ingredients.

[0053] Figure 5 This is the sensitivity test result of sheep-derived components.

[0054] Figure 6 Actual photo of the integrated microfluidic chip and sample processing tube.

[0055] Figure 7 This is the test result of actual pork samples.

[0056] Figure 8 This is the test result of actual horse meat samples.

[0057] Figure 9 This is the test result of actual duck meat samples.

[0058] Figure 10 Figure for the detection result of the actual sample of beef.

[0059] Figure 11 Figure for the detection result of the actual sample of mutton.

[0060] Figure 12 Figure for the detection result of the actual sample of pork ham.

[0061] Figure 13 Figure for the detection result of the actual sample of roast duck.

[0062] Figure 14 Figure for the detection result of the actual sample of sauced beef.

[0063] Figure 15 Figure for the detection result of the actual sample of milk.

[0064] Figure 16 Figure for the detection result of the actual sample of hand-grabbed mutton.

[0065] Figure 17 Figure for the detection result of the actual sample of pork of Comparative Example 1.

[0066] Figure 18 Figure for the detection result of the actual sample of horse meat of Comparative Example 1.

[0067] Figure 19 Figure for the detection result of the actual sample of duck meat of Comparative Example 1.

[0068] Figure 20 Figure for the detection result of the actual sample of beef of Comparative Example 1.

[0069] Figure 21 Figure for the detection result of the actual sample of mutton of Comparative Example 1. DETAILED DESCRIPTION

[0070] In order to make the technical means, creative features, purposes and effects of the present application easy to understand, the present application is further illustrated below in combination with specific examples, but the following examples are only preferred examples of the present application, not all. Based on the examples in the embodiments, other examples obtained by those skilled in the art without creative labor are within the protection scope of the present application. In the following examples, if not otherwise specified, the operation methods used are all conventional operation methods, the equipment used is all conventional equipment, and the equipment materials used in each example are all the same.

[0071] The microfluidic chip device for nucleic acid detection of the present invention is detailed in patent 202110055537.8, the magnetic bead drying method is detailed in patent 202110222434.6, the sample processing tube is detailed in patent 202320319535.X, and the overall device, i.e., the PCR reaction device, is detailed in patent 202110057507.0. The actual picture of the integrated microfluidic chip and sample processing tube is as follows Figure 6 shown.

[0072] In the following examples, pork, horse, duck, beef and mutton were purchased from Beijing market.

[0073] Example 1 Primer probe combination for rapid on-site detection of pig, horse, duck, cattle, and sheep-derived ingredients

[0074] Primer and probe sequences were designed based on the species-specific sequences of pigs, horses, ducks, cattle, and sheep.

[0075] Among them, the primer probe sequence of pig is:

[0076] pig-F:5'-CAACTCTAATATCCACACGA-3'(SEQ ID NO:1);

[0077] pig-R:5'-TCGAAGTACTTTAATGGGA-3'(SEQ ID NO:2);

[0078] pig-P:5'-TTCTGAGCACTGTCCGTA-3' (SEQ ID NO:3).

[0079] The primer and probe sequences for horse are:

[0080] Equus-F:5'-AACACTAGCCCTCATAAGCAT-3' (SEQ ID NO:4);

[0081] Equus-R:5'-TACTGCGAATTCGAGGATAGTTA-3' (SEQ ID NO:5);

[0082] Equus-P: 5'-CTCAACAGCCCTTATTACGTT-3' (SEQ ID NO: 6).

[0083] The primer and probe sequences for duck are:

[0084] Anas-F:5'-TACTGGCTATGCACTACACC-3'(SEQ ID NO:7);

[0085] Anas-R: 5'-TGAGTCAGCCATATTGGACG-3' (SEQ ID NO: 8);

[0086] Anas-P: 5'-CCTCAGTAGCCAACACATGC-3' (SEQ ID NO: 9).

[0087] The primer probe sequences of bovine are:

[0088] Bos-F: 5'-TCGCGTGTCCTAAAGGGGTA-3' (SEQ ID NO: 10);

[0089] Bos-R: 5'-ACATTCGGAAGCCCTAAAGCC-3' (SEQ ID NO: 11);

[0090] Bos-P: 5'-CTGCCGATGGATGTGTTCAGAG-3' (SEQ ID NO: 12).

[0091] The primer probe sequences of sheep are:

[0092] Capra-F: 5'-ATCTGCCTATTCATACATATCG-3' (SEQ ID NO: 13);

[0093] Capra-R: 5'-GTAAAACATAGCCTATGAATGCTG-3' (SEQ ID NO: 14);

[0094] Capra-P: 5'-TAATCCTCCTGCTCGCGACA-3' (SEQ ID NO: 15);

[0095] Ovis-F: 5'-CATTGTTTTATTCCCTAGCC-3' (SEQ ID NO: 16);

[0096] Ovis-R: 5'-TAATTGAAGTATTCACTGTTGGA-3' (SEQ ID NO: 17);

[0097] Ovis-P: 5'-ATTATTTCCCACATCAAACCGACT-3' (SEQ ID NO: 18).

[0098] The primer probe compositions of the pig, horse, duck, bovine, and sheep components described in the application are used for detecting the pig, horse, duck, bovine, and sheep components in combination or individually.

[0099] In the examples, the Bos probe is labeled with ATTO 425, the quenching group is SQ1, the Capra and Ovis probes are labeled with FAM, the quenching group is MGB, the Pig probe is labeled with HEX, the quenching group is MGB, the Anas probe is labeled with CY5, the quenching group is BHQ2, and the Equus probe is labeled with CY5.5, the quenching group is BHQ3.

[0100] Example 2: A microfluidic chip kit for detecting porcine, equine, anatine, bovine, and ovine-derived components

[0101] (1) The kit includes the primer probe composition described in Example 1, with the final concentrations shown in Table 1 below.

[0102] Table 1: Primer probe concentrations

[0103] Primer Probe Name Final Concentration Bos-F, Bos-R 0.4 μΜ, 0.4 μΜ Bos-P 0.2 μΜ Capra-F, Capra-R 0.4 μΜ, 0.4 μΜ Capra-P 0.2 μΜ Ovis-F, Ovis-R 0.4 μΜ, 0.4 μΜ Ovis-P 0.2 μΜ Pig-F, Pig-R 0.4 μΜ, 0.4 μΜ Pig-P 0.2 μΜ Anas-F, Anas-R 0.4 μΜ, 0.4 μΜ Anas-P 0.2 μΜ Equus-F, Equus-R 0.4 μΜ, 0.4 μΜ Equus-P 0.2 μΜ

[0104] (2) The kit also includes purification reagents and air-drying reagents.

[0105] 1) The purification reagents include 450 μL of lysis solution, 450 μL of washing solution, and 850 μL of elution solution, 15 μL of magnetic beads, the lysis solution and washing solution include 4.2 M guanidine hydrochloride, 0.28 M sodium acetate pH 4.7, 1.4% Triton X-100, and 30% 1,3-butanediol, the elution solution includes 10 mM Tris-HCl pH 8.5, the purification reagents are added to the sample layer in the chip, and the magnetic bead drying method is described in patent 202110222434.6.

[0106] 2) The 50 μL air-drying reagent system is shown in Table 2 below.

[0107] Table 2: Air-drying reagent system

[0108] Source Component Volume 4x Air-Dryable qPCR Mix 12.5 μL Primer Probe Mix 2.5 μL

[0109] The reagents in Table 2 above are added to the reaction chamber of the integrated microfluidic chip, the chip is placed in a forced air drying oven, and the air-drying operation is performed according to the instructions of the 4x Air-Dryable qPCR Mix.

[0110] Example 3: Sample DNA preparation

[0111] (1) 10 mg of porcine, equine, anatine, bovine, and ovine meat was weighed separately;

[0112] (2) The sample DNA was extracted using QIAGEN QIAamp DNA Mini Kit, and the operation steps were performed according to the instructions;

[0113] (3) Qubit quantification, the concentrations of the five samples are as follows:

[0114] Table 3 Sample DNA quantification results

[0115] Sample Concentration Pig 9.84 ng / μL Horse 24.3 ng / μL Duck 26.7 ng / μL Cow 12 ng / μL Sheep 20 ng / μL

[0116] Preparation of sample processing tube in Example 4

[0117] (1) Install the sample processing tube;

[0118] (2) Add about 1 g of corundum to the tube, and add 3 mL of sample pretreatment solution (50 mM Tris-HCl (pH 8.0), 700 mM NaCl, 10 mM EDTA, 1% SDS) to the tube;

[0119] (3) Place the tube in the tooling position of the precision hand press, and seal it with aluminum foil at 160°C.

[0120] Pig, horse, duck, cow, sheep detection method in Example 5

[0121] (1) Tear off the aluminum foil protective film of the sample processing tube, place it vertically, add the meat or meat product to be detected into the sample processing tube, and tighten the tube cap;

[0122] (2) Keep the tube body upright, rub 5-10 times to make the sample fully contact with the grinding material and ensure sufficient grinding;

[0123] (3) Open the outer packaging, take out the reagent compartment and reaction layer of the chip, tear off the sealing tape on the back of the reagent compartment, hold the reagent compartment and reaction layer with the head of the reaction layer vertically upward, press hard to pierce the reagent compartment to the upper and lower layers of the chip, and complete the assembly;

[0124] (4) Unscrew the sample compartment cover, add 2-3 drops (50 μL) of sample grinding solution to the sample compartment, and tighten the sample compartment cover;

[0125] (5) Press and hold the device power button for more than 3 seconds to turn it on, and click the "Detection" button on the home page;

[0126] (6) Wait for 3 seconds, see the red light flashing in the scanning window, and scan the chip two-dimensional code;

[0127] (7) According to the screen prompt, scan the sample information or skip;

[0128] (8) According to the screen prompt, open the cabin door, insert the chip, and close the cabin door;

[0129] (9) Click the "Run" button to start detection;

[0130] (10) After the operation is completed, view the detection results.

[0131] The sample processing tube processes the sample for about 1 minute; the nucleic acid extraction and purification on the integrated microfluidic chip takes about 12 minutes; the real-time quantitative PCR reaction program on the integrated microfluidic chip is: 95℃ for 1 minute; (95℃ for 5 seconds, 60℃ for 8 seconds), 40 cycles, and the total time of 40 cycles is 22 minutes. The whole process time from sample processing, nucleic acid extraction and purification to amplification detection result of pork, horse meat, duck meat, beef and mutton is about 35 minutes.

[0132] Experimental Example 1 Sensitivity detection

[0133] Pork, horse, duck, beef and mutton DNA samples with DNA content of 100 ng, 10 ng and 1 ng were prepared for nucleic acid amplification detection test, and the system source components are shown in Table 2, and the detection results are shown in Table 4, and the detection curve is shown in Figure 1 - Figure 5 .

[0134] Table 4 Sensitivity detection results

[0135]

[0136]

[0137] From Table 4 above, it can be seen that the primer composition and the kit thereof have good sensitivity, and can detect pork, horse, duck, beef and mutton DNA samples with a content of 1 ng.

[0138] Experimental Example 2 Specificity detection

[0139] The negative quality control (deionized water), donkey meat (100 mg), rabbit meat (100 mg), goose meat (100 mg), fish meat (100 mg), pigeon meat (100 mg), quail meat (100 mg) and chicken meat (100 mg) were detected according to the above method, and the detection Ct value is shown in Table 5.

[0140] Table 5 Specificity detection results

[0141] Sample Target Ct Value Target Detection Result NTC — Not Detected Donkey Meat — Not Detected Rabbit Meat — Not Detected Goose Meat — Not Detected Fish Meat — Not Detected Pigeon Meat — Not Detected Quail Meat — Not Detected Chicken Meat — Not Detected

[0142] From Table 5, it can be seen that the primer probe combination and the kit thereof have good specificity.

[0143] Experimental Example 3 Actual sample detection

[0144] Pork (1 g), horse meat (1 g), duck meat (1 g), beef (100 mg), mutton (100 mg), pork ham (1 g), roasted duck (1 g), sauced beef (1 g), milk (50 μL) and hand-grabbed mutton (1 g) were used for experiments.

[0145] The above extracted samples were detected according to the system and steps of the above-mentioned Examples 1-5, and the detection results are shown in Table 6. Figure 7 - Figure 16 As shown in Table 6, the detection Ct values are as follows.

[0146] Table 6 Detection results of meat and meat product samples

[0147]

[0148]

[0149] As can be seen from Table 6, the pig, horse, duck, cattle, and sheep-derived component primer probe composition of the present application can detect real samples of meat and products, and can be used for rapid identification on site.

[0150] Comparative Example 1

[0151] Comparative Example 1 and Examples 1-4 and Experimental Example 1 only differ in primer probe sequences, and the rest are the same. The primer probe sequences of Comparative Example 1 are as follows:

[0152] Among them, the primer probe sequence of pig is:

[0153] pig-F: 5'-ATCTACATGATTCATTACAATTAC-3' (SEQ ID NO: 19);

[0154] pig-R: 5'-CTATGTTTTTGAGTTTTGAGTTCG-3' (SEQ ID NO: 20);

[0155] pig-P: 5'-ATCTCAAACTACTCATACCCA-3' (SEQ ID NO: 21).

[0156] The primer probe sequence of horse is:

[0157] Equus-F: 5'-TGTAGCCCTAGCCGTGCGGCTAACC-3' (SEQ ID NO: 22);

[0158] Equus-R: 5'-TAGGATGATAAACGTAATAAGGGCTG-3' (SEQ ID NO: 23);

[0159] Equus-P: 5'-CGCCGGACACCTCCTAATACACCTC-3' (SEQ ID NO: 24).

[0160] The primer probe sequence of duck is:

[0161] Anas-F: 5'-GGCCACACAAATCCTCACAG-3' (SEQ ID NO: 25);

[0162] Anas-R: 5'-TGTGTTGGCTACTGAGGAGAAA-3' (SEQ ID NO: 26);

[0163] Anas-P: 5'-CCTACTGGCTATGCACTACACCGCAGAC-3' (SEQ ID NO: 27).

[0164] The primer probe sequence of bovine is:

[0165] Bos-F: 5'-CCGATGGATGTGTTCAGAGCT-3' (SEQ ID NO: 28);

[0166] Bos-R: 5'-GCCAAATGTCTGGGTGTAGATACC-3' (SEQ ID NO: 29);

[0167] Bos-P: 5'-TGGGCTTTAGGGCTTCCGAATGTGAA-3' (SEQ ID NO: 30).

[0168] The primer probe sequence of sheep is:

[0169] Capra-F: 5'-GGTCTATATTATGGATCATATACC-3' (SEQ ID NO: 31);

[0170] Capra-R: 5'-CTATGAATGCTGTGGCCATTGT-3' (SEQ ID NO: 32);

[0171] Capra-P: 5'-TAGAAACATGAAACATCGGAGTAATCCTCCT-3' (SEQ ID NO: 33).

[0172] The real samples of pigs, horses, ducks, bovine and sheep were put in, and the experimental results of Example 3 and Comparative Example 1 were as shown in Table 6, and the Ct value results were as shown in Table 7. Figure 17 - Figure 21

[0173] Table 7 Ct value of Comparative Example 1

[0174] Sample Example 3 Ct Value Comparative Example 1 Ct Value Pork 22.73 31.19 Horse Meat 20.75 Not Detected Duck Meat 18.88 32.19 Beef 22.58 30.82 Sheep Meat 18.78 31.08

[0175] From the results, it can be seen that the Ct value is delayed to a certain extent, the fluorescence value is lower, and there is no amplification, which indicates that the detection sensitivity is low. ​

[0176] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A primer-probe combination for on-site detection of pig, horse, duck, cattle, and sheep-derived ingredients, characterized in that: The primer-probe combination includes: primer pairs and probes for detecting sequences of pig-derived components as shown in SEQ ID NOs: 1-3; primer pairs and probes for detecting sequences of horse-derived components as shown in SEQ ID NOs: 4-6; primer pairs and probes for detecting sequences of duck-derived components as shown in SEQ ID NOs: 7-9; primer pairs and probes for detecting sequences of bovine-derived components as shown in SEQ ID NOs: 10-12; and primer pairs and probes for detecting sequences of sheep-derived components as shown in SEQ ID NOs: 13-18.

2. Use of the primer-probe combination according to claim 1 in the preparation of products for detecting ingredients derived from pigs, horses, ducks, cattle, and sheep.

3. The use according to claim 2, characterized in that The products include independent detection reagents, chips and test kits.

4. A kit for on-site detection of ingredients derived from pigs, horses, ducks, cattle, and sheep, characterized in that: The kit comprises the primer-probe combination according to claim 1.

5. The kit according to claim 4, characterized in that The test kit is an integrated closed microfluidic chip test kit.

6. The kit according to claim 5, characterized in that The integrated closed microfluidic chip kit includes purification reagents and air-dried qPCR reagents.

7. A method for on-site detection of ingredients derived from pigs, horses, ducks, cattle, and sheep, characterized in that: The method comprises the following steps: (1) preparing the primer-probe combination described in claim 1 into an integrated closed microfluidic kit; (2) adding a sample containing ingredients from pigs, horses, ducks, cattle, or sheep into a sample processing tube for processing to obtain a liquid to be tested; (3) dropping the liquid to be tested into the integrated closed microfluidic reagent kit; (4) Place the test kit containing the liquid to be tested on the testing equipment for testing and analysis.

8. The method according to claim 7, characterized in that The step (2) specifically comprises: adding a sample pre-treatment solution and abrasive particles into the sample processing tube; The sample pretreatment solution includes Tris-HCl (pH 8.0), NaCl, EDTA and SDS; The sample described in step (2) is pork, horse, duck, beef, mutton or their products.

9. The method according to claim 7, characterized in that The step (4) specifically includes: 1) Turn on the rapid detection device; 2) Scan the QR code; 3) Inserting the integrated microfluidic chip kit; 4) Run the test; 5) End and check the test results and amplification curve.

10. Use of the primer-probe combination according to claim 1, the kit according to any one of claims 4 to 6, or the method according to any one of claims 7 to 9 in detecting components derived from pigs, horses, ducks, cattle, sheep, their derivatives, and their products.

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