Primer composition and detection method for detecting pathogenic Campylobacter based on loop-mediated isothermal amplification technology

Abstract

The present invention discloses a primer combination and detection method for detecting pathogenic Campylobacter based on loop-mediated isothermal amplification technology, belonging to the field of biological detection technology for pathogenic bacteria. The detection method provided by the present invention can quickly and accurately detect whether a sample contains pathogenic Campylobacter jejuni and Campylobacter coli. Specifically, the detection and reading of the test results can be completed within 20 minutes, which is significantly shorter than the currently commonly used real-time fluorescence quantitative PCR method. It has important application value in public health, food safety, animal husbandry and veterinary medicine, and entry-exit inspection and quarantine.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection of pathogenic bacteria, and particularly relates to a primer composition and a detection method for detecting pathogenic Campylobacter based on loop-mediated isothermal amplification technology. Background Art

[0002] Foodborne pathogens are one of the main causes of foodborne illness, a core issue in global food safety, and have become a major public health concern threatening human health. In recent years, foodborne pathogenic disease incidents have occurred frequently both domestically and internationally, with pathogenic bacterial contamination in the food chain and the resulting foodborne illnesses becoming commonplace.

[0003] Campylobacter is a foodborne pathogen that is common to both humans and animals. It is widely distributed in the animal kingdom and is transmitted to humans through food and water. It can cause diarrhea, gastroenteritis and extraintestinal infections. Campylobacter ) mainly include Campylobacter jejuni ( C.jejuni ) and Campylobacter coli ( C. coli ) and more. Campylobacter jejuni is a zoonotic pathogen widely found in various meat products, such as pork, beef, chicken, and duck. It is currently the culprit behind the most reported outbreaks of gastrointestinal illnesses, including collective diarrhea. Infection is primarily transmitted through ingestion of contaminated poultry, food, dairy products, or water. Typical clinical symptoms include acute, self-limited gastroenteritis. Severe cases can include bloody or purulent stools, meningitis, pyelonephritis, and miscarriage in pregnant women. It is listed by the World Health Organization as the most common foodborne illness. Campylobacter coli is another common zoonotic foodborne pathogen that causes gastrointestinal infections. Therefore, convenient, rapid, and accurate detection of Campylobacter jejuni and Campylobacter coli is crucial for preventing the spread of related diseases.

[0004] Currently, detection methods for Campylobacter include traditional culture, enzyme-linked immunosorbent assay (ELISA), and quantitative fluorescence PCR. Traditional culture involves inoculating a sample onto a specific culture medium and cultivating it under microaerobic conditions. Identification is based on colony morphology and biochemical characteristics. This method requires high sample pretreatment, specialized technicians and equipment, and is prone to false-negative results. ELISA utilizes the specific binding of antigens and antibodies to detect antigens or antibodies in a sample using enzyme-labeled antibodies. While it can simultaneously test a large number of samples and is suitable for large-scale screening, it can produce false-positive or false-negative results, has low sensitivity and specificity, requires a dedicated microplate reader, and places high demands on antibody quality. The performance of test kits varies significantly between manufacturers. Quantitative fluorescence PCR is the most widely used molecular biology detection method. It monitors the progress of the PCR reaction in real time and quantitatively detects the target Campylobacter through changes in the fluorescence signal. It has good sensitivity and specificity, but is slow, requires specialized equipment, reagents, and technicians, and is expensive.

[0005] Based on this, the development of a rapid, sensitive, specific and accurate detection method for Campylobacter jejuni and Campylobacter coli is of great significance for food safety testing, clinical diagnosis and epidemiological investigation.

[0006] Isothermal amplification is a nucleic acid amplification technology currently widely used in scientific research and clinical testing. It amplifies trace amounts of DNA or RNA templates by 100,000 to 1 million times. By adding special fluorescent dyes during the amplification process, the product increase process can be visually depicted in real time. Therefore, it is often used to observe the presence or amount of genes. Among the many isothermal amplification technologies, the most commonly used is loop-mediated isothermal amplification (LAMP) technology. Compared with the well-known PCR technology, LAMP has the following advantages: (1) It is fast, with the entire process completed in 15-20 minutes, and the amplification multiple is comparable to the 90-minute PCR amplification process, which is particularly advantageous in time-sensitive scenarios such as emergency and outdoor testing; (2) It has lower template purity requirements than the PCR system and is compatible with fast and simple lysis methods; (3) It has better specificity and low commercialization cost; (4) The reagents can be freeze-dried and easily stored.

[0007] Currently, existing methods for detecting Campylobacter jejuni based on loop-mediated isothermal amplification (LMAP), such as CN106367516 A, are available. However, the detection process described in this method takes up to an hour and relies on gel electrophoresis to present results, which can easily lead to aerosol contamination and is not suitable for clinical diagnosis. Furthermore, the C. jejuni detection method disclosed in CN106916906 A uses visual or instrumental color interpretation based on the color change in the amplification pool after amplification. Specifically, the indicator used is neutral red, which changes color between pH 6.4 and 8.0 (from red to yellow). Before the isothermal amplification reaction, the reaction solution is alkaline and yellow in color. Once the amplification reaction occurs, as the amplification product accumulates, the solution pH becomes acidic, causing the color to change from yellow to red. This method has low accuracy in determining amplification results, and is prone to errors.

[0008] In view of this, the present invention provides a method for detecting Campylobacter jejuni and Campylobacter coli based on loop-mediated isothermal amplification technology. The method can achieve rapid and accurate judgment and is suitable for public health, food safety, animal husbandry and veterinary medicine, and entry-exit inspection and quarantine. Summary of the Invention

[0009] The loop-mediated isothermal amplification (LAMP) reaction is a novel nucleic acid amplification technology based on chain displacement cycling. The reaction is performed at a constant temperature of 60-65°C. LAMP uses four to six primers, each recognizing six to eight regions on the target gene, giving the reaction greater specificity. Because the LAMP reaction produces a large amount of a white magnesium pyrophosphate precipitate as a byproduct, the amplified product can be directly identified by visual observation or turbidimetry without electrophoresis analysis. The present invention incorporates a fluorescent dye into the amplification system, allowing for more intuitive observation of the amplification process using a fluorescent thermostated amplification instrument or a fluorescent PCR instrument, enabling accurate interpretation of the results.

[0010] Therefore, LAMP primer design is crucial for accurate detection of Campylobacter jejuni and Campylobacter coli. Typically, LAMP primers are determined by the following method: screening the target gene sequence to be detected; performing a Blast comparison on the target gene sequence similarity; using online software to design multiple primer pairs targeting six regions of the target gene (F3c, F2c, and F1c at the 3' end of the target gene, and B1, B2, and B3 at the 5' end of the target gene); and finally, screening to obtain the optimal LAMP primers.

[0011] One object of the present invention is to provide a set of LAMP primer compositions for detecting Campylobacter jejuni and Campylobacter coli based on loop-mediated isothermal amplification technology; a second object of the present invention is to provide the use of the LAMP primer set in the preparation of products for detecting and screening Campylobacter jejuni and Campylobacter coli; a third object of the present invention is to provide a kit or microfluidic chip containing the LAMP primer set; and a fourth object of the present invention is to provide a method for detecting Campylobacter jejuni and Campylobacter coli based on loop-mediated isothermal amplification technology.

[0012] The purpose of the present invention is achieved through the following technical solutions:

[0013] In a first aspect, the present invention provides a set of loop-mediated isothermal amplification primer compositions, characterized in that the primer composition comprises a LAMP primer set for detecting Campylobacter jejuni and a LAMP primer set for detecting Campylobacter coli; the LAMP primer set for detecting Campylobacter jejuni consists of primers shown in SEQ ID NO: 1 to SEQ ID NO: 4; the LAMP primer set for detecting Campylobacter coli consists of primers shown in SEQ ID NO: 5 to SEQ ID NO: 8.

[0014] In a specific embodiment of the present invention, the LAMP primer set for detecting Campylobacter jejuni is as follows:

[0015] .

[0016] In a specific embodiment of the present invention, the LAMP primer set for detecting Campylobacter coli is as follows:

[0017] .

[0018] In a second aspect, the present invention provides a use of the loop-mediated isothermal amplification primer composition according to the first aspect of the present invention in at least one of the following:

[0019] 1) Use in the preparation of products for detecting and / or assisting in the detection of Campylobacter;

[0020] 2) Application in the preparation of drugs for screening or assisting in screening for treating Campylobacter-related diseases

[0021] The Campylobacter is Campylobacter jejuni and Campylobacter coli, and the Campylobacter-related diseases refer to diseases caused by Campylobacter jejuni and Campylobacter coli, including but not limited to diarrhea and gastroenteritis.

[0022] The products include but are not limited to reagents, kits, test strips, membrane strips, chips or detection platforms.

[0023] In a third aspect, the present invention provides a kit, characterized in that the kit comprises the loop-mediated isothermal amplification primer composition described in the first aspect of the present invention.

[0024] The kit is a rapid test product for detecting Campylobacter jejuni and Campylobacter coli based on loop-mediated isothermal amplification technology. It can quickly and accurately detect whether the sample to be tested contains Campylobacter jejuni and Campylobacter coli. The kit also includes other conventional reagents required for loop-mediated isothermal amplification technology.

[0025] In some embodiments of the present invention, the other conventional reagents include Bst enzyme, reverse transcriptase, dNTPs, ultrapure water, buffer, and magnesium ions.

[0026] In some embodiments of the present invention, the kit further includes a sample lysis solution, a sample diluent, and a sample DNA extraction reagent / kit, which can be purchased from commercial sources or prepared by the user.

[0027] In one embodiment of the present invention, the sample lysate contains a surfactant, wherein the surfactant is composed of one or more of NP-40, Triton X-100, PEG 6000, Tween-20, Tween-80, and sodium dodecyl sulfate (SDS). The mass concentration of each surfactant component in the sample lysate is between 0.2% and 4.3%.

[0028] In order to read the amplification results efficiently and accurately, the kit also includes a fluorescent dye, which is a dye with a fluorescent group commonly used by those skilled in the art, and has a fluorescence excitation wavelength and emission wavelength between 485-530 nm. In a specific embodiment, the fluorescent dye is Eva Green.

[0029] Preferably, the loop-mediated isothermal amplification primer composition, fluorescent dye and other conventional reagents in the kit are packaged separately.

[0030] In a preferred embodiment of the present invention, the loop-mediated isothermal amplification primer combination, fluorescent dye, and other conventional reagents in the kit are stored in the form of lyophilized beads in a vacuum-packed aluminum foil bag. Specifically, the lyophilized beads are prepared by the following method:

[0031] S1) preparing a buffer containing Bst polymerase, reverse transcriptase, fluorescent dye, and dNTPs, and adding a loop-mediated isothermal amplification primer combination to form a LAMP amplification reaction solution;

[0032] S2) dropping the LAMP amplification reaction solution into liquid nitrogen to form a sphere;

[0033] S3) Place in freeze-drying equipment and freeze-dry overnight to form freeze-dried beads.

[0034] In the LAMP amplification reaction solution described in step S1), primer P k1 、P k2 、P j1 、P j2 The final concentration of primer P is 0.1-0.2 μM. k3 、P k4 、P j3 、P j4 The final concentration is 0.3-1.0 μM.

[0035] Furthermore, the kit also includes a positive control, which is a plasmid containing a Campylobacter jejuni or Campylobacter coli gene.

[0036] Furthermore, the kit also includes a negative control, which is pure water treated with diethyl pyrocarbonate (DEPC).

[0037] In a fourth aspect, the present invention provides a chip, including a microfluidic chip, characterized in that the microfluidic chip is composed of 2-4 reaction detection areas, each reaction detection area includes an injection pool, a distribution pool, a capillary microvalve and an amplification pool connected in sequence, and the amplification pool is coated with the loop-mediated isothermal amplification primer composition described in the first aspect of the present invention.

[0038] In a specific embodiment of the present invention, the loop-mediated isothermal amplification primer composition is fixed in the amplification pool by a vacuum drying method.

[0039] Those skilled in the art know that the injection pool is used to load the reaction liquid, the distribution pool is used to evenly distribute the reaction liquid to the amplification pool, and the amplification pool is used to perform loop-mediated isothermal amplification reaction (LAMP). The capillary microvalve controls the movement of the fluid in the microfluidic chip by utilizing the blocking effect of the capillary on the liquid.

[0040] In a fifth aspect, the present invention provides a method for detecting Campylobacter jejuni and Campylobacter coli based on loop-mediated isothermal amplification technology that is not intended for disease treatment or diagnosis, characterized in that the method comprises the following steps:

[0041] S1: Extract genomic DNA or RNA from the sample to be tested, reverse transcribe the RNA into cDNA, and combine the DNA and cDNA as sample DNA;

[0042] S2: Add the sample DNA to the loop-mediated isothermal amplification reaction solution and amplify at 65°C for 20-25 minutes;

[0043] S3: Read the fluorescence value every 20-30 seconds and judge the result based on the amplification curve.

[0044] The loop-mediated isothermal amplification reaction solution in step S2 is a buffer solution containing Bst polymerase, reverse transcriptase, fluorescent dye and dNTPs. Furthermore, the loop-mediated isothermal amplification reaction solution also includes freeze-dried beads prepared according to the loop-mediated isothermal amplification reaction solution.

[0045] The sample to be tested is selected from all samples that may contain Campylobacter jejuni and / or Campylobacter coli and need to be tested, including but not limited to chicken, pork, and feces.

[0046] The technical solution provided by the present invention has the following beneficial technical effects:

[0047] The kit or microfluidic chip for detecting Campylobacter jejuni and Campylobacter coli based on loop-mediated isothermal amplification technology provided by the present invention can quickly and accurately detect whether the sample to be tested contains pathogenic Campylobacter jejuni and Campylobacter coli, and has important application value in public health, food safety, animal husbandry and veterinary medicine, and entry-exit inspection and quarantine. In practical applications, the detection method provided by the present invention can complete the detection and read the test results within 20 minutes. The detection time is significantly shorter than the real-time fluorescence quantitative PCR method currently commonly used, and it is also of great significance for assisting in guiding treatment and medication. At the same time, the detection method provided by the present invention can also be used for regional epidemiological surveys and epidemic monitoring to study the prevalence of pathogenic Campylobacter jejuni and Campylobacter coli. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Fluorescence amplification curves of positive samples of Campylobacter jejuni and Campylobacter coli detection kits;

[0049] Figure 2 Gradient amplification curve of Campylobacter jejuni quality control product;

[0050] Figure 3 Gradient amplification curve of Campylobacter coli quality control product;

[0051] Figure 4 Campylobacter jejuni-specific test results;

[0052] Figure 5 Campylobacter coli-specific test results;

[0053] Figure 6 Amplification curve of the primer set (Campylobacter coli-2) for Campylobacter coli samples;

[0054] Figure 7 Amplification curve of the primer set (Campylobacter coli-3) for Campylobacter jejuni samples. DETAILED DESCRIPTION

[0055] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

[0056] Example 1 Design of LAMP primer composition

[0057] The target gene sequences to be detected were determined based on the genomes of Campylobacter jejuni and Campylobacter coli in the database, and a Blast comparison was performed on the target gene sequences to ensure that the selected target genes had an intraspecies similarity of more than 95%, were highly conserved, and could be used for mutant strain detection, thereby screening out the best 1-2 target genes. The loop-mediated isothermal amplification (LAMP) primer design online tool, PrimerExplore V4, was then used to design multiple pairs of LAMP primer sets for the six different regions of the screened target genes (i.e., the F3c, F2c, and F1c regions at the 3' end of the target gene, and the B1, B2, and B3 regions at the 5' end of the target gene). The 2-3 LAMP primer sets with the best theoretical parameters and the highest success rate were screened for experimental verification.

[0058] According to the above method, the optimal LAMP primer set for detecting Campylobacter jejuni obtained by the present invention consists of the sequences shown in SEQ ID NOs: 1-4; the optimal LAMP primer set for detecting Campylobacter coli obtained by the present invention consists of the sequences shown in SEQ ID NOs: 5-8.

[0059] Table 1 LAMP primer composition

[0060] .

[0061] Example 2 Kit for detecting Campylobacter jejuni and Campylobacter coli based on LAMP technology

[0062] The kit comprises the following parts:

[0063] (1) Lyophilized beads;

[0064] (2) Sample lysis buffer, sample diluent, negative quality control product, and positive quality control product;

[0065] (3) Boxes for separating and centrally packaging these reagent bottles or tubes.

[0066] The freeze-dried beads were prepared by the following method:

[0067] S1) preparing a buffer containing Bst polymerase, reverse transcriptase, fluorescent dye and dNTPs, adding a loop-mediated isothermal amplification primer combination to form a LAMP amplification reaction solution; in the LAMP amplification reaction solution, primer P k1 、P k2 、P j1 、P j2 The final concentration of primer P is 0.1-0.2 μM. k3 、P k4 、P j3 、P j4 The final concentration is 0.3-1.0 μM;

[0068] S2) dropping the LAMP amplification reaction solution into liquid nitrogen to form a sphere;

[0069] S3) Place in freeze-drying equipment and freeze-dry overnight to form freeze-dried beads.

[0070] Example 3 Method for detecting Campylobacter jejuni and Campylobacter coli based on LAMP technology

[0071] The detection steps are as follows:

[0072] S1: Use a stool swab as the specimen to be tested. Place the collected stool swab in a dry, dedicated small box. It can be sent directly for testing or stored in a 4°C refrigerator for 7 natural days.

[0073] S2: Place the swab into a plastic tube containing sample lysis solution, break the swab, cover the tube, and shake vigorously for 20 seconds;

[0074] S3: Take out one drop, add it to the plastic tube containing the diluent, and shake for 20 seconds to mix;

[0075] S4: Take one drop of the diluent or pipette 25 μl into the reaction tube and mix with the lyophilized beads (prepared in Example 2) in the tube. Directly pipette 25 μl of the negative and positive quality control products into the reaction tube.

[0076] S5: Place the reaction tube on an oscillator for 10 seconds, centrifuge briefly, and then place it in a dedicated fluorescence device for amplification (fluorescence thermostat amplification instrument or fluorescence PCR instrument);

[0077] S6: Program settings: 65°C, 30 seconds to read FAM fluorescence, 40 cycles, total duration 20 minutes;

[0078] S7: After amplification, carefully remove the reaction tube, wrap it with gloves, tie a knot and discard it to avoid aerosol contamination;

[0079] S8: Result determination: 1) If there is no S-shaped amplification curve, the sample is determined to be negative; 2) If the amplification curve is clearly S-shaped, it is determined to be a positive sample, such as Figure 1 shown.

[0080] Example 4 Sensitivity Detection

[0081] A certain amount of plasmid containing gene fragments of Campylobacter jejuni and Campylobacter coli was dissolved in an appropriate amount of ultrapure water. The OD value was measured and the concentration was calculated using the formula OD260×50µg / ml. The number of copies per ml was calculated based on the molar mass of the plasmid. The plasmid was then diluted with ultrapure water to 3×10 3 copies / ml, 1×10 3 copies / ml, 3×10 2 copies / ml, 1×10 2 copies / ml.

[0082] In the reaction tube, 25µl of plasmid template of various gradients was added, and each gradient was repeated three times. Together with the negative control, the experiment was performed on a Macrostone SLAN-96S fluorescence quantitative PCR instrument using a temperature control program of 65°C for 30 seconds (FAM fluorescence was read) and 40 cycles.

[0083] The experimental results are as follows Figure 2 、 Figure 3 As shown, the FAM channel, three replicates, can see 3 × 10 3 copies / ml, 1×10 3 copies / ml, 3×10 2 copies / ml, 1×10 2 All replicates of the four concentrations of 1×10 copies / ml showed positive results, while the negative control showed negative results. This fully demonstrates that the sensitivity of the LAMP method for detecting Campylobacter jejuni and Campylobacter coli can reach 1×10 2 copies / ml.

[0084] Example 5 Specificity Detection

[0085] Campylobacter fetus ( Campylobacter foetus ), Campylobacter conciseus ( Campylobacter concisus ), Campylobacter salivarius ( Campylobacter sputorum ) and 25μl of DNA extracted from Campylobacter jejuni and Campylobacter coli were added to the reaction tube containing freeze-dried beads for detecting Campylobacter jejuni genes. Only Campylobacter jejuni DNA was amplified, showing a positive curve ( Figure 4 ); When they were added to the reaction tube for detecting freeze-dried beads of Campylobacter coli genes, only the DNA of Campylobacter coli was amplified, and the DNA of the other four Campylobacter species were all negative ( Figure 5 ), indicating that the specificity of the kit was consistent with expectations.

[0086] LAMP primer set screening example

[0087] In order to quickly and accurately amplify the target gene site, the present invention uses primer design software to design LAMP primers. However, preliminary experimental results show that the primers designed by the software can theoretically amplify the target gene, but they may not meet the requirements of the present invention in terms of specificity and sensitivity. To this end, we not only performed base substitutions on the sequences of the two paired primers, but also repeatedly tested the amplification effects of different primer pairs to obtain good specificity and sensitivity. The following is a brief description of the primer screening process using Campylobacter coli as an example.

[0088] For Campylobacter coli, the present invention selected the highly specific GlyA gene as the target gene. LAMP primers were designed using online LAMP primer design software. To increase the probability of successful amplification, the present invention selected three sets of primers with the best overall parameters. One set of primers is shown in Table 1, and the other two sets of primers are shown in Table 2:

[0089] Table 2 LAMP primer sets for Campylobacter coli

[0090] .

[0091] Although the primer set with the best theoretical parameters and the highest success rate was used, the amplification results were still unsatisfactory. The primer set (Campylobacter coli-2) had a poor amplification efficiency for each Campylobacter coli template ( Figure 6 ), indicating that the primer set designed by the software cannot achieve the purpose of amplifying the target gene. In the specific experiment of the primer set (Campylobacter coli-3), it has no effect on Campylobacter jejuni ( Campylobacter jejuni ) showed a significant increase ( Figure 7 ), indicating that the software-designed primer set has poor specificity and cannot achieve the gene detection purpose of the present invention. The present inventors analyzed that the aforementioned nonspecific amplification occurs because Campylobacter coli and Campylobacter jejuni are closely related. Whole-genome alignments show that the genomes of Campylobacter jejuni and Campylobacter coli are both 1.6-1.7 Mb, with approximately 93%-95% nucleotide identity (ANI). Differential genes account for only 5% of the total genome, making them evolutionarily closely related sister species. Therefore, primer design requires careful optimization of detection specificity. These results also confirm that not all primers designed by the software are suitable, and LAMP primer sets 2 and 3 both have deficiencies in sensitivity or specificity. Through repeated testing and adjustments, the present inventors ultimately determined that the primer set shown in SEQ ID NOs: 5-8 in Table 1 is the optimal LAMP primer for detecting Campylobacter coli.

[0092] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A loop-mediated isothermal amplification primer composition, characterized in that The primer composition consists of a LAMP primer set for detecting Campylobacter jejuni and a LAMP primer set for detecting Campylobacter coli; the LAMP primer set for detecting Campylobacter jejuni consists of primers shown in SEQ ID NO: 1 to SEQ ID NO: 4; the LAMP primer set for detecting Campylobacter coli consists of primers shown in SEQ ID NO: 5 to SEQ ID NO:

8.

2. Use of the loop-mediated isothermal amplification primer composition according to claim 1 in the preparation of a product for detecting or assisting in the detection of Campylobacter, wherein the Campylobacter is Campylobacter jejuni and Campylobacter coli.

3. The use according to claim 2, characterized in that The products include reagents, test kits, test strips, membrane strips, chips or detection platforms.

4. A kit, characterized in that The kit comprises the loop-mediated isothermal amplification primer composition according to claim 1.

5. The kit according to claim 4, characterized in that The kit also includes other conventional reagents required for loop-mediated isothermal amplification technology, and the other conventional reagents include Bst enzyme, reverse transcriptase, dNTPs, ultrapure water, buffer, and magnesium ions.

6. The kit according to claim 5, characterized in that The kit also includes a fluorescent dye; The loop-mediated isothermal amplification primer composition, fluorescent dye and other conventional reagents in the kit are packaged separately; or, The loop-mediated isothermal amplification primer composition, fluorescent dye and other conventional reagents in the kit are stored in a vacuum-packed aluminum foil bag in the form of freeze-dried beads.

7. The kit according to claim 6, characterized in that The freeze-dried beads were prepared by the following method: S1) preparing a buffer containing Bst polymerase, reverse transcriptase, fluorescent dye, and dNTPs, and adding a loop-mediated isothermal amplification primer combination to form a LAMP amplification reaction solution; S2) dropping the LAMP amplification reaction solution into liquid nitrogen to form a sphere; S3) Place in freeze-drying equipment and freeze-dry overnight to form freeze-dried beads.

8. The kit according to claim 6, characterized in that The kit also includes a positive control and a negative control; the positive control is a plasmid containing a Campylobacter jejuni or Campylobacter coli gene; and the negative control is pure water treated with diethyl pyrocarbonate.

9. Chips, including microfluidic chips, characterized in that The microfluidic chip consists of 2-4 reaction detection areas, each reaction detection area includes an injection pool, a distribution pool, a capillary microvalve and an amplification pool connected in sequence, and the amplification pool is coated with the loop-mediated isothermal amplification primer composition according to claim 1.

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