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

Abstract

The invention discloses a primer composition for detecting pathogenic campylobacter based on a loop-mediated isothermal amplification technology and a detection method, and belongs to the technical field of biological detection of pathogenic bacteria. The detection method provided by the invention can quickly and accurately detect whether a sample to be detected contains pathogenic campylobacter jejuni and campylobacter colons or not, specifically, detection can be completed within 20 minutes, a detection result can be read, and the detection time is obviously shorter than that of a real-time fluorescent quantitative PCR method which is commonly adopted at present; the method has important application value in public health, food safety, animal husbandry and veterinary medicine and entry and exit inspection and quarantine.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection of pathogenic bacteria, and specifically 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 diseases, a core issue of global food safety, and have become an important public health issue that threatens people's health. In recent years, foodborne pathogen disease incidents have occurred frequently at home and abroad, and pathogenic bacteria contamination in the food chain and the foodborne diseases caused by them are common.

[0003] Campylobacter is a foodborne pathogen that 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 jejuni ( C. jejuni ) and Campylobacter coli ( C. coli ) and so on. Campylobacter jejuni is a zoonotic pathogen widely found in various meat products, such as pork, beef, chicken, and duck. It is currently the culprit for the outbreak of gastrointestinal diseases such as collective diarrhea. It is mainly infected by ingesting poultry, food, dairy products or water contaminated by it. The main clinical symptoms are acute, self-limited gastroenteritis. In severe cases, bloody stools, purulent stools, meningitis, pyelonephritis, and miscarriage in pregnant women may occur. It is listed as the most common foodborne disease by the World Health Organization. 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 of great significance for preventing the spread of related diseases.

[0004] Currently, the detection methods for Campylobacter mainly include traditional culture method, enzyme-linked immunosorbent assay (ELISA), and fluorescence quantitative PCR method. The traditional culture method involves inoculating the sample onto a specific culture medium and culturing it under microaerophilic conditions, followed by identification based on colony morphology, biochemical characteristics, etc. This method has high requirements for sample pretreatment and requires professional technicians and equipment, making it prone to false negative results. The ELISA method utilizes the specific binding of antigen and antibody and detects the antigen or antibody in the sample through enzyme-labeled antibody. It can detect a large number of samples simultaneously and is suitable for large-scale screening. However, it has false positive or false negative results, low sensitivity and specificity, requires a dedicated microplate reader, and has high requirements for the quality of antibodies. There are significant performance differences among kits from different manufacturers. The fluorescence quantitative PCR method is currently the most widely used molecular biology detection method. By real-time monitoring the PCR reaction process, it quantitatively detects the target Campylobacter through changes in fluorescence signals. It has good sensitivity and specificity, but has a slow detection speed, requires professional equipment, reagents, and technicians, and has a high detection cost.

[0005] Based on this, developing a rapid, sensitive, specific, and accurate detection method for Campylobacter jejuni and Campylobacter coli is of great significance for food safety detection, 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 the number of trace DNA or RNA templates by 100,000 to one million times. During the amplification process, by adding special fluorescent dyes, the increase process of the product can be vividly depicted in real time, so it is often used to observe the presence or amount of genes. Among 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) Fast, the entire process can be completed in 15 - 20 minutes, and the amplification multiple is equivalent to that of a 90-minute PCR amplification process, with outstanding advantages in time-sensitive scenarios such as emergency and outdoor testing; (2) Has lower requirements for the purity of the template than the PCR system and can be compatible with rapid and simple lysis methods; (3) Has better specificity and low commercialization cost; (4) The reagent can be freeze-dried and is easy to store.

[0007] Currently, there are methods for detecting Campylobacter jejuni based on loop-mediated isothermal amplification technology in the prior art, such as CN106367516 A. However, the detection process of the method described therein takes up to 1 hour, and the results are presented by gel electrophoresis, which is prone to aerosol contamination and is not suitable for clinical diagnosis. In addition, for the detection method of Campylobacter jejuni disclosed in the prior art CN106916906 A, the visual judgment or color judgment using equipment is carried out through the color change in the amplification pool after the amplification ends. Specifically, the indicator used is neutral red, and the color change range is between pH 6.4 - 8.0 (from red to yellow). Before the isothermal amplification reaction occurs, since the reaction solution is alkaline, the color is yellow. When the amplification reaction occurs, as the amplification products accumulate continuously, the pH environment of the solution becomes acidic, so the color will change from yellow to red. The accuracy of judging the amplification results by this method is low, and the possibility of judgment error is relatively large.

[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, which can achieve rapid and accurate judgment and is applicable to 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 new nucleic acid amplification technology based on strand displacement autocycling reaction. The reaction is carried out under the constant temperature condition of 60 - 65°C. LAMP has 4 - 6 primers, which respectively recognize six to eight regions on the target gene, making the LAMP reaction have higher specificity. Since a large amount of by-product - white magnesium pyrophosphate precipitate will be produced in the LAMP reaction, the amplification products can be judged directly by naked eye observation or turbidimeter without electrophoresis analysis. In the present invention, a fluorescent dye is added to the amplification system, and the amplification situation is observed more intuitively through a fluorescence isothermal amplification instrument or a fluorescence PCR instrument, and the results are accurately judged.

[0010] Therefore, the design of LAMP primers is the key to accurately detecting Campylobacter jejuni and Campylobacter coli. Generally, the LAMP primers are determined by the following method: screening the target gene sequence to be detected; performing Blast comparison on the similarity of the target gene sequence; designing multiple pairs of primers for six regions of the target gene (F3c region, F2c region and F1c at the 3' end of the target gene, and B1 region, B2 region and B3 region at the 5' end of the target gene) using online software; and screening to obtain the best 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 application 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; 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 object of the present invention is achieved by the following technical solutions: In the first aspect, the present invention provides a set of loop-mediated isothermal amplification primer compositions, characterized in that the primer compositions include 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 the primers shown in SEQ ID NO: 1 - SEQ ID NO: 4; the LAMP primer set for detecting Campylobacter coli consists of the primers shown in SEQ ID NO: 5 - SEQ ID NO: 8.

[0013] In a specific embodiment of the present invention, the LAMP primer set for detecting Campylobacter jejuni is as follows: 。

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

[0015] In the second aspect, the present invention provides an application of the loop-mediated isothermal amplification primer composition described in the first aspect of the present invention in at least one of the following: 1) Application in the preparation of products for detecting and / or assisting in the detection of Campylobacter; 2) Application in the preparation of drugs for screening or assisting in screening for the treatment of Campylobacter-related diseases 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.

[0016] The products include but are not limited to reagents, kits, test papers, membrane strips, chips or detection platforms.

[0017] In the third aspect, the present invention provides a kit, characterized in that the kit includes the loop-mediated isothermal amplification primer composition described in the first aspect of the present invention.

[0018] The kit is a rapid detection product for detecting Campylobacter jejuni and Campylobacter coli based on loop-mediated isothermal amplification technology, which 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.

[0019] In some embodiments of the present invention, the other conventional reagents include Bst enzyme, reverse transcriptase, dNTPs, ultrapure water, buffer, and magnesium ions.

[0020] In some embodiments of the present invention, the kit also includes sample lysate, sample diluent, and sample DNA extraction reagent / kits, which can be purchased through commercial channels or prepared by oneself.

[0021] In a specific embodiment of the present invention, the sample lysate contains a surfactant, which 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 component of the surfactant in the sample lysate is between 0.2% and 4.3%.

[0022] In order to efficiently and accurately read the amplification results, the kit also includes a fluorescent dye, which is a dye with a fluorescent group commonly used by those skilled in the art, and the fluorescence excitation wavelength and emission wavelength are both between 485 - 530 nm. In a specific embodiment, the fluorescent dye is Eva Green.

[0023] Preferably, the loop-mediated isothermal amplification primer composition, fluorescent dye, and other conventional reagents in the kit are independently packaged.

[0024] In a preferred embodiment of the present invention, 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. Specifically, the freeze-dried beads are prepared by the following method: S1) Prepare a buffer containing Bst polymerase, reverse transcriptase, fluorescent dye, and dNTPs, and add the loop-mediated isothermal amplification primer composition to form a LAMP amplification reaction solution; S2) Drop the LAMP amplification reaction solution into liquid nitrogen to form spheres; S3) Place it in a freeze-drying device and freeze-dry overnight to form freeze-dried beads.

[0025] In the LAMP amplification reaction solution described in step S1), primer P k1 , P k2 , P j1 , P j2The final concentration is 0.1 - 0.2 μM, and primers P k3 , P k4 , P j3 , P j4 The final concentration is 0.3 - 1.0 μM.

[0026] Furthermore, the kit further includes a positive control, which is a plasmid containing the gene of Campylobacter jejuni or Campylobacter coli.

[0027] Furthermore, the kit further includes a negative control, which is pure water treated with diethyl pyrocarbonate (DEPC).

[0028] 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 regions, and each reaction detection region includes a sample injection pool, a distribution pool, a capillary microvalve, and an amplification pool that are connected in sequence. The amplification pool is coated with the loop-mediated isothermal amplification primer composition described in the first aspect of the present invention.

[0029] 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.

[0030] Those skilled in the art know that the sample injection pool is used to load the reaction solution, the distribution pool is used to evenly distribute the reaction solution into the amplification pool, the amplification pool is used to perform loop-mediated isothermal amplification reaction (LAMP), and the capillary microvalve controls the movement of the fluid in the microfluidic chip by utilizing the blocking effect of the capillary on the liquid.

[0031] 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 for the purpose of disease treatment and diagnosis, characterized in that the method includes the following steps: S1: Extract the genomic DNA or RNA of the sample to be tested, reverse transcribe the RNA into cDNA, and combine the DNA and cDNA as the sample DNA; S2: Add the sample DNA into the loop-mediated isothermal amplification reaction solution, and amplify at 65 °C for 20 - 25 min; S3: Read the fluorescence value every 20 - 30 s, and judge the result through the amplification curve.

[0032] The loop-mediated isothermal amplification reaction solution described 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.

[0033] The sample to be tested is selected from all samples that may contain Campylobacter jejuni and / or Campylobacter coli and need to be detected, including but not limited to chicken, pork, and feces.

[0034] The technical solution provided by the present invention has the following beneficial technical effects: The kit or microfluidic chip for detecting Campylobacter jejuni and Campylobacter coli based on the 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 can be completed and the detection results can be read within 20 minutes according to the detection method provided by the present invention. The detection time is significantly shorter than the currently commonly used real-time fluorescence quantitative PCR method, 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 investigations and epidemic monitoring to study the prevalence of pathogenic Campylobacter jejuni and Campylobacter coli. Description of the Drawings

[0035] Figure 1 Fluorescence amplification curve of positive samples in the kit for detecting Campylobacter jejuni and Campylobacter coli; Figure 2 Gradient amplification curve of Campylobacter jejuni quality control product; Figure 3 Gradient amplification curve of Campylobacter coli quality control product; Figure 4 Specific detection result of Campylobacter jejuni; Figure 5 Specific detection result of Campylobacter coli; Figure 6 Amplification curve of Campylobacter coli sample by primer set (Campylobacter coli - 2); Figure 7 Amplification curve of Campylobacter jejuni sample by primer set (Campylobacter coli - 3). Detailed Embodiments

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only partial embodiments of the present invention, not all of them. All other embodiments obtained by those of ordinary skill in the art without making creative efforts based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0037] Example 1 Design of LAMP Primer Composition Determine the target gene sequence to be detected according to the genomes of Campylobacter jejuni and Campylobacter coli in the database, perform Blast comparison on the similarity of the target gene sequences to ensure that the similarity of the selected target genes within the species is above 95%, with high conservation, and can be used for the detection of mutant strains, and screen out the best 1-2 target genes; then use the online tool PrimerExplore V4 for loop-mediated isothermal amplification (LAMP) primer design to design multiple pairs of LAMP primer sets for 6 different regions of the selected target genes (i.e., the F3c region, F2c region, and F1c at the 3' end of the target gene, and the B1 region, B2 region, and B3 region at the 5' end of the target gene); select 2-3 groups of LAMP primers with the best theoretical parameters and the highest success rate for experimental verification.

[0038] According to the above method, the best LAMP primer set for detecting Campylobacter jejuni screened by the present invention is composed of the sequences shown in SEQ ID NO: 1-4; the best LAMP primer set for detecting Campylobacter coli screened by the present invention is composed of the sequences shown in SEQ ID NO: 5-8.

[0039] Table 1 LAMP primer composition 。

[0040] Example 2 Kit for detecting Campylobacter jejuni and Campylobacter coli based on LAMP technology The kit includes the following parts: (1) Lyophilized beads; (2) Sample lysis solution, sample dilution solution, negative control product, positive control product; (3) A packaging box for separating and centrally packaging these reagent bottles or tubes.

[0041] The lyophilized beads are prepared by the following method: S1) Prepare a buffer solution containing Bst polymerase, reverse transcriptase, fluorescent dye, and dNTPs, and add the loop-mediated isothermal amplification primer composition to form a LAMP amplification reaction solution; in the LAMP amplification reaction solution, the final concentrations of primers P k1 、P k2 、P j1 、P j2 are 0.1-0.2 μM, and the final concentrations of primers P k3 、P k4 、P j3 、P j4 are 0.3-1.0 μM; S2) Drop the LAMP amplification reaction solution into liquid nitrogen to form spheres; S3) Place it in a freeze-drying device and freeze-dry overnight to form lyophilized beads.

[0042] Example 3 Method for Detecting Campylobacter jejuni and Campylobacter coli Based on LAMP Technology The detection operation steps are as follows: S1: Use a fecal swab as the specimen to be tested. Put the sampled fecal swab into a dry special small box, and it can be directly sent for inspection or stored in a 4°C refrigerator for 7 natural days; S2: Put the swab into a plastic tube containing sample lysis solution, break the swab, cover the tube cap, and shake it strongly with an oscillator for 20 seconds; S3: Take out one drop and add it to a plastic tube containing dilution solution, and shake it for 20 seconds to mix evenly; S4: Take out one drop from the dilution solution or pipette 25 μl, add it to the reaction tube, and mix it with the freeze-dried beads (prepared in Example 2) in the tube. For negative and positive quality control products, directly pipette 25 μl and add it; S5: Place the reaction tube on an oscillator and shake it for 10 seconds, then perform instantaneous centrifugation and put it into a dedicated fluorescence device for amplification (fluorescence thermostatic amplifier or fluorescence PCR instrument); S6: Program setting: Read FAM fluorescence at 65°C for 30 seconds, 40 cycles, with a total duration of 20 minutes; S7: After the amplification is completed, carefully take out the reaction tube, wrap it with gloves, tie a knot and discard it to avoid aerosol contamination; S8: Result determination: 1) If there is no S-shaped curve in the amplification, it is determined that the sample is negative; 2) If the amplification curve is significantly S-shaped, it is determined as a positive sample, as Figure 1 shown.

[0043] Example 4 Sensitivity Detection Take a certain amount of plasmid containing the gene fragments of Campylobacter jejuni and Campylobacter coli and dissolve it in an appropriate amount of ultrapure water. By measuring the OD value, calculate its concentration using the formula OD260×50 μg / ml, and calculate the number of copies per milliliter according to the molar mass of the plasmid. Then dilute it successively with ultrapure water to 3×10 3 copies / ml, 1×10 3 copies / ml, 3×10 2 copies / ml, 1×10 2 copies / ml.

[0044] In the reaction tube, add 25 μl of plasmid templates at various gradients, with three replicates for each gradient. Together with the negative control, use a temperature control program of 65°C for 30 seconds (read FAM fluorescence), 40 cycles, and conduct the experiment on a Hongshi SLAN-96S fluorescence quantitative PCR instrument.

[0045] The experimental results are as Figure 2 、 Figure 3As shown, the FAM channel has three replicates, and it can be seen that 3×10 3 copies / ml, 1×10 3 copies / ml, 3×10 2 copies / ml, 1×10 2 copies / ml. All replicates at these four concentrations were positive, while the negative control showed negative. This fully demonstrates that when using the LAMP method to detect Campylobacter jejuni and Campylobacter coli, the sensitivity can reach 1×10 2 copies / ml.

[0046] Example 5 Specificity Detection Add 25 μl of DNA extracted from Campylobacter fetus ( Campylobacter foetus ), Campylobacter concisus ( Campylobacter concisus ), Campylobacter salivarius ( Campylobacter sputorum ), as well as Campylobacter jejuni and Campylobacter coli to the reaction tube containing the freeze-dried beads for detecting the gene of Campylobacter jejuni. Only the DNA of Campylobacter jejuni was amplified, showing a positive curve ( Figure 4 ); and when they were added to the reaction tube containing the freeze-dried beads for detecting the gene of Campylobacter coli, only the DNA of Campylobacter coli was amplified, and the DNA of the other four Campylobacter species showed negative ( Figure 5 ), indicating that the specificity of the kit is consistent with the expectation.

[0047] Examples of Screening LAMP Primer Sets In order to amplify the target gene locus quickly and accurately, the present invention uses primer design software to design LAMP primers. However, the preliminary experimental results show that the primers designed by the software can theoretically amplify the target gene, but may not necessarily meet the requirements of the present invention in terms of specificity and sensitivity. Therefore, we not only performed base substitutions on the sequences of the paired two primers, but also repeatedly tested the amplification effects of different primer pairs to obtain better specificity and sensitivity. Taking Campylobacter coli as an example, the primer screening process is briefly described below.

[0048] For Campylobacter coli, the present invention selects the GlyA gene with strong specificity as the target gene and designs LAMP primers through an online LAMP primer design software. In order to increase the success probability of amplification, the present invention selects three sets of primers with the best comprehensive parameters. One set of primers is shown in Table 1, and the information of the other two sets of primers is shown in Table 2: Table 2 LAMP Primer Sets for Campylobacter coli .

[0049] Although the primer sets with the best theoretical parameters and the highest success rate were used, the amplification results were still not ideal. The primer set (Campylobacter coli - 2) had 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 significant effect on Campylobacter jejuni ( Campylobacter jejuni ) showed a significant increase ( Figure 7 ), indicating that the specificity of the primer set designed by the software is poor, and the purpose of detecting genes in the present invention cannot be achieved. The technical personnel of the present invention analyzed that the reason for the above-mentioned non-specific amplification is that Campylobacter coli and Campylobacter jejuni are very close in affinity. Whole genome comparison shows that the whole genomes of Campylobacter jejuni and Campylobacter coli are both 1.6-1.7Mb, the nucleotide homology (ANI) is about 93%-95%, and the differential genes only account for 5% of the total genome. They are very similar sister species of the same genus in evolution, so the detection specificity needs to be specially optimized in primer design. The above results also confirm that not all primers designed by the software can be used, and LAMP primer sets 2 and 3 have defects in sensitivity or specificity. The present invention finally obtained the primer set shown in SEQ ID NO: 5-8 in Table 1 as the best LAMP primer for detecting Campylobacter coli through repeated testing and adjustment.

[0050] 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 the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Loop-mediated isothermal amplification primer composition, characterized in that, The primer composition includes 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 - SEQ ID NO: 4; the LAMP primer set for detecting Campylobacter coli consists of primers shown in SEQ ID NO: 5 - SEQ ID NO:

8.

2. Application of the loop-mediated isothermal amplification primer composition according to claim 1 in at least one of the following: 1) Application in the preparation of products for detecting and / or assisting in the detection of Campylobacter; 2) Application in the preparation of drugs for screening or assisting in screening drugs for treating Campylobacter-related diseases; The Campylobacter refers to Campylobacter jejuni and Campylobacter coli, and the Campylobacter-related diseases refer to diseases caused by Campylobacter jejuni and Campylobacter coli, including diarrhea and gastroenteritis.

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

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

5. The kit according to claim 4, characterized in that, The kit further includes other conventional reagents required for the 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, wherein, The kit also includes a fluorescent dye; The loop-mediated isothermal amplification primer composition, fluorescent dye and other conventional reagents in the kit are separately and independently packaged; 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 are prepared by the following method: S1) Prepare a buffer containing Bst polymerase, reverse transcriptase, fluorescent dye and dNTPs, and add the loop-mediated isothermal amplification primer composition to form a LAMP amplification reaction solution; S2) Drop the LAMP amplification reaction solution into liquid nitrogen to form spheres; S3) Place it in a freeze-drying device 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 the gene of Campylobacter jejuni or Campylobacter coli; the negative control is pure water treated with diethyl pyrocarbonate.

9. A chip, including a microfluidic chip, characterized in that, The microfluidic chip consists of 2 - 4 reaction detection zones, and each reaction detection zone includes a sample 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.

10. A method for detecting Campylobacter jejuni and Campylobacter coli based on loop-mediated isothermal amplification technology without the purpose of disease treatment and diagnosis, characterized in that, The method includes the following steps: S1: Extract genomic DNA or RNA from the sample to be tested, reverse transcribe RNA into cDNA, and combine DNA and cDNA as sample DNA; S2: Add the sample DNA to the loop-mediated isothermal amplification reaction solution and amplify at 65°C for 20 - 25 min; S3: Read the fluorescence value every 20 - 30 s and judge the result through the amplification curve.

Images