Primer probe combination, kit and use for detecting foodborne Arcobacter

Abstract

The present invention discloses a primer-probe combination, a kit and a use for detecting foodborne Arcobacter, and belongs to the technical field of pathogenic Arcobacter detection. The present invention provides a triple amplification system based on fluorescent quantitative PCR, and screens a primer-probe combination with good specificity and high sensitivity for detecting Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii, while reducing the operation steps and the required reagents, improving the detection efficiency and economy, and providing a powerful tool for the rapid diagnosis and prevention of foodborne Arcobacter-related diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of pathogenic Arcobacter detection, and particularly relates to a primer-probe combination, a kit and uses for detecting foodborne Arcobacter. Background Art

[0002] Foodborne diseases are one of the major challenges facing global public health. These diseases are usually caused by ingesting food or water contaminated by pathogens. With the diversification of human dietary sources and methods, food safety issues caused by foodborne parasitic diseases are becoming increasingly prominent.

[0003] Arcobacter is a Gram-negative short rod, which is considered to be an emerging enteric pathogen and zoonosis, and has gradually attracted the attention of researchers. The size of Arcobacter is (0.2-0.9)×(0.5-3)μm. It is a Gram-negative, non-spore, non-capsulated curved short rod, usually S-shaped or spiral-shaped, and can reach 20μm in length. Arcobacter was formerly known as Campylobacter-like. It is a new Gram-negative pathogen that is similar in morphology to Campylobacter and is closely related. At present, 11 species of Arcobacter have been found, the most common of which are Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii, which are closely related to human and animal diseases. Arcobacter is widely distributed in nature. Its pathogenicity is comparable to that of Campylobacter, and it is easier to survive than Campylobacter, which has attracted the attention of many researchers.

[0004] Studies have found that the most important source of human infection of Arcobacter is food. The bacteria can be detected in products such as cattle, pigs, turkeys, and geese, among which poultry is considered to be the largest reservoir. In addition, the bacteria are also present in large quantities in various water bodies, including drinking water, lake water, surface water, groundwater, sewage, etc. Arcobacter can enter the human large intestine through water bodies, causing a series of intestinal diseases and endangering the health of humans and animals. The results of the study showed that Arcobacter is the dominant bacterial group in urban sewage and activated sludge in urban sewage chemical biological flocculation tanks, and is pathogenic.

[0005] The main symptoms of Arcobacter infection are persistent diarrhea, accompanied by abdominal pain, stomach cramps, nausea, vomiting, and fever. Eating contaminated food or drinking contaminated water is the most common way for people to be infected with Arcobacter. Therefore, early detection or rapid and accurate treatment through detection after infection is an effective measure to prevent or respond to Arcobacter infection incidents.

[0006] Traditional detection technology for Arcobacter is mainly isolation and culture. The isolation and culture method needs to go through the identification process of bacterial enrichment culture, purification identification, biochemical detection, etc., which has the problems of cumbersome operation, long time consumption and low sensitivity. With the continuous development of molecular biology technology, more and more rapid and specific detection methods have emerged, and its main representative is polymerase chain reaction (PCR). However, ordinary PCR detection technology requires agarose gel electrophoresis to determine the size of the amplified product, which is time-consuming and easy to damage the health of the detection personnel. Fluorescence quantitative PCR (quantitative PCR, qPCR) is one of the widely used molecular diagnostic tools. The method usually uses fluorescent dyes to mark and track PCR products, and monitors the reaction process online in real time. Not only is the detection method simple, but also the cost is low.

[0007] Based on the above background, the present invention provides a triple amplification system based on fluorescent quantitative PCR, which screens out primer-probe combinations with good specificity and high sensitivity for the detection of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii, while reducing the number of operating steps and required reagents, while improving the detection efficiency and economy, providing a powerful tool for the rapid diagnosis and prevention of foodborne Arcobacter-related diseases. Summary of the invention

[0008] The first object of the present invention is to provide a primer-probe combination for detecting foodborne Arcobacter based on fluorescent quantitative PCR, wherein the primers and probes can realize specific amplification and detection of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii in the same system; the second object of the present invention is to provide a use of the primer-probe combination in the preparation of a product for detecting foodborne Arcobacter, specifically, the present invention provides a detection kit, wherein the kit comprises the primer-probe combination as described above; another object of the present invention is to provide a detection method for foodborne Arcobacter.

[0009] The present invention includes the following technical solutions:

[0010] In a first aspect, the present invention provides a primer-probe combination for detecting foodborne Arcobacter, characterized in that the primer-probe combination consists of upstream and downstream primers and probes shown in 1)-3):

[0011] 1) The upstream and downstream primers for detecting Arcobacter brucei are shown in SEQ ID NO: 10 and SEQ ID NO: 11, and the probe is shown in SEQ ID NO: 12;

[0012] 2) The upstream and downstream primers for detecting psychrophilic Arcobacter are shown in SEQ ID NO: 13 and SEQ ID NO: 14, and the probe is shown in SEQ ID NO: 15;

[0013] 3) The upstream and downstream primers for detecting Arcobacter skrjavinii are shown in SEQ ID NO: 16 and SEQ ID NO: 17, and the probe is shown in SEQ ID NO: 18;

[0014] The Arcobacter described in the present invention is a combination of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii.

[0015] Furthermore, the 5' end of the probe is labeled with a fluorescent reporter group, and the fluorescent reporter signals of the probes do not interfere with each other; the 3' end of the probe is labeled with a fluorescent quencher group.

[0016] The "signals do not interfere with each other" described in the present invention means that the fluorescent groups used in the above-mentioned probes are different and will not affect each other's detection, that is, different channels can be used for detection. For example, FAM, HEX, ROX, and Cy5 can be used. The absorbance values ​​of these groups are not close, and different channels can be selected. The signals of different channels will not interfere with each other; in addition, the three signals of each fluorescent channel can be distinguished by adding different probe concentrations to distinguish the fluorescence values, thereby forming different clustering signals, and further distinguished by clustering analysis software, without mutual interference.

[0017] In a specific embodiment of the present invention, the fluorescent reporter groups labeled at the 5' end of the probes for detecting the three foodborne Arcobacter species are selected from FAM, HEX and ROX, respectively.

[0018] In a second aspect, the present invention provides a use of the primer-probe combination described in the first aspect of the present invention in preparing a product for detecting foodborne Arcobacter.

[0019] The products for detecting foodborne Arcobacter include but are not limited to reagents, test kits, chips, test papers, membrane strips or detection platforms.

[0020] In a third aspect, the present invention provides a kit for detecting foodborne Arcobacter, the kit comprising the primer-probe combination described in the first aspect of the present invention.

[0021] Furthermore, the kit also includes an enzyme mixture, a negative quality control product, a positive quality control product, and a PCR buffer.

[0022] The enzyme mixture contains at least one of high-fidelity Taq enzyme, UDG enzyme, magnesium ions and dNTPs.

[0023] The negative quality control product of the present invention is pure water treated with diethyl pyrocarbonate (DEPC).

[0024] The positive quality control product described in the present invention is a plasmid containing the specific sequences of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii described in the present invention, and the specific sequences are known to the technicians of the present invention.

[0025] In a fourth aspect, the present invention provides a method for detecting foodborne Arcobacter that is not intended for disease diagnosis and treatment, the method comprising the following steps:

[0026] (1) Extracting DNA or RNA from the sample to be tested;

[0027] (2) configuring a PCR amplification system containing the primer-probe combination described in the first aspect of the present invention to carry out a PCR amplification reaction;

[0028] (3) Determine whether the corresponding foodborne Arcobacter exists in the sample to be tested and the initial concentration of the foodborne Arcobacter according to the amplification curve.

[0029] In the amplification system described in step (2), the final concentration of the upstream primer is 0.1-0.2 μM, the final concentration of the downstream primer is 0.2-0.3 μM, and the final concentration of the probe is 0.1-0.15 μM.

[0030] The sample to be tested described in the present invention is any substance that may contain foodborne Arcobacter, including but not limited to food, feces, oral swabs, and anal swabs.

[0031] The primer-probe combination provided by the present invention is based on real-time fluorescence quantitative PCR technology, and the PCR product amplification amount is detected in real time by detecting the strength of the fluorescent signal, and Arcobacter, such as Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii, are detected and quantitatively analyzed simultaneously, and the detection sensitivity is high. Further, the present invention first eliminates the primer group that nonspecific amplification and primer dimer appear in the triple PCR system, and then optimizes the primers with low sensitivity, and finally screens to select primer pairs and probes with good specificity and high sensitivity. The primer and probe combination provided by the present invention can be further prepared into a detection kit for real-time and rapid detection of foodborne Arcobacter. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Amplification plot showing non-specific amplification with the initial primers.

[0033] Figure 2 Amplification curves with lower sensitivity appeared during the optimization process.

[0034] Figure 3 Amplification curves from stool sample experiments.

[0035] Figure 4 In the specific detection experiment, only the amplification curve of the Arcobacter brucei gene template was added.

[0036] Figure 5 In the specific detection experiment, only the amplification curve of the psychrophilic Arcobacter gene template was added.

[0037] Figure 6 In the specific detection experiment, only the amplification curve of the Arcobacter stuartii gene template was added.

[0038] Figure 7 In the specific detection experiment, only the amplification curve of Campylobacter jejuni gene template was added.

[0039] Figure 8 Figure 1. Results of sensitivity test for Arcobacter brucei.

[0040] Fig. 9 Results of sensitivity test on psychrophilic Arcobacter.

[0041] Fig.10 Figure 1. Results of sensitivity test for Arcobacter stuartii. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Example 1 Design and optimization process of primers and probes

[0044] The design and optimization process of the primer-probe combination involved in the present invention is as follows:

[0045] First, according to the prior art, the three foodborne Arcobacter genes or DNA fragments described in the present invention are obtained as candidate amplification genes; multiple pairs of spare primers are designed for all candidate amplification genes or DNA fragments using premier6 software; all primer schemes are subjected to Blast analysis in Genbank to obtain primer schemes with higher specificity as experimental verification schemes. The present invention selects multiple candidate amplification genes for each Arcobacter, and then designs multiple pairs of spare primers for each candidate amplification gene. After Blast analysis, 1-2 experimental verification schemes appear for one candidate amplification gene of each Arcobacter). Then, each experimental verification scheme is verified by single-plex PCR to determine the availability of primers, including specificity evaluation and sensitivity evaluation, and the spare primers with poor effects are eliminated. The specific process is as follows:

[0046] (1) The nucleotide information of the primer probe initially designed by the present invention is shown in the following table:

[0047] Table 1 Primer-probe combinations for detecting foodborne Arcobacter

[0048]

[0049]

[0050] Since the three Arcobacter genes described in the present invention are very similar, it is not easy to select specific regions, which often leads to poor specificity in the design of multiple primers. The prior art usually chooses to use 16S rRNA or 23S rRNA gene sequences for species identification, but considering that their homology is higher than 96%, it is extremely difficult to design, and the subsequent experimental system is also difficult to construct. When performing the initial primer design, the present invention breaks through the conventional design ideas, that is, selects gene sequences other than 16S rRNA and 23S rRNA genes as candidate amplification genes, and designs the primers and probes shown in Table 1. Even so, the primers initially designed by the present invention still have the problem of non-specific amplification, as shown in the following example. Figure 1As shown, when only the gene template of Arcobacter brucei is added, the amplification of Arcobacter psychrophilus will be brought about. At this time, the amplification curves for the gene templates of Arcobacter psychrophilus and Arcobacter stuartii are normal.

[0051] (2) The technicians of the present invention screened and optimized the amplification primers and probes for Arcobacter brucei and Arcobacter psychrophilus in Table 1. After many attempts, the present invention obtained the primer-probe combination described in Table 2.

[0052] Table 2 Primer-probe combinations for detecting foodborne Arcobacter

[0053]

[0054]

[0055] The specific amplification was verified by adding gene templates of Arcobacter brucei, Arcobacter psychrophilus, and Arcobacter stuartii. The results showed that the primer-probe combination provided in Table 2 could specifically amplify Arcobacter brucei, Arcobacter psychrophilus, and Arcobacter stuartii. However, when the copy number of the gene template was as low as 2×10 2 When the number of copies / ml was 1.5, the number of Arcobacter stuartii did not show an S-shaped curve ( Figure 2 ), indicating that the primers have poor amplification sensitivity for Arcobacter skrjavinii.

[0056] (3) The technicians of the present invention continued to design, screen and optimize the primers and probes for Arcobacter skrjavinii. Finally, the present invention preferably obtained the primers and probes described in Table 3, and the nucleotide information thereof is shown in the following table:

[0057] Table 3 Primer probe combinations for detecting foodborne Arcobacter

[0058]

[0059] (4) The validated primers in Table 3 were mixed for multiplex PCR to verify the usability of the primers during co-amplification. The results showed that the primer-probe combination provided in Table 3 would not form primer dimers. Therefore, the primer-probe combination shown in Table 3 is the best primer-probe combination suitable for the simultaneous detection of Arcobacter brucei, Arcobacter psychrophilus, and Arcobacter sternbergi.

[0060] In the above table, "F" represents the upstream primer; "R" represents the downstream primer; and "P" represents the probe.

[0061] Furthermore, the 5' end of the probe in the above table is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a fluorescent quencher group. Specifically,

[0062] The probes for detecting Arcobacter brucei are:

[0063] 5'-FAM-CTGGTGATAAAATCTTAGGTATGG-BHQ1-3';

[0064] The probes for detecting psychrophilic Arcobacter are:

[0065] 5'-HEX-TGCCTTGCTTGGCTGCTTCTATGGTATT-BHQ1-3';

[0066] Probes for detecting Arcobacter stetzeri:

[0067] 5'-ROX-TTGTATTGTGAGCATAAACTGTTTTT-BHQ2-3';

[0068] Example 2 Kit for detecting foodborne Arcobacter

[0069] The present invention provides a kit as shown in the following table:

[0070] Table 4 Kit ingredients

[0071]

[0072] In the above kit, the final concentration of the upstream primer in the primer-probe mixture is selected from 0.1-0.2 μM, the final concentration of the downstream primer is selected from 0.2-0.3 μM, and the final concentration of the probe is selected from 0.1-0.15 μM. Those skilled in the art can prepare the enzyme mixture by conventional techniques or purchase it through commercial channels.

[0073] Example 3 Detection method of foodborne Arcobacter

[0074] (1) Obtaining samples for testing

[0075] (2) Extraction of DNA from samples to be tested

[0076] The genomic DNA in the sample to be tested is extracted using a commercially available DNA extraction kit or a method well known to those skilled in the art.

[0077] (3) PCR amplification reaction

[0078] a) mixing the primer-probe mixture and the enzyme mixture in the kit provided in Example 2 at a volume ratio of 1:5 to form a PCR amplification system, and dispensing into reaction tubes, wherein the final concentration of the upstream primer is 0.1 μM, the final concentration of the downstream primer is 0.2 μM, and the final concentration of the probe is 0.1 μM;

[0079] b) Add the nucleic acid extract of the sample to be tested (or negative or positive quality control product) to the reaction tube, cover the tube tightly, centrifuge it momentarily, put it into the thermal amplification instrument, perform PCR amplification reaction according to the amplification program shown in the table below, and read the fluorescence in the corresponding channels of FAM, HEX, and ROX respectively.

[0080] Table 5 PCR amplification program

[0081]

[0082] (4) Result determination

[0083] If there is no S-shaped curve in the amplification, the sample is judged to be negative;

[0084] If the amplification curve shows an obvious S-shape, it is judged as a positive sample.

[0085] The positive and negative quality control products provided in the kit are used for quality control in clinical trials, and the operation method is the same as that of the samples to be tested. The quality control standards are as follows:

[0086] The amplification curve of the negative control product has no obvious S-shaped growth; the amplification curve of the positive control product is S-shaped;

[0087] The above requirements must be met simultaneously in the same experiment, otherwise, the experiment will be considered invalid and must be repeated.

[0088] Example 4 Detection of fecal samples

[0089] (1) Sample collection, transportation and storage

[0090] Place the stool sample to be tested in a special dry small box. The storage period should not exceed one month. If necessary, transport it at room temperature.

[0091] (2) Sample processing

[0092] a) Take an appropriate amount of sample and place it in a centrifuge tube containing 300 μl Trizol and 100 μl chloroform. Fold the tube cap, shake vigorously for 20 seconds, let it stand for 3 minutes, and then centrifuge at 12,000 rpm for 2 minutes;

[0093] b) Carefully remove the colorless upper layer of liquid (be careful not to touch the middle flocculent layer), transfer it to a newly sterilized 1.5 ml centrifuge tube, then add 10 μl of magnetic beads (mix thoroughly and then aspirate), mix thoroughly with an oscillator, centrifuge at 8,000 rpm for 1 minute, and carefully discard all the liquid;

[0094] c) Add 400 μl of 75% DEPC ethanol, shake thoroughly to mix, centrifuge at 8,000 rpm for 1 minute, and drain as much liquid as possible (use a pipette to drain the residual liquid to avoid touching the precipitated particles);

[0095] d) Place the centrifuge tube containing the precipitate in a fume hood to air-dry for 15 minutes (you can also use an open heater to dry at 60°C for 5 minutes to prevent cross-contamination of samples), then add 30μl of nuclease-free H2O, fully resuspend with an appropriate pipette, let it stand for 1 minute, then centrifuge at 8,000rpm for 1 minute. The processed samples can be directly used for PCR detection or temporarily stored at -20°C. If stored for more than one month, they can be stored at -70°C.

[0096] The PCR amplification reaction was carried out using the method provided in Example 3, and the detection results were as follows: Figure 3 As shown in the figure, it can be seen that the three types of foodborne Arcobacter in the fecal samples detected in this embodiment are all positive, and in the same detection system, the primer probe combination provided by the present invention can achieve good specificity of common amplification without mutual interference.

[0097] Example 5 Kit-Specific Detection

[0098] Only Arcobacter brucei gene template was added to the PCR amplification system, and the other Arcobacter templates were not added. PCR amplification was performed according to the method provided in Example 3. The results are as follows Figure 4 As shown, it can be seen that only the FAM channel shows an S-shaped amplification curve in the amplification results, and no amplification occurs in other channels.

[0099] In the PCR amplification system, only the psychrophilic Arcobacter gene template was added, and the other Arcobacter templates were not added. PCR amplification was performed according to the method provided in Example 3. The results are as follows: Figure 5 As shown, it can be seen that only the HEX channel shows an S-shaped amplification curve in the amplification results, and no amplification occurs in other channels.

[0100] In the PCR amplification system, only the gene template of Arcobacter stuartii was added, and the other Arcobacter templates were not added. PCR amplification was performed according to the method provided in Example 3. The results are as follows: Figure 6 As shown, it can be seen that only the ROX channel shows an S-shaped amplification curve in the amplification results, and no amplification occurs in other channels.

[0101] The above results indicate that the kit and detection method provided by the present invention can well distinguish the three pathogenic Arcobacter described in the present invention.

[0102] Campylobacter jejuni, which is relatively close to the three Arcobacter species described in the present invention, was selected as the bacteria to be tested. The Campylobacter jejuni gene template was added to the amplification system, and PCR amplification was performed according to the method provided in Example 3. The results are as follows: Figure 7 As shown, the amplification curve has no obvious S-shaped growth, indicating that the primer-probe combination provided by the present invention has no cross reaction with Campylobacter jejuni, which is close to the genus Arcobacter, and has good specificity.

[0103] Example 6 Kit Sensitivity Detection

[0104] Take a certain amount of positive plasmids containing the three types of Arcobacter described in the present invention and dissolve them in an appropriate amount of ultrapure water. OD value is measured and calculated according to the formula OD 260 ×50μg / ml was used to calculate its concentration, and the number of copies per ml was calculated based on the molar mass of the plasmid, and then diluted with ultrapure water to 5×10 3 copies / ml, 1×10 3 copies / ml, 2×10 2 copies / ml, 5×10 1 Since each plasmid contains 1 copy of each Vibrio gene fragment, the concentration of each Arcobacter species is the same.

[0105] In the reaction tube, add 10 μl of enzyme mixture and 0.5 μl of primer probe mixture. After preliminary mixing, add 10 μl of plasmid template of various gradients respectively. Each gradient is repeated three times. The temperature control program of 50°C for 2 minutes, 95°C for 3 minutes, followed by 40 cycles (95°C for 10 seconds, 58°C for 35 seconds) is adopted. The experiment is carried out on a Macrostone SLAN-96S five-channel fluorescence quantitative PCR instrument.

[0106] The results of the sensitivity test of Arcobacter brucei are as follows Figure 8 Shown, FAM channel, three replicates, 5 × 10 1 copies / ml is negative; the sensitivity test results of psychrophilic Arcobacter are as follows Fig. 9 Shown, HEX channel, three replicates, 5 × 10 1 Copies / ml is negative; the sensitivity test results of Arcobacter stuartii are as follows Fig.10 Shown, ROX channel, three replicates, 5 × 10 1 In summary, the sensitivity of Arcobacter brucei, Arcobacter psychrophilus and Arcobacter sternii can reach 2×10 2 copies / ml.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned 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 primer-probe combination for detecting foodborne Arcobacter, characterized in that: The primer-probe combination consists of the upstream and downstream primers and probes shown in 1)-3) composition: 1) The upstream and downstream primers for detecting Arcobacter brucei are shown in SEQ ID NO: 10 and SEQ ID NO: 11, and the probe is shown in SEQ ID NO: 12; 2) The upstream and downstream primers for detecting psychrophilic Arcobacter are shown in SEQ ID NO: 13 and SEQ ID NO: 14, and the probe is shown in SEQ ID NO: 15; 3) The upstream and downstream primers for detecting Arcobacter stutzeri are shown in SEQ ID NO: 16 and SEQ ID NO: 17, and the probe is shown in SEQ ID NO: 18; The arcobacterium is Arcobacter brucei ( Arcobacter butzleri ), psychrophilic Arcobacter ( Arcobacter cryaerophilus ) and Arcobacter stutzeri ( Arcobacter skirrowii ) combination.

2. The primer-probe combination according to claim 1, characterized in that The 5' end of the probe is labeled with a fluorescent reporter group, and the fluorescent reporter signals of the probes do not interfere with each other; the 3' end of the probe is labeled with a fluorescent quencher group.

3. The primer-probe combination according to claim 2, characterized in that The fluorescent reporter groups labeled at the 5' end of the probes used to detect three foodborne Arcobacter species were selected from FAM, HEX and ROX, respectively.

4. Use of the primer-probe combination according to any one of claims 1 to 3 in preparing a product for detecting foodborne Arcobacter.

5. The use according to claim 4, characterized in that The products for detecting foodborne Arcobacter include reagents, test kits, chips, test papers, membrane strips or detection platforms.

6. A kit for detecting foodborne Arcobacter, characterized in that: The kit comprises the primer-probe combination according to any one of claims 1 to 3.

7. The kit according to claim 6, characterized in that The kit also includes an enzyme mixture, a negative quality control product, a positive quality control product, and a PCR buffer.

8. A method for detecting foodborne Arcobacter for purposes other than disease diagnosis and treatment, the method comprising the following steps: (1) Extract DNA or RNA from the sample to be tested; (2) preparing a PCR amplification system containing the primer-probe combination according to any one of claims 1 to 3 to carry out a PCR amplification reaction; (3) Determine whether the corresponding foodborne Arcobacter exists in the sample to be tested and the initial concentration of the foodborne Arcobacter based on the amplification curve.

9. The method according to claim 8, characterized in that In the amplification system, the final concentration of the upstream primer is 0.1-0.2µM, the final concentration of the downstream primer is 0.2-0.3µM, and the final concentration of the probe is 0.1-0.15µM.

10. The method according to claim 8, characterized in that The sample to be tested is any substance that may contain foodborne Arcobacter, including food and feces.

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