Holding type listeria monocytogenes specific molecular target and rapid detection method thereof

Abstract

The invention discloses a persistent listeria monocytogenes specific molecular target and a rapid detection method thereof, and belongs to the technical field of microbiological detection. The molecular target is obtained through bioinformatics analysis and screening, is further screened through specific experimental verification, and is respectively specific genome sequence fragments of six retention types of listeria monocytogenes CC5, CC8, CC31, CC87, CC121 and CC204. A corresponding specific primer group is designed based on the target, sample DNA is amplified through PCR, a product is subjected to visual analysis in combination with gel electrophoresis, and whether the target retention type strain exists or not can be judged according to the size of a band. The detection method has the advantages of being short in detection time, low in cost, simple and convenient to operate and high in specificity, results are accurate, judgment is easy, practicability is high, the limitation that traditional culture and identification periods are long, and phenotypic analysis steps are tedious is broken through, and the detection method is suitable for rapid screening of the persistent listeria monocytogenes in samples of food, environment and the like.

Description

Technical Field

[0001] This invention belongs to the field of microbial testing technology, specifically relating to specific molecular targets of persistent Listeria monocytogenes and their rapid detection methods. Background Technology

[0002] With the continuous improvement of living standards, food safety has become a focus of social concern. Among these concerns, foodborne pathogen contamination is one of the main factors causing food safety problems. Listeria monocytogenes (…) Listeria monocytogenes As an important foodborne pathogen, *Clostridium difficile* is characterized by its strong environmental adaptability and high tolerance. This bacterium can survive and reproduce in a wide temperature range of 4–45℃ and in highly acidic environments. It is a zoonotic pathogen and one of the most deadly foodborne infections, primarily transmitted to humans through contaminated food or infected animals. Human infection can cause gastroenteritis, and in severe cases, can lead to life-threatening diseases such as meningitis and sepsis. Infection in pregnant women can result in serious consequences such as miscarriage and stillbirth.

[0003] The survival of Listeria monocytogenes in the food processing environment is closely related to persistent strains. Persistent strains refer to subpopulations that survive treatment with antibiotics or other bactericidal methods. These strains exhibit high environmental tolerance and can recover and reproduce under suitable conditions, leading to recurrent infections and seriously affecting clinical treatment and prognosis. Some Listeria monocytogenes strains carry specific functional genes and phenotypic characteristics, making them highly adaptable to adverse environmental stresses and difficult to eradicate completely with conventional disinfection measures. Furthermore, persistent Listeria monocytogenes can form biofilms on the surfaces of food processing equipment, exhibiting strong resistance to adverse environments, disinfectants, and antibiotics, thereby increasing the risk of listeriosis in humans through food chain contamination. Therefore, establishing rapid and accurate detection methods for persistent Listeria monocytogenes is of great significance for ensuring food safety and public health.

[0004] Currently, the detection and typing of Listeria monocytogenes face a dual challenge. In the initial identification stage, traditional culture methods, while accurately identifying down to the species level, are time-consuming, cumbersome, and costly, making them unsuitable for rapid screening. Furthermore, these methods only provide a qualitative "presence or absence" result and cannot further identify persistent subtypes. Multilocus sequence typing (MLST) is a commonly used technique for identifying persistent subtypes of Listeria monocytogenes. Based on species classification, multiple housekeeping genes (typically seven) are amplified and sequenced. Alleles of each housekeeping gene pair are matched against a database to determine the persistent type. While this method achieves accurate typing, it is time-consuming, costly, and cumbersome, failing to meet the practical needs of enterprise internal control and regulatory enforcement. To improve detection efficiency, molecular biology detection technologies, such as PCR, have been applied in recent years due to their advantages of speed, sensitivity, and ease of operation, leading to their use in pathogen screening. However, existing research in the field of PCR detection of persistent Listeria monocytogenes still has shortcomings: on the one hand, it mainly focuses on a few persistent types such as CC8, CC121, and CC87 / CC88, while for other persistent types (such as CC5, CC31, and CC204), there is a lack of specific molecular targets, primers, and methods, forming a detection blind spot; on the other hand, existing gene sequencing methods are expensive, complex to operate, and have long detection times, resulting in low efficiency and high cost in practical applications.

[0005] In conclusion, developing a molecular detection scheme that can systematically cover multiple important persistent Listeria monocytogenes, including CC5, CC31, and CC204, and achieve rapid, simple, and highly specific identification, is of great scientific significance and urgent application value for improving the food safety monitoring system and achieving precise risk intervention. Summary of the Invention

[0006] To address the shortcomings of existing molecular detection methods for persistent Listeria monocytogenes, such as incomplete target coverage, lack of specific detection methods for CC5, CC31, and CC204 strains, and the technical problems of fragmented, costly, complex, and inefficient identification of multiple important persistent strains, this invention aims to provide a combination of specific molecular targets for persistent Listeria monocytogenes and a rapid detection method thereof.

[0007] To achieve the above objectives, the present invention employs the following technical solution: The present invention provides a molecular target combination for detecting persistent Listeria monocytogenes, wherein the nucleotide sequence of the molecular target combination is any one or a combination of SEQ ID NO.1 to SEQ ID NO.13; wherein the persistent Listeria monocytogenes is any one or a combination of CC5, CC8, CC31, CC87, CC121 and CC204.

[0008] Molecular targets with nucleotide sequences as shown in SEQ ID NO.1~SEQ ID NO.4 are used to identify persistent CC5 Listeria monocytogenes; molecular targets with nucleotide sequences as shown in SEQ ID NO.5 and SEQ ID NO.6 are used to identify persistent CC8 Listeria monocytogenes; molecular targets with nucleotide sequences as shown in SEQ ID NO.7 and SEQ ID NO.8 are used to identify persistent CC31 Listeria monocytogenes; molecular targets with nucleotide sequences as shown in SEQ ID NO.9 are used to identify persistent CC87 Listeria monocytogenes; molecular targets with nucleotide sequences as shown in SEQ ID NO.10 and SEQ ID NO.11 are used to identify persistent CC121 Listeria monocytogenes; and molecular targets with nucleotide sequences as shown in SEQ ID NO.12 and SEQ ID NO.13 are used to identify persistent CC204 Listeria monocytogenes.

[0009] The present invention provides a set of specific primers for detecting the combination of said molecular targets, the primer set comprising specific primer pairs with nucleotide sequences as shown in SEQ ID NO.14 to SEQ ID NO.41.

[0010] The primer set specifically includes: primer pairs SEQ ID NO.14 and SEQ ID NO.15 for amplifying SEQ ID NO.1; primer pairs SEQ ID NO.16 and SEQ ID NO.17 for amplifying SEQ ID NO.2; primer pairs SEQ ID NO.18 and SEQ ID NO.19 for amplifying SEQ ID NO.3; primer pairs SEQ ID NO.20 and SEQ ID NO.21 for amplifying SEQ ID NO.4; primer pairs SEQ ID NO.22 and SEQ ID NO.23 for amplifying SEQ ID NO.5; primer pairs SEQ ID NO.24 and SEQ ID NO.25 for amplifying SEQ ID NO.6; primer pairs SEQ ID NO.26 and SEQ ID NO.27 for amplifying SEQ ID NO.7; primer pairs SEQ ID NO.28 and SEQ ID NO.29 for amplifying SEQ ID NO.8; primer pairs SEQ ID NO.30 and SEQ ID NO.31 for amplifying SEQ ID NO.9; and primer pairs SEQ ID NO.10 and SEQ ID NO.15 for amplifying SEQ ID NO.10. Primers NO.32 and SEQ ID NO.33, or SEQ ID NO.34 and SEQ ID NO.35; primer pairs SEQ ID NO.36 and SEQ ID NO.37 for amplifying SEQ ID NO.11; primer pairs SEQ ID NO.38 and SEQ ID NO.39 for amplifying SEQ ID NO.12; and primer pairs SEQ ID NO.40 and SEQ ID NO.41 for amplifying SEQ ID NO.13.

[0011] The application of the molecular target combination for detecting persistent Listeria monocytogenes or the specific primer set described herein in the preparation of products for detecting persistent Listeria monocytogenes.

[0012] The product in question is a reagent kit.

[0013] This invention provides a kit for detecting persistent Listeria monocytogenes, comprising the aforementioned primer set.

[0014] The kit also contains all the necessary components for polymerase chain reaction, and each pair of primers in the primer set can be configured for singleton or multiplex PCR reaction systems.

[0015] This invention provides a method for identifying various persistent Listeria monocytogenes, comprising the following steps: (1) Using sample DNA as a template, perform PCR amplification reaction using at least one primer pair from the primer set described in claim 3 or 4; (2) Detect the amplification products. By analyzing whether there is a specific amplification band corresponding to the target persistent Listeria monocytogenes, it can be determined whether the sample contains and what kind of persistent Listeria monocytogenes it contains.

[0016] The PCR amplification reaction system includes template DNA, upstream primer, and downstream primer.

[0017] Preferably, the PCR reaction system is 20 μL, comprising: PCR reaction mix, template DNA 2 μL, and 1.0 μL each of 10 μM upstream and downstream primers.

[0018] The PCR amplification reaction program is as follows: pre-denaturation at 95℃ for 2-10 min; followed by 30-40 cycles, each cycle including denaturation at 95℃ for 15-30 s, annealing at 55-60℃ for 30-90 s, extension at 72℃ for 30-90 s; and a final extension at 72℃ for 5-10 min after the cycle.

[0019] In step (2), the specific amplification bands and their corresponding retention types are as follows: 279 bp, 364 bp, 165 bp and 243 bp bands correspond to CC5 type; 286 bp and 211 bp bands correspond to CC8 type; 184 bp and 209 bp bands correspond to CC31 type; 259 bp band corresponds to CC87 type; 156 bp, 149 bp and 118 bp bands correspond to CC121 type; and 214 bp and 212 bp bands correspond to CC204 type.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The specific molecular targets (SEQ ID NO.1 to SEQ ID NO.13) provided by this invention are highly specific and can accurately identify six persistent Listeria monocytogenes. For the first time, they systematically cover the detection gaps in the prior art (especially CC5, CC31, and CC204), providing the most core material basis for the comprehensive and accurate identification of the main persistent Listeria monocytogenes in the food processing environment.

[0021] The primer set provided by this invention transforms target combinations into operable detection tools. The 28 specific primer sequences, which are strictly matched to the targets, ensure high specificity and high success rate in subsequent PCR detection. The public disclosure of the primer sequences allows those skilled in the art to directly synthesize and use them, avoiding the complex primer design and optimization process, and lowering the technical threshold and application costs.

[0022] The detection method provided by this invention uses a pair of primers for simultaneous detection, overcoming the shortcomings of traditional culture methods and low efficiency of gene sequencing. The PCR detection method based on this target ensures specific results and easy interpretation, while significantly shortening the detection time and reducing the detection cost, making it highly practical. Attached Figure Description

[0023] Figure 1 The image shows the electrophoresis results of the specificity evaluation of the PCR detection method for persistent CC5 Listeria monocytogenes (SEQ ID NO.1) in Example 2. In the image, M is the DL2000 DNA standard marker, 1-6 are the target persistent CC5 Listeria monocytogenes, 7-58 are non-target persistent Listeria monocytogenes, 59-82 are other Listeria strains and non-Listeria strains, and NC is the negative control.

[0024] Figure 2 The image shows the electrophoresis results of the specificity evaluation of the PCR detection method for persistent CC5 Listeria monocytogenes (SEQ ID NO.2) in Example 2. In the image, M is the DL2000 DNA standard marker, 1-6 are the target persistent CC5 Listeria monocytogenes, 7-58 are non-target persistent Listeria monocytogenes, 59-82 are other Listeria strains and non-Listeria strains, and NC is the negative control.

[0025] Figure 3 The image shows the electrophoresis results of the specificity evaluation of the PCR detection method for persistent CC5 Listeria monocytogenes (SEQ ID NO.3) in Example 2. In the image, M is the DL2000 DNA standard marker, 1-6 are the target persistent CC5 Listeria monocytogenes, 7-58 are non-target persistent Listeria monocytogenes, 59-82 are other Listeria strains and non-Listeria strains, and NC is the negative control.

[0026] Figure 4 The image shows the electrophoresis results of the specificity evaluation of the PCR detection method for persistent CC5 Listeria monocytogenes (SEQ ID NO.4) in Example 2. In the image, M is the DL2000 DNA standard marker, 1-6 are the target persistent CC5 Listeria monocytogenes, 7-58 are non-target persistent Listeria monocytogenes, 59-82 are other Listeria strains and non-Listeria strains, and NC is the negative control.

[0027] Figure 5This is an electrophoresis result of the specificity evaluation of the PCR detection method for persistent CC8 Listeria monocytogenes (SEQ ID NO.5) in Example 3. In this image, M is the DL2000 DNA standard marker, 7-13 are the target persistent CC8 Listeria monocytogenes, 1-6 and 13-58 are non-target persistent Listeria monocytogenes, 59-82 are other Listeria strains and non-Listeria strains, and NC is the negative control.

[0028] Figure 6 This is an electrophoresis result of the specificity evaluation of the PCR detection method for persistent CC8 Listeria monocytogenes (SEQ ID NO. 6) in Example 3. In this diagram, M is the DL2000 DNA standard marker, 7-13 are the target persistent CC8 Listeria monocytogenes, 1-6 and 13-58 are non-target persistent Listeria monocytogenes, 59-82 are other Listeria strains and non-Listeria strains, and NC is the negative control.

[0029] Figure 7 This is an electrophoresis result of the specificity evaluation of the PCR detection method for persistent CC31 Listeria monocytogenes (SEQ ID NO. 7) in Example 4. In this diagram, M is the DL2000 DNA standard marker, 14-15 are the target persistent CC31 Listeria monocytogenes, 1-13 and 15-58 are non-target persistent Listeria monocytogenes, 59-82 are other Listeria strains and non-Listeria strains, and NC is the negative control.

[0030] Figure 8 This is an electrophoresis result of the specificity evaluation of the PCR detection method for persistent CC31 Listeria monocytogenes (SEQ ID NO. 8) in Example 4. In this image, M is the DL2000 DNA standard marker, 14-15 are the target persistent CC31 Listeria monocytogenes, 1-13 and 15-58 are non-target persistent Listeria monocytogenes, 59-82 are other Listeria strains and non-Listeria strains, and NC is the negative control.

[0031] Figure 9 This is an electrophoresis result of the specificity evaluation of the PCR detection method for persistent CC87 Listeria monocytogenes (SEQ ID NO. 9) in Example 5. In this image, M is the DL2000 DNA standard marker, 16-26 are the target persistent CC87 Listeria monocytogenes, 1-16 and 27-58 are non-target persistent Listeria monocytogenes, 59-82 are other Listeria strains and non-Listeria strains, and NC is the negative control.

[0032] Figure 10This is an electrophoresis result of the specificity evaluation of the PCR detection method for persistent Listeria monocytogenes CC121 (SEQ ID NO.10) in Example 6. In this diagram, M is the DL2000 DNA standard marker, 27-31 are the target persistent Listeria monocytogenes CC121, 1-26 and 32-58 are non-target persistent Listeria monocytogenes, 59-82 are other Listeria strains and non-Listeria strains, and NC is the negative control.

[0033] Figure 11 This is an electrophoresis result of the specificity evaluation of the PCR detection method for persistent Listeria monocytogenes CC121 (SEQ ID NO.10) in Example 6. In this diagram, M is the DL2000 DNA standard marker, 27-31 are the target persistent Listeria monocytogenes CC121, 1-26 and 32-58 are non-target persistent Listeria monocytogenes, 59-82 are other Listeria strains and non-Listeria strains, and NC is the negative control.

[0034] Figure 12 This is an electrophoresis result of the specificity evaluation of the PCR detection method for persistent Listeria monocytogenes CC121 (SEQ ID NO.11) in Example 6. In this diagram, M is the DL2000 DNA standard marker, 27-31 are the target persistent Listeria monocytogenes CC121, 1-26 and 32-58 are non-target persistent Listeria monocytogenes, 59-82 are other Listeria strains and non-Listeria strains, and NC is the negative control.

[0035] Figure 13 This is an electrophoresis result of the specificity evaluation of the PCR detection method for persistent CC204 Listeria monocytogenes (SEQ ID NO.12) in Example 7. In this diagram, M is the DL2000 DNA standard marker, 32 is the target persistent CC204 Listeria monocytogenes, 1-31 and 33-58 are non-target persistent Listeria monocytogenes, 59-82 are other Listeria strains and non-Listeria strains, and NC is the negative control.

[0036] Figure 14 This is an electrophoresis result of the specificity evaluation of the PCR detection method for persistent CC204 Listeria monocytogenes (SEQ ID NO.13) in Example 7. In the figure, M is the DL2000 DNA standard marker, 32 is the target persistent CC204 Listeria monocytogenes, 1-31 and 33-58 are non-target persistent Listeria monocytogenes, 59-82 are other Listeria strains and non-Listeria strains, and NC is the negative control. Detailed Implementation

[0037] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0038] It should be noted that the terms "first," "second," etc., used in the specification and claims are only used to distinguish similar objects and do not necessarily describe a specific order or sequence; it should be understood that these terms can be used interchangeably where appropriate. Furthermore, the terms "comprising," "having," and any variations thereof are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to the expressly listed steps or units, but may include other steps or units not expressly listed or inherent to it.

[0039] From June 2011 to July 2016, a total of 4,300 food samples, including ready-to-eat foods, aquatic products, edible fungi, frozen foods, vegetables, meat products, and dairy products, were systematically collected from supermarkets and farmers' markets in 43 representative cities / regions. All samples were strictly isolated and identified as Listeria monocytogenes according to national standards (GB 4789.30-2010 / 2016) and ultimately stored in glycerol at -80°C. The persistent strains CC5, CC8, CC31, CC87, CC121, and CC204 used for target screening and validation in this invention were obtained through molecular typing and screening from this strain library, which has broad geographical and food category representativeness. All validation strains used in this embodiment were purchased from Guangdong Huankai Microbial Technology Co., Ltd. or Guangdong Provincial Microbial Culture Collection Center (GDMCC).

[0040] Example 1 This embodiment provides a method for discovering novel molecular targets specific to persistent Listeria monocytogenes. The specific steps are as follows: Genome sequences were downloaded from the GenBank database of NCBI (https: / / www.ncbi.nlm.nih.gov / genome) (up to January 2026). A total of 1921 persistent Listeria monocytogenes strain genome sequences and different persistent Listeria monocytogenes whole genome DNA sequences (all whole genome sequences have been uploaded to the "Foodborne Listeria Monocytogenes Whole Genome Database" of the Institute of Microbiology, Guangdong Academy of Sciences) were downloaded. Comparative genomics analysis was performed using Roary v3.11.2, a typical tool for large-scale prokaryotic pan-genome construction. All genome sequences used for analysis were analyzed using Prokka v1.11. After annotation with Prokka, the gff annotation files of all Listeria monocytogenes strains were copied and pan-genome analysis was performed. The generated gene_presence_absence.csv file is the pan-genome matrix file. Then, the presence / absence profiles of all genes in the pan-genome matrix file were converted to 1 / 0 form for the purpose of mining specific targets related to persistent Listeria monocytogenes. The target genes were present in 100% of the genome sequences of all target strains and absent in the genome sequences of other non-target strains. After obtaining these target genes, BLAST alignment was performed on the whole genome of Listeria monocytogenes in a public database for further screening. Genome sequences that were specifically present in the target persistent type and completely absent in all other non-target persistent types and closely related Listeria species were finally obtained as specific candidate targets for persistent Listeria monocytogenes. The gene sequences are shown in Tables 1-3, as shown in SEQ ID NO.1 to SEQ ID NO.13.

[0041] Table 1: Specific target nucleotide sequences of persistent Listeria monocytogenes

[0042] Table 2: Specific target nucleotide sequences of persistent Listeria monocytogenes

[0043] Table 3: Specific target nucleotide sequences of persistent Listeria monocytogenes

[0044] Example 2 This embodiment provides the establishment of a rapid detection method for a novel molecular target specific to persistent CC5 Listeria monocytogenes. Primers were designed based on the novel specific molecular target of persistent CC5 Listeria monocytogenes to form a rapid detection method. The specific operation is as follows: (1) Primer design: Based on the base sequences of the new specific molecular target (SEQ ID NO.1 to SEQ ID NO.4), specific amplification primers were designed using Oligo 7. The primer sequences are shown in Table 4. Table 4: Primer sequences for specific PCR detection of persistent Listeria monocytogenes CC5

[0045] (2) Establishment of detection methods Step 1: Extracting DNA template The specific steps are as follows: the persistent CC5 Listeria monocytogenes is cultured overnight in BHI liquid medium, and bacterial genomic DNA is extracted using the TIANGEN Bacterial Genomic DNA Extraction Kit (DP302) as a template for testing.

[0046] Step 2: PCR detection system and amplification procedure The PCR detection system is 20 μL, which includes 10 μL of PCR reaction mix, 2 μL of template DNA, 1 μL each of 10 μM primers, and double-distilled water to bring the total system to 20 μL; The PCR detection conditions were as follows: pre-denaturation at 95℃ for 2-10 min; followed by 30-40 cycles, each cycle consisting of denaturation at 95℃ for 15-30 s, annealing at 55-60℃ for 30-90 s, extension at 72℃ for 30-90 s; after the cycles, a final extension at 72℃ for 5-10 min was performed, and the sample was stored at -20℃.

[0047] Step 3: Interpretation of PCR test results Gel electrophoresis was used to detect the amplification products. The presence of single amplification bands at positions 279bp, 364bp, 165bp, and 243bp was determined. If a single amplification band was present, the sample contained persistent CC5 Listeria monocytogenes. If no single amplification band was observed, the sample did not contain persistent CC5 Listeria monocytogenes.

[0048] Step 4: Specificity evaluation results of the PCR detection method Six persistent CC5 Listeria monocytogenes strains, 52 non-target persistent Listeria monocytogenes (including CC8, CC31, and CC87), four other Listeria monocytogenes (including Listeria innocense and Listeria sieboldii), and 20 non-listeria monocytogenes (including Cronobacter sakazakii and Staphylococcus aureus) were collected and subjected to PCR detection according to the above method. The results are as follows. Figures 1-4As shown, this detection method only showed specific amplification bands for persistent CC5 Listeria monocytogenes; other persistent Listeria monocytogenes and non-Listeria monocytogenes strains did not show specific bands. The strains used and the detection results are shown in Tables 5-6. In the table results column, "+" indicates positive and "-" indicates negative.

[0049] Table 5: Results of the specificity evaluation test of the persistent CC5 Listeria monocytogenes strain of this invention

[0050] Table 6: Results of the specificity evaluation test of the persistent CC5 Listeria monocytogenes strain of this invention

[0051] In summary, this detection method has high specificity and can accurately identify persistent CC5 Listeria monocytogenes. It shows no cross-reactivity with other closely related persistent Listeria monocytogenes, Listeria monocytogenes of different species, and common foodborne pathogens.

[0052] Example 3 This embodiment provides the establishment of a rapid detection method for novel specific molecular targets (SEQ ID NO.5~SEQ ID NO.6) of persistent CC8 Listeria monocytogenes. Primers were designed based on the novel specific molecular targets of persistent CC8 Listeria monocytogenes to form a rapid detection method. The specific operation is as follows: (1) Primer design Specific amplification primers were designed based on the base sequences of the novel specific molecular target (SEQ ID NO.5–SEQ ID NO.6). The primer sequences are shown in Table 7. Table 7: Primer sequences for specific PCR detection of persistent CC8 Listeria monocytogenes

[0053] (2) Establish detection methods Step 1: Extracting DNA template The specific steps are as follows: the persistent CC8 Listeria monocytogenes is cultured overnight in BHI liquid medium, and bacterial genomic DNA is extracted using the TIANGEN Bacterial Genomic DNA Extraction Kit (DP302) as a template for testing.

[0054] Step 2: PCR detection system and amplification procedure The PCR detection system is 20 μL, which includes 10 μL of PCR reaction mix, 2 μL of template DNA, 1 μL each of 10 μM primers, and double-distilled water to bring the total system to 20 μL; The PCR detection conditions were as follows: pre-denaturation at 95℃ for 2-10 min; followed by 30-40 cycles, each cycle consisting of denaturation at 95℃ for 15-30 s, annealing at 55-60℃ for 30-90 s, extension at 72℃ for 30-90 s; after the cycles, a final extension at 72℃ for 5-10 min was performed, and the sample was stored at -20℃.

[0055] Step 3: Interpretation of PCR test results Take 5 μL of PCR product and perform 2.0% agarose gel electrophoresis. Detect the amplification product by gel electrophoresis and determine whether there is a single amplification band at the 286bp and 211bp positions. If there is, it indicates that the sample contains persistent CC8 Listeria monocytogenes; if no corresponding single amplification band appears, the sample does not contain persistent CC8 Listeria monocytogenes.

[0056] Step 4: Specificity evaluation results of the PCR detection method Seven persistent CC8 Listeria monocytogenes strains, 51 non-target persistent Listeria monocytogenes strains including CC5, CC31, and CC87, four other Listeria monocytogenes strains including Listeria innocense and Listeria sieboldii, and 20 non-listeria monocytogenes strains including Cronobacter sakazakii and Staphylococcus aureus were collected and PCR detection was performed according to the above method. The results are as follows. Figures 5-6 As shown, this detection method only exhibits specific amplification bands for persistent CC8 Listeria monocytogenes; other persistent and non-listeria monocytogenes strains do not show specific bands. The strains used and the detection results are shown in Tables 8-9. In the tables, "+" indicates a positive result, and "-" indicates a negative result. This indicates that the method is highly specific for persistent CC8 Listeria monocytogenes and shows no cross-reactivity.

[0057] Table 8: Results of the specificity evaluation test of the persistent CC8 Listeria monocytogenes strain of the present invention

[0058] Table 9: Results of the specificity evaluation test for the persistent CC8 Listeria monocytogenes strain of this invention

[0059] In summary, the PCR detection method provided in this embodiment can accurately and specifically identify persistent CC8 Listeria monocytogenes.

[0060] Example 4 This embodiment provides the establishment of a rapid detection method for a novel molecular target specific to persistent Listeria monocytogenes CC31. Primers were designed based on the novel specific molecular target of persistent Listeria monocytogenes CC31 to form a rapid detection method. The specific operation is as follows: (1) Primer design Specific amplification primers were designed based on the base sequences of the novel specific molecular target (SEQ ID NO.7–SEQ ID NO.8). The primer sequences are shown in the table below. Table 6: Primer sequences for specific PCR detection of persistent Listeria monocytogenes CC31

[0061] (2) Construction of detection method Step 1: Extracting DNA template The specific steps are as follows: Listeria monocytogenes CC31 is cultured overnight in BHI liquid medium, and bacterial genomic DNA is extracted using the TIANGEN Bacterial Genomic DNA Extraction Kit (DP302) as a template for testing.

[0062] Step 2: PCR detection system and amplification procedure The PCR detection system is 20 μL, which includes 10 μL of PCR reaction mix, 2 μL of template DNA, 1 μL each of 10 μM primers, and double-distilled water to bring the total system to 20 μL; The PCR detection conditions were as follows: pre-denaturation at 95℃ for 2-10 min; followed by 30-40 cycles, each cycle consisting of denaturation at 95℃ for 15-30 s, annealing at 55-60℃ for 30-90 s, extension at 72℃ for 30-90 s; after the cycles, a final extension at 72℃ for 5-10 min was performed, and the sample was stored at -20℃.

[0063] Step 3: Interpretation of PCR test results Gel electrophoresis was used to detect the amplification products. The presence of single amplification bands at positions 184bp and 209bp was determined. If a single amplification band was present, the sample contained persistent CC31 Listeria monocytogenes. If no single amplification band was present, the sample did not contain persistent CC31 Listeria monocytogenes.

[0064] Step 4: Specificity evaluation results of the PCR detection method Two persistent Listeria monocytogenes strains (CC31), 56 non-target persistent Listeria monocytogenes strains (CC5, CC8, CC87, etc.), four other Listeria monocytogenes strains (Listeria innocense, Listeria sieboldii, etc.), and 20 non-listeria monocytogenes strains (Cronobacter sakazakii, Staphylococcus aureus, etc.) were collected and PCR detection was performed according to the above method. The results are as follows. Figures 7-8 As shown, this detection method only showed specific amplification bands for the persistent CC31 Listeria monocytogenes strain; other persistent Listeria monocytogenes and non-Listeria monocytogenes strains did not show specific bands. The strains used and the detection results are shown in Tables 10-11. In the table's detection result column, "+" indicates positive and "-" indicates negative.

[0065] Table 10: Results of the test evaluation of the specificity of the retained CC31 Listeria monocytogenes strain of the present invention

[0066] Table 11: Results of the test for the specificity evaluation of Listeria monocytogenes using the CC31 retention type of the present invention

[0067] Example 5 This embodiment provides the establishment of a rapid detection method for a novel molecular target specific to persistent Listeria monocytogenes CC87. Primers were designed based on the novel specific molecular target of persistent Listeria monocytogenes CC87 to form a rapid detection method. The specific operation is as follows: (1) Primer design Specific amplification primers were designed based on the novel specific molecular target base sequence SEQ ID NO.9. The primer sequences are shown in Table 12. Table 12: Primer sequences for specific PCR detection of persistent Listeria monocytogenes CC87

[0068] (2) Construction of detection method Step 1: Extracting DNA template The specific steps are as follows: Listeria monocytogenes CC87 is cultured overnight in BHI liquid medium, and bacterial genomic DNA is extracted using the TIANGEN Bacterial Genomic DNA Extraction Kit (DP302) as a template for testing.

[0069] Step 2: PCR detection system and amplification procedure The PCR detection system is 20 μL, which includes 10 μL of PCR reaction mix, 2 μL of template DNA, 1 μL each of 10 μM primers, and double-distilled water to bring the total system to 20 μL; The PCR detection conditions were as follows: pre-denaturation at 95℃ for 2-10 min; followed by 30-40 cycles, each cycle consisting of denaturation at 95℃ for 15-30 s, annealing at 55-60℃ for 30-90 s, extension at 72℃ for 30-90 s; after the cycles, a final extension at 72℃ for 5-10 min was performed, and the sample was stored at -20℃.

[0070] Step 3: Interpretation of PCR test results Gel electrophoresis was used to detect the amplification products. The presence of a single amplification band at the 259bp position was determined. If a single amplification band was present, it indicated that the sample contained persistent CC87 Listeria monocytogenes. If no single amplification band was present, the sample did not contain persistent CC87 Listeria monocytogenes.

[0071] Step 4: Specificity evaluation results of the PCR detection method Eleven persistent Listeria monocytogenes strains (CC87), 47 non-target persistent Listeria monocytogenes strains (CC5, CC8, CC121, etc.), four other Listeria monocytogenes strains (Listeria innocense, Listeria sieboldii, etc.), and 20 non-listeria monocytogenes strains (Cronobacter sakazakii, Staphylococcus aureus, etc.) were collected and subjected to PCR detection according to the above method. The results are as follows: Figure 9 As shown, this detection method only showed specific amplification bands for the persistent CC87 Listeria monocytogenes strain; other persistent Listeria monocytogenes and non-Listeria monocytogenes strains did not show specific bands. The strains used and the detection results are shown in Tables 13-14. In the table's detection result column, "+" indicates positive and "-" indicates negative.

[0072] Table 13: Results of the test for the specificity evaluation of Listeria monocytogenes of the persistent CC87 strain of the present invention

[0073] Table 14: Results of the test for the specificity evaluation of Listeria monocytogenes of the persistent CC87 strain of the present invention

[0074] Example 6 This embodiment provides the establishment of a rapid detection method for a novel molecular target specific to persistent Listeria monocytogenes CC121. Primers were designed based on the novel specific molecular target of persistent Listeria monocytogenes CC121 to form a rapid detection method. The specific operation is as follows: (1) Primer design Specific amplification primers were designed based on the novel specific molecular target base sequences SEQ ID NO.10~SEQ ID NO.11. The primer sequences are shown in Table 15. Table 15: Primer sequences for specific PCR detection of Listeria monocytogenes CC121

[0075] (2) Construction of detection method Step 1: Extracting DNA template The specific steps are as follows: Listeria monocytogenes CC121 is cultured overnight in BHI liquid medium, and bacterial genomic DNA is extracted using the TIANGEN Bacterial Genomic DNA Extraction Kit (DP302) as a template for testing.

[0076] Step 2: PCR detection system and amplification procedure The PCR detection system is 20 μL, which includes 10 μL of PCR reaction mix, 2 μL of template DNA, 1 μL each of 10 μM primers, and double-distilled water to bring the total system to 20 μL; The PCR detection conditions were as follows: pre-denaturation at 95℃ for 2-10 min; followed by 30-40 cycles, each cycle consisting of denaturation at 95℃ for 15-30 s, annealing at 55-60℃ for 30-90 s, extension at 72℃ for 30-90 s; after the cycles, a final extension at 72℃ for 5-10 min was performed, and the sample was stored at -20℃.

[0077] Step 3: Interpretation of PCR test results Gel electrophoresis was used to detect the amplification products. The presence of single amplification bands at positions 156bp, 149bp, and 118bp was determined. If a single amplification band was present, the sample contained persistent CC121 Listeria monocytogenes. If no single amplification band was present, the sample did not contain persistent CC121 Listeria monocytogenes.

[0078] Step 4: Specificity evaluation results of the PCR detection method Five persistent Listeria monocytogenes strains (CC121), 53 non-target persistent Listeria monocytogenes strains (CC5, CC8, CC121, etc.), four other Listeria monocytogenes strains (Listeria innocense, Listeria sieboldii, etc.), and 20 non-listeria monocytogenes strains (Cronobacter sakazakii, Staphylococcus aureus, etc.) were collected and subjected to PCR detection according to the above method. The results are as follows: Figures 10-12 As shown, this detection method only showed specific amplification bands for the persistent CC121 Listeria monocytogenes strain; other persistent Listeria monocytogenes and non-Listeria monocytogenes strains did not show specific bands. The strains used and the detection results are shown in Tables 16 and 17. In the table's detection result column, "+" indicates positive and "-" indicates negative.

[0079] Table 16: Results of the test for the specificity evaluation of Listeria monocytogenes using the persistent CC121 strain of the present invention

[0080] Table 17: Results of the test evaluation of the specificity of the retained CC121 Listeria monocytogenes strain of the present invention

[0081] Example 7 This embodiment provides the establishment of a rapid detection method for a novel molecular target specific to persistent Listeria monocytogenes CC204. Primers were designed based on the novel specific molecular target of persistent Listeria monocytogenes CC204 to form a rapid detection method. The specific operation is as follows: (1) Primer design Specific amplification primers were designed based on the novel specific molecular target base sequences SEQ ID NO.12~SEQ ID NO.13. The primer sequences are shown in Table 18. Table 18: Primer sequences for specific PCR detection of Listeria monocytogenes CC204

[0082] (2) Construction of detection method Step 1: Extracting DNA template The specific steps are as follows: Listeria monocytogenes CC204 was cultured overnight in BHI liquid medium, and bacterial genomic DNA was extracted using the TIANGEN Bacterial Genomic DNA Extraction Kit (DP302) as a template for testing.

[0083] Step 2: PCR detection system and amplification procedure The PCR detection system is 20 μL, which includes 10 μL of PCR reaction mix, 2 μL of template DNA, 1 μL each of 10 μM primers, and double-distilled water to bring the total system to 20 μL; The PCR detection conditions were as follows: pre-denaturation at 95℃ for 2-10 min; followed by 30-40 cycles, each cycle consisting of denaturation at 95℃ for 15-30 s, annealing at 55-60℃ for 30-90 s, extension at 72℃ for 30-90 s; after the cycles, a final extension at 72℃ for 5-10 min was performed, and the sample was stored at -20℃.

[0084] Step 3: Interpretation of PCR test results Gel electrophoresis was used to detect the amplification products. The presence of single amplification bands at positions 214bp and 212bp was determined. If a single amplification band was present, the sample contained persistent CC204 Listeria monocytogenes. If no single amplification band was present, the sample did not contain persistent CC204 Listeria monocytogenes.

[0085] Step 4: Specificity evaluation results of the PCR detection method One persistent Listeria monocytogenes strain (CC121), 57 non-target persistent Listeria monocytogenes strains (including CC5, CC8, and CC121), four other Listeria monocytogenes strains (including Listeria innocense and Listeria sieboldii), and 20 non-listeria monocytogenes strains (including Cronobacter sakazakii and Staphylococcus aureus) were collected and subjected to PCR detection according to the above method. The results are as follows. Figures 13-14 As shown, this detection method only showed specific amplification bands for the persistent CC204 Listeria monocytogenes strain; other persistent Listeria monocytogenes and non-Listeria monocytogenes strains did not show specific bands. The strains used and the detection results are shown in Tables 19-20. In the table results column, "+" indicates positive and "-" indicates negative.

[0086] Table 19: Results of the test for the specificity evaluation of Listeria monocytogenes in the persistent CC204 strain of this invention

[0087] Table 20: Results of the test for the specificity evaluation of Listeria monocytogenes in the persistent CC204 strain of this invention

[0088] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A molecular target combination for detecting persistent Listeria monocytogenes, characterized in that, The nucleotide sequences of the molecular target combination are any one or a combination of SEQ ID NO.1 to SEQ ID NO.13; the persistent Listeria monocytogenes is any one or a combination of CC5, CC8, CC31, CC87, CC121 and CC204.

2. The molecular target combination according to claim 1, characterized in that, Molecular targets with nucleotide sequences as shown in SEQ ID NO.1~SEQ ID NO.4 are used to identify persistent Listeria monocytogenes CC5; molecular targets with nucleotide sequences as shown in SEQ ID NO.5 and SEQ ID NO.6 are used to identify persistent Listeria monocytogenes CC8; molecular targets with nucleotide sequences as shown in SEQ ID NO.7 and SEQ ID NO.8 are used to identify persistent Listeria monocytogenes CC31; molecular targets with nucleotide sequences as shown in SEQ ID NO.9 are used to identify persistent Listeria monocytogenes CC87; molecular targets with nucleotide sequences as shown in SEQ ID NO.10 and SEQ ID NO.11 are used to identify persistent Listeria monocytogenes CC121; and molecular targets with nucleotide sequences as shown in SEQ ID NO.12 and SEQ ID NO.13 are used to identify persistent Listeria monocytogenes CC204.

3. A set of specific primers for detecting the molecular target combination of claim 1 or 2, characterized in that, The primer set contains specific primer pairs with nucleotide sequences as shown in SEQ ID NO.14 to SEQ ID NO.

41.

4. The primer set according to claim 3, characterized in that, The primer set specifically includes: primer pairs SEQ ID NO.14 and SEQ ID NO.15 for amplifying SEQ ID NO.1; primer pairs SEQ ID NO.16 and SEQ ID NO.17 for amplifying SEQ ID NO.2; primer pairs SEQ ID NO.18 and SEQ ID NO.19 for amplifying SEQ ID NO.3; primer pairs SEQ ID NO.20 and SEQ ID NO.21 for amplifying SEQ ID NO.4; primer pairs SEQ ID NO.22 and SEQ ID NO.23 for amplifying SEQ ID NO.5; primer pairs SEQ ID NO.24 and SEQ ID NO.25 for amplifying SEQ ID NO.6; primer pairs SEQ ID NO.26 and SEQ ID NO.27 for amplifying SEQ ID NO.7; primer pairs SEQ ID NO.28 and SEQ ID NO.29 for amplifying SEQ ID NO.8; primer pairs SEQ ID NO.30 and SEQ ID NO.31 for amplifying SEQ ID NO.9; and primer pairs SEQ ID NO.10 and SEQ ID NO.15 for amplifying SEQ ID NO.

10. Primers NO.32 and SEQ ID NO.33, or SEQ ID NO.34 and SEQ ID NO.35; primer pairs SEQ ID NO.36 and SEQ ID NO.37 for amplifying SEQ ID NO.11; primer pairs SEQ ID NO.38 and SEQ ID NO.39 for amplifying SEQ ID NO.12; and primer pairs SEQ ID NO.40 and SEQ ID NO.41 for amplifying SEQ ID NO.

13.

5. The use of the molecular target combination for detecting persistent Listeria monocytogenes as described in claim 1 or 2, or the specific primer set as described in claim 3 or 4, in the preparation of products for detecting persistent Listeria monocytogenes.

6. The application according to claim 5, characterized in that, The product in question is a reagent kit.

7. A kit for detecting persistent Listeria monocytogenes, characterized in that, The primer set comprising the primer set of claim 3 or 4, and further comprising all the necessary components for polymerase chain reaction, wherein each pair of primers in the primer set can be configured for singleton or multiplex PCR reaction systems.

8. A method for identifying multiple persistent forms of Listeria monocytogenes, characterized in that, Includes the following steps: (1) Using sample DNA as a template, perform PCR amplification reaction using at least one primer pair from the primer set described in claim 3 or 4; (2) Detect the amplification products. By analyzing whether there is a specific amplification band corresponding to the target persistent Listeria monocytogenes, it can be determined whether the sample contains and what kind of persistent Listeria monocytogenes it contains.

9. A method for identifying multiple persistent types of Listeria monocytogenes according to claim 8, characterized in that, The PCR amplification reaction system contains template DNA, upstream primer, and downstream primer.

10. A method for identifying multiple persistent types of Listeria monocytogenes according to claim 8, characterized in that, The PCR amplification reaction program is as follows: pre-denaturation at 95℃ for 2-10 min; followed by 30-40 cycles, each cycle including denaturation at 95℃ for 15-30 s, annealing at 55-60℃ for 30-90 s, extension at 72℃ for 30-90 s; and a final extension at 72℃ for 5-10 min after the cycle.

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