Genetic marker for identifying biofilm phenotype of Listeria monocytogenes and application of genetic marker

Abstract

The invention belongs to the field of genetic markers, and particularly relates to a genetic marker for identifying the biofilm phenotype of Listeria monocytogenes and application of the genetic marker. The invention provides a plurality of genetic markers for identifying the biofilm phenotype of the listeria monocytogenes, the biofilm phenotype of the listeria monocytogenes can be identified, and meanwhile, corresponding detection primers are provided. Results of embodiments show that the marker provided by the invention can be used for biofilm phenotype identification of Listeria monocytogenes. The invention provides a genetic marker for rapidly judging the biofilm phenotype of the listeria monocytogenes, and can provide a basis for optimization of a disinfection process in the food industry and precise prevention and control of the listeria monocytogenes.

Description

Technical Field

[0001] The present invention belongs to the field of genetic markers, and in particular relates to a genetic marker for identifying the biofilm phenotype of Listeria monocytogenes and an application thereof. Background Art

[0002] Listeria monocytogenes is a foodborne pathogen that can persist and spread in food and food industry equipment. Listeria monocytogenes has extremely strong environmental tolerance and can survive in adverse environments such as low temperature, low osmotic pressure and disinfectants. The environmental tolerance of Listeria monocytogenes is mainly related to its ability to form biofilms, and about 60% of food poisoning incidents are related to the formation of biofilms by foodborne pathogens. Bacteria in the biofilm state are 100-1000 times more resistant to disinfectants than planktonic bacteria. In the food industry, strains with strong biofilm-forming ability are considered to have stronger environmental adaptability. Listeria monocytogenes with this phenotype are more difficult to remove, have a higher probability of contaminating food, and are more likely to pose a serious threat to food safety.

[0003] At present, the determination method of biofilm phenotype is mainly based on physical and chemical indicators. For example, the 96-well plate crystal violet staining method is the most commonly used method for bacterial biofilm determination due to its simple operation and low cost. However, this method is time-consuming and cannot quickly indicate the biofilm formation ability of Listeria monocytogenes isolates in the food industry. Therefore, there is an urgent need for a marker that can quickly and efficiently identify the biofilm phenotype. Summary of the invention

[0004] The object of the present invention is to provide genetic markers for identifying the biofilm phenotype of Listeria monocytogenes.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a genetic marker for identifying the biofilm phenotype of Listeria monocytogenes, wherein the genetic marker is a gene and / or a plasmid, and the gene is one or more of vip, srmB, uvrB, smc_4, inlH, degA, lmo0447, ssbA, recT, cycB, blaI and mntR.

[0007] The present invention also provides the use of the above genetic markers in identifying the biofilm phenotype of Listeria monocytogenes.

[0008] The present invention also provides a detection substance for identifying the phenotype of Listeria monocytogenes biofilm, wherein the detection substance can detect the above genetic marker.

[0009] Preferably, the detection substance includes primers, the sequences of the primers are shown in SEQ ID NO.1-26, and the sequences of the primers for detecting vip genes are shown in SEQ ID NO.1-2.

[0010] The primer sequence for detecting the srmB gene is SEQ ID NO.3-4,

[0011] The primer sequences for detecting the uvrB gene are SEQ ID NO.5-6,

[0012] The primer sequences for detecting the smc_4 gene are SEQ ID NO.7-8,

[0013] The primer sequences for detecting the mntR gene are SEQ ID NO.9-10,

[0014] The primer sequences for detecting the inlH gene are SEQ ID NO.11-12,

[0015] The primer sequences for detecting the degA gene are SEQ ID NO.13-14,

[0016] The primer sequences for detecting the cycB gene are SEQ ID NO.15-16,

[0017] The primer sequences for detecting the lmo0447 gene are SEQ ID NO.17-18,

[0018] The primer sequences for detecting the blaI gene are SEQ ID NO.19-20,

[0019] The primer sequences for detecting the ssbA gene are SEQ ID NO.21-22,

[0020] The primer sequences for detecting the recT gene are SEQ ID NO.23-24,

[0021] The primer sequences for detecting plasmids are SEQ ID NO.25-26,

[0022] The present invention also provides a kit for detecting the biofilm phenotype of Listeria monocytogenes, wherein the kit comprises the above-mentioned primers.

[0023] The present invention also provides a method for identifying the phenotype of Listeria monocytogenes biofilm, the method comprising identifying whether the above-mentioned marker exists.

[0024] Preferably, the identification method also includes multiple sequence locus typing.

[0025] The present invention also provides the application of the above genetic markers in optimizing the disinfection process in the food industry.

[0026] The present invention also provides the use of the above genetic markers in the prevention and control of Listeria monocytogenes, which can achieve accurate prevention and control of different phenotypes.

[0027] Beneficial effects:

[0028] The present invention uses genetic markers to quickly identify the Listeria monocytogenes biofilm phenotype.

[0029] The vip gene in the marker of the present invention can determine the biofilm forming ability of the strain, and has a high accuracy rate for determining strong, medium and weak biofilm phenotypes. The vip gene combined with srmB, cycB and uvrB genes can increase the accuracy rate of biofilm phenotype determination to 85.7% to 100.0%.

[0030] The marker of the present invention can improve the detection accuracy by combining with the multi-sequence site typing method, and the phenotype determination accuracy of the vip gene on specific ST8, ST87 and ST121 is respectively improved to 87.5%-90.0%, 100.0% and 100.0%. The vip gene is not suitable for determining the biofilm phenotype of STs strains such as ST9, ST155 and ST91, while the accuracy of the smc_4, srmB-inlH, inlH and ssbA genes in determining the above biofilm phenotypes is 88.9%-100.0%, 75%-100.0%, 100.0% and 100.0% respectively.

[0031] The marker disclosed in the present invention can be used as a screening index for rapidly evaluating the biofilm ability of Listeria monocytogenes. The present invention provides a genetic marker for rapidly determining the biofilm phenotype of Listeria monocytogenes, and can provide a basis for optimizing the disinfection process in the food industry and accurately preventing and controlling Listeria monocytogenes. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0033] Figure 1 This is a graph showing the results of marker detection. DETAILED DESCRIPTION

[0034] The invention provides genetic markers for identifying the biofilm phenotype of Listeria monocytogenes, and provides corresponding identification materials, kits and methods.

[0035] The method adopted by the present invention is a method for rapidly identifying the biofilm phenotype of Listeria monocytogenes by using genetic markers. The present invention provides a method for identifying the biofilm phenotype of Listeria monocytogenes with the vip gene as a preliminary genetic marker, and improves the accuracy of identifying the biofilm phenotype of Listeria monocytogenes by combining the vip gene with 1-3 genes (srmB, cycB, uvrB) or STs (ST8, ST87, ST121). In addition, the smc_4, srmB-inlH, inlH, and ssbA genes can be used for the rapid identification of the biofilm phenotypes of ST9, ST155, ST91, and the remaining STs strains.

[0036] Multilocus sequence typing (MLST) is based on the MLST method for Listeria monocytogenes provided by the Pasteur Institute in France (https: / / bigsdb.pasteur.fr / listeria / primers-used / ). Specifically, the PCR products are sent to Shanghai Sangon Biotech Co., Ltd. for bidirectional sequencing, and the sequencing sequences are compared with the database to determine the allele numbers of each housekeeping gene. The combination of the 7 allele numbers of abcZ-bglA-cat-dapE-dat-ldh-lhkA is the ST type of the strain.

[0037] The discrimination criteria for strong biofilm phenotype, medium biofilm phenotype, and weak biofilm phenotype are as follows: Referring to the method of "STEPANOVIC S, VUKOVIC D, DAKIC I, SAVIC B, SVABIC-VLAHOVIC M. A MODIFIED MICROTITER-PLATE TEST FOR QUANTIFICATION OF STAPHYLOCOCCAL BIOFILMFORMATION. JMICROBIOL METHODS. 2000;40(2):175-179. DOI:10.1016 / S0167-7012(00)00122-6", the biofilm formation ability of each strain is analyzed. The average OD595 value of the negative control plus 3 times the standard deviation (s) is the ODc benchmark. OD < ODc indicates no biofilm formation, 1ODc < OD < 2ODc indicates weak biofilm formation ability, 2ODc < OD < 4ODc indicates medium biofilm formation ability, and OD > 4ODc indicates strong biofilm formation ability.

[0038] The genetic marker screening method for identifying the biofilm phenotype of Listeria monocytogenes in the present invention includes the following steps:

[0039] (1) MLST and biofilm-forming ability of 103 Listeria monocytogenes isolates were identified by multiple sequence loci typing and 96-well plate crystal violet staining. The results showed that the 103 strains were divided into 22 ST types, of which 48.5% of the strains showed a strong biofilm phenotype, 41.7% of the strains showed a moderate biofilm phenotype, and 9.7% of the strains showed a weak biofilm phenotype.

[0040] (2) Based on the ST type and biofilm-forming ability of the isolated strains, 12 representative strains (including 4 strains with strong, medium, and weak biofilm phenotypes) were selected for whole-genome next-generation sequencing. Comparative genomics analysis was used to explore the genetic factors associated with the biofilm phenotype.

[0041] (3) Genetic factors related to the biofilm phenotype were discovered, including 13 genetic markers: vip, srmB, uvrB, smc_4, mntR, inlH, degA, cycB, lmo0447, blaI, ssbA, recT genes and plasmids. The primer information is shown in Table 1.

[0042] Table 1. Genetic marker primer information

[0043]

[0044]

[0045] The gene functions of the above markers are as follows:

[0046] (1) The vip gene plays a crucial role in the pathogenicity of Listeria monocytogenes and is a necessary factor for the bacteria to enter host cells. It promotes the invasion of Listeria monocytogenes by mediating the binding of bacteria to host cell surface receptors. At present, there is a lack of research on the correlation between the vip gene and the ability to form biofilms.

[0047] (2) The SrmB protein encoded by the srmB gene is mainly involved in the synthesis of DEAD-box RNA-dependent ATPase, which is mainly involved in the assembly of large ribosomal subunits.

[0048] (3) The uvrB gene is a key regulatory factor encoding nucleotide excision repair proteins. This gene belongs to the SOS system, which is an inducible DNA damage repair system that plays a vital role in the biofilm formation ability and pathogenicity of bacteria. Studies have shown that the uvrB gene is involved in the biofilm formation ability of Listeria monocytogenes, and the lack of the uvrB gene will lead to a decrease in the biofilm formation ability.

[0049] (4) The smc gene is involved in the condensation and separation of cell chromosomes, thereby regulating bacterial growth. Studies have shown that the smc gene is involved in the biofilm formation of Streptococcus mutans.

[0050] (5) The function of the mntR gene is unknown.

[0051] (6) As a member of the virulence gene cluster of Listeria monocytogenes, the inlH gene plays a very important role in host adhesion and invasion. Some studies have shown that the inlH gene is related to the formation of biofilm of Listeria monocytogenes.

[0052] (7) The degA gene in thermophilic Bacillus mainly encodes a transcriptional repressor that responds to and regulates the inositol degradation operon iolX. However, the role of the degA gene in the biofilm-forming ability of Listeria monocytogenes is still unclear.

[0053] (8) The cycB gene belongs to the ABC transporter 5a subfamily and is responsible for encoding sugar-binding lipoprotein.

[0054] (9) The lmo0447 gene is a member of the stress resistance island SSI-1 gene cluster. Several studies have shown that the SSI-1 resistance island is associated with tolerance to low pH and high salt concentrations, thereby helping Listeria monocytogenes survive under stress and serving as a marker of persistent tolerance.

[0055] (10) The function of the blaI gene is unknown.

[0056] (11) The ssbA gene encodes a DNA-binding protein, and the loss of this gene reduces the ability of S. mutans to form biofilms.

[0057] (12) recT genes are an important type of transcriptional regulatory factor that regulates various genes that play important functions in cellular metabolism and provides the host with a variety of environmental adaptation advantages, including biofilm formation and virulence.

[0058] (13)Plasmid (repA) is responsible for encoding the plasmid replication protein of Listeria monocytogenes.

[0059] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0060] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0061] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0062] In the examples, "-" indicates that the corresponding gene is not carried, and "+" indicates that the corresponding gene is carried.

[0063] Example 1

[0064] Based on 13 genetic markers including vip, srmB, uvrB, smc_4, inlH, degA, lmo0447, ssbA, recT, cycB, blaI and mntR genes and plasmids, PCR technology was used to verify the presence of relevant genetic markers in 103 strains and identify the biofilm phenotype of the strains.

[0065] The specific results are as follows:

[0066] Among the 103 strains, strains with strong biofilm phenotypes generally did not carry the vip gene. - The gene was able to identify 86.0% of strong biofilm strains. When combined with srmB + and cycB + When the combination of , the accuracy rate is further improved to 93.0%.

[0067] For VIP - The moderate and weak biofilm strains in the group can be detected by srmB + srmB - and cycB - The accuracy rates of the two methods were 85.7% and 100.0% respectively (see Figure 1 A).

[0068] In VIP + The accuracy of gene pairing for identifying moderate and weak biofilm strains was 83.7% and 70.0%, respectively. + and srmB + After the genes were added, the accuracy rates increased to 91.7% and 85.7% respectively. + In the group, uvrB - The accuracy of the gene for determining the strong biofilm phenotype strain was 100.0% (see Figure 1 A).

[0069] Example 2

[0070] The biofilm phenotype of the strain was identified based on 13 genetic markers including vip, srmB, uvrB, smc_4, inlH, degA, lmo0447, ssbA, recT, cycB, blaI and mntR genes and plasmids combined with multiple sequence locus typing. The sequence types (ST types) included: ST8, ST87, ST121, ST9, ST155, ST91 and other STs.

[0071] In ST8, VIP - The accuracy of genetic detection of strong biofilm strains was 87.5%, which was comparable to lmo0447 + , cycB + 、degA + After gene combination, the accuracy rate is increased to 100.0%. - recT in the group + The accuracy of gene pair determination of medium and weak biofilm phenotype strains was 100.0% (see Figure 1 B).

[0072] VIP + The accuracy of the gene indicative of moderate biofilm was 90.0%, while blaI - The accuracy of the gene can be further improved to 100.0%. + The gene can indicate strong biofilm strains 100.0%. It is worth noting that all ST8 strong biofilm strains carry plasmids (see Figure 1 B).

[0073] In ST87, VIP - Able to identify 100.0% of strong biofilm strains, while vip + 100.0% of the medium biofilm strains were identified (see Figure 1 E).

[0074] In ST121, VIP - 100.0% of strong biofilm strains were identified, vip + 100.0% of the moderate and weak biofilm strains were identified (see Figure 1 G).

[0075] In ST9, ST155, ST91 and other ST strains,

[0076] In ST9, smc_4 - The accuracy of detecting strong and medium biofilm strains was 88.9% and 100.0%, respectively, while smc_4 + 100.0% of weak biofilm strains can be identified (see Figure 1 C).

[0077] In ST155, srmB + and inlH + The combination of mntR + The gene can indicate 75.0% of weak biofilm strains (see Figure 1 D).

[0078] In ST91, inlH - indicated 100.0% of the biofilm strains, while inlH + This indicates 100.0% of strong biofilm strains (see Figure 1 F).

[0079] In other STs, ssbA - The gene can indicate 100.0% of weak biofilm strains, while ssbA + The gene can indicate 100.0% of strong and moderate biofilm strains (see Figure 1 H).

[0080] From the above examples, it can be seen that the genetic markers provided by the present invention can be used alone or in combination with multiple sequence loci typing to identify the biofilm phenotype of Listeria monocytogenes.

[0081] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A genetic marker for identifying the biofilm phenotype of Listeria monocytogenes, characterized in that: The genetic marker is a gene and / or a plasmid, and the gene is one or more of vip, srmB, uvrB, smc-4, inlH, degA, lmo0447, ssbA, recT, cycB, blaI and mntR.

2. Use of the genetic marker according to claim 1 in identifying the biofilm phenotype of Listeria monocytogenes.

3. A detection substance for identifying the phenotype of Listeria monocytogenes biofilm, characterized in that: The detection substance can detect the genetic marker according to claim 1.

4. The detection substance according to claim 3, characterized in that The detection substance includes a primer, and the sequence of the primer is shown in SEQ ID NO.1-26. The primer sequence for detecting the vip gene is SEQ ID NO.1-2, The primer sequence for detecting the srmB gene is SEQ ID NO.3-4, The primer sequences for detecting the uvrB gene are SEQ ID NO.5-6, The primer sequences for detecting the smc_4 gene are SEQ ID NO.7-8, The primer sequences for detecting the mntR gene are SEQ ID NO.9-10, The primer sequences for detecting the inlH gene are SEQ ID NO.11-12, The primer sequences for detecting the degA gene are SEQ ID NO.13-14, The primer sequences for detecting the cycB gene are SEQ ID NO.15-16, The primer sequences for detecting the lmo0447 gene are SEQ ID NO.17-18, The primer sequences for detecting the blaI gene are SEQ ID NO.19-20, The primer sequences for detecting the ssbA gene are SEQ ID NO.21-22, The primer sequences for detecting the recT gene are SEQ ID NO.23-24, The primer sequences for detecting the plasmid are SEQ ID NO.25-26.

5. A kit for detecting the biofilm phenotype of Listeria monocytogenes, characterized in that: The kit comprises the primers according to claim 4.

6. A method for identifying the phenotype of Listeria monocytogenes biofilm, characterized in that: The method comprises identifying the presence or absence of the genetic marker of claim 1.

7. The method according to claim 6, characterized in that The identification method also includes multiple sequence locus typing.

8. Use of the genetic markers according to claim 1 in optimizing the disinfection process in the food industry.

9. Use of the genetic marker according to claim 1 in the prevention and control of Listeria monocytogenes.

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