Marker, TaqMan primer probe set, LAMP primer set, kit, detection method, recombinant plasmid and application for simultaneously detecting multiple bacteria

Abstract

The invention discloses a marker, a TaqMan primer probe group, an LAMP (loop-mediated isothermal amplification) primer group, a kit, a detection method, a recombinant plasmid and application for simultaneously detecting six bacteria. The marker is a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence which is derived from 16S rRNA gene in bacillus cereus and has more than 95% of homology with the SEQ ID NO: 1. The invention provides a TaqMan primer probe group and an LAMP (Loop-Mediated Isothermal Amplification) primer group. The TaqMan primer probe group and the LAMP primer group are combined with a corresponding TaqMan PCR (Polymerase Chain Reaction) or LAMP method, so that the rapid detection of six bacteria, namely bacillus cereus, rose kocuria, micrococcus, kocuria rootrophila, escherichia coli and staphylococcus aureus, can be realized at the same time. A final detection result proves that the detection method provided by the invention has high sensitivity and accuracy when being used for detecting six bacteria.

Description

Technical Field

[0001] The present application relates to the technical field of bacterial detection, and specifically to markers, TaqMan primer probe sets, LAMP primer sets, kits, detection methods, recombinant plasmids and applications for simultaneously detecting multiple bacteria. Background Art

[0002] In the fields of food, medicine, medical devices, etc., excessive bacteria and other microorganisms in the production environment are an important cause of product contamination. In the field of cell therapy, products are often unable to undergo terminal sterilization or sterilization filtration, and require higher sterility assurance and process control of the process. For example, the autologous CAR-T cell therapy products that are currently being developed / marketed have large individual differences, small single-batch production, short shelf life of some products, and urgent clinical needs, and they are in urgent need of rapid release. Traditional sterility inspection methods take at least two weeks, which has become one of the biggest shortcomings restricting the release time. Therefore, a faster and more flexible method is needed for bacterial quality control.

[0003] Genetic testing can improve the speed and sensitivity of bacterial detection, but the bacterial detection technology based on genetic testing on the market mainly performs single detection on bacterial species. The microecological environment in the production workshop or laboratory environment is relatively complex, and there are many species of bacteria that cause bacterial contamination. Among them, Bacillus cereus, Coxiella rosea, Micrococcus, Coxiella rhizophila, Escherichia coli, Staphylococcus aureus and other species are common bacteria in the production environment. If multiple bacterial species are tested and verified one by one, the entire detection method will be complicated, time-consuming, and cause high detection costs.

[0004] Therefore, the development of rapid and simultaneous detection products and methods for multiple bacteria has become a market need. Summary of the invention

[0005] In view of this, the purpose of the present application is to provide a marker, a TaqMan primer probe set, a LAMP primer set, a kit, a detection method, a recombinant plasmid and an application for simultaneously detecting multiple bacteria. The marker is a fragment of a 16S rRNA gene, which can be used to simultaneously detect six bacteria: Bacillus cereus, Coxiella rosea, Micrococcus, Coxiella rhizophila, Escherichia coli, and Staphylococcus aureus. For the marker sequence, a TaqMan primer probe set and a loop-mediated isothermal amplification (LAMP) primer set were designed respectively, and a nucleic acid amplification experiment was performed. The final experimental results show that the marker sequence and the TaqMan primer probe set and the LAMP primer set can be used for the detection of at least six bacteria, and have the advantages of good detection accuracy, high sensitivity, and fast detection speed.

[0006] In order to achieve or at least partially achieve the above objectives, the present application provides the following technical solutions:

[0007] In a first aspect, the present application provides a gene marker for detecting multiple bacteria, for simultaneously detecting at least 6 bacteria including Bacillus cereus, Coxsackia rosea, Micrococcus, Coxsackia rhizophila, Escherichia coli, and Staphylococcus aureus, wherein the gene marker is selected from at least one of the following:

[0008] (1) the nucleotide sequence shown in SEQ ID NO: 1;

[0009] (2) A nucleotide sequence derived from the 16S rRNA gene of Bacillus cereus and having more than 95% homology with SEQ ID NO: 1.

[0010] In a second aspect, the present application provides a TaqMan primer probe set, which is used to specifically amplify at least one of the aforementioned gene markers, and the TaqMan primer probe set includes primers with nucleotide sequences as shown in SEQ ID NO:2 and SEQ ID NO:4 and a probe as shown in SEQ ID NO:4.

[0011] In a third aspect, the present application provides a LAMP primer set, which is used to specifically amplify at least one of the aforementioned gene markers, and the LAMP primer set includes the following primers:

[0012] F3: nucleotide sequence as shown in SEQ ID NO:5;

[0013] B3: nucleotide sequence as shown in SEQ ID NO:6;

[0014] FIP: nucleotide sequence as shown in SEQ ID NO:7;

[0015] BIP: nucleotide sequence as shown in SEQ ID NO:8;

[0016] LF: nucleotide sequence as shown in SEQ ID NO:9;

[0017] LB: The nucleotide sequence is shown in SEQ ID NO:10.

[0018] In a fourth aspect, the present application provides a kit comprising the aforementioned TaqMan primer probe set and / or LAMP primer set.

[0019] In a fifth aspect, the present application provides a method for simultaneously detecting at least 6 bacteria including Bacillus cereus, Coxiella rosea, Micrococcus, Coxiella rhizophila, Escherichia coli, and Staphylococcus aureus, wherein the method is TaqMan PCR and uses the aforementioned TaqMan primer probe set.

[0020] In a sixth aspect, the present application provides another method for simultaneously detecting at least 6 bacteria including Bacillus cereus, Coxiella rosea, Micrococcus, Coxiella rhizophila, Escherichia coli, and Staphylococcus aureus, wherein the method is LAMP and uses the aforementioned LAMP primer set.

[0021] In a seventh aspect, the present application also provides a recombinant plasmid, wherein the target gene fragment of the recombinant plasmid is the nucleotide sequence of the aforementioned gene marker.

[0022] In an eighth aspect, the present application provides the use of the aforementioned gene markers in the preparation of multi-bacteria detection products.

[0023] The markers, TaqMan primer probe sets, LAMP primer sets, kits, detection methods, recombinant plasmids and applications provided in this application for simultaneous detection of multiple bacteria have at least the following beneficial effects compared with the prior art:

[0024] 1. The gene markers, TaqMan primer probe sets, and LAMP primer sets provided in this application for detection have strong specificity and high sensitivity, which are conducive to improving detection accuracy, and the detection method is simple, time-saving, and low in detection cost. The TaqMan primer probe set and LAMP primer set have good compatibility effects when used in bacterial detection methods.

[0025] 2. The kit product provided in this application can simultaneously detect six types of bacteria, including Bacillus cereus, Coxsackie syndrome, Micrococcus, Coxsackie syndrome, Escherichia coli, and Staphylococcus aureus, and has the advantages of good sensitivity, high accuracy, and a wide linear reporting range. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The colony morphology of 6 common laboratory bacteria screened out in the examples of this application are Bacillus cereus, Coxsackie rosea, Micrococcus, Coxsackie rhizophila, Escherichia coli, and Staphylococcus aureus.

[0027] Figure 2 This is the TaqMan fluorescent quantitative PCR standard curve drawn for the example of this application.

[0028] Figure 3 This is the amplification result of TaqMan PCR compatibility detection in the embodiment of this application.

[0029] Figure 4 This is the sensitivity test result of TaqMan PCR in the example of this application.

[0030] Figure 5 This is the result of LAMP compatible detection amplification in the embodiment of the present application.

[0031] Figure 6This is the sensitivity result of LAMP detection of simulated samples in the embodiment of the present application.

[0032] Figure 7 This is the sensitivity result of the TaqMan PCR detection simulation sample in the embodiment of this application. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0034] Those skilled in the art will appreciate that, unless otherwise stated, the "said", "the", and "the aforementioned" used in the text of the present application may also include plural forms. It should be further understood that the term "comprising" used in the specification of the present application means the presence of the features, steps, and operations, but does not exclude the presence or addition of one or more other features, integers, and steps.

[0035] In the text of this application, the terms "first", "second" and the like are used to distinguish between identical or similar items with substantially the same functions and effects. This is only for the purpose of clearly describing the technical solutions of the embodiments of this application, and shall not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features.

[0036] Those skilled in the art will appreciate that the experimental methods in the examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0037] The present application example establishes two methods for rapid detection of common laboratory bacteria based on 16S rRNA gene. Six common laboratory bacteria, including Bacillus cereus, Coxiella rosea, Micrococcus, Coxiella rhizophila, Escherichia coli, and Staphylococcus aureus, are obtained by separation and purification of common laboratory bacteria. Homology comparison and analysis are performed based on the gene sequences of the known 16S rRNA genes of each bacterium in GenBank, and highly conserved regions are selected to design a pair of degenerate primers and a degenerate probe for TaqMan PCR detection. LAMP degenerate primers are designed for detection using LAMP online primer design software Primer Explorer V5 (http: / / primerexplorer.jp / e / index.html), and the two methods are verified for their compatibility using genomic DNA of six common laboratory bacteria, Coxiella rosea, Micrococcus, Coxiella rhizophila, Bacillus cereus, Escherichia coli, and Staphylococcus aureus. This highly conserved region can be used as a marker for detecting the above six bacteria. The final experimental results of TaqMan PCR and LAMP methods showed that the marker sequence, TaqMan primer probe set, and LAMP primer set can be used for the detection of six bacteria, with the characteristics of good detection accuracy, high sensitivity, and fast detection speed.

[0038] Based on this, an embodiment of the present application provides a genetic marker for detecting multiple bacteria, which is used to simultaneously detect at least 6 bacteria including Bacillus cereus, Coxsackie syndrome, Micrococcus, Coxsackie syndrome, Escherichia coli, and Staphylococcus aureus, wherein the genetic marker is selected from the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence derived from the 16S rRNA gene in Bacillus cereus and having more than 95% homology with SEQ ID NO: 1. In the present embodiment, six bacteria, namely, Bacillus cereus, Coxsackia rosea, Micrococcus, Coxsackia rhizophila, Escherichia coli, and Staphylococcus aureus, were screened out from common bacteria in the laboratory. The DNAMAN V6 software was used to perform homology comparison analysis on the gene sequences of the 16S rRNA genes of the six bacteria in GenBank, and a highly conserved region of the 16S rRNA gene of Bacillus cereus was screened out. The sequence is shown in SEQ ID NO: 1, and the sequence has a high homology in the 16S rRNA genes of the other five bacteria. Therefore, the nucleotide sequence shown in SEQ ID NO: 1 can be used as a marker for simultaneously detecting at least the above six bacteria. At the same time, the marker can also be a nucleotide sequence derived from a highly conserved region of the 16S rRNA gene of Bacillus cereus and having a homology of more than 95% with SEQ ID NO: 1, preferably a nucleotide sequence having a homology of more than 98% with SEQ ID NO: 1, and more preferably a nucleotide sequence having a homology of more than 99% with SEQ ID NO: 1.

[0039] In certain embodiments, the nucleotide sequence derived from a highly conserved region of the 16S rRNA gene of Bacillus cereus and having homology to the SEQ ID NO:1 can be expressed as a nucleotide sequence having codon degeneracy with the nucleotide sequence shown in SEQ ID NO:1, i.e., a nucleotide sequence obtained by synonymously replacing the nucleotide sequence shown in SEQ ID NO:1, which can also be used as a marker for simultaneously detecting at least the above 6 bacteria.

[0040] Based on this, the embodiment of the present application provides a TaqMan primer probe set, which is used to specifically amplify at least one of the aforementioned gene markers, and the TaqMan primer probe set includes primers with nucleotide sequences such as SEQ ID NO: 2 and SEQ ID NO: 3 and a probe as shown in SEQ ID NO: 4. Through these primers and probes, SEQ ID NO: 1 or a homologous nucleotide sequence thereof can be specifically amplified and detected, thereby realizing the detection of at least the above 6 bacteria at the same time.

[0041] Based on this, an embodiment of the present application provides a LAMP primer set, which is used to specifically amplify at least one of the aforementioned gene markers, and the LAMP primer set includes the following three groups of primers: the first group of primers are external primers, including F3: the nucleotide sequence is shown in SEQ ID NO:5, B3: the nucleotide sequence is shown in SEQ ID NO:6; the second group of primers are internal primers, including FIP: the nucleotide sequence is shown in SEQ ID NO:7, BIP: the nucleotide sequence is shown in SEQ ID NO:8; the third group of primers are loop primers, including LF: the nucleotide sequence is shown in SEQ ID NO:9, LB: the nucleotide sequence is shown in SEQID NO:10.

[0042] Based on this, the embodiment of the present application provides a kit, which includes the aforementioned TaqMan primer probe set and / or LAMP primer set. In these kits, the TaqMan primer probe set and LAMP primer set contained therein can independently realize the simultaneous and rapid detection of six bacteria, namely Bacillus cereus, Coxiella rosea, Micrococcus, Coxiella rhizophila, Escherichia coli, and Staphylococcus aureus. The specific judgment principle is that when the test result is positive, it is determined that at least one of the above six bacteria exists; when the test result is negative, it indicates that at least one of the above six bacteria does not exist.

[0043] Based on this, an embodiment of the present application provides a method for simultaneously detecting at least 6 bacteria including Bacillus cereus, Coxiella rosea, Micrococcus, Coxiella rhizophila, Escherichia coli, and Staphylococcus aureus. The method is TaqMan PCR and uses the aforementioned TaqMan primer probe set.

[0044] Based on this, the embodiments of the present application also provide another method for simultaneously detecting at least 6 bacteria including Bacillus cereus, Coxiella rosea, Micrococcus, Coxiella rhizophila, Escherichia coli, and Staphylococcus aureus. The method is LAMP and uses the aforementioned LAMP primer set.

[0045] The above two detection methods can be used alone or in combination. TaqMan PCR can realize quantitative detection of bacteria, and its cost is lower than LAMP method, which is a qualitative bacteria detection method with fast detection speed and low detection cost. The appropriate detection method can be selected according to the actual situation.

[0046] Based on this, an embodiment of the present application further provides a recombinant plasmid, wherein the target gene fragment of the recombinant plasmid is the nucleotide sequence of the aforementioned gene marker.

[0047] Based on this, the embodiments of the present application provide the use of the aforementioned gene markers in the preparation of multi-bacteria detection products.

[0048] The technical solution of the present application and the technical effects achieved are described in detail below through more specific embodiments.

[0049] 1. Isolation and purification of common bacteria in the laboratory

[0050] Place LB culture plates in different corners of the laboratory, collect bacteria, and culture at 37°C overnight. Use a sterile pipette to pick up a single colony and inoculate it into LB liquid culture medium. Culture and shake at 37°C and 220 rpm for 12 to 14 hours, then take 1 ml of the bacterial solution and send it to Beijing Qingke Biotechnology Co., Ltd. (Wuhan Branch) for strain identification.

[0051] Finally, the colony morphology of six common laboratory bacteria, including Bacillus cereus, Coxsackie rosea, Micrococcus, Coxsackie rhizophila, Escherichia coli, and Staphylococcus aureus, was screened out. Figure 1 shown.

[0052] 2. Primer Design and Synthesis

[0053] According to the principle of primer design, a pair of degenerate primers was designed for the sequence shown in SEQ ID NO: 1 using the primer design software Primer 5.0:

[0054] 16S rRNA-F: nucleotide sequence as shown in SEQ ID NO: 2;

[0055] 16S rRNA-R: nucleotide sequence as shown in SEQ ID NO: 3;

[0056] The degenerate primers were synthesized by Beijing Zixi Biotechnology Co., Ltd.

[0057] 3. Design and Synthesis of Probes

[0058] A degenerate probe was designed in the middle of the above degenerate primer using Oligo 7 software: TaqMan Probe: The nucleotide sequence is shown in SEQ ID NO: 4, the 5' end of the probe is labeled with a FAM fluorescent group, and the 3' end is labeled with a MGB quenching group. The degenerate probe was synthesized by Beijing Zixi Biotechnology Co., Ltd.

[0059] 4. Extraction of Bacterial Genomes

[0060] Referring to the instructions, the FastPure Blood / Cell / Tissue / Bacteria DNAIsolation MiniKit extraction kit was used to extract the DNA genomes of the above 6 bacteria, and the NanoDropTM One / OneC micro-volume UV-Vis spectrophotometer was used to detect the concentration and purity (Note: when the bacterial solution is G- bacterial solution, the genome extraction step can be carried out according to the instructions in the kit; when the bacterial solution is G+ bacterial solution, the bacteria must first be treated with lysozyme at 37°C for 30 minutes when extracting the genome, and then follow the instructions in the kit).

[0061] 5. Construction of positive plasmid and drawing of standard curve

[0062] The conserved gene fragment of 16S rRNA gene (nucleotide sequence as the target fragment shown in SEQ ID NO: 1) was obtained by PCR amplification, amplified by high-purity Taq DNA polymerase and purified by PCR product. The target fragment was connected to the vector using pMD19-T and transferred into Escherichia coli DH5α for amplification and identification. The concentration of the extracted recombinant positive plasmid was 393.2 ng / μL by ultra-micro spectrophotometer. The copy number of the recombinant positive plasmid was calculated using the following formula: copy number (copies / μL) = 6.02×10 23 × positive plasmid concentration / MW, where MW is the molecular weight of the positive plasmid, which can be obtained by multiplying the number of bases by the average base molecular weight. The final calculated positive plasmid copy number is 1.23×10 11 The positive plasmid was diluted 10-fold in a gradient manner, and 6 dilution concentrations were selected, with the concentrations being 5.7×10 6 , 5.7×10 5 , 5.7×10 4 , 5.7×10 3 , 5.7×10 2 , 1.14×10 2 Each dilution concentration was repeated 3 times, and a standard curve was drawn according to the cycle threshold value (Ct value).

[0063] 6.qPCR detection

[0064] The StepOnePlusTM real-time fluorescence quantitative PCR system (Thermo Fisher Scientific, USA) was used for qPCR, and the quantitative reagent used was Taq Pro U+Multiple Probe qPCR Mix (Novozyme Biotech, Inc.). The reaction system is shown in Table 1:

[0065] Table 1

[0066] Reaction system volume 2×Taq Pro U+Multiple Probe qPCR Mi 10μL 10 μmol / L upstream primer 1μL 10 μmol / L downstream primer 1μL 10μmol / L probe 0.5μL Template (positive plasmid) 5μL <![CDATA[ddH2O]]> Make up to 20 μL

[0067] The operating program was as follows: contamination digestion at 37°C for 2 min; pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, extension at 72°C and fluorescence detection for 30 s, for a total of 42 cycles.

[0068] 7. qPCR compatibility, sensitivity (LOD), and lower limit of quantification (LLOQ) testing

[0069] Compatibility test: The genomic DNA of common bacteria in the laboratory was extracted using a bacterial genomic DNA extraction kit and used as a template for fluorescent quantitative PCR reaction. A negative control was set up and fluorescent quantitative PCR was performed using the optimal reaction conditions to evaluate its compatibility.

[0070] Sensitivity test: The positive plasmid was diluted 10 times in a gradient from 5.7×10 6 The copies / μL was diluted to 5.7×10 copies / μL, and 5.0 μL was taken as template for detection using the qPCR system to determine the minimum detection amount of the reaction. ddH2O was used as template as a negative control.

[0071] Quantitative lower limit detection: The working concentration is 1.14×10 2 The lower limit of quantification was determined by measuring the number of copies / μL of the standard plasmid.

[0072] 8.LAMP primer design and reaction system

[0073] According to the conserved gene fragment of 16S rRNA gene (nucleotide sequence as shown in SEQ ID NO: 1), a set of compatible LAMP primers was designed, including two external primers: F3: nucleotide sequence as shown in SEQ ID NO: 5, B3: nucleotide sequence as shown in SEQ ID NO: 6; two internal primers, including FIP: nucleotide sequence as shown in SEQ ID NO: 7, BIP: nucleotide sequence as shown in SEQ ID NO: 8; two loop primers, including LF: nucleotide sequence as shown in SEQ ID NO: 9, LB: nucleotide sequence as shown in SEQ ID NO: 10; the primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. (Wuhan Branch).

[0074] Prepare 20 μL of the reaction system as shown in Table 2 below:

[0075] Table 2

[0076] Reaction system volume HS-Bst4.0LyoMixB 7.5μL HS-Bst4.0LyoMixA 1μL 10×LAMP Primer Mix 2μL 50×LHNB Dye 0.4μL Template (positive plasmid) 9.1μL

[0077] The composition of 10×LAMP Primer Mix reagent (100 μL) is shown in Table 3:

[0078] Table 2

[0079] composition volume Primer F3 2μL Primer B3 2μL Primer FIP 16μL Primer BiP 16μL Primer LF 4μL Primer LB 4μL <![CDATA[ddH2O]]> 56μL

[0080] The above reaction system was mixed and reacted at 65°C for 45 minutes. The LHNB dye in the product was mixed with Mg before the reaction started. 2+ The combination produces a deep violet color. As the LAMP reaction proceeds, the generated PPi binds to the Mg in the system. 2+ , making the LHNB appear light green.

[0081] 9. LAMP compatibility test

[0082] The established LAMP method was used to amplify common bacteria in the laboratory, with ddH2O as a negative control, and the compatibility of the method was verified by observing the reaction color.

[0083] 10. Simulated sample testing

[0084] Extract genomic DNA from 1 mL of Bacillus cereus culture, elute with 50 μL Elution Buffer, then take 5 μL of genomic DNA and dilute it by 10 times. Use DNA with different dilutions as template for LAMP sensitivity detection. At the same time, perform TaqMan PCR test with the same template to compare the difference in sensitivity between TaqMan PCR and LAMP methods.

[0085] result

[0086] (1) Preparation of positive plasmid standard curve: Figure 2 is the standard curve of TaqMan fluorescence quantitative PCR. As can be seen from the figure, the standard curve has a good linear relationship, with a slope of -3.4271 and a correlation coefficient R 2 =0.9992, the standard curve equation is Y=-3.4271X+42.71, where Y is the Ct value of the amplified sample, and X is the starting copy number of the sample DNA. Substituting the Ct value of the sample into the formula can calculate the starting copy number of the sample DNA, and the quantitative detection of the sample can be achieved.

[0087] (2) TaqMan PCR compatibility test: The genomic DNA of six common laboratory bacteria, Escherichia coli, Staphylococcus aureus, Coxsackie syndrome, Micrococcus, Coxsackie rhizophila, and Bacillus cereus, were used as templates for fluorescence quantitative PCR reaction, and negative controls were set up for fluorescence quantitative PCR detection. The test results were as follows: Figure 3 As shown, from Figure 3It can be seen that the six bacterial base tests were all positive, and the negative control test results were negative, indicating that the method has good specificity.

[0088] (3) TaqMan PCR sensitivity test: Fluorescence quantitative PCR test was performed using standard plasmids of different dilutions as templates. The results are shown in the following table. Figure 4 As shown, from Figure 4 It can be seen that the fluorescence quantitative PCR results showed that the minimum detection amount was 5.7×10 copies / μL.

[0089] (4) TaqMan PCR standard curve quantitative lower limit detection: Fluorescence quantitative PCR detection was performed using the standard mass of each dilution as a template. 2 When the standard sample of 100 copies / μL was tested, its deviation = (actual copy number-theoretical copy number) / theoretical copy number was 9.18%, and the coefficient of variation (standard deviation / average CT value) was 0.4% (as shown in Table 3 below), indicating that the fluorescent quantitative PCR detection using TaqMan PCR primers and probes has good accuracy and stability.

[0090] Table 3

[0091]

[0092] (5) LAMP compatibility test: Using the established LAMP detection system, LAMP amplification was performed with genomic DNA of different bacterial species at the optimal temperature. The amplification results were as follows: Figure 5 As shown, all six bacterial samples had obvious positive results, while the negative control had no color change, which indicated that the established LAMP detection method had good compatibility.

[0093] (6) Simulated sample detection: The genomic DNA of Bacillus cereus was diluted to different concentrations and amplified using the LAMP detection method. As the template concentration decreased, the dilution was 10 -8 No color change (such as Figure 6 The TaqMan PCR method was used to amplify templates of different dilution concentrations. The results showed that at a dilution of 10 -10 Positive test results can still be obtained (eg Figure 7 As shown), it is shown that the TaqMan PCR primers and probes provided in the examples of the present application have a higher detection sensitivity for the above 6 common bacteria in laboratories.

[0094] In summary, the marker sequence provided in the present application and the TaqMan primer probe set and LAMP primer designed based on the marker sequence can be used to detect at least 6 common laboratory bacteria including Bacillus cereus, Coxiella rosea, Micrococcus, Coxiella rhizophila, Escherichia coli, and Staphylococcus aureus, and were verified by TaqMan PCR and LAMP detection methods, both showing high sensitivity and high accuracy.

[0095] The present application is described in detail above. Specific examples are used in this article to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the present application and its core ideas. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. A gene marker for detecting multiple bacteria, characterized in that: The gene marker is used to simultaneously detect at least six types of bacteria including Bacillus cereus, Coxsackia rosea, Micrococcus, Coxsackia rhizophila, Escherichia coli, and Staphylococcus aureus, and the gene marker is selected from at least one of the following: (1) the nucleotide sequence shown in SEQ ID NO: 1; (2) A nucleotide sequence derived from the 16S rRNA gene of Bacillus cereus and having more than 95% homology with SEQ ID NO:

1.

2. A TaqMan primer probe set, characterized in that: The TaqMan primer probe set is used to specifically amplify at least one of the gene markers described in claim 1, and the TaqMan primer probe set includes primers with nucleotide sequences as shown in SEQ ID NO: 2 and SEQ ID NO: 3 and a probe as shown in SEQ ID NO:

4.

3. A LAMP primer set, characterized in that: The LAMP primer set is used to specifically amplify at least one of the gene markers in claim 1, and the LAMP primer set includes the following primers: F3: nucleotide sequence as shown in SEQ ID NO:5; B3: nucleotide sequence as shown in SEQ ID NO:6; FIP: nucleotide sequence as shown in SEQ ID NO:7; BIP: nucleotide sequence as shown in SEQ ID NO:8; LF: nucleotide sequence as shown in SEQ ID NO:9; LB: The nucleotide sequence is shown in SEQ ID NO:

10.

4. A kit, characterized in that: It comprises the TaqMan primer probe set according to claim 2 and / or the LAMP primer set according to claim 3.

5. A method for simultaneously detecting six bacteria, namely, Bacillus cereus, Coxsackie spp., Micrococcus, Coxsackie rhizophila, Escherichia coli, and Staphylococcus aureus, characterized in that: The method is TaqMan PCR, which uses the TaqMan primer probe set described in claim 2.

6. A method for simultaneously detecting six bacteria, namely, Bacillus cereus, Coxsackie spp., Micrococcus, Coxsackie rhizophila, Escherichia coli, and Staphylococcus aureus, characterized in that: The method is LAMP, and the LAMP primer set according to claim 3 is used.

7. A recombinant plasmid, characterized in that: The target gene fragment of the recombinant plasmid is the nucleotide sequence of the gene marker according to claim 1.

8. Use of the gene marker according to claim 1 in the preparation of a multi-bacteria detection product.

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