Primer probe group for detecting bacillus thermoamyloliquefaciens, kit and application
By designing a primer probe set for specific genes of Bacillus atherosclerosis and combining with fluorescence quantitative PCR detection methods, the problem of difficulty in detecting the bacteria quickly and accurately in the prior art is solved, and the detection effect of high specificity, stability and sensitivity is achieved.
Patent Information
- Application Number
- CN202311407255.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-26
- Publication Date
- 2025-04-29
AI Technical Summary
The prior art is difficult to detect Bacillus thermophage quickly and accurately, especially in food and dairy products.
A primer probe set was designed to target genes in the genome of Bacillus amylophagecta that are responsible for encoding cytosine methyltransferase, and combined with fluorescence quantitative PCR detection methods to achieve specific detection of the bacteria.
It realizes rapid and accurate detection of Bacillus thermophage, has high specificity, stability and sensitivity, and is suitable for food testing.
Smart Images
Figure CN119899902A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a primer probe set, a kit and application for detecting thermomylophagic Bacillus, and relates to the technical field of food detection. Background Art
[0002] Thermomylophagic Bacillus is a Gram-negative bacterium of the genus Bacillus, which has extremely strong heat resistance. It can survive not only in food raw materials and raw milk, but also in sterilized finished milk and other dairy products. Thermomylophagic Bacillus can produce lipolytic enzymes and β-galactosidase, which may cause corruption and acidification of dairy products. Therefore, it is particularly important to detect Thermomylophagic Bacillus. How to quickly and accurately detect Thermomylophagic Bacillus has become a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention
[0003] The present invention designs a primer probe set for the gene encoding cytosine methyltransferase in the thermomylophagous Bacillus genome, and establishes a fluorescent quantitative PCR detection method based on the primer probe set, which can achieve specific detection of thermomylophagous Bacillus with good repeatability and high sensitivity.
[0004] The present invention also provides a kit comprising the primer probe set, and application of the primer probe set and the kit in detecting thermoamylophagous Bacillus.
[0005] A first aspect of the present invention provides a primer probe set for detecting thermomylophagic Bacillus, comprising a first primer, a second primer and a probe;
[0006] The nucleotide sequence of the first primer is shown in SEQ ID NO: 1;
[0007] The nucleotide sequence of the second primer is shown in SEQ ID NO: 2;
[0008] The nucleotide sequence of the probe is shown in SEQ ID NO:3.
[0009] In a specific embodiment, the 5' end of the probe is connected to a fluorescent reporter group, and the 3' end of the probe is connected to a fluorescent quencher group.
[0010] In a specific embodiment, the fluorescent reporter group is FAM, and the fluorescent quencher group is NFQ.
[0011] The second aspect of the present invention provides use of any of the above-mentioned primer probe sets in the preparation of a product for detecting thermoamylophagous Bacillus.
[0012] The third aspect of the present invention provides a kit for detecting thermomylophagic Bacillus, comprising any one of the primer probe sets described above.
[0013] In a specific embodiment, the kit further comprises a positive standard and a blank control;
[0014] The positive standard is a recombinant plasmid containing a target gene, and the GeneID of the target gene is 72730835; the blank control is enzyme-free water.
[0015] A fourth aspect of the present invention provides use of the primer probe set or any of the above-mentioned kits in detecting thermoamylophagous Bacillus.
[0016] A fifth aspect of the present invention provides a method for detecting thermomylophagic Bacillus, comprising the following steps:
[0017] Step 1, extracting DNA from the sample to be tested;
[0018] Step 2: Perform PCR amplification on the DNA of the sample to be tested using any of the primer probe sets described above or any of the kits described above. After the amplification, obtain the Ct value of the sample to be tested according to the amplification curve; at the same time, perform PCR amplification on the positive standard and the blank control using the same method. After the amplification, obtain the Ct value of the positive standard and the Ct value of the blank control according to the amplification curve;
[0019] Step 3: When the Ct value of the positive standard is ≤35 and the Ct value of the blank control is >35, the amplification result of the sample to be tested is judged to be valid;
[0020] When the Ct value of the sample to be tested is ≤35, it is determined that the sample to be tested contains thermomylophagic Bacillus, and when the Ct value is greater than 35, it is determined that the sample to be tested does not contain thermomylophagic Bacillus.
[0021] In a specific embodiment, the method further comprises: drawing a standard curve, and substituting the Ct value of the sample to be tested into the standard curve for calculation to obtain the concentration of Bacillus amylophagous in the sample to be tested, specifically comprising the following steps:
[0022] The target gene was amplified using the DNA of Bacillus thermomylovora as a template, and the GeneID of the target gene was 72730835;
[0023] The target gene is connected to a plasmid to construct a recombinant plasmid, the recombinant plasmid that has been sequenced correctly is configured to obtain a series of standard products with a concentration gradient, and the copy number of the recombinant plasmid in the standard product is calculated;
[0024] Using any of the above-mentioned primer probe sets or any of the above-mentioned kits to perform PCR amplification on a series of concentration gradient standard products respectively, and after the amplification, obtaining Ct values corresponding to the series of concentration gradient standard products according to the amplification curve;
[0025] A standard curve was obtained by plotting the Ct value as the ordinate and the logarithmic value of the copy number of the recombinant plasmid as the abscissa.
[0026] In a specific embodiment, the conditions for PCR amplification are: pre-denaturation stage: 95°C, 3 minutes; denaturation: 95°C, 5 seconds; annealing and extension: 60°C, 40 seconds; repeating the denaturation to annealing and extension steps for a total of 40 cycles.
[0027] The primer probe set provided by the present invention and the fluorescent quantitative PCR detection method established based on the primer probe set have high specificity and good stability, and the sensitivity can reach 16.7pg / μL, which is suitable for the detection of thermomylophagic Bacillus. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.
[0029] Figure 1 The standard curve is obtained by PCR amplification of a series of recombinant plasmid standards with a concentration gradient;
[0030] Figure 2 This is a graph showing the specificity of the primer-probe set provided by the present invention;
[0031] Figure 3 This is a graph showing the experimental results of the sensitivity of the primer probe set provided by the present invention to Bacillus thermomylovora;
[0032] Figure 4 This is a graph showing the repeatability experimental results of the primer probe set provided by the present invention on thermoamylophagous Bacillus. DETAILED DESCRIPTION
[0033] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0034] In the present invention, "fluorescence quantitative PCR" refers to a method in which a fluorescent group is added to the PCR amplification reaction system, and the fluorescent signal of each cycle product in the amplification reaction is detected in real time, and finally the unknown template is quantitatively analyzed through a standard curve. Take the probe method fluorescence quantitative PCR as an example: during PCR amplification, a specific fluorescent probe is added at the same time as a pair of primers, and a fluorescent reporter group and a fluorescent quencher group are respectively labeled at both ends of the probe. At the beginning, the probe is completely bound to any single strand of DNA, and the fluorescent signal emitted by the reporter group is absorbed by the quencher group, and no fluorescent signal can be detected; during PCR amplification, the Taq enzyme cleaves and degrades the probe, separating the reporter fluorescent group and the quencher fluorescent group, so that the fluorescent monitoring system can receive the fluorescent signal, that is, every time a DNA chain is amplified, a fluorescent molecule is formed, so that the accumulation of the fluorescent signal is completely synchronized with the formation of the PCR product.
[0035] In the present invention, "Ct value" represents the cycle threshold of the sample, that is, the corresponding cycle number after the fluorescence signal of the product amplification during the PCR process reaches the set threshold. For example, Ct=19 means that the product amplified to the 19th cycle reached the set fluorescence threshold.
[0036] In the present invention, "FAM" is a fluorescent reporter group, which can be excited to an excited state when exposed to ultraviolet light or blue-violet light, and emit fluorescence when returning to the ground state from the excited state. Those skilled in the art can connect it to the 5' end of the probe according to conventional technical means.
[0037] In the present invention, "NFQ" is a non-fluorescent quenching group, which is used to quench the fluorescent signal generated by the fluorescent reporter group. Those skilled in the art can connect it to the 3' end of the probe according to conventional technical means.
[0038] In the present invention, "MGB" is a binder that can be inserted into the minor groove of DNA. MGB can stabilize the hybridization probe and increase the melting temperature (Tm). Those skilled in the art can connect it to the 3' end of the probe according to conventional technical means.
[0039] Example 1
[0040] In this example, the gene encoding cytosine methyltransferase rsmI (GeneID: 72730835) was used as the target fragment, and upstream and downstream primers and TaqMan probes were designed, as shown in Table 1.
[0041] Table 1 Primer probe set provided in Example 1
[0042]
[0043] In Table 1, the TaqMan probe shown in Sequence 3 has a fluorescent reporter group FAM connected to its 5' end and NFQ and MGB connected to its 3' end.
[0044] Example 2
[0045] This embodiment provides a detection method for thermomylophagic Bacillus based on the qPCR method, which specifically includes the following steps:
[0046] 1. Extract the microbial genomic DNA of the sample to be tested. The sample to be tested can be raw milk, finished milk, dairy products, and other foods to be tested. DNA extraction can be performed using a conventional DNA extraction kit in the art.
[0047] For example, the bacteria / fungus / yeast DNA extraction kit may be Lifefeng DK621-01, and the food DNA extraction kit may be Magen DJL17-01.
[0048] A positive standard and a blank control were prepared, wherein the positive standard was a plasmid containing the target fragment, and the blank control was enzyme-free water.
[0049] 2. Perform PCR amplification reaction on the test samples, positive standard and blank control prepared in step 1 respectively.
[0050] 2.1. Primers: The primer probe set provided in Example 1 of the present invention was used.
[0051] 2.2. PCR reaction system: The PCR reaction system was prepared according to the configuration shown in Table 2.
[0052] Table 2 PCR reaction system
[0053]
[0054]
[0055] 2.3 PCR amplification conditions: pre-denaturation: 95°C, 3 min; denaturation: 95°C, 5 sec; annealing and extension: 60°C, 40 sec; repeat denaturation to annealing and extension steps for a total of 40 cycles.
[0056] 3. Interpretation of results: After the PCR is completed, the cycle curves of the sample to be tested, the positive standard, and the blank control are obtained, and the corresponding Ct value is obtained according to the cycle curve. When the Ct value of the positive standard is ≤35 and the Ct value of the blank control is >35, it means that the amplification curve of the sample to be tested is valid; when the Ct value of the sample to be tested is ≤35, it is determined that the sample to be tested contains Bacillus amyloliquefaciens, and when the Ct value is >35, it is determined that the sample to be tested does not contain Bacillus amyloliquefaciens.
[0057] In order to achieve the quantitative detection of thermomylophagous Bacillus, this embodiment also constructs a standard curve to achieve the quantitative detection of thermomylophagous Bacillus in the sample to be tested, which specifically includes the following steps:
[0058] 1. Preparation of standard: Using DNA extracted from thermoamylophagous Bacillus as a template, design primers and amplify the target fragment. After gel excision, recovery and purification of the PCR product, connect it to the pUC57 plasmid to construct a recombinant pUC57 plasmid; introduce the recombinant pUC57 plasmid into competent Escherichia coli cells, screen positive single colonies, extract the plasmid and sequence it to confirm its correctness.
[0059] 2. Calculate the copy number based on the plasmid concentration
[0060] The concentration of positive plasmid was determined by Bio-Drop ulite+ ultra-micro nucleic acid protein analyzer to be 94.862ng / uL, the base number of plasmid was 2694bp, the target band size was 92bp, the average molecular weight of each base was 660Da / bp, and the number of gene copies per microliter of sample was estimated according to the formula: sample copies / ul = Avogadro constant (6.02×10 23 )×plasmid concentration ng / ul×10 -9 / (660×number of bases in the recombinant plasmid), where the number of bases in the recombinant plasmid = the number of bases in the vector sequence + the number of bases in the inserted sequence.
[0061] According to this method, the target gene copy number was calculated to be 3.11×10 10 Copies / uL.
[0062] 3. Standard curve drawing
[0063] The recombinant plasmid was diluted to 10 10 Copies / ul~10 2 Copies / ul, and 9 concentration gradient standards were obtained. Real-time fluorescence quantitative PCR was performed according to the above PCR reaction system and amplification conditions. After the reaction was completed, the Ct value was obtained according to the amplification curve, and the Ct value was used as the ordinate and the logarithm of the number of recombinant plasmid copies was used as the abscissa to obtain the Figure 1 The standard curve is shown. Figure 1 It can be seen that when the recombinant plasmid concentration is 10 10 Copies / ul~10 2 Copies / ul, the logarithm of the plasmid copy number has a very good linear relationship with the Ct value, and the correlation coefficient R 2 =0.9997. Standard curve regression equation: y=-3.3405×Log(x)+43.656, amplification efficiency is 99.23%, y is Ct value, x is the copy number of plasmid standard.
[0064] according to Figure 1 The standard curve shown in the figure can be used to quantitatively detect Bacillus amylophagous by substituting the Ct value of the sample to be tested into the standard curve.
[0065] The specificity, sensitivity and repeatability of this method were tested below.
[0066] 1. Specificity experiment
[0067] The DNA extracted from the thermomylophagic Bacillus isolated and purified in the laboratory was used as the positive control, and the DNA extracted from the thermomylophagic Bacillus was used as the positive control. The DNA extracted from the thermomylophagic Bacillus was used as the positive control. The DNA extracted from the thermomylophagic Bacillus was used as the positive control. The DNA extracted from the thermomylophagic Bacillus was used as the positive control. The DNA extracted from the thermomylophagic Bacillus was used as the positive control. The DNA extracted from the thermomylophagic Bacillus was used as the positive control. The DNA extracted from the thermomylophagic Bacillus was used as the positive control. The DNA extracted from the thermomylophagic Bacillus was used as the positive control. The DNA extracted from the thermomylophagic Bacillus was used as the positive control. The DNA extracted from the thermomylophagic Bacillus was used as the positive control. The DNA extracted from the thermomylophagic Bacillus was used as the positive control. DNA extracts of Bacillus, Listeria monocytogenes, beta-hemolytic Streptococcus, Pseudomonas alcaligenes, Pseudomonas fluorescens, Candida parapsilosis, Rhodotorula colloidinum, Candida pseudopsilosis, Candida boidinii, Aspergillus niger, Aspergillus polydorum, Aureobasidium pullulans, Aspergillus fumigatus, Penicillium chrysogenum, Mucor circinelloides, Aspergillus terreus, Cladosporium buddingii, Penicillium globulum, and Candida albicans were used as negative controls, and ddH2O was used as blank control. Real-time fluorescence quantitative PCR was performed using the above-mentioned PCR detection method, and a total of three parallel experiments were designed.
[0068] The names and numbers of the above microorganisms are shown in Table 3.
[0069] Among them, the PCR amplification curve is as follows Figure 2 As shown, specific amplification signals were detected only in the reaction tube of Bacillus thermomylovora, and no fluorescent signals accumulated in other reaction tubes, indicating that this method has good specificity.
[0070] Table 3 Specificity test results
[0071]
[0072]
[0073]
[0074] 2. Sensitivity test
[0075] DNA was extracted from pure culture of Bacillus thermomylovora using a DNA extraction kit, and the DNA concentration was measured to be 167 ng / ml using the Bio-Dropulite+ ultra-micro nucleic acid protein analyzer. The DNA was diluted 10 times in a gradient manner with sterile water, and the serially diluted DNA was used as a template for PCR amplification to determine the sensitivity of the detection system.
[0076] The results are as follows Figure 3 As shown, the sensitivity of the designed primers is relatively high, reaching 0.0167 ng / μL.
[0077] 3. Repeatability experiment
[0078] The DNA standard plasmid was tested by quantitative PCR five times to analyze the repeatability. Figure 4 As shown, it can be seen that the detection method has good repeatability, and the repeated CV value is 0.057, which is less than 2%.
[0079] In summary, the primer probe set provided by the present invention and the fluorescent quantitative PCR detection method established based on the primer probe set have high specificity and good repeatability, and the sensitivity can reach 16.7pg / μL, which is suitable for the detection of thermomylophagic Bacillus.
[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A primer probe set for detecting thermomylophagic Bacillus, characterized in that: comprising a first primer, a second primer and a probe; The nucleotide sequence of the first primer is shown in SEQ ID NO: 1; The nucleotide sequence of the second primer is shown in SEQ ID NO: 2; The nucleotide sequence of the probe is shown in SEQ ID NO:
3.
2. The primer probe set according to claim 1, characterized in that: The 5' end of the probe is connected to a fluorescent reporter group, and the 3' end of the probe is connected to a fluorescent quencher group.
3. The primer probe set according to claim 2, characterized in that: The fluorescent reporter group is FAM, and the fluorescent quencher group is NFQ.
4. Use of the primer probe set according to any one of claims 1 to 3 in the preparation of a product for detecting thermoamylophagous Bacillus.
5. A kit for detecting thermomylophagic Bacillus, characterized in that: The invention comprises the primer-probe set according to any one of claims 1 to 3.
6. The kit according to claim 5, characterized in that The kit also includes a positive standard and a blank control; The positive standard is a recombinant plasmid containing a target gene, and the GeneID of the target gene is 72730835; the blank control is enzyme-free water.
7. Use of the primer-probe set according to any one of claims 1 to 3 or the kit according to any one of claims 5 to 6 in detecting Bacillus thermomylophagous.
8. A method for detecting thermomylophagic Bacillus, characterized in that: The steps include: Step 1, extracting DNA from the sample to be tested; Step 2, using the primer probe set described in any one of claims 1 to 3 or the kit described in any one of claims 5 to 6 to perform PCR amplification on the DNA of the sample to be tested, and after the amplification is completed, the Ct value of the sample to be tested is obtained according to the amplification curve; at the same time, the positive standard and the blank control are PCR amplified by the same method, and after the amplification is completed, the Ct value of the positive standard and the Ct value of the blank control are respectively obtained according to the amplification curve; Step 3: When the Ct value of the positive standard is ≤35 and the Ct value of the blank control is >35, the amplification result of the sample to be tested is judged to be valid; When the Ct value of the sample to be tested is ≤35, it is determined that the sample to be tested contains thermomylophagic Bacillus, and when the Ct value is greater than 35, it is determined that the sample to be tested does not contain thermomylophagic Bacillus.
9. The method according to claim 8, characterized in that The method further comprises: drawing a standard curve, and substituting the Ct value of the sample to be tested into the standard curve for calculation to obtain the concentration of Bacillus amylophagous in the sample to be tested, which specifically comprises the following steps: The target gene was amplified using the DNA of Bacillus thermomylovora as a template, and the GeneID of the target gene was 72730835; The target gene is connected to a plasmid to construct a recombinant plasmid, the recombinant plasmid that has been sequenced correctly is configured to obtain a series of standard products with a concentration gradient, and the copy number of the recombinant plasmid in the standard product is calculated; Using the primer probe set according to any one of claims 1 to 3 or the kit according to any one of claims 5 to 6, respectively, PCR amplification is performed on a series of standard samples with a concentration gradient, and after the amplification, the Ct values corresponding to the series of standard samples with a concentration gradient are obtained according to the amplification curve; A standard curve was obtained by plotting the Ct value as the ordinate and the logarithmic value of the copy number of the recombinant plasmid as the abscissa.
10. The method according to claim 8 or 9, characterized in that: The conditions of the PCR amplification are: pre-denaturation stage: 95°C, 3 minutes; denaturation: 95°C, 5 seconds; annealing and extension: 60°C, 40 seconds; repeating the denaturation to annealing and extension steps for a total of 40 cycles.