Specific primers, probes and detection methods for real-time fluorescence quantitative PCR detection of residual host DNA in Escherichia coli

Abstract

The invention provides specific primers, probes and a detection method for real-time fluorescence quantitative PCR detection of residual Escherichia coli host DNA, and utilizes real-time fluorescence quantitative PCR (qPCR for short) technology to detect residual Escherichia coli host DNA.

Description

[0001] Reference to electronically submitted sequence listings

[0002] This application incorporates by reference the sequence listing of the file "230478-Sequence Listing-ST26.xml" with a size of 16,384 bytes, which was created on September 26, 2023 together with this application. 1. Technical Field

[0003] The invention relates to the field of molecular detection, and in particular to specific primers, probes and a detection method for detecting residual Escherichia coli host DNA using real-time fluorescence quantitative PCR. 2. Background technology

[0004] In recent years, with the rapid development of gene and cell therapy, plasmid DNA, as a carrier of cell and gene therapy, has been widely used in products such as DNA vaccine products, viral gene therapy products, cell gene therapy products, stem cell therapy products and mRNA. Residual DNA in host cells has been the focus of attention of domestic and foreign drug regulatory agencies because of its special potential safety risks and potential tumorigenicity and / or infectiousness. Therefore, Escherichia coli, as the most widely used and successful expression system, has received increasing attention as an important indicator for product process optimization and finished product release in DNA products. The high-sensitivity host DNA detection kits in Escherichia coli available on the market are expensive and have high experimental costs, both domestically and abroad. With the increase in the number of samples for the detection of residual host DNA in Escherichia coli, it has gradually become a trend to develop a low-cost detection kit for residual host DNA in Escherichia coli with the same detection accuracy. The present invention utilizes the commercial low-cost Escherichia coli genomic DNA reference materials and real-time fluorescence quantitative PCR premix available on the market, and designs multiple sets of specific primers and probes based on the most conserved 16s rRNA sequence in Escherichia coli, forming a set of detection methods that can quickly and low-costly detect Escherichia coli host DNA residues in DNA products.

[0005] In order to evaluate the residual amount of host cell DNA, there are currently DNA probe hybridization method, fluorescent dye method, DNA binding protein immune threshold method and real-time fluorescence quantitative PCR (Quantitative Real-time PCR, referred to as qPCR) method. At present, if the spot size is evaluated by naked eye, the DNA probe hybridization method can be semi-quantitatively determined. Only in combination with precision systems such as imagers can more accurate quantitative detection be achieved. The experimental operation is complicated, the method is not very stable, and the detection time is relatively long. The residual DNA amount of the fluorescent dye method is in the range of 1.25 to 80 ng / mL, with good linearity, but its sensitivity does not reach the 3pg / mL detection limit in this patent. At the same time, its fluorescent signal is easily interfered and has poor specificity. The DNA binding protein immune threshold method uses the non-specific sequence immunodetection technology of DNA antibodies, which cannot specifically identify the host residual DNA sequence, and is easily contaminated by the environment and the operator's DNA, resulting in high readings and inaccurate results. The qPCR method, which is the only standard method recommended by the United States Pharmacopeia (USP) as the host residual DNA in biological products, has the advantages of sequence specificity, sensitivity, accuracy and precision. 3. Summary of the invention

[0006] The present invention provides a forward qPCR primer and a reverse qPCR primer, wherein: the nucleic acid sequence of the forward qPCR primer is the nucleic acid sequence provided in SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:7; the nucleic acid sequence of the reverse qPCR primer is the nucleic acid sequence provided in SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8.

[0007] In certain embodiments, the present invention provides a nucleic acid-specific probe, the nucleic acid sequence of which is the nucleic acid sequence provided in SEQ ID NO:3, SEQ ID NO:6, or SEQ ID NO:9.

[0008] In certain embodiments, the present invention provides a forward qPCR primer, a reverse qPCR primer and a nucleic acid-specific probe, wherein the nucleic acid sequence of the forward qPCR primer is the nucleic acid sequence provided in SEQ ID NO: 1; the nucleic acid sequence of the reverse qPCR primer is the nucleic acid sequence provided in SEQ ID NO: 2; the nucleic acid sequence of the nucleic acid probe is SEQ ID NO: 3, or a nucleic acid sequence provided in a complementary sequence thereof.

[0009] In certain embodiments, the present invention provides a forward qPCR primer, a reverse qPCR primer and a nucleic acid-specific probe, wherein the nucleic acid sequence of the forward qPCR primer is the nucleic acid sequence provided in SEQ ID NO:4; the nucleic acid sequence of the reverse qPCR primer is the nucleic acid sequence provided in SEQ ID NO:5; the nucleic acid sequence of the nucleic acid probe is SEQ ID NO:6, or a nucleic acid sequence provided in a complementary sequence thereof.

[0010] In certain embodiments, the present invention provides a forward qPCR primer, a reverse qPCR primer and a nucleic acid-specific probe, wherein the nucleic acid sequence of the forward qPCR primer is the nucleic acid sequence provided in SEQ ID NO:7; the nucleic acid sequence of the reverse qPCR primer is the nucleic acid sequence provided in SEQ ID NO:8; the nucleic acid sequence of the nucleic acid probe is the nucleic acid sequence provided in SEQ ID NO:9, or a nucleic acid sequence provided in a complementary sequence thereof.

[0011] In certain embodiments, the nucleic acid probe is a nucleic acid-specific probe. In certain embodiments, the 5′ end of the nucleic acid-specific probe is modified with a first fluorescent group or a fluorescent quenching group. In certain embodiments, the 3′ end of the nucleic acid-specific probe is modified with a second fluorescent group or a fluorescent quenching group.

[0012] In certain embodiments, the 5′ end of the nucleic acid-specific probe is modified with a 6-carboxyfluorescein (6-FAM) fluorescent group. In certain embodiments, the 5′ end of the nucleic acid-specific probe is modified with a hexachloro-6-methylfluorescein (HEX) fluorescent group. In certain embodiments, the 5′ end of the nucleic acid-specific probe is modified with a tetrachloro-6-carboxyfluorescein (TET) fluorescent group. In certain embodiments, the 5′ end of the nucleic acid-specific probe is modified with a 2,7-dimethyl-4,5-dichloro-6-carboxyfluorescein (6-JOE) fluorescent group. In certain embodiments, the 5′ end of the nucleic acid-specific probe is modified with a 6-carboxytetramethylrhodamine (TAMRA) fluorescent group. In certain embodiments, the 5′ end of the nucleic acid-specific probe is modified with a 3H-indocyanine (Cy3) fluorescent group. In certain embodiments, the 5′ end of the nucleic acid-specific probe is modified with a 5H-indocyanine Cy5 fluorescent group. In certain embodiments, the 3′ end of the nucleic acid probe nucleic acid-specific probe is modified with a 6-carboxytetramethyl rhodamine (TAMRA) fluorescent group. In certain embodiments, the 3′ end of the nucleic acid-specific probe is modified with a black hole quencher 1 (Black Hole Quencher 1, BHQ1). In certain embodiments, the 3′ end of the nucleic acid-specific probe is modified with a black hole quencher 2 (Black HoleQuencher 2, BHQ2). In certain embodiments, the 3′ end of the nucleic acid-specific probe is modified with a black hole quencher 3 (BlackHole Quencher 1, BHQ3).

[0013] In certain embodiments, the present invention provides a qPCR component, comprising a forward qPCR primer and a reverse qPCR primer; wherein the nucleic acid sequence of the forward qPCR primer is a nucleic acid sequence provided in SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7; and the nucleic acid sequence of the reverse qPCR primer is a nucleic acid sequence provided in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. In certain embodiments, the qPCR component further comprises a nucleic acid probe, and the nucleic acid sequence of the nucleic acid probe is a nucleic acid sequence provided in SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 9.

[0014] In certain embodiments, the qPCR components further comprise genomic DNA derived from Escherichia coli (E. Coli) used as a positive control, the sequence comprising the nucleic acid sequence provided in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, and the genomic DNA of Escherichia coli (E. Coli) is derived from the reference sequence number GCF_000005845.2 of the National Center for Biotechnology Information (NCBI) of the United States.

[0015] In certain embodiments, the present invention provides a kit comprising any one of the qPCR components and instructions for use. 3.1 Description of the Figures

[0016] Figure 1 The figure shows the amplification curve of the specific embodiment 3 of the present invention when the primers and probes are used for amplification, and from left to right are the six concentration levels of standard solutions prepared for the positive control: the concentration of the standard solution 6 is: 300000 pg / mL; the concentration of the standard solution 5 is: 30000 pg / mL; the concentration of the standard solution 4 is: 3000 pg / mL; the concentration of the standard solution 3 is: 300 pg / mL; the concentration of the standard solution 2 is: 30 pg / mL; the concentration of the standard solution 1 is: 3 pg / mL amplification curve; wherein ΔRn represents the fluorescence increment, that is, the increment of the amplified product, and the cycle threshold (Cycle threshold, abbreviated Ct) represents the cycle number.

[0017] Figure 2 The standard curve of the specific real-time example 3 of the present invention using primers and probe amplification is shown, wherein the horizontal axis represents the logarithmic value of the concentration of the standard prepared from the positive control of genomic DNA from Escherichia coli (E.Coli), and the vertical axis represents the cycle threshold (Cycle threshold, abbreviated Ct) indicating the cycle number. 4. Specific implementation methods

[0018] This article provides specific primers, probes and detection methods for real-time fluorescence quantitative PCR detection of Escherichia coli host DNA residues. Implementation plan

[0019] 1. A forward qPCR primer and a reverse qPCR primer, wherein: The nucleic acid sequence of the forward qPCR primer is the nucleic acid sequence provided in SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 6; The nucleic acid sequence of the reverse qPCR primer is the nucleic acid sequence provided in SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8.

[0020] 2. A nucleic acid-specific probe, the nucleic acid sequence of which is the nucleic acid sequence provided in SEQ ID NO:3, SEQ ID NO:6, or SEQ ID NO:9.

[0021] 3. A forward qPCR primer, a reverse qPCR primer and a nucleic acid-specific probe, wherein: The nucleic acid sequence of the forward qPCR primer is the nucleic acid sequence provided by SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 6; The nucleic acid sequence of the reverse qPCR primer is the nucleic acid sequence provided in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8; The nucleic acid sequence of the nucleic acid probe is SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 9, or a nucleic acid sequence provided in a complementary sequence thereof.

[0022] 4. The nucleic acid-specific probe according to Embodiment 2, or the forward qPCR primer, reverse qPCR primer and nucleic acid probe according to claim 3, wherein the nucleic acid probe is a nucleic acid-specific probe, the 5′ end is modified with a first fluorescent group, and the 3′ end is modified with a second fluorescent group or a fluorescence quenching group.

[0023] 5. The nucleic acid-specific probe according to embodiment 2, or the forward qPCR primer, reverse qPCR primer and nucleic acid probe according to claim 3, wherein the nucleic acid probe is a nucleic acid-specific probe with a fluorescent group modified at the 5′ end such as 6-carboxyfluorescein (6-FAM), and a fluorescent group modified at the 3′ end. The end is modified with a fluorescent group such as 6-carboxytetramethylrhodamine (TAMRA) or a quenching group such as Black Hole Quencher 1 (BHQ1).

[0024] 6. A qPCR component comprising the forward qPCR primer defined in embodiment 1 or 3, and the reverse qPCR primer defined in embodiment 1 or 3.

[0025] 7. The qPCR components according to embodiment 6, further comprising the nucleic acid probe defined in embodiment 2-4, or 5.

[0026] 8. The qPCR components according to embodiment 6 or 7, further comprising genomic DNA derived from Escherichia coli (E. Coli) used as a positive control, the sequence of which comprises the nucleic acid sequence provided in SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

[0027] 9. A kit comprising the qPCR components according to any one of embodiments 6 to 8 and instructions for use.

[0028] 10. A kit comprising six concentration levels of standard solutions prepared by the positive control described in Embodiment 7, which are: The concentration of standard solution 1 is: 3pg / mL; The concentration of standard solution 2 is: 30 pg / mL; The concentration of standard solution 3 is: 300 pg / mL; The concentration of standard solution 4 is: 3000 pg / mL; The concentration of standard solution 5 is: 30000 pg / mL; The concentration of standard solution 6 is: 300000 pg / mL. 4.2. Definitions

[0029] Real-time fluorescence quantitative PCR refers to a method in which a fluorescent group is added to the PCR amplification reaction system, 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. Real-time fluorescence quantitative PCR refers to Quantitative Real-time PCR, abbreviated as qPCR.

[0030] ΔRn refers to the increase in fluorescence signal produced under specific qPCR conditions.

[0031] The cycle threshold, abbreviated as Ct, refers to the cycle number when the fluorescence signal reaches the set threshold during qPCR amplification.

[0032] The threshold generally refers to 10 times the fluorescence signal of 3 to 15 cycles of qPCR reaction and can be set at any position in the exponential amplification stage of the fluorescence signal.

[0033] Absorbance is optical density, abbreviated as OD. Absorbance is a term in physics and chemistry, which refers to the logarithm to the base 10 of the ratio of the incident light intensity before the light passes through a solution or a substance to the transmitted light intensity after the light passes through the solution or a certain substance. 4.3 Experiments

[0034] The examples provided below are for illustrative purposes only and are not intended to limit the present invention unless otherwise specified. Therefore, the present invention should not be considered limited to the following examples, but should be understood to include any and all obvious variations as a result of the teachings provided herein.

[0035] It should be understood that modifications that do not substantially affect the activity of the various embodiments of the invention may also be encompassed within the definition of the invention provided herein. Therefore, the following examples are intended to illustrate the invention but not to limit the invention. 4.3.1. Example 1: Preparation of Escherichia coli genomic DNA standards

[0036] The positive control E. coli genomic DNA standard can be derived from a commercial E. coli genomic DNA reference or prepared by a bacterial genomic DNA mini-purification kit, and the component is E. coli genomic DNA. The following is an example of Takara's MiniBEST Bacteria Genomic DNA Extraction Kit (Code No. 9763).

[0037] Take 0.5 mL of Luria-Bertani medium (LB medium) and culture overnight until the bacterial cell volume reaches 3-4 OD / mL. Transfer to a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 2 minutes, and discard the supernatant.

[0038] Add 180 μL of Buffer GL (from Takara kit, Code No. 9763), 20 μL of Proteinase K (20 mg / ml, from Takara kit, Code No. 9763) and 10 μL of RNase A (10 mg / ml, from Takara kit, Code No. 9763), shake well and incubate at 56°C. Incubate in a water bath for 10 minutes, at which point the solution should be transparent and clear.

[0039] Add 200 μL of Buffer GB (from Takara kit, Code No. 9763) and 200 μL of 100% ethanol, and mix thoroughly by pipetting.

[0040] Install the centrifuge column on the collection tube, transfer the solution into the centrifuge column, centrifuge at 12000 rpm for 2 minutes, and discard the filtrate.

[0041] 500 μL of Buffer WA (from Takara kit, Code No. 9763) was added to the centrifugal column, centrifuged at 12000 rpm for 1 minute, and the filtrate was discarded.

[0042] Add 700 μL of Buffer WB (from Takara kit, Code No. 9763) to the centrifugal column, centrifuge at 12000 rpm for 1 minute, discard the filtrate, and repeat this operation once.

[0043] Place the spin column on the collection tube and centrifuge at 12000 rpm for 2 minutes.

[0044] Place the centrifuge column in a new 1.5 mL centrifuge tube, add 50 μL of Elution Buffer (from Takara kit, Code No. 9763) to the center of the centrifuge column membrane, let stand at room temperature for 5 minutes, and centrifuge at 12000 rpm for 2 minutes to elute the DNA.

[0045] The content and purity of the extracted genomic DNA were detected by UV spectrophotometry (260nm / 280nm) and agarose gel electrophoresis, and then the DNA was packaged and stored at -80°C. 4.3.2. Example 2: Fluorescent labeling of specific nucleic acid probes

[0046] The specific nucleic acid probes (SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12) described in the present invention may have labeling groups at the 5' end and 3' end as shown in the following table:

[0047] Table 1 4.3.3. Example 3: Use of Real-time Fluorescence Quantitative qPCR Detection Kit for Residual Escherichia coli Host DNA

[0048] Experimental instrument: ABI QuantStudio 5 real-time fluorescence quantitative PCR instrument

[0049] Preparation of standard curve of positive control Escherichia coli genomic DNA reference: Positive control Escherichia coli genomic DNA reference (can be derived from the United States Pharmacopeia, The united states pharmacopoeia, abbreviated as USP) is used to prepare standard solutions of 6 concentration levels using TE buffer (composed of 10mM Tris-HCl, 1mM EDTA pH=8.0): the concentration of standard solution 1 is 3pg / mL; the concentration of standard solution 2 is 30pg / mL; the concentration of standard solution 3 is 300pg / mL; the concentration of standard solution 4 is 3000pg / mL; the concentration of standard solution 5 is 30000pg / mL; the concentration of standard solution 6 is 300000pg / mL.

[0050] The composition of the qPCR single-well reaction system (20 μL) is as follows: 10 μL qPCR premix (main components: 400 μM deoxyadenine triphosphate-dATP, 400 μM deoxyguanine triphosphate-dGTP, 400 μM deoxythymine triphosphate-dTTP, 400 μM deoxycytosine triphosphate-dGTP; 4 mM Mg 2+ ; 0.1U / μL hot start Taq DNA polymerase; 20mM Tris-HCL buffer, pH8.3), 0.4μL of 10μM forward specific primer (SEQ ID NO:5), 0.4μL of 10μM reverse specific primer (SEQ ID NO:6), 0.4μL of 10μM specific probe (SEQ ID NO:7), 4μL TE buffer (components are 10mMTris-HCl, 1mM EDTA pH=8.0) and 5μL DNA template.

[0051] The qPCR reaction category consists of a non-template control and a positive control standard solution. The DNA template of the non-template control is TE buffer. The positive control standard solution, such as Figure 1 Shown: From left to right are the standard solutions of 6 concentration levels prepared for the positive control: the concentration of standard solution 6 is: 300000 pg / mL; the concentration of standard solution 5 is: 30000 pg / mL; the concentration of standard solution 4 is: 3000 pg / mL; the concentration of standard solution 3 is: 300 pg / mL; the concentration of standard solution 2 is: 30 pg / mL; the concentration of standard solution 1 is: 3 pg / mL amplification curve; double replicate wells.

[0052] On the real-time fluorescence quantitative PCR instrument, create a blank new program and select the absolute quantitative detection template. Create a new detection probe, the fluorescent group can be selected as FAM, and the quenching fluorescent group can be selected as TAMRA. Set the reaction program as follows: pre-denaturation at 95℃ for 5 minutes; PCR stage: 95℃ for 15 seconds, 60℃ for 40 seconds (number of cycles is 45).

[0053] The test results of the kit are as follows (threshold 0.2):

[0054] Method system suitability: Cycle threshold (Ct) of non-template control was not detected and 37.583; Figure 2 As shown, described, the standard curve R 2is 0.999; the amplification efficiency is 96.401%; the relative standard deviation (RSD) of each standard point duplicate well is between 0.1% and 0.9%; the cycle threshold (Ct) of the lowest point of the standard is 34.216.

[0055] Specificity: Specificity refers to the ability of the analytical method to correctly determine the analyte in the presence of other components. The specificity of the kit method is examined by the results of the non-template control. The results show that the cycle threshold (Cycle threshold, abbreviated Ct) of the non-template control of the standard dilution is lower than the cycle threshold (Cyclethreshold, abbreviated Ct) of the lowest point of the standard, indicating that the method is specific.

[0056] Linearity: refers to the ability of the linear test result to be directly proportional to the concentration of the analyte in the sample within the designed range. In this experiment, within the range of 3 to 300,000 pg / mL, the logarithmic values ​​of the six concentration levels of standard solutions prepared by the positive control, 3pg / mL, 30pg / mL, 300pg / mL, 3000pg / mL, 30000pg / mL, and 300,000pg / mL, were used as the horizontal coordinate LgX, and the number of cycles experienced by the fluorescence signal to reach the exponential threshold line (0.2) set in the S-type amplification curve, namely the cycle threshold (Cycle threshold, abbreviated Ct) Y, was used as the vertical coordinate for linear fitting. The standard curve result is Y = -3.411LgX + 35.709, r 2 is 0.999.

[0057] Accuracy and range: Accuracy refers to the degree to which the result determined by the established method is close to the true value or reference value, and is generally expressed as recovery rate. The accuracy of the method is examined by examining the back-calculation accuracy of each calibration point. The calibration back-calculation accuracy of the kit method is in the range of 92% to 111%. Therefore, the range of this method is 3 to 300,000 pg / mL.

[0058] The detection method of Escherichia coli host DNA residue in Example 3 has good system adaptability, and the specificity, linearity, range and accuracy of the method are investigated, and the lowest point of the standard curve is 3pg / mL. The primers and probes of the present invention have high specificity and sensitivity, and the detection range is 3pg / mL to 300000pg / mL, which meets the market demand for the detection of Escherichia coli host DNA residues. The design of the primers and probes of the present invention is based on the very conservative 16srRNA (SEQ ID NO: 13) gene sequence in Escherichia coli, which can more accurately reflect the real Escherichia coli host DNA residues. 4.3.4. Example 4: Detection of E. coli host DNA residues in samples from different plasmid purification process steps

[0059] The experimental instruments, the preparation of the standard curve of the positive control Escherichia coli genomic DNA reference product, the composition of the single-well reaction system of qPCR and the reaction procedure were the same as those in Example 3.

[0060] Sample treatment: Dilute the samples from different process steps with TE buffer to an appropriate plasmid concentration, such as 100 ng / mL, to obtain the test solution.

[0061] The qPCR reaction categories consist of non-template control, standard curve and test solution. The DNA template of the non-template control is TE buffer, and the standard concentrations of the standard curve points of the positive control are 3pg / mL, 30pg / mL, 300pg / mL, 3000pg / mL, 30000pg / mL, 300000pg / mL; all prepared samples are double-well. Detection is carried out after loading.

[0062] Experimental Results

[0063] Table 2 Example 4 By accurately quantifying the residual amount of E. coli host DNA in samples of different purification process steps in plasmid production, effective process optimization guidance and plasmid product release can be carried out. 4.4. Sequence Listing Table 3

Claims

1. A forward qPCR primer and a reverse qPCR primer, wherein: a) the nucleic acid sequence of the forward qPCR primer is the nucleic acid sequence provided in SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 6; b) The nucleic acid sequence of the reverse qPCR primer is the nucleic acid sequence provided in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO:

8.

2. A nucleic acid-specific probe, the nucleic acid sequence of which is the nucleic acid sequence provided in SEQ ID NO:3, SEQ ID NO:6, or SEQ ID NO:

9.

3. A forward qPCR primer, a reverse qPCR primer and a nucleic acid-specific probe, wherein: The nucleic acid sequence of the forward qPCR primer is the nucleic acid sequence provided by SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 6; The nucleic acid sequence of the reverse qPCR primer is the nucleic acid sequence provided in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8; The nucleic acid sequence of the nucleic acid probe is SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 9, or a nucleic acid sequence provided in a complementary sequence thereof.

4. The nucleic acid-specific probe according to claim 2, or the forward qPCR primer, reverse qPCR primer and nucleic acid probe according to claim 3, wherein the nucleic acid probe is a nucleic acid-specific probe, the 5′ end is modified with a first fluorescent group, and the 3′ end is modified with a second fluorescent group or a fluorescence quenching group.

5. The nucleic acid-specific probe according to claim 2, or the forward qPCR primer, reverse qPCR primer and nucleic acid probe according to claim 3, wherein the nucleic acid probe is a nucleic acid-specific probe with a fluorescent group modified at the 5′ end such as 6-carboxyfluorescein (6-FAM), and a fluorescent group modified at the 3′ end such as 6-carboxytetramethylrhodamine (TAMRA) or a quencher modified such as Black HoleQuencher 1 (BHQ1).

6. A qPCR component comprising the forward qPCR primer defined in claim 1 or 3, and the reverse qPCR primer defined in claim 1 or 3.

7. The qPCR components according to claim 6, further comprising the nucleic acid probe defined in claim 2-4, or 5.

8. The qPCR components according to claim 6 or 7, further comprising genomic DNA derived from Escherichia coli (E. Coli) used as a positive control, the sequence of which comprises the nucleic acid sequence provided in SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:

12.

9. A kit comprising the qPCR components according to any one of claims 6 to 8 and instructions for use.

10. A kit comprising 6 concentration levels of standard solutions prepared by the positive control described in claim 7, which are: The concentration of standard solution 1 is: 3pg / mL; The concentration of standard solution 2 is: 30 pg / mL; The concentration of standard solution 3 is: 300 pg / mL; The concentration of standard solution 4 is: 3000 pg / mL; The concentration of standard solution 5 is: 30000 pg / mL; The concentration of standard solution 6 is: 300000 pg / mL.

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