CN112592965B - E.coli host DNA residue detection kit adopting TaqMan probe method - Google Patents
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Abstract
The invention relates to an E.coli host DNA residue detection kit of a TaqMan probe method, which comprises the following components: e.coli DNA Control, DNA Dilution Buffer,2x MaxDetect qPCR Mix,10xE.coli Detection Mix; the invention also relates to the application and the using method of the kit. The kit comprises a DNA reference E.coli DNA Control for preparing a standard curve, is traced to a national standard, and can rapidly and specifically detect DNA residues of E.coli host cells with fg level by using a Taqman probe quantitative PCR method. The method is suitable for different sample types from the intermediate product to the final product of the biological pharmacy, the detection result is accurate and reliable, the precision is high, and the detectable concentration range is 0.3 ng/mu L-30 fg/mu L.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to an E.coli host DNA residue detection kit of a TaqMan probe method.
Background
TaqMan fluorescent probe is an oligonucleotide probe, the 5 'end of which carries a fluorescent group, such as FAM, TET, VIC, HEX, and the 3' end of which carries a quenching group, such as TAMRA, BHQ, and the like. During PCR amplification, a specific fluorescent probe is added simultaneously with the addition of the primer. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; during PCR amplification, the 5'-3' exonuclease activity of Taq enzyme can degrade the probe by enzyme digestion, so that the reporter group and the quenching group are separated, and the fluorescence monitoring system can receive a fluorescence signal. I.e., one fluorescent molecule is formed for each amplified DNA strand, achieving complete synchronization of fluorescent signal accumulation and PCR product formation.
Development of TaqMan Probe method
By introducing a fluorescent labeled probe based on the 5'-3' exonuclease activity of TaqDNA polymerase, the real-time quantitative PCR system is remarkably improved. The use of these fluorescent probes has led to the development of a real-time quantitative method, which allows the detection of specific amplification products only, avoiding false positives where common fluorescent quantitative PCR dyes would bind primer dimers or non-specific amplification products. In addition, development of fluorescent labeled probes also eliminates the post-PCR processing procedure for probe degradation analysis.
Working principle of TaqMan probe method
TaqMan reagents detect specific PCR products as they accumulate during PCR cycles by using fluorescent probes. The working principle is as follows:
1. constructing a segment of oligonucleotide probe: a fluorescent dye reporter group is marked at the 5 'end, and a quenching dye group is marked at the 3' end. The proximity of the quenching group significantly reduces the fluorescence emitted by the reporter dye group by steric Fluorescence Resonance Energy Transfer (FRET) when the probe remains intact.
2. If the target sequence is present, the probe anneals downstream of one of the primer binding sites and the excision is completed as the primer extends through the 5'-3' exonuclease activity of the TaqDNA polymerase.
3. Excision of the probe will:
separating the reporter dye group from the quencher dye group, and enhancing the signal of the reporter dye group.
The probe is removed from the strand of interest and the primer continues to extend along the end of the template strand. Thus, the intervention of the probe does not inhibit the whole PCR process.
4. With each cycle, more reporter dye molecules are cleaved from the respective probes and the fluorescence intensity increases with the number of amplified fragments synthesized.
Advantages and disadvantages of TaqMan probe method
The advantages of the TaqMan method are as follows:
specific hydrolysis (hybridization) between the probe and the target molecule (target) is required to generate a fluorescent signal
Probes can be labeled with significantly different reporter dyes, amplified and detected for two different sequences in one reaction tube
And the PCR post-treatment is not needed, so that the analysis workload is reduced and the material cost is saved.
The main disadvantage of the TaqMan method is that different probes need to be synthesized according to different sequences.
Detection type Using TaqMan Probe method
One-step RT-PCR for RNA quantification;
two-step RT-PCR for RNA quantification;
DNA/cDNA quantification;
allele detection;
positive and negative detection by IPC.
Application of TaqMan probe method
The TaqMan probe method fluorescence quantitative PCR is suitable for pathogen detection, host nucleic acid residue detection, disease drug resistance gene research, drug efficacy assessment and genetic disease diagnosis. The novel TaqMan-MGB probe is also used for analyzing gene mutation (SNP), and is expected to become a first technical platform for gene diagnosis and personalized medicine analysis.
From the above, the TaqMan probe method has good specificity and high accuracy, and is an extremely sensitive nucleic acid detection method. Along with the vigorous development of the biopharmaceutical industry, in order to improve the standard of industrial production process, the TaqMan probe method fluorescence quantitative PCR is widely applied to the detection of E.coli host cell DNA residues, and the current situation that the imported kit is high in price and the domestic kit is easy to pollute is that a kit with high detection precision and no exogenous interference is urgently needed.
Based on the detection kit, the E.coli host DNA residue detection kit of the TaqMan probe method is developed, and the DNA residue of the E.coli host cell with the fg level can be rapidly, specifically and accurately detected, so that the detection kit has the advantages of having exogenous interference or not and higher precision compared with the domestic same product.
Disclosure of Invention
The first object of the present invention is to provide a primer pair for detecting E.coli host DNA residues, which aims at overcoming the defects of the prior art.
The second object of the invention is to provide a reaction system for detecting E.coli host DNA residues, which aims at overcoming the defects of the prior art.
A third object of the present invention is to provide the use of the reaction system as described above, in view of the deficiencies of the prior art.
A fourth object of the present invention is to provide a method for detecting e.coli host DNA residues, which addresses the deficiencies of the prior art.
The fifth purpose of the invention is to provide a kit for detecting E.coli host DNA residues by using a TaqMan probe method, which has stronger anti-interference capability and higher precision, aiming at the defects of the prior art.
A sixth object of the present invention is to provide a method of using the kit as described above, which addresses the shortcomings of the prior art.
In order to achieve the first object, the invention adopts the following technical scheme:
a primer pair for detecting E.coli host DNA residues is a forward primer shown in a sense primer of 5'-GGAATCGCTAGTAATCGTGGAT-3' (SEQ ID NO: 1) and a reverse primer shown in an anti primer of 5'-AGTCATGAATCACAAAGTGGTAAGC-3' (SEQ ID NO: 2).
In order to achieve the second purpose, the invention adopts the following technical scheme:
a reaction system for detecting E.coli host DNA residues is a real-time fluorescent quantitative PCR reaction system and comprises a primer pair and a probe shown as probe 5'-AATGCCACGGTGAATACGTTCCCG-3' (5 '-FAM,3' -BHQ 1) (SEQ ID NO: 3).
In order to achieve the third object, the present invention adopts the following technical scheme:
the application of the reaction system in preparing a kit for detecting E.coli host DNA residues is provided.
Preferably, the E.coli host DNA residue detection is applicable to, but not limited to, therapeutic protein drugs, recombinant vaccines or monoclonal antibodies for E.coli expression or production.
In order to achieve the fourth object, the present invention adopts the following technical scheme:
a method for detecting e.coli host DNA residues, said method comprising: and (3) extracting DNA from the sample to be detected by using the reaction system, performing real-time fluorescence quantitative PCR, and calculating the amount of E.coli DNA in the sample to be detected according to the obtained standard curve.
Preferably, the sample to be tested can be, but is not limited to, a therapeutic protein drug expressed or produced by E.coli, a recombinant vaccine or a monoclonal antibody.
In order to achieve the fifth object, the present invention adopts the following technical scheme:
an E.coli host DNA residue detection kit of TaqMan probe method,the kit comprises E.coli DNA Control, DNA Dilution Buffer,2x MaxDetect qPCR Mix,10x E.coli Detection Mix; the DNADilution Buffer contains NaN 3 The 10x E.coli Detection Mix contains the primer pair and the probe shown in SEQ ID NO. 3.
Preferably, the E.coli DNA Control concentration is 30 ng/. Mu.L.
In order to achieve the sixth object, the present invention adopts the following technical scheme:
the use method of the kit comprises the following steps:
a) After DNA is extracted from the sample by the pretreatment kit, the sample is diluted to a proper concentration by DNA Dilution Buffer;
b) E.coli DNA Control was diluted with a DNA Dilution Buffer ten-fold gradient to yield 5 concentrations of standard: 300 pg/. Mu.L, 30 pg/. Mu.L, 3 pg/. Mu.L, 300 fg/. Mu.L, 30 fg/. Mu.L;
c) Preparing a PCR reaction mixed solution according to the quantity to be detected, sequentially adding a positive Control (water), 10x E.coli Detection Mix,2x MaxDetect qPCR Mix, uniformly mixing, and split charging into 0.2mL of eight-connecting tubes or 96-well plates serving as fluorescent quantitative PCR;
d) Sequentially adding the diluted sample in the negative control, a) and the diluted standard substance in b) into the subpackaged PCR reaction mixed solution, uniformly mixing, and then placing into a quantitative PCR instrument for detection;
e) And drawing a standard curve according to the detection result, and calculating the concentration of E.coli host DNA contained in the sample to be detected.
Preferably, naN in DNA Dilution Buffer 3 The content ratio of (2) is 1/10000.
The invention has the advantages that:
the invention provides an E.coli host DNA residue detection kit for a TaqMan probe method. The TaqMan probe and the primer designed by the detection kit can specifically detect the target sequence of E.coli multi-site, and can not be interfered by exogenous genome common in biopharmaceutical processes such as human, CHO or Pichia pastoris. In the similar products, the accuracy is good, the precision is high, the specificity is better, and no exogenous genome interference exists.
Drawings
Fig. 1 is a schematic diagram of the present invention.
FIG. 2 is an amplification curve of 5 standards in example 1.
FIG. 3 is a standard curve of 5 standards in example 1.
Fig. 4 is a standard curve.
FIG. 5 is a comparison of the results of the detection of genomic contamination of E.coli reference and E.coli reference mixed with human, CHO or Pichia respectively in example 1.
Detailed Description
The invention is further described below in conjunction with the detailed description. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications of the present invention may be made by those skilled in the art after reading the description of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto.
The general idea of using the E.coli host DNA residue detection kit of the TaqMan probe method to detect the E.coli host DNA residue is as follows: and (3) extracting DNA of a sample to be detected in the process flow, diluting the DNA to a proper concentration range, diluting an E.coli DNA Control reference substance into a standard substance with 5 concentration gradients by using DNA Dilution Buffer, detecting qPCR (quantitative polymerase chain reaction) of the sample, the standard substance and a negative Control by using the existing PCR reaction reagent in the kit, and then calculating the concentration of E.coli host DNA in the sample to be detected according to a standard curve.
Example 1 Effect experiment
1. Reagent preparation
1) E.coli DNA Control preparation
The genome of E.coli cells was first extracted using the "bacterial genomic DNA extraction kit" of the Tiangen organism (DP 302-02).
The E.coli DNA national standard (lot number: 270027-201101) and the E.coli genome extracted as described above were then diluted to a concentration of 30 ng/. Mu.L required for E.coli DNA Control and further diluted to a concentration gradient required for 5 standards for standard curve preparation. And (3) comparing the extracted E.coli genome with an E.coli DNA national standard by using Ct value of an amplification curve after qPCR operation, thereby obtaining E.coli DNA Control.
2) DNA Dilution Buffer preparation
TABLE 1
| Concentration of reagent stock solution | Final concentration |
| 2M Tris HCl(pH7.5) | 1mM |
| 1M EDTA | 0.1 |
| 10％NaN 3 | 0.01％ |
3) 2x MaxDetect qPCR Mix preparation
The product B532953 SGExcel GoldStar TaqMan qPCR premix (containing ROX) is packaged according to the packaging specification.
4) 10x E.coli Detection Mix preparation
TABLE 2
Sequence synthesis is carried out according to the sequences, and the final concentration of sense/anti-primer and probe is 10 mu M, and the product is preserved at-20 ℃ for standby.
2. DNA sample preparation
1) The sample to be tested was diluted with 1xPBS to the appropriate concentration range.
2) And extracting DNA in the sample to be detected by adopting an EkesaiMaxExract universal host DNA residual sample pretreatment kit (MB 000-0011).
3. Gradient dilution E.coli DNA Control preparation standard substance
For e.coli, 5 standards of dilution gradient need to be prepared: 300 pg/. Mu.L, 30 pg/. Mu.L, 3 pg/. Mu.L, 300 fg/. Mu.L, 30 fg/. Mu.L.
1) The 6 low adsorption 1.5mL centrifuge tubes were labeled E0, E1, E2, E3, E4, E5. E0 is an intermediate dilution transition duct, and the rest E1 to E5 sequentially correspond to the above 5 standards of dilution gradient.
2) To each of the 6 centrifuge tubes, 90. Mu. L DNA Dilution Buffer was added.
3) E.coli DNA Control (30 ng/. Mu.L) was removed from the-20deg.C refrigerator, gently vortexed and mixed, and then centrifuged rapidly to collect the liquid from the tube lid and tube wall to the bottom of the centrifuge tube.
4) 10. Mu.L of E.coli DNA Control was added to the E0 tube, thoroughly vortexed with the existing 90. Mu. L DNA Dilution Buffer, and all the liquid was rapidly centrifuged to the bottom.
5) Then carrying out 5 times of 10-fold gradient dilution according to the following table, and operating the same as the step 4.
TABLE 3 Table 3
4. Preparation of PCR reaction mixture
1) The number of samples to be tested and the number of controls are first determined.
2) After the desired reagents were completely thawed at room temperature, they were mixed and centrifuged rapidly.
3) A PCR reaction mixture was prepared according to the reagents and corresponding volumes in the following tables.
TABLE 4 Table 4
| Reagent(s) | 1 required volume in 30. Mu.L reaction |
| Negative Control(water) | 2μL |
| 10xE.coli Detection Mix | 3μL |
| 2x MaxDetect qPCR Mix | 15μL |
| Total | 20μL |
5. Preparing a standard curve and sample testing
1) Based on the number of standard and sample, the number of wells required for the reaction is calculated, typically three wells per template (one well cannot be made when the standard curve is first measured).
Reaction well number= (standard curve of 5 concentration gradients+1 template-free control ntc+1 negative quality control neg+number of TS samples to be tested+number of ERC recovered by corresponding labeling of samples to be tested) ×3
Ntc= No Template Control (DNA dilution DNADilution Buffer or negative control water)
Neg= Negative Extraction Control (sample pretreatment of sample matrix solution or PBS with sample to be tested, purified solution NEG)
Ts=test Sample (Sample to be tested)
Erc= Extraction Recovery Control (e.g. 30pg e.coll standard DNA is added to the sample to be tested, and then the sample is pretreated together with the same batch of sample to be tested, and the obtained purified solution is labeled to recover ERC)
3) The following example 96-well plate layout
TABLE 5
3) mu.L of the PCR reaction mixture was added to each well.
4) mu.L of the above template DNA was added to the corresponding well.
5) After the tube cover or the sealing film of the 96-well plate is covered, the tube cover or the sealing film of the 96-well plate is evenly mixed and rapidly centrifuged, and then the tube cover or the sealing film of the 96-well plate is detected by a machine.
6. Quantitative PCR program setting and loading machine
The following procedure was Agilentent Technologies Stratagenes Mx3000P, software MxPro:
1) The software is turned on.
2) Clicking Setup first selects the Plate Setup button to set the layout on the 96-well panel.
3) The reporter fluorophore was selected to be FAM and the quencher fluorophore was none (Applied Biosystems 7500 qPCR instrument detects reference fluorescence as ROX).
4) The Thermal Profile Setup button is then selected to set the cycling program for the PCR.
5) Click RUN for quantitative PCR assay.
7. Quantitative PCR data analysis
The following procedure was Agilent Technologies Stratagene Mx3000P, software MxPro:
1) Clicking on Analyze, in the Amplification Plot panel of Results, can initially see if the amplification curve is normal in morphology.
2) Clicking on Setup sets the Well type column of Standard curve wells as Standard in the Plate Setup panel and assigns 3000, 300, 30, 3, 0.3 (meaning total amount of DNA per Well in pg) in the Standard Quantity column respectively and is named E1, E2, E3, E4, E5 in the corresponding showwell Names column.
3) In the Plate Setup panel, the column of Well type without template control NTC wells is set as NTC, the column of Well type with negative quality control NEG wells, sample wells to be tested, sample ERC wells is set as ungnow, and named NTC, NEG, TS, ERC in the corresponding column of showw Well Names.
4) Clicking on Analyze, R2, slope (Slope), intercept (Intercept) and Efficiency (Efficiency) of the Standard Curve can be read in the Standard Curve panel of Results.
5) In the Text Report panel of Results, the quality column can read the detection values of the template-free control NTC, the negative quality control NEG, the sample to be detected and the sample ERC, and the unit is pg/10 mu L. The units may be subsequently converted to pg/μl or pg/mL in the detection report.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and additions to the present invention may be made by those skilled in the art without departing from the principles of the present invention and such modifications and additions are to be considered as well as within the scope of the present invention.
SEQUENCE LISTING
<110> Ikesai biotechnology (Tai House) Co., ltd
<120> E.coli host DNA residue detection kit of TaqMan probe method
<130> /
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213> artificial sequence
<400> 1
ggaatcgcta gtaatcgtgg at 22
<210> 2
<211> 25
<212> DNA
<213> artificial sequence
<400> 2
agtcatgaat cacaaagtgg taagc 25
<210> 3
<211> 24
<212> DNA
<213> artificial sequence
<400> 3
aatgccacgg tgaatacgtt cccg 24
Claims (11)
1. The reaction system for detecting the E.coli host DNA residues is characterized by being a real-time fluorescent quantitative PCR reaction system and comprising a primer pair consisting of a forward primer shown in SEQ ID NO. 1 and a reverse primer shown in SEQ ID NO. 2 and a probe shown in SEQ ID NO. 3.
2. Use of the reaction system of claim 1 for the preparation of a kit for the detection of e.coli host DNA residues.
3. The use according to claim 2, wherein said e.coli host DNA residue detection is suitable for e.coli expression or production of therapeutic protein drugs.
4. The use according to claim 3, wherein the therapeutic protein drug is a recombinant vaccine or a monoclonal antibody.
5. A method for detecting e.coli host DNA residues, comprising: the reaction system of claim 1 is used for extracting DNA from a sample to be detected, then real-time fluorescence quantitative PCR is carried out, and the amount of E.coli DNA in the sample to be detected is calculated according to the obtained standard curve.
6. The method of claim 5, wherein the sample to be tested is a therapeutic protein drug expressed or produced by E.coli.
7. The method of claim 6, wherein the therapeutic protein drug is a recombinant vaccine or monoclonal antibody.
8. The E.coli host DNA residue detection kit of the TaqMan probe method is characterized by comprising E.coli DNA Control, DNA Dilution Buffer and 2x MaxDetect qPCR Mix,10x E.coliDetection Mix; the DNA Dilution Buffer contains NaN 3 The 10x E.coli DetectionMix contains the primer pair as set forth in claim 1 and a probe as shown in SEQ ID NO. 3.
9. The kit of claim 8, wherein the E.coli DNA Control concentration is 30 ng/. Mu.L.
10. The method of using the kit of claim 8, comprising the steps of: a) After DNA is extracted from the sample by the pretreatment kit, the sample is diluted to a proper concentration by DNA Dilution Buffer; b) E.coli DNA Control was diluted with a DNA Dilution Buffer ten-fold gradient to yield 5 concentrations of standard: 300 pg/. Mu.L, 30 pg/. Mu.L, 3 pg/. Mu.L, 300 fg/. Mu.L, 30 fg/. Mu.L; c) Preparing a PCR reaction mixed solution according to the quantity to be detected, sequentially adding a positive Control (water), 10xE.coli Detection Mix,2x MaxDetect qPCR Mix, uniformly mixing, and split charging into 0.2mL of eight-connecting tubes or 96-well plates serving as fluorescent quantitative PCR; d) Sequentially adding the diluted sample in the negative control, a) and the diluted standard substance in b) into the subpackaged PCR reaction mixed solution, uniformly mixing, and then placing into a quantitative PCR instrument for detection; e) And drawing a standard curve according to the detection result, and calculating the concentration of E.coli host DNA contained in the sample to be detected.
11. The method of claim 10, wherein the NaN is DNA Dilution Buffer 3 The content ratio of (2) is 1/10000.
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