CN116716415A - Primer probe for quantitative detection of DNA residual content of insect cells, method and application thereof - Google Patents
Primer probe for quantitative detection of DNA residual content of insect cells, method and application thereof Download PDFInfo
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- -1 eclipse Chemical compound 0.000 claims description 2
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims description 2
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Abstract
The invention relates to a primer and a probe for quantitatively detecting the DNA residual content of insect cells and a method for detecting the DNA residual content of insect Sf9 cells by the primer and the probe. The invention also relates to a kit comprising the primer and the probe. The primer, the probe, the detection method and the kit have higher specificity and sensitivity, and can well meet the detection requirements in the field. The primer, the probe, the detection method and the kit can be used for efficiently, rapidly, sensitively and accurately detecting the level and the content of residual DNA of the insect Sf9 cells, can keep high sensitivity and accuracy under various detection environments and conditions, can be widely applied to quality control of gene therapy products, vaccines and protein products produced by an insect cell-baculovirus expression system, and has high application value.
Description
Technical Field
The invention relates to the field of gene monitoring, in particular to a method for detecting the DNA residual content of insect cells and quantitatively detecting the DNA residual content of the insect cells and application thereof.
Background
DNA remaining in host cells not only affects the purity and safety of the biologic, but also presents a potential carcinogenicity, thus requiring stringent control and monitoring. The fluorescent quantitative PCR technology can perform specific detection aiming at different host DNA cells, and after twenty years of development, the real-time fluorescent quantitative PCR technology (qPCR) has high sensitivity and high specificity.
Real-time fluorescent quantitative PCR (qPCR) technology has been widely used for diagnosis of various infectious diseases in clinical disease diagnosis and evaluation of therapeutic effects; detecting clinical disease diagnosis animal diseases; food safety operations such as food-borne microorganisms, food allergens, transgenic research and the like. qPCR is mainly based on the use of intercalating dyes or fluorescent probes (such as TaqMan), one can compare DNA levels of multiple samples by monitoring the fluorescence intensity during PCR. The fluorescent signal in the probe method is only derived from the target sequence, is not influenced by nonspecific amplification and primer dimer, and is far superior to the cheap dye method in terms of specificity and accuracy.
TaqMan probes are divided into two types according to the difference of fluorescence quenching groups marked at the 3' end of the TaqMan probes; common TaqMan probes and TaqMan MGB probes. The quenching group of the MGB probe adopts a Non-fluorescence quenching group (Non-Flourescent Quencher), does not generate fluorescence, and can greatly reduce the intensity of background signals. Meanwhile, MGB (Minor Groove Binder) modification groups are also connected to the probe, so that the Tm value of the probe can be improved by about 10 ℃. Therefore, the MGB probe can be shorter than the common probe in design, thereby not only reducing the synthesis cost, but also greatly improving the success rate of probe design. Because short probes are easier to design than long probes in cases where the DNA base composition of the template is not reasonable. Experiments prove that the TaqMan MGB probe can be distinguished to be more ideal for the templates rich in A/T.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a primer and a probe for detecting residual DNA of insect cells, and a quantitative detection method and application of the residual DNA content. The primer and the probe can be used for stably, accurately and specifically detecting the residual DNA of the insect cells, and the detection is performed by real-time fluorescent quantitative PCR (Q-PCR).
The first object of the invention is to provide a primer and a probe for detecting residual DNA of insect cells, wherein the sequences of the primer and the probe are as follows:
an upstream primer: 5'-AAAAGATAGAAACCAACCTGGCTTAC-3' (SEQ ID NO: 1);
a downstream primer: 5'-CGACCTCGATGTTGGATTAAGAT-3' (SEQ ID NO: 2);
and (3) probe: 5'-CCGGTTTGAACTCAG-3' (SEQ ID NO: 3).
Further, the 5 'end of the probe is connected with a fluorescence report group, the 3' end of the probe is connected with a fluorescence quenching group, the fluorescence report group is selected from any one of FAM, JOE, HEX or VIC, and the fluorescence quenching group is selected from any one of TAMRA, eclipse, BHQ or MGB.
Further, the fluorescence reporting group is FAM, and the fluorescence quenching group is MGB.
The second object of the invention is to provide a method for detecting the residual quantity of Sf9 cell DNA in a sample, which is used for detecting the sample to be detected by adopting the primer and the probe.
Further, the method comprises the steps of:
(1) Treating a sample to be detected, and extracting DNA in the sample as a template for standby;
(2) Preparing a reaction system, wherein the reaction system comprises: template, upstream primer, downstream primer, probe, polymerase and the like;
(3) Real-time fluorescent quantitative PCR reactions were performed.
Further, the real-time fluorescence quantitative PCR reaction system is as follows:
further, the reaction conditions of the real-time fluorescent quantitative PCR are as follows:
the first step: 95 ℃ 30sec,1 cycle;
and a second step of: (95 ℃ C. For 5sec,60 ℃ C. For 30 sec), 40 cycles.
Furthermore, the method also comprises the step of establishing a standard curve, wherein the positive quantitative standard substance is diluted by 10 times of gradient, and the positive quantitative standard substance is used as a template for real-time fluorescence quantitative PCR detection, so that the standard curve is established.
Further, the result judgment is to calculate the residual quantity of Sf9 cell DNA in the sample to be tested according to a standard curve.
A third object of the present invention is to provide a kit for detecting the residual amount of Sf9 cell DNA in a sample, the kit comprising the primers and probes of any of claims 1-3, the kit further comprising real-time fluorescent quantitative PCR amplification reagents, positive quantitative standards and/or negative controls.
Compared with the prior art, the invention has the beneficial effects that:
1. the primer, the probe, the detection method and the kit have very high specificity and sensitivity, also have very high amplification efficiency, can stably, accurately, sensitively and rapidly detect the Sf9 cell DNA residues, and has the lowest detectable concentration of 10 copies/mu l in real-time fluorescence quantitative qPCR, while the lowest detectable concentration of 1.0x10 in conventional qPCR 4 The copies/. Mu.l, demonstrated that the real-time fluorescent quantitative qPCR sensitivity established in this study was higher than that of conventional PCR.
2. The primer, the probe, the detection method and the kit can be widely applied to quality control of gene therapy products, vaccines and protein products produced by an insect cell-baculovirus expression system, and have high application value.
Drawings
FIG. 1 shows the identification of the digestion of pMD18-T-sf9 plasmid;
FIG. 2 is a kinetic profile of real-time fluorescent quantitative RT-PCR;
FIG. 3 is a real-time fluorescent quantitative RT-PCR standard curve;
FIG. 4 is a real-time fluorescent quantitative RT-PCR kinetic profile (specificity test);
FIG. 5 is a conventional PCR reaction (sensitivity test);
Detailed Description
The following examples are only for the purpose of illustrating the invention and are not intended to limit the scope of the invention.
The reagent materials mentioned in the examples below are all common materials on the market, and the actual preparation is carried out by conventional methods, and the methods not described in detail in the examples are all conventional in the art.
Materials and methods involved in embodiments of the invention
1. Test article
The test sample is recombinant protein inactivated vaccine obtained by infecting insect cells (sf 9 and High Five) with baculovirus expressing recombinant protein, separating recombinant protein or virus-like particles from cell culture liquid after cytopathic effect, and inactivating the virus-like particles.
2. Standard genomic DNA
After the sf9 cells are subjected to expansion culture, cell DNA is extracted according to the instruction of a blood/cell/tissue genome DNA extraction kit, and the DNA concentration and purity are measured by a Nanodrop2000 ultraviolet spectrophotometer.
3. Main reagent and instrument
Viral nucleic acid DNA/RNA extraction kits (column extraction) were purchased from pre-biosystems, ltd; the pMD18-T vector was purchased from Dalianbao bioengineering Co., ltd; DL2000 maker was purchased from beijing full gold biotechnology limited; plasmid Mini Kit I purchased from OMEGA company; agarose gel DNA recovery kit was purchased from beijing tiangen biology company; the ApexHF HS DNA polymerase premix and Pro Taq HS premix probe method qPCR kit are purchased from Hunan Ai Kerui bioengineering Co., ltd; conventional PCR apparatus (Bio-RAD C1000 TouchTM Thermal cycler gradient PCR apparatus); fluorescent quantitative PCR instrument (BA qTOWER3 AJ EN 03.19); bio-Rad Chemi DocTM XRS + gel imager.
Example 1 determination of amplified target fragment and primer probe
According to the literature, the above sf9 cell 16sRNA was selected as an amplification target fragment for residual DNA detection, and primers were designed which can have excellent sensitivity and specificity. The length of the product obtained by the amplification of the primer pair is 571bp.
The Sf9-F/R primer sequences are as follows:
Sf9-F：5’-GGGCTGCAGTATATTGACTGTACAAAGGTA-3’(SEQ ID NO:1)
Sf9-R：5’-CATTATTTAATAAATTTAATTTAAATATTTGATCCTTTCGTACT-3’(SEQ ID NO:2)
example 2 preparation of standards
Using the extracted genome of insect cells as a template, the gene sequence fragment of sf9 16srRNA of insect cells was amplified using the sf9-F/R primer pair designed in example 1.
The PCR reaction system (50. Mu.l) is shown in Table 1:
TABLE 1 PCR reaction System
TABLE 2 PCR reaction procedure
The PCR products were detected by 1% agarose gel electrophoresis. As shown in FIG. 1, the target band of 571bp was found, the size of the obtained fragment was consistent with the expected 571bp, the pMD18-T vector was ligated after the target band was recovered, the ligation product was transformed into DH 5. Alpha. Competent cells, single colony shaking culture was performed for 12 hours, plasmids were extracted using OMEGA miniprep kit, plasmids were identified by PCR, positive plasmids were sent to the Protoengineering Co., ltd. For sequencing, sequence comparison confirmed successful construction of pMD18-T-sf9 plasmids, the correct plasmids were assayed for concentration by a spectrophotometer, and stored at-15℃or below for use.
Example 3 establishment of a real-time fluorescent quantitative PCR Standard Curve Using standards
The recombinant pMD18-T-sf9 of example 2 was taken, the total length of the standard was 3263bp, the plasmid concentration was measured by a DNA Aquent spectrophotometer, and the mass unit was converted into the number of molecules according to the formula on the molecular cloning test guidelines. The recombinant plasmid is diluted to 10 by using sterilized water 0 、10 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 、10 9 And 10 10 COPIES/. Mu.l. Fluorescent quantitative PCR primers and probes were designed:
F1:AAAAGATAGAAACCAACCTGGCTTAC
R1:CGACCTCGATGTTGGATTAAGAT
P1:FAM-CCGGTTTGAACTCAG-MGB
templates were diluted in different ratios and qPCR reactions were performed multiple times, the qPCR reaction system (20 μl) is shown in Table 3:
TABLE 3PCR reaction System
TABLE 4qPCR reaction procedure
In the reaction plateau phase, the number of different starting templates is not directly proportional to the amount of product, but the number of starting templates is inversely proportional to the CT value experienced by the reaction reaching the threshold. Amplification kinetics Curve results show 10 1 Specific amplification curves were not seen for either, 1 or the negative control. The logarithmic values of plasmid DNA of different concentrations at which the curve is most stable are taken as abscissa, i.e.10 3 ～10 10 COPIES/. Mu.l. Linear regression is plotted with the measured CT values as ordinate,standard curves for real-time fluorescent quantitative RT-qPCR were obtained as shown in fig. 2 and 3.
The standard curve shows that in the measured concentration range, the logarithm of the standard concentration and the corresponding CT value show obvious linear relation, the regression curve slope is-3.52, and the correlation coefficient R 2 0.993. The regression equation for the standard curve is: y= -4.0234x+46.703, where y represents Ct value and x represents logarithm of molecular number.
Example 4 methodological verification of methods for measuring residual amounts of DNA in insect cells sf9
1. Specificity verification
In the inactivated vaccine prepared by baculovirus expression system, baculovirus genome is contained in addition to sf9 cell genome, and in order to verify the specificity of primer probe designed in example 1, bacmid1 plasmid of auxiliary baculovirus genome prepared by self in laboratory and Bacmid2 plasmid containing target gene and auxiliary baculovirus genome are added into sf9 cell genome in the amount of 1ul and concentration of 1×10 8 The copies/. Mu.l was subjected to qPCR amplification.
TABLE 5 detection of CT values by qPCR of different baculovirus genomic plasmids
The results in Table 5 show that the amplification CT values of the added DNA and the non-added DNA show no significant difference, which means that the primers and probes do not generate nonspecific amplification with other DNA components, thus the primers designed in example 1 do not amplify other DNA except sf9 cell DNA, and the primers and probes designed in example 1 have high specificity and good specificity.
At the same time, 8X 10 is taken 5 HF, CHO, ST and PK-15, extracting genome as template according to the instruction of blood/cell/tissue genome DNA extraction kit, setting negative control, fluorescent quantitative qPCR amplification under optimized reaction condition, and parallel test.
TABLE 6 qPCR detection of CT values for different cells
The results in Table 6 and FIG. 4 show that the HF cell genome produced nonspecific amplification, the CT value was 10.93.+ -. 0.098, and no amplification curve was found for other cell genomes, demonstrating good primer specificity.
2. Results of sensitivity experiments
A10-fold gradient dilution was performed on the standard positive plasmid obtained in example 2, and conventional PCR detection was performed using sf9-F and sf9-R primers. The results are shown in FIG. 5, which shows that the lowest concentration detectable by conventional PCR is 1.0X10 4 The copies/. Mu.l, demonstrated that the real-time fluorescent quantitative qPCR sensitivity established in this study was much higher than that of conventional PCR.
The PCR reaction system (25. Mu.l) is shown in Table 7:
TABLE 7PCR reaction System
TABLE 8PCR reaction procedure
3. Results of the repeatability experiments
At 10 5 、10 6 、10 7 3 dilutions of the standard substance are used as templates for carrying out repeated tests in batches and between batches, 3 repeats are set for each dilution, 3 real-time fluorescence quantitative qPCR tests are carried out, and the results show that the variation coefficient CV (%) of repeated results in batches and between batches is smaller than 3%, which indicates that the method has better repeatability and stability.
TABLE 9 statistical results of real-time fluorescent quantitative PCR repeatability detection
Claims (10)
1. A set of primers and probes for detecting residual amount of insect Sf9 cell DNA, wherein the sequences of the primers and probes are as follows:
an upstream primer: 5'-AAAAGATAGAAACCAACCTGGCTTAC-3' (SEQ ID NO: 1);
a downstream primer: 5'-CGACCTCGATGTTGGATTAAGAT-3' (SEQ ID NO: 2);
and (3) probe: 5'-CCGGTTTGAACTCAG-3' (SEQ ID NO: 3).
2. The primer and probe of claim 1, wherein the probe has a fluorescent reporter group attached to the 5 'end and a fluorescent quencher group attached to the 3' end, wherein the fluorescent reporter group is selected from any one of FAM, JOE, HEX and VIC, and the fluorescent quencher group is selected from any one of TAMRA, eclipse, BHQ and MGB.
3. The primer and probe of claim 2, wherein the fluorescent reporter group is FAM and the fluorescent quencher group is MGB.
4. A method for detecting residual amount of Sf9 cell DNA in a sample, characterized in that the primer and probe according to any one of claims 1-3 are used for detecting the sample to be detected.
5. The method according to claim 4, characterized in that the method comprises the steps of:
(1) Treating a sample to be detected, and extracting DNA in the sample as a template for standby;
(2) Preparing a reaction system, wherein the reaction system comprises: templates, upstream primers, downstream primers, probes, polymerase;
(3) Real-time fluorescent quantitative PCR reactions were performed.
6. The method of claim 5, wherein the real-time fluorescent quantitative PCR reaction system is:
7. the method of claim 5, wherein the reaction conditions of the real-time fluorescent quantitative PCR are:
the first step: 95 ℃ 30sec,1 cycle;
and a second step of: (95 ℃ C. For 5sec,60 ℃ C. For 30 sec), 40 cycles.
8. The method of claim 5, further comprising the step of establishing a standard curve by diluting the positive quantitative standard with a 10-fold gradient and performing real-time fluorescent quantitative PCR detection using the diluted positive quantitative standard as a template.
9. The method according to claim 5, wherein the result determination is to calculate the residual amount of Sf9 cell DNA in the sample to be tested based on a standard curve.
10. A kit for detecting residual amount of Sf9 cell DNA in a sample, characterized in that the kit comprises the primer and probe of any one of claims 1-3, the kit further comprising real-time fluorescent quantitative PCR amplification reagents, positive quantitative standards and/or negative controls.
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| CN117568493A (en) * | 2024-01-16 | 2024-02-20 | 苏州良辰生物医药科技有限公司 | Reagent and kit for identifying insect cell line |
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| CN117568493A (en) * | 2024-01-16 | 2024-02-20 | 苏州良辰生物医药科技有限公司 | Reagent and kit for identifying insect cell line |
| CN117568493B (en) * | 2024-01-16 | 2024-04-12 | 苏州良辰生物医药科技有限公司 | Reagent and kit for identifying insect cell line |
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