CN113528706A - Reagent for detecting replication-competent lentivirus by three-channel double-quenching probe and detection method thereof - Google Patents
Reagent for detecting replication-competent lentivirus by three-channel double-quenching probe and detection method thereof Download PDFInfo
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Abstract
The invention discloses a reagent for detecting a reproducible lentivirus by a three-channel double-quenching probe, which comprises a first primer pair, a second primer pair and a third primer pair, wherein the first primer pair is a VSV-G primer and a probe pair; the second primer probe pair is a gag primer probe pair; the third primer pair is an RPP30 primer pair. The invention also discloses a detection method of the replication-competent lentivirus, which comprises the following steps: s1 cell culture; s2, preparing a sample to be tested; s3, preparing a standard curve sample; s4 determining the required reaction hole number; s5, preparing a required mixed solution of qPCR reaction solution and a primer probe; s6, adding the mixed solution, the standard substance, the diluent, the negative quality control NCS, the sample to be detected and the sample ERC into the reaction hole according to requirements; s7, controlling the reaction process of each reaction hole; s8 the Ct values and the concentrations of the three targets for each sample were obtained using analytical software. The invention can reduce the background signal value, improve the final signal value, has high detection sensitivity and is more beneficial to the detection of low concentration.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a reagent for detecting replication-competent lentivirus (RCL) by a three-channel double-quenching probe and a detection method thereof.
Background
With the rapid development of CAR-T (chimeric antigen receptor T cell immunotherapy) cellular drugs, lentiviral vectors are commonly used to efficiently introduce chimeric genes into T cells during the production of CAR-T, and despite the replication deficiency of lentiviral vectors, the potential risk of replicating lentiviruses (RCL) may result if homologous or non-homologous recombination occurs between the shuttle plasmid, packaging plasmid and T cells or between the lentiviral vector and endogenous reverse transcription elements of T cells during the production of lentiviruses.
The FDA proposed detection method includes: 1) detecting RCL-related proteins; 2) and detecting the RCL specific DNA sequence in the sample by a real-time quantitative qPCR method. The standard cell co-culture method for detecting RCL has a long period, and about 6 weeks or more is needed to obtain an experimental result.
The TaqMan probe method is a highly specific quantitative PCR technique, and is characterized in that the 3'→ 5' exonuclease activity of Taq enzyme is utilized to cleave a probe, thereby generating a fluorescent signal. Since the probe is specifically bound to the template, the intensity of the fluorescent signal represents the amount of template. FDA now allows rapid detection of RCL status in products using TaqMan probes, i.e., hydrolysis probes (hydrolysisprobes) qPCR methods.
In the prior art, a patent with publication number CN110117675A discloses a reagent and a method for real-time fluorescence quantitative PCR detection of RCL, and discloses a reagent combination including a first primer and a first probe for specific amplification of VSV-G gene, wherein the first primer pair includes a first upstream primer and a first downstream primer, a second primer pair and a second probe for specific amplification of VSV-G gene, the second primer pair includes a second upstream primer and a second downstream primer, a third primer pair and a third probe for specific amplification of reference gene, and the third primer pair includes a third upstream primer and a third downstream primer. The internal reference of the internal reference gene is an internal reference, and the expression of the internal reference gene in each tissue and cell is relatively constant, and the internal reference gene is used as a reference when detecting the expression level change of the gene. The method has the functions of correcting the sample loading amount and experimental errors in the sample loading process and ensuring the accuracy of experimental results. Thus, in this document, two primer probe pairs specifically amplifying the VSV-G gene are essentially used. The reference also discloses a RCL detection method, which comprises: providing a DNA sample to be detected, and carrying out real-time fluorescence quantitative PCR on the DNA sample to be detected by using the reagent combination; and calculating the Cq value and the VSV-G gene copy number of the DNA sample to be detected, thereby judging whether the RCL is contained in the sample. The method is a TaqMan probe method. In the same amplification system, a first primer pair and a first probe for specifically amplifying the VSV-G gene and a third primer pair and a third probe for specifically amplifying the reference gene are utilized to carry out real-time fluorescence quantitative PCR on the DNA sample to be detected.
It has been found that, in the above-mentioned patent documents, the reagent and method for real-time quantitative fluorescence PCR detection of RCL have three pairs of primers and probes, but the first pair of primers and probes and the second pair of primers and probes are primer-probe pairs for specifically amplifying the VSV-G gene, and the third pair of primers and probes are reference genes for specifically amplifying the VSV-G gene, and the three pairs of primers and probes are essentially single-channel detection methods, and have problems of low amplification efficiency, poor specificity, poor accuracy, and the like.
Disclosure of Invention
The invention aims to provide a reagent for detecting replication-competent lentivirus by using a three-channel double-quenching probe and a detection method thereof, which can solve the problems of low amplification efficiency, poor specificity, low accuracy and the like of the conventional reagent for detecting replication-competent lentivirus by using the three-channel double-quenching probe and the detection method thereof in the background art.
In order to achieve the purpose, the invention is realized by the following technical scheme: the reagent for detecting the reproducible lentivirus by the three-channel double-quenching probe is characterized in that: the kit comprises a first primer pair, a second primer pair and a third primer pair, wherein the first primer pair is a VSV-G primer and probe pair, and the gene of the VSV-G forward primer is SEQ ID NO: 1, the gene of the reverse primer is SEQ ID NO: 2, the gene of the corresponding probe is SEQ ID NO: 3; the second primer pair is a gag primer pair, and the gene of the gag forward primer is SEQ ID NO: 4, the gene of the reverse primer is SEQ ID NO: 5, the gene of the corresponding probe is SEQ ID NO: 6; the third primer pair is an RPP30 primer pair, and the gene of the RPP30 forward primer is SEQ ID NO: 7, the gene of the reverse primer is SEQ ID NO: 8, the gene of the corresponding probe is SEQ ID NO: 9.
the further scheme is that the probes are double-quenching probes, and on the basis of a 5 'nuclease hydrolysis probe of the double-quenching probes, a common quenching group is arranged at the 3' end of the probe, and an intermediate quenching group is additionally arranged in the middle of the probe.
In a further scheme, the intermediate quenching group is one of a ZEN quenching group, a TAO quenching group, an DUQ quenching group or a DBQ quenching group.
In a further embodiment, the concentrations of the three primer pairs are 200nM to 400nM for the first primer pair, 200nM to 400nM for the second primer pair, and 200nM to 400nM for the third primer pair, respectively. .
The invention also aims to provide a detection method for detecting the replication-competent lentivirus by using the three-channel double-quenching probe, which comprises the following steps:
s1 cell culture, which comprises the following steps: infecting 293T cells with lentivirus and culturing for at least 5 passages;
s2 preparing a sample to be tested, specifically: extracting a sample by using a DNA extraction kit;
diluting an S3 standard substance, and preparing a standard curve sample;
s4, determining the number of reaction holes according to the standard curve to be detected, the blank control and the number of samples to be detected;
s5, preparing a mixed solution of a corresponding amount of qPCR reaction solution and primer probes according to the required number of reaction holes, shaking and centrifuging for later use, placing each reagent on ice for melting, and slightly shaking and mixing uniformly;
s6, adding a corresponding amount of mixed solution of qPCR reaction solution and primer probes into each reaction hole, and adding a standard substance, a diluent, a negative quality control NCS, a sample to be detected and a sample ERC into different reaction holes according to requirements;
s7, controlling the reaction process of each reaction hole, firstly carrying out UNG enzyme warm bath, then activating polymerase, carrying out PCR circulation for a plurality of cycles, then carrying out DNA denaturation treatment, and then carrying out annealing and extension treatment;
and S8, inputting the concentrations of the standard products into analysis software, and automatically outputting the Ct value of each sample and the concentrations of the three targets by the software according to the standard curve.
In step S1, the lentivirus infects 293T cells and cultures the cells for at least 5 generations, because the number of generations is small, the content of impurities is high, and thus the cells are cultured for at least 5 generations to remove the influence of impurities on the detection.
Further, in step S4, the method for calculating the number of reaction wells includes: reaction well number = (standard curve of 6 concentration gradients +1 no-template control NTC +1 negative quality control NCS + number of samples to be tested × 2) × 3. The reason why the sample to be detected is x 2 is that each sample to be detected should be detected with ERC of the sample at the same time.
In a further embodiment, in step S5, the mixture of qPCR reaction solution and primer Probe includes AceQ U + Universal Probe Master Mix premix, VSV-G/gag/RPP30-F, VSV-G/gag/RPP30-R, and VSV-G/gag/RPP 30-P.
The reagent for detecting the reproducible lentivirus by the three-channel double-quenching probe and the detection method thereof have the following advantages: 1) the three-channel double-quenching probe detection reagent for detecting the reproducible lentivirus adopts three pairs of primer probe pairs, can detect the reproducible lentivirus by real-time fluorescence quantitative PCR, and has high amplification efficiency, good specificity and high accuracy; 2) The detection method for detecting the reproducible lentivirus by the three-channel double-quenching probe adopts the double-quenching probe, and adds an intermediate quenching group in the middle of the probe besides a common quenching group at the 3' end of the probe, so that the background signal can be reduced, the fluorescence intensity can be improved, and the detection sensitivity can be improved.
Drawings
FIG. 1 is a schematic diagram of an amplification curve for detection of VSV-G using the FAM channel.
In the figure, curve 1 is an amplification curve using a conventional detection method described in the background art, and curve 2 is an amplification curve using a detection method of the present invention.
FIG. 2 is a graph showing an amplification curve for gag detection using a VIC channel.
In the figure, curve 3 is an amplification curve obtained by the conventional detection method described in the background art, and curve 4 is an amplification curve obtained by the detection method of the present invention.
FIG. 3 is a schematic representation of the amplification curve for detecting RPP30 using the CY5 signal channel.
In the figure, curve 5 is an amplification curve obtained by the conventional detection method described in the background art, and curve 6 is an amplification curve obtained by the detection method of the present invention.
FIG. 4 is a schematic of an amplification curve for low concentration VSV-G detection using the FAM channel.
In the figure, curve 7 is an amplification curve obtained by the conventional detection method described in the background art, and curve 8 is an amplification curve obtained by the detection method of the present invention.
FIG. 5 is a graph showing the amplification curve for low concentration gag detection using the VIC channel. In the figure, curve 9 is an amplification curve obtained by the conventional detection method described in the background art, and curve 10 is an amplification curve obtained by the detection method of the present invention.
FIG. 6 is a schematic of amplification curves for low concentration RPP30 detection using the CY5 signal channel. In the figure, curve 11 is an amplification curve using a conventional detection method described in the background art, and curve 12 is an amplification curve using a detection method of the present invention.
Detailed Description
The technical solutions of the present invention are described clearly and completely by the following embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A reagent for detecting replication-competent lentivirus by a three-channel double-quenching probe comprises a first primer probe pair, a second primer probe pair and a third primer probe pair, wherein the first primer probe pair is a VSV-G primer and probe pair, and the gene of the VSV-G forward primer is SEQ ID NO: 1, the gene of the reverse primer is SEQ ID NO: 2, the gene of the corresponding probe is SEQ ID NO: 3; the second primer pair is a gag primer pair, and the gene of the gag forward primer is SEQ ID NO: 4, the gene of the reverse primer is SEQ ID NO: 5, the gene of the corresponding probe is SEQ ID NO: 6; the third primer pair is an RPP30 primer pair, and the gene of the RPP30 forward primer is SEQ ID NO: 7, the gene of the reverse primer is SEQ ID NO: 8, the gene of the corresponding probe is SEQ ID NO: 9. the concentrations of the three primer pairs are respectively 200 nM-400 nM for the first primer pair, 200 nM-400 nM for the second primer pair, and 200 nM-400 nM for the third primer pair.
The probes of the three primer probe pairs are double-quenching probes, and on the basis of a 5 'nuclease hydrolysis probe of the double-quenching probes, besides a common quenching group is arranged at the 3' end of the probe, an intermediate quenching group is additionally added in the middle of the probe, wherein the intermediate quenching group is one of a ZEN quenching group, a TAO quenching group, an DUQ quenching group or a DBQ quenching group. In one embodiment, the intermediate quencher is located about 9bp from the 5' fluorophore.
A method for detecting replication-competent lentivirus by a three-channel double-quenching probe comprises the following steps:
s1 cell culture, which comprises the following steps: infecting 293T cells with lentivirus and culturing for at least 5 passages;
s2 preparing a sample to be tested, specifically: extracting a sample by using a DNA extraction kit;
diluting an S3 standard substance, and preparing a standard curve sample;
s4, determining the number of reaction holes according to the standard curve to be detected, the blank control and the number of samples to be detected, wherein the method for calculating the number of the reaction holes comprises the following steps: the number of reaction wells = (standard curve of 6 concentration gradients +1 no-template control NTC +1 negative quality control NCS + number of samples to be tested × 2) × 3, where the number of the samples to be tested × 2 is (6 +1+1+3 × 2) × 3=42 because ERC of each sample to be tested should be simultaneously tested during the test, for example, 3 samples to be tested are used;
s5, preparing a mixed solution of the qPCR reaction solution and the primer probes in a corresponding amount according to the required number of reaction holes, for example, if the number of the reaction holes is 42, preparing the mixed solution in a corresponding amount, shaking and centrifuging the mixed solution for later use, placing the reagents on ice to melt, and slightly shaking and mixing the reagents uniformly;
s6, adding a corresponding amount of mixed solution of qPCR reaction solution and primer probes into each reaction hole, and adding a standard substance, a diluent, a negative quality control NCS, a sample to be detected and a sample ERC into different reaction holes according to requirements; taking 42 reaction wells as an example, dividing the reaction wells into three groups of experiments, wherein 14 reaction wells are used in each group of experiments, and standard curve reagents with different concentration gradients are added into 6 reaction wells; adding a diluent into 1 reaction well to form a template-free control NTC; adding negative quality control NCS into 1 reaction hole; samples to be detected are added into the three reaction holes respectively, and the ERC samples are added into the other three reaction holes.
S7, controlling the reaction process of each reaction hole, and firstly carrying out UNG enzyme warm bath at 37 ℃ for 2 minutes; then activating the polymerase at 95 ℃ for 5 minutes; PCR is circulated for 40 cycles, and then DNA denaturation treatment is carried out at the temperature of 95 ℃ for 10 seconds; annealing and extending at 60 deg.c for 30 sec;
and S8, inputting the concentrations of the standard products into analysis software, and automatically outputting the Ct value of each sample and the concentrations of the three targets by the software according to the standard curve.
In the step S5, the mixture of the qPCR reaction solution and the primer Probe includes AceQ U + Universal Probe Master Mix premix, VSV-G/gag/RPP30-F, VSV-G/gag/RPP30-R, and VSV-G/gag/RPP 30-P. .
In the above step S3, the DNA extraction kit may be any of various kits, as long as it has the function of extracting DNA of a lentivirus infecting 293T cells.
Comparative example 1
The detection of VSV-G, gag and RPP30 were carried out in sequence using the detection method of the present invention and the detection method of the prior art, and the detection results are shown in FIGS. 1-3. As shown in FIG. 1, an amplification curve for detecting VSV-G by using FAM channel, FIG. 2, an amplification curve for detecting gag by using VIC channel, and FIG. 3, an amplification curve for detecting RPP30 by using CY5 channel, wherein the abscissa is cycle number and the ordinate is fluorescence intensity, it can be seen from FIGS. 1 to 3 that the detection method of the present invention has the advantages of lower background signal value, higher final signal value, good detection sensitivity and is more favorable for low concentration detection.
Comparative example 2
The detection method of the invention and the detection method of the prior art are adopted to carry out dilution detection test, and low-concentration samples are detected at the same time, and the detection results are shown in figures 4-6. The specific process is that the standard substance is diluted to 10 copies/mu L, and the detection method of the invention and the detection method of the prior art are adopted to simultaneously carry out the dilution detection experiment. As shown in FIGS. 4-5, when the prior art is used to detect low concentration samples, the detection signal values of VSV-G and gag are low, the repeatability is poor, the CT value is delayed, the quantification is inaccurate, when the RPP30 detection is carried out, the detection signal values are low, undetected and the detection probability is low, while when the detection method of the present invention is used to detect, the detection signal values are high, both can be detected, the repeatability is strong, the quantitative detection is accurate, and the CT value is not delayed.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
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Claims (7)
1. A reagent for detecting replication-competent lentivirus by a three-channel double-quenching probe is characterized in that: the kit comprises a first primer pair, a second primer pair and a third primer pair, wherein the first primer pair is a VSV-G primer and probe pair, and the gene of the VSV-G forward primer is SEQ ID NO: 1, the gene of the reverse primer is SEQ ID NO: 2, the gene of the corresponding probe is SEQ ID NO: 3; the second primer pair is a gag primer pair, and the gene of the gag forward primer is SEQ ID NO: 4, the gene of the reverse primer is SEQ ID NO: 5, the gene of the corresponding probe is SEQ ID NO: 6; the third primer pair is an RPP30 primer pair, and the gene of the RPP30 forward primer is SEQ ID NO: 7, the gene of the reverse primer is SEQ ID NO: 8, the gene of the corresponding probe is SEQ ID NO: 9.
2. the reagent for detecting replication-competent lentivirus by using the three-channel double-quenching probe as claimed in claim 1, wherein: the probes are double-quenching probes, and on the basis of a 5 'nuclease hydrolysis probe of the double-quenching probes, a common quenching group is arranged at the 3' end of the probe, and an intermediate quenching group is additionally arranged in the middle of the probe.
3. The reagent for detecting replication-competent lentivirus by using the three-channel double-quenching probe as claimed in claim 2, wherein: the intermediate quenching group is one of a ZEN quenching group, a TAO quenching group, an DUQ quenching group or a DBQ quenching group.
4. The reagent for detecting replication-competent lentivirus by using the three-channel double-quenching probe as claimed in claim 1, wherein: the concentrations of the three primer probe pairs are respectively 200 nM-400 nM for the first primer probe pair, 200 nM-400 nM for the second primer probe pair, and 200 nM-400 nM for the third primer probe pair.
5. A method for detecting a replication-competent lentivirus using a three-channel double-quenching probe comprising the reagent according to any one of claims 1 to 4, wherein: which comprises the following steps:
s1 cell culture, which comprises the following steps: infecting 293T cells with lentivirus and culturing for at least 5 passages;
s2 preparing a sample to be tested, specifically: extracting a sample by using a DNA extraction kit;
diluting an S3 standard substance, and preparing a standard curve sample;
s4, determining the number of reaction holes according to the standard curve to be detected, the blank control and the number of samples to be detected;
s5, preparing a mixed solution of a corresponding amount of qPCR reaction solution and primer probes according to the required number of reaction holes, shaking and centrifuging for later use, placing each reagent on ice for melting, and slightly shaking and mixing uniformly;
s6, adding a corresponding amount of mixed solution of qPCR reaction solution and primer probes into each reaction hole, and adding a standard substance, a diluent, a negative quality control NCS, a sample to be detected and a sample ERC into different reaction holes according to requirements;
s7, controlling the reaction process of each reaction hole, firstly carrying out UNG enzyme warm bath, then activating polymerase, carrying out PCR circulation for a plurality of cycles, then carrying out DNA denaturation treatment, and then carrying out annealing and extension treatment;
and S8, inputting the concentrations of the standard products into analysis software, and automatically outputting the Ct value of each sample and the concentrations of the three targets by the software according to the standard curve.
6. The method for detecting replication-competent lentivirus by using a three-channel double-quenching probe according to claim 5, wherein the method comprises the following steps: in step S4, the method for calculating the number of reaction wells includes: reaction well number = (standard curve of 6 concentration gradients +1 no-template control NTC +1 negative quality control NCS + number of samples to be tested × 2) × 3.
7. The method for detecting replication-competent lentivirus by using a three-channel double-quenching probe according to claim 5, wherein the method comprises the following steps: in the step S5, the mixture of the qPCR reaction solution and the primer Probe comprises AceQ U + Universal Probe Master Mix premix, VSV-G/gag/RPP30-F, VSV-G/gag/RPP30-R and VSV-G/gag/RPP 30-P.
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| CN114807440A (en) * | 2022-04-13 | 2022-07-29 | 福建省农业科学院畜牧兽医研究所 | Primer and probe for pigeon rotavirus A type fluorescent quantitative PCR detection and kit thereof |
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