CN112877475A - Primer probe combination, kit and detection method for detecting RCL - Google Patents
Primer probe combination, kit and detection method for detecting RCL Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
The invention provides a primer probe combination, a kit and a detection method for detecting RCL, belonging to the technical field of virus detection, wherein the primer probe combination comprises a primer pair and a probe, the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID No.1, the nucleotide sequence of a downstream primer of the primer pair is shown as SEQ ID No.2, and the nucleotide sequence of the probe is shown as SEQ ID No. 3. The primer probe combination provided by the invention has the advantages that the detection result does not exceed 8h from sample preparation to detection, the detection period is shortened, and the lower limit of detection on RCL is 5 copies. When the RCL copy number in the sample is more than 5 copies, the positive coincidence rate is 100 percent; when the copy number in the sample is 5 copies, the positive coincidence rate is 95%.
Description
Technical Field
The invention relates to the technical field of virus detection, in particular to a primer probe combination, a kit and a detection method for detecting RCL.
Background
Lentiviral particles are currently widely used in clinical therapeutic research as a gene vector tool, including gene and cell therapies. However, these viral particles are replication-defective lentiviruses due to engineering processes during production.
However, since replication-competent virus particles (RCL) may be theoretically generated during the production of lentiviral particles, in 2006, the U.S. promulgated Guidance on RCL for gene therapy products, namely FDA (food AND DRUG administration) RCR Guidance, was issued, AND in 2010, the U.S. FDA suggested that RCL detection was required for lentiviral-vector clinical cells AND gene therapy products.
Currently, the FDA recommended assay protocol is a cell-based assay protocol, with the overall assay protocol testing time exceeding 6 weeks, but many phase I/II clinical trials currently require fresh cell products to be administered to subjects. Obviously, the cell-based RCL detection scheme cannot meet the current demand, so that the establishment of a short-cycle detection scheme is urgently needed to replace the current cell-based RCL detection method.
Disclosure of Invention
In view of the above, the present invention aims to provide a primer probe combination, a kit and a detection method for RCL, wherein the primer probe combination provided by the present invention can obtain a detection result within 8 hours from sample preparation to detection, and the detection period is shortened.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a primer probe combination for detecting RCL, which comprises a primer pair and a probe, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID No.1, the nucleotide sequence of a downstream primer of the primer pair is shown as SEQ ID No.2, and the nucleotide sequence of the probe is shown as SEQ ID No. 3.
Preferably, the kit further comprises an internal reference primer pair and an internal reference probe, wherein the nucleotide sequence of an upstream primer of the internal reference primer pair is shown as SEQ ID No.4, the nucleotide sequence of a downstream primer of the internal reference primer pair is shown as SEQ ID No.5, and the nucleotide sequence of the internal reference probe is shown as SEQ ID No. 6.
The invention also provides a kit of RCL, which comprises the primer probe combination in the technical scheme.
The invention also provides a kit for detecting RCL, which comprises the primer probe combination in the technical scheme.
The invention also provides a method for detecting RCL, which comprises the following steps:
1) extracting RNA of a sample to be detected, and reversely transcribing the RNA into cDNA;
2) carrying out fluorescent quantitative PCR on the cDNA obtained in the step 2) by using the primer probe combination in the technical scheme, and taking the cDNA extracted from the Jurkat cell as background control;
when the 3 Ct values of the sample to be detected and the background control are all determined to be underwent, the RCL is not contained in the sample to be detected, and the detection result is negative;
when the 3 Ct values of the sample to be detected are all larger than the Ct value of the background control, the sample to be detected is negative;
when 2 Ct values of the sample to be detected are larger than the Ct value of the background control and 1 Ct value of the sample to be detected is smaller than the Ct value of the background control, the sample to be detected is negative;
when 2 Ct values of the sample to be detected are smaller than the Ct value of the background control and 1 Ct value of the sample to be detected is larger than the Ct value of the background control, the sample to be detected is positive in detection result;
and when the 3 Ct values of the sample to be detected are all smaller than the Ct value of the background control, the detection result of the sample to be detected is positive.
Preferably, the system of the fluorescent quantitative PCR of step 2) comprises 2 XQuantNova Probe PCR Mix 15. mu.L, double distilled water 4.85. mu.L, 10. mu.M concentration of 1. mu.L of upstream and downstream primers of the primer set of claim 1, 10. mu.M concentration of 0.5. mu.L of the Probe of claim 1, 10. mu.M concentration of 1. mu.L of upstream and downstream primers of the internal reference primer set, 0.5. mu. L, ROX 0.15.15. mu.L of the internal reference Probe, and 4. mu.L of cDNA per 30. mu.L.
Preferably, the running program of the fluorescence quantitative PCR comprises: 2min at 95 ℃; 95 ℃ for 5s, 60 ℃ for 30s, 40 cycles.
Preferably, double distilled water is used as a negative control.
Preferably, the RNA obtained in the step 1) is used as an RNA sample control.
Preferably, a plasmid containing the VSVG gene and the RPP30 gene is used as a positive control.
The invention provides a primer probe combination, a kit and a detection method for detecting RCL, wherein the primer probe combination comprises a primer pair and a probe, the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID No.1, the nucleotide sequence of a downstream primer of the primer pair is shown as SEQ ID No.2, and the nucleotide sequence of the probe is shown as SEQ ID No. 3.
The results of the embodiments of the present invention show that: the primer probe combination provided by the invention is adopted to detect for no more than 8 hours from sample preparation to computer detection, the detection period is shortened, and the lower limit of detection on RCL is 5 copies. When the RCL copy number in the sample is more than 5 copies, the positive coincidence rate is 100 percent; when the copy number in the sample is 5 copies, the positive coincidence rate is 95%.
Drawings
FIG. 1 is a VSVG gene standard graph;
fig. 2 is a RPP30 standard graph.
Detailed Description
The invention provides a primer probe combination of RCL, which comprises a primer pair and a probe, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID No.1, the nucleotide sequence of a downstream primer of the primer pair is shown as SEQ ID No.2, and the nucleotide sequence of the probe is shown as SEQ ID No. 3. In the invention, the primer probe combination is designed aiming at a conserved sequence of a Vesicular stomatitis virus fusogenic envelope G glycoprotein (VSVG) gene.
SEQ ID No.1：TGCACACTTTCTGAGAAGGAGAGACA；
SEQ ID No.2：CAAGACTACAAACACATGCAGTAAT；
SEQ ID No. 3: FAM-ACATTCACCTTCCATGCAGATA-MGB is preferably labeled with FAM at the 5' end when used, but is not limited to FAM, and may be VIC or HEX.
In the invention, the primer probe combination also preferably comprises an internal reference primer pair and an internal reference probe, wherein the nucleotide sequence of an upstream primer of the internal reference primer pair is shown as SEQ ID No.4, the nucleotide sequence of a downstream primer of the internal reference primer pair is shown as SEQ ID No.5, and the nucleotide sequence of the internal reference probe is shown as SEQ ID No. 6. In the present invention, the reference primer pair and the reference probe are designed for a highly conserved region of the RPP30 gene.
SEQ ID No.4：CTCATTGAAAGCCTCAGATACCGCGA；
SEQ ID No.5：CCTCTGAAAGCACAGTAGTACA；
SEQ ID No. 6: cy5-GCGCCGATACTTCAATCAATAC-MGB is preferably labeled with Cy5 when reused, but is not limited to Cy5, and may be VIC or HEX.
The invention also provides a kit for detecting RCL, which comprises the primer probe combination in the technical scheme. In the invention, the kit comprises a primer pair and a probe, and the nucleotide sequence is shown as SEQ ID No. 1-6.
The invention also provides a method for detecting RCL, which comprises the following steps:
1) extracting RNA of a sample to be detected, and reversely transcribing the RNA into cDNA;
2) carrying out fluorescent quantitative PCR on the cDNA obtained in the step 2) by using the primer probe combination in the technical scheme, and taking the cDNA extracted from the Jurkat cell as background control;
when the 3 Ct values of the sample to be detected and the background control are all determined to be underwent, the detection result of the sample to be detected is negative, and no RCL exists;
when the 3 Ct values of the sample to be detected are all larger than the Ct value of the background control, the sample to be detected is negative in detection result;
when 2 Ct values of the sample to be detected are larger than the Ct value of the background control and 1 Ct value of the sample to be detected is smaller than the Ct value of the background control, the sample to be detected is negative in detection result;
when 2 Ct values of the sample to be detected are smaller than the Ct value of the background control and 1 Ct value of the sample to be detected is larger than the Ct value of the background control, the sample to be detected is positive in detection result;
and when the 3 Ct values of the sample to be detected are all smaller than the Ct value of the background control, the sample to be detected is positive in detection result.
And when the detection result is positive, the retest is required, the detection result is still positive, and the retest is required to be carried out by a cell culture method by a third-party detection mechanism.
In the present invention, the sample to be tested preferably includes a cell therapy product or peripheral blood. The method for processing the sample to be detected is not particularly limited, and the method can be carried out by a person skilled in the art according to conventional processing.
In the present invention, the system of the quantitative fluorescence PCR preferably comprises, per 30. mu.L, 2 XQuantiNova Probe PCR Mix 15. mu.L, water 4.85. mu.L, 1. mu.L of each of the upstream and downstream primers of the primer set described in the above-mentioned embodiment at a concentration of 10. mu.M, 0.5. mu.L of the Probe described in the above-mentioned embodiment at a concentration of 10. mu.M, 1. mu.L of each of the upstream and downstream primers of the internal reference primer set at a concentration of 10. mu.M, 0.5. mu. L, ROX 0.15.15. mu.L of the internal reference Probe, and 4. mu.L of cDNA. In the present invention, the running program of the fluorescence quantitative PCR preferably includes: 2min at 95 ℃; 95 ℃ for 5s, 60 ℃ for 30s, 40 cycles.
In the invention, double distilled water is preferably used as a negative control, and no amplification is caused in the negative control, which indicates that the reaction system is free from pollution. The RNA obtained is preferably used as an RNA sample control, no amplification is carried out, and the fact that no plasmid containing VSVG gene is left in a sample to be detected is shown. In the present invention, a plasmid containing the VSVG gene and the RPP30 gene is preferably used as a positive control, and the method for constructing the VSVG gene and the RPP30 gene into the plasmid is not particularly limited, and those skilled in the art can construct the plasmid according to the conventional techniques. In the present invention, the plasmid preferably comprises a pEAZY plasmid.
In order to further illustrate the present invention, the following detailed description of the invention is given in conjunction with examples, which should not be construed to limit the scope of the invention.
TABLE 1 major kit sources
Example 1
1) Plasmid template Standard preparation
The VSVG and RPP30 gene fragment was amplified, the RPP30 gene was ligated by blunt end to pEAZY plasmid vector, designated pEAZY-P, which was ligated to VSVG gene by double digestion with restriction enzymes SalI and NheI, completing the double gene ligation to this vector, but not limited to pEAZY plasmid vector. To refine the copy number of the plasmid standards, the standards were quantitated precisely using Bio-rad QX200, and finally diluted to 109copy/μ l, subpackaged and frozen at-80 ℃ to avoid repeated freeze thawing.
2) Control sample preparation
RNA of Jurkat cells was extracted using the MiniBEST Plant RNA Extraction Kit and then reverse-transcribed into cDNA for use as a background control at a final cDNA concentration of 200 ng/. mu.l, and the control samples used included background control BC (background control), no Template control NTC (No Template control), RNA control before reverse transcription, and positive control PC (Positive control), where PC was 20copy plasmid Template standard + the aforementioned reverse-transcribed 100ng cDNA.
TABLE 2 control sample information
3) Fluorescent quantitative PCR detection method
The fluorescent quantitative PCR reaction detection reaction system is shown in Table 3.
TABLE 3 PCR reaction System
The reaction system was run on an ABI 7500 instrument with the operating program as in table 4.
TABLE 4 PCR run program
| Step (ii) of | Time | Temperature (. degree.C.) |
| Initial activation | 2min | 95 |
| Denaturation of the material | 5s | 95 |
| Bonding and extension | 30s | 60 |
| Number of |
40 |
4) Determination of detection result
The control amount of RNA added is equivalent to the amount before cDNA inversion, for example: mu.l of RNA was added, and if 2. mu.l was inverted to 10. mu.l of cDNA, 10. mu.l of cDNA was added. Therefore, the sample to be detected, BC and NTC controls need to be compared with the Ct value of the detection sample respectively. Confirmation is performed in 3 steps as follows:
NTC has no amplification, which indicates that the reaction system has no pollution;
RNA contrast is not amplified, which indicates that VSVG plasmid residue is not existed in the sample to be detected;
the BC reference is shown in table 5:
TABLE 5 BC comparison
Establishment of standard curve by double fluorescent quantitative PCR
RCL is detected by adopting a single-particle double PCR method, the plasmid template standard substance is diluted by 10 times in a gradient way, and the gradient dilution range is 106copy/μl-100copy/. mu.l, each target sample was repeated 3 times. The reaction system is shown in Table 1, the reaction conditions are shown in Table 3, and after amplification, a standard curve aiming at VSVG and RPP30 is established, and the standard curve meets the screening gold standard of fluorescent quantitative PCR (the amplification efficiency is 90-110%, and the linear relation R is299.90% or more) amplification efficiencies were 100.35%, 101.65%, respectively, and linear relationships were 99.95% and 99.92%, respectively, and the standard curves are shown in fig. 1 and fig. 2, respectively.
By adopting the primer and the probe, negative control BC and NTC and RNA control detection results are all underended. The primers and probes designed by the invention have good specificity.
Example 2
Detection sensitivity and reliability verification
To verify the reliability of the method, the positive and negative detection rates of the method were verified separately. The specific method for designing the positive detection rate is that 80, 40, 20, 10 and 5copy plasmids are respectively added into cDNA of the Juckat cell, other methods are the same as the example 1, the detection result shows that the lower limit of the detection sensitivity is 5copy, the coincidence rate of the positive detection higher than 5 copies is 100%, when the sample contains 5 copies, the detection rate is 95%, and the specific information is as follows:
TABLE 6 sample Positive detection Rate
The negative detection coincidence rate is designed as follows: only 100ng of cDNA was detected in each reaction, and the virus was repeated 20 times, with a 100% negative match.
In addition, the detection result can be obtained in the same day after the sample is prepared and the sample is tested on the machine for no more than 8 hours, and the detection period of the traditional cell-based detection method is more than 3 weeks, so the period of the detection method established by the invention is far shorter than that of the traditional method.
Example 3
To verify the reproducibility of the method, an intra-batch repeat and an inter-batch repeat were performed, respectively
The in-batch repetition is that plasmid standards with different concentrations are repeated 3 times at each concentration, the Ct value and the Coefficient of Variation (C.V.) of the detection are shown in Table 6, the results of the other methods are the same as the example 1, and the results show that the CV of the in-batch repeated Coefficient of Variation is not more than 1.5 percent, and the inter-batch repeated Coefficient of Variation is not more than 4 percent, which indicates that the probe, the primer and the method have very good repeatability.
TABLE 7 Day 0RPP30 results repeated in batches
TABLE 8 Day0 VSVG in-batch repeat results
The batch-to-batch repeats were amplified with the probes and primers of the present invention at Day1, Day3, and Day7, respectively, to verify the reliability of the batch-to-batch repeats of the method, and the results are shown in table 9.
TABLE 9 VSVG batch-to-batch repeat results
| Day one | The third day | The seventh day | Mean value | Coefficient of variation C.V% |
| 16.57 | 17.19 | 16.62 | 16.79 | 2.07％ |
| 19.22 | 20.05 | 19.48 | 19.58 | 2.16％ |
| 22.42 | 23.85 | 22.79 | 23.02 | 3.23％ |
| 26.59 | 26.90 | 25.84 | 26.44 | 2.05％ |
| 30.37 | 31.46 | 30.75 | 30.86 | 1.80％ |
| 33.62 | 34.82 | 32.43 | 33.62 | 3.55％ |
| 35.45 | 36.46 | 35.79 | 35.90 | 1.43％ |
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be construed as the protection scope of the present invention.
Sequence listing
<110> Beijing ancient cooking peptide source Biotechnology Ltd
Jiao Shunchang
<120> primer probe combination, kit and detection method for detecting RCL
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Claims (10)
1. A primer probe combination for detecting RCL is characterized by comprising a primer pair and a probe, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID No.1, the nucleotide sequence of a downstream primer of the primer pair is shown as SEQ ID No.2, and the nucleotide sequence of the probe is shown as SEQ ID No. 3.
2. The primer probe combination of claim 1, further comprising an internal reference primer pair and an internal reference probe, wherein the nucleotide sequence of the upstream primer of the internal reference primer pair is shown as SEQ ID No.4, the nucleotide sequence of the downstream primer of the internal reference primer pair is shown as SEQ ID No.5, and the nucleotide sequence of the internal reference probe is shown as SEQ ID No. 6.
3. A kit for detecting RCL, comprising the primer-probe combination of claim 1.
4. A kit for detecting RCL, comprising the primer-probe combination of claim 2.
5. A method of detecting RCL, comprising the steps of:
1) extracting RNA of a sample to be detected, and reversely transcribing the RNA into cDNA;
2) performing fluorescent quantitative PCR on the cDNA obtained in the step 2) by using the primer probe combination of claim 4, and taking the cDNA extracted from Jurkat cells as a background control;
when the 3 Ct values of the sample to be detected and the background control are all determined to be underwent, the RCL is not contained in the sample to be detected, and the detection result is negative;
when the 3 Ct values of the sample to be detected are all larger than the Ct value of the background control, the sample to be detected is negative;
when 2 Ct values of the sample to be detected are larger than the Ct value of the background control and 1 Ct value of the sample to be detected is smaller than the Ct value of the background control, the sample to be detected is negative;
when 2 Ct values of the sample to be detected are smaller than the Ct value of the background control and 1 Ct value of the sample to be detected is larger than the Ct value of the background control, the sample to be detected is positive in detection result;
and when the 3 Ct values of the sample to be detected are all smaller than the Ct value of the background control, the detection result of the sample to be detected is positive.
6. The detection method according to claim 5, wherein the system of the fluorescent quantitative PCR of step 2) comprises 2 XQuantiNova Probe PCR Mix 15. mu.L, 4.85. mu.L of double distilled water, 1. mu.L of each of the upstream and downstream primers of the primer set of claim 1 at a concentration of 10. mu.M, 0.5. mu.L of the Probe of claim 1 at a concentration of 10. mu.M, 1. mu.L of each of the upstream and downstream primers of the internal reference primer set at a concentration of 10. mu.M, 0.5. mu. L, ROX 0.15. mu.L of the internal reference Probe, and 4. mu.L of cDNA per 30. mu.L.
7. The detection method according to claim 5 or 6, wherein the running program of the fluorescence quantitative PCR comprises: 2min at 95 ℃; 95 ℃ for 5s, 60 ℃ for 30s, 40 cycles.
8. The detection method according to claim 5, wherein double distilled water is used as a negative control.
9. The detection method according to claim 5, wherein the RNA obtained in the step 1) is used as an RNA sample control.
10. The method of detecting according to claim 5, wherein a plasmid containing the VSVG gene and the RPP30 gene is used as a positive control.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113528706A (en) * | 2021-07-13 | 2021-10-22 | 苏州博腾生物制药有限公司 | Reagent for detecting replication-competent lentivirus by three-channel double-quenching probe and detection method thereof |
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| CN111910015A (en) * | 2019-05-08 | 2020-11-10 | 深圳宾德生物技术有限公司 | Probe, primer pair, fluorescent quantitative PCR kit and method for detecting replication type lentivirus |
| CN111918972A (en) * | 2018-01-31 | 2020-11-10 | 朱诺治疗学股份有限公司 | Methods and reagents for assessing the presence or absence of replication competent viruses |
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Patent Citations (5)
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| WO2014011996A1 (en) * | 2012-07-13 | 2014-01-16 | The Trustees Of The University Of Pennsylvania | Methods of assessing the suitability of transduced t cells for administration |
| CN111918972A (en) * | 2018-01-31 | 2020-11-10 | 朱诺治疗学股份有限公司 | Methods and reagents for assessing the presence or absence of replication competent viruses |
| CN110117675A (en) * | 2018-02-05 | 2019-08-13 | 上海赛比曼生物科技有限公司 | A kind of reagent and method of real-time fluorescence quantitative PCR detection RCL |
| CN109295261A (en) * | 2018-11-21 | 2019-02-01 | 温州医科大学附属第医院 | It is a kind of for detecting the primer, probe and detection method of slow virus RCL |
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| CN113528706A (en) * | 2021-07-13 | 2021-10-22 | 苏州博腾生物制药有限公司 | Reagent for detecting replication-competent lentivirus by three-channel double-quenching probe and detection method thereof |
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