Primer pair for detecting replicable virus RCL and application thereof

Abstract

The invention discloses a primer pair for detecting replicable virus RCL and application of the primer pair. The primer pair comprises a forward primer and a reverse primer; the sequence of the forward primer is as shown in SEQ ID NO: 1, and the sequence of the reverse primer is as shown in SEQ ID NO: 2. According to the primer pair disclosed by the invention, the more accurate detection on the number of cells in tissues is realized, the detection sensitivity can reach 10 < 2 > Copies / mu L, a probe does not need to be designed, and the primer pair is high in sensitivity, economical and convenient to use.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a primer pair for detecting replication-competent virus (RCL) and its application. Background Art

[0002] Although in the preparation process of cell therapy, most retroviruses and lentiviruses are designed as replication-defective viral vectors, and multiple plasmids are co-transfected to express lentiviruses or γ-retroviruses, there is still a possibility of generating replication-competent virus (RCL) revertants. RCL poses a serious risk to the human body and is one of the main safety risks of immune cell therapy products. Regulatory agencies in various countries recommend that not only should virus reversion be fully considered during product design and quality research, but also long-term monitoring should be carried out in later research to exclude the risk of RCL. Regulations require that when genetic manipulation is carried out using lentiviral vectors, RCL, as an important safety risk concern, in addition to using sensitive indicator cell culture methods to detect RCL in the virus (supernatant, cells at the end of the virus production period) during the production process, the final cell product should also be tested for RCL release using a rapid method that has been validated methodologically during release inspection. There is an urgent need for a rapid, highly sensitive, and economically convenient experimental method for detecting products. Summary of the Invention

[0003] The technical problem to be solved by the present invention is the defect in the prior art of lacking a rapid, highly sensitive, and economically convenient detection method for replication-competent virus (RCL). The present invention provides a primer pair for detecting replication-competent virus (RCL) and its application, achieving a relatively accurate detection of the number of cells in tissues, and the detection sensitivity can reach 10 2 Copies / μL.

[0004] The present invention analyzes the content of replicable virus by a validated qPCR method. In the PCR reaction system, an excessive amount of SYBR fluorescent dye (SYBR Green, i.e., TB Green) is added. After the SYBR fluorescent dye is non-specifically incorporated into double-stranded DNA, it emits a fluorescent signal, while the SYBR dye molecules that are not incorporated into the strand do not emit any fluorescent signal, thus ensuring that the increase in fluorescent signal is completely synchronous with the increase in PCR products. SYBR binds only to double-stranded DNA, so the specificity of the PCR reaction can be determined through the melting curve. This method designs primers based on the sequence of the specific gene of the replicable virus (RCL) VSVG. When there is contamination by the replicable virus (RCL), specific amplification by PCR will specifically replicate and amplify the target DNA, and then the fluorescent dye will specifically bind to produce a fluorescent signal, and the corresponding fluorescent value will be detected by the instrument. Finally, a method for quantitative analysis of the unknown template is carried out through a standard curve. SYBR Green I is the most commonly used DNA-binding dye in fluorescence quantitative PCR and binds non-specifically to double-stranded DNA. In the free state, SYBR Green emits weak fluorescence, but once it binds to double-stranded DNA, its fluorescence increases by 1000 times. Therefore, the total fluorescent signal emitted by a reaction is proportional to the amount of double-stranded DNA present and will increase with the increase in amplification products. Although it has high requirements for primer specificity, it has the characteristics of not requiring probe design, high sensitivity, being able to perform melting curve analysis, and being economical and convenient to use. In this experiment, TB GREEN is used as the dye label, and the higher the content of the target gene in the nucleic acid, the faster the significant increase in fluorescence will be observed.

[0005] In the initial cycles of PCR, the fluorescent signal hardly changes, which is thus defined as the baseline in the amplification curve, and the increase in fluorescence beyond the baseline is the detection of the cumulative target molecules. The fixed fluorescence threshold line is automatically set by the instrument on the logarithmic growth phase of the amplification curve. Ct (threshold cycle) is the number of cycles experienced when the fluorescent signal in each reaction well reaches the set threshold. Ct has a linear relationship with the logarithm of the initial copy number of the test sample, so the test sample can be quantified through the standard curve.

[0006] The present invention solves the above technical problems through the following technical solutions:

[0007] The first aspect of the present invention provides a primer pair targeting the VSV-G gene, and the primer pair includes a forward primer and a reverse primer; the sequence of the forward primer is as shown in SEQ ID NO:1, and the sequence of the reverse primer is as shown in SEQ ID NO:2.

[0008] The second aspect of the present invention provides a kit for detecting replicable virus, characterized in that the kit includes the primer pair as described in the first aspect.

[0009] In some embodiments of the present invention, the kit further comprises a non-specific double-stranded nucleic acid fluorescent chimeric dye.

[0010] In some specific embodiments of the present invention, the non-specific double-stranded nucleic acid fluorescent chimeric dye is TB GREEN dye.

[0011] The third aspect of the present invention provides an application of a primer pair as described in the first aspect in the preparation of a kit for detecting replicable viruses.

[0012] The fourth aspect of the present invention provides an application of a primer pair as described in the first aspect, or a kit as described in the second aspect, in the preparation of a reagent for detecting replicable viruses.

[0013] The fifth aspect of the present invention provides a method for detecting replicable viruses, which comprises the step of contacting a primer pair as described in the first aspect, or a kit as described in the second aspect, with a sample to be tested to carry out an amplification reaction.

[0014] In some embodiments of the present invention, the method further comprises extracting the DNA of the sample to be tested.

[0015] In some embodiments of the present invention, the method is for non-diagnostic purposes, such as detecting environmental samples in a laboratory.

[0016] In some embodiments of the present invention, the method further comprises the step of adding a non-specific double-stranded nucleic acid fluorescent chimeric dye to the reaction system before or after the amplification reaction occurs.

[0017] In some embodiments of the present invention, the method further comprises the step of detecting the fluorescence signal in the amplification reaction; optionally, it further comprises the step of comparing the fluorescence signal detected in the amplification reaction with a standard curve.

[0018] In some embodiments of the present invention, the standard curve is obtained by the following method:

[0019] The VSV-G gene standard stock solution (1×10 10 copies / μL) is serially diluted 10-fold to 1×10 1 ~1×10 9 copies / μL for a total of 9 gradients to obtain standard solution; adding the primer pair as described in the first aspect to the standard solution to carry out an amplification reaction, and obtaining the Ct value of the amplification reaction through the fluorescence intensity; constructing a linear curve with the starting copy number of each concentration standard as the abscissa and the Ct value as the ordinate.

[0020] Based on the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0021] The reagents and raw materials used in the present invention are all commercially available.

[0022] The positive and progressive effects of the present invention are as follows:

[0023] The primer pair provided by the present invention can quickly and highly sensitively detect replicable viruses, has strong specificity, and generates fluorescence signals by adding fluorescent dyes, without the need to design probes, is economical and convenient to use. Description of the Drawings

[0024] Figure 1 It is a standard curve graph of VSVG. Detailed Embodiments

[0025] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. The experimental methods without specific conditions in the following examples are carried out according to conventional methods and conditions, or selected according to the product instructions.

[0026] Example 1

[0027] 1. Primers

[0028] The primers used in the experiment are the upstream and downstream primers VSVG primers (F / R) as follows:

[0029]

[0030] 2. Key Reagents

[0031] Name Manufacturer / Brand Product Item Number Specification Nuclease-Free Water Solarbio R1600 500 mL / bottle Real-Time Fluorescent Quantitative PCR Premix TaKaRa RR420 1 mL / vial EasyDilution Diluent TaKaRa 9160 1 mL / vial VSVG Primer GenScript NA NA VSVG Positive Control Shanghai Genechem Co., Ltd. NA NA

[0032] 3. Key Instruments

[0033]

[0034] 4. Key Consumables

[0035]

[0036] 5. Detection Steps

[0037] 5.1 Sample Preparation

[0038] Test sample (Sample): Genomic DNA (gDNA) of the sample cells to be tested. (For the gRNA of the cells, refer to Pengyu Huang et al., Direct Reprogramming of Human Fibroblasts to Functional and Expandable Hepatocytes, Cell Stem Cell 14, 370 - 384. The cells were cultured to extract gRNA, and the Chinese National Institute for Food and Drug Control conducted the replication - competent lentivirus test on the above - mentioned cell gRNA (cell culture method). The results showed that the detection results of the three methods, namely P24 antigen detection, psi - gas detection, and VSVG detection, were all negative. The inspection report number was SH202100742).

[0039] Negative control (Negative control sample, NCS): Genomic DNA (gDNA) of UCF cells (UCF cells are fibroblasts and have not been transfected with a lentiviral vector).

[0040] No template control (NTC): Nuclease - free water.

[0041] Positive control (Positive control sample, PCS): 1×10 3 copies / μL VSVG plasmid standard.

[0042] 5.2 Primer preparation

[0043] Add nuclease - free water according to the dilution concentration provided on the tube wall of the primer (dry powder): 10 μmol / L, 500 μL of water.

[0044] 5.3 Preparation of positive control

[0045] Positive control stock solution.

[0046] First, dilute the VSVG plasmid dry powder with nuclease - free water to 1×10 10 copies / μL to obtain the positive control stock solution. The dilution method is as follows:

[0047] For the mass m (ng) of the positive control and the total length L (bp) of the plasmid vector, the amount of substance M (mol) can be calculated. The calculation formula is as follows:

[0048] m = n×M, where:

[0049] m is the plasmid mass provided by the manufacturer;

[0050] n is the average molecular weight of the VSVG positive control;

[0051] L is the number of base pairs of the VSVG positive control, which is 4246 bp. The average molecular weight of each base pair is 660 Da. Therefore, the average molecular weight (n) of the VSVG positive control is 4246 × 660 = 2.80×10 6 .

[0052] Convert the amount of substance M (mol) to the number of copies according to the following formula. The calculation formula is as follows:

[0053] Number of copies = M × NA, where:

[0054] NA is Avogadro's constant, equal to 6.02×10 23 .

[0055] The prepared VSVG positive control preservation solution can be directly used for detection and should meet the following requirements:

[0056] Reagent Name Specification (copies / μL) Storage Condition Validity Period VSVG Positive Control <![CDATA[1×10 10 > -30°C or below 1 year

[0057] 5.4 Positive control dilution

[0058] Take the VSVG positive control preservation solution and dilute it to the corresponding concentration according to the method in Table 1 with the EasyDilution diluent:

[0059] Table 1

[0060]

[0061]

[0062] 5.5 Cell and tissue DNA extraction

[0063] (1) Extract DNA from cell samples. After collecting the cells into a centrifuge tube, centrifuge and discard the supernatant; resuspend the cells with pre-cooled 1×PBS using a pipette, centrifuge and discard the supernatant; then add 1 mL of Ezol lysis solution using a pipette to lyse the cells.

[0064] (2) Add 0.2 mL of chloroform using a pipette, carefully cover the tube cap, shake vigorously for 10 s, and let it stand at room temperature for 3 min;

[0065] (3) Centrifuge at 4°C and 12,000 g for 20 min in a refrigerated high-speed centrifuge, discard the supernatant, add 300 μL of isopropanol to the middle and lower organic phases, vortex for 5 s or invert and mix for 5 s, and place on ice for 3 min;

[0066] (4) Use a pipette to aspirate 700 μL of the sample and any possible precipitate, add it to the centrifugal column, gently cover the lid, centrifuge at 8,000 g at room temperature for 15 s using a high-speed centrifuge, and discard all the flow-through liquid using a pipette;

[0067] (5) Transfer the remaining sample to the centrifuge column, gently cover the lid, centrifuge at 8,000 g for 15 s at room temperature using a high-speed centrifuge, and discard all the flow-through liquid using a pipette.

[0068] (6) Add 700 μL of 1×WB to the centrifuge column, gently cover the lid, centrifuge at 8,000 g for 15 s at room temperature using a high-speed centrifuge, and discard all the flow-through liquid using a pipette. Repeat this step 3 times.

[0069] (7) Increase the centrifuge speed to 12,000 xg and centrifuge for 3 min without adding 1×WB.

[0070] (8) Transfer the centrifuge column to a new 1.5 mL centrifuge tube, add 50 μL of sterile water dropwise to the center using a pipette, gently cover the tube lid, let it stand at room temperature for 2 min, and centrifuge at 4℃ and 10,000 g for 3 min in a refrigerated high-speed centrifuge to elute the DNA. Note: Extraction can also be performed using an equivalent "Genomic DNA Extraction Kit".

[0071] 5.6 Preparation of the reaction system

[0072] Before preparing the reaction system, the number of reaction wells required for the experiment should be calculated first:

[0073] Number of reaction wells (N0) = (number of standards + number of controls + number of test samples) × number of parallel wells;

[0074] To make up for the losses during pipetting, 5 or 8 should be added to the number of reaction wells during actual preparation (add 5 when the number of reaction wells < 48; add 8 when the number of reaction wells ≥ 48), and the resulting value is recorded as the preparation number (N). The preparation ratios are shown in Table 2:

[0075] Table 2

[0076]

[0077] Addition of template

[0078] Add the corresponding templates to the wells where the amplification system has already been added, 2 μL per well (the templates include the gDNA of the test article, positive control, and negative control, and nuclease-free water for the blank control).

[0079] Seal the 96-well plate with a PCR sealing film and centrifuge briefly using a microplate centrifuge.

[0080] 5.7 Real-time fluorescence quantitative PCR amplification

[0081] The reaction program is shown in Table 3:

[0082] Table 3

[0083]

[0084] 5.8 Data analysis

[0085] Experimental validity

[0086] Negative control (NTC): CT value ≥ 30;

[0087] Negative control (NCS): CT value ≥ 30;

[0088] Positive control (PCS): CT value < 30;

[0089] 5.9 Experimental results

[0090] Based on the CT value of the experimental results, judge the negativity / positivity of the sample. The judgment criteria are as follows:

[0091] Sample Ct value ≥ 30, negative;

[0092] Sample Ct value < 30, positive.

[0093] 6. Verification implementation process

[0094] The experimental method is shown in 5 Experimental procedures.

[0095] 6.1 Specificity

[0096] The gDNA of cells not transfected with the lentiviral vector is used as a negative control to prove that the primers of VSVG cannot amplify specific fragments in cell lines without virus packaging;

[0097] Using DEPC-H 2 O as a blank control to prove that the VSVG primers will not be interfered by the reaction system;

[0098] 6.1.1 Solution preparation

[0099] Test samples: 3 batches of test samples (Batch1, Batch2, Batch3), extract gDNA for subsequent experiments;

[0100] Positive control: Use a plasmid standard of 1.00E+07 copies / μL as a control product;

[0101] Negative control: Extract gDNA from 1 batch of negative cells for subsequent experiments;

[0102] Blank control: DEPC water is used as a blank control.

[0103] 6.1.2 Method / Procedure

[0104] Take the above solutions and detect them according to the experimental method in 5. Set 3 parallel wells for each sample and detect once.

[0105] 6.1.3 The results are recorded in Table 4 and Figure 1 。

[0106] Table 4 Specificity verification results

[0107]

[0108] 6.2 Acceptance criteria

[0109] For the blank control and negative control, the results should be negative, without obvious amplification curves, and the Ct value ≥ 30;

[0110] For the test samples: The results of 3 batches of test sample cells should be negative, without obvious amplification curves, and the Ct value ≥ 30;

[0111] For the positive control (using the plasmid standard of 1.00E+07 copies / μL at the highest point of the standard curve as the control sample), the results should be positive, with obvious amplification curves, and the Ct value < 30;

[0112] The RSD between the same test samples ≤ 30%.

[0113] 6.2.1 Conclusion

[0114] Blank control (DEPC-H 2 O) Ct value: All are ≥ 30 between 30.66 and 33.80;

[0115] Negative control Ct value: All are ≥ 30 between 34.74 and 35.37;

[0116] Ct values of 3 batches of test sample cells: All are ≥ 30 between 32.52 and 37.69;

[0117] Positive control (using the plasmid standard of 1.00E+07 copies / μL at the highest point of the standard curve as the control sample) Ct value: All are < 30 between 13.78 and 13.85;

[0118] The RSD between the same test samples is between 0 and 8%, all ≤ 30%.

[0119] 6.3 Detection limit

[0120] The detection limit refers to the lowest concentration or amount of the analyte with known concentration that can be reliably quantified in the test. In this verification, the known concentration of VSVG plasmid standard was used for repeated detection, and the concentration corresponding to the recovery rate between 50 - 200% and RSD ≤ 30% was taken as the detection limit.

[0121] 6.3.1 Solution preparation:

[0122] The VSVG plasmid standard was serially diluted, and the low-concentration standard solutions (1.00E+03 copies / μL, 1.00E+02 copies / μL, 1.00E+01 copies / μL) were assayed 9 times. Calculate the RSD value and recovery rate. The lowest concentration corresponding to RSD ≤ 30% and recovery rate between 50% and 200% is the detection limit.

[0123] Prepare as shown in Table 5 below:

[0124] Table 5

[0125]

[0126]

[0127] 6.3.2 Method / Procedure:

[0128] Take the above solutions and detect them according to the experimental method in 5, with 3 parallels for each concentration and repeated 3 times.

[0129] 6.4 The results are recorded in Table 6 below.

[0130] Table 6 Verification Results of Detection Limit

[0131]

[0132] Note: *1 is abnormal data and has been excluded.

[0133] 6.4.1 Acceptance Criteria

[0134] For the detection results of three low-concentration plasmid standards, the lowest concentration with at least 95% of the results having a Ct value < 30 is the detection limit;

[0135] The results of the blank control and negative control are negative;

[0136] The recovery rate is between 50% and 200%;

[0137] The RSD between the same low-concentration plasmid standards ≤ 30%.

[0138] 6.4.2 Conclusion

[0139] Ct value of the blank control (DEPC-H2O): between 30.66 and 33.80, all ≥ 30;

[0140] Ct value of the negative control: between 34.74 and 35.37, all ≥ 30;

[0141] The detection limit is 1.00E+02 copies / μL (n = 9, 100% CT value < 30), the recovery rate = 110% is between 50% and 200%, and the RSD = 15% ≤ 30%.

[0142] Compliance with requirements: Yes □ No

[0143] 6.5 Durability

[0144] Use the same solution as that used in the specificity verification, and have two experimenters perform the detection. The RSD ≤ 30%, demonstrating the influence value among different personnel.

[0145] 6.5.1 Solution Preparation

[0146] Use the same solution as that used in the specificity verification. For the specific preparation method, see 6.1 Specificity Verification Solution Preparation.

[0147] 6.5.2 Method / Procedure

[0148] Take the above solution, and have two testers (Experimenter 1 and Experimenter 2) perform the detection according to the experimental method in 5 respectively. There are 3 parallels for each concentration, and the detection is performed once.

[0149] Record the results in Table 7 below.

[0150] Table 7 Verification Results of Different Personnel

[0151]

[0152] 6.5.4 Acceptance Criteria

[0153] The results of the blank control and the negative control should both be negative.

[0154] Positive control: Plasmid standard (using the plasmid standard at the highest point of the standard curve, 1.00E+0 7 copies / μL as the reference substance) The result should be positive.

[0155] The RSD ≤ 30% among different personnel for the same test sample.

[0156] 6.5.5 Conclusion

[0157] Blank control (DEPC-H 2 O) Ct value: between 32.69 and 33.08, all ≥ 30;

[0158] Negative control Ct value: between 33.57 and 35.00, all ≥ 30;

[0159] Test sample cell Ct value: between 33.84 and 36.03, all ≥ 30;

[0160] Positive control (using the plasmid standard at the highest point of the standard curve, 1.00E+07 copies / μL as the reference substance) Ct value: between 13.19 and 13.81, all < 30;

[0161] The RSD between different personnel for the same test sample is between 0% and 6%, all ≤ 30%.

[0162] Whether it meets the requirements: Yes □ No

[0163] 6.6 System suitability

[0164] The results generated during the verification all meet the acceptance criteria for system suitability listed in Table 8.

[0165] System suitability acceptance criteria

[0166] Table 8

[0167] Parameter Acceptance Criteria <![CDATA[Coefficient of determination (R 2 )]]> <![CDATA[R 2 ≥0.98]]> Slope of Standard Curve -3.1～-3.8 Blank Control No obvious amplification curve and Ct≥30 Negative Control No obvious amplification curve and Ct≥30 Positive Control There is an obvious amplification curve, and Ct < 30

[0168] 7. Verification summary

[0169] After verification of specificity, detection limit, and durability, the detection of replicable virus RCL (qPCR dye method) can be applied to the detection of replicable virus RCL in test samples, as shown in Table 9.

[0170] Table 9 Verification summary table

[0171]

[0172]

Claims

1. A primer pair targeting the VSV - G gene, characterized in that, the primer pair comprises a forward primer and a reverse primer; the sequence of the forward primer is shown as SEQ ID NO:1, and the sequence of the reverse primer is shown as SEQ ID NO:

2.

2. A kit for detecting replicable viruses, characterized in that, the kit includes the primer pair according to claim 1; preferably, the kit further includes a non - specific double - stranded nucleic acid fluorescent chimeric dye.

3. The kit according to claim 2, characterized in that, the non - specific double - stranded nucleic acid fluorescent chimeric dye is TBGREEN dye.

4. Use of the primer pair according to claim 1 in the preparation of a kit for detecting replicable viruses.

5. Use of the primer pair according to claim 1, or the kit according to claim 2 or 3 in the preparation of a reagent for detecting replicable viruses.

6. A method for detecting replicable viruses, characterized in that, it includes the step of contacting the primer pair according to claim 1, or the kit according to claim 2 or 3 with a sample to be tested to carry out an amplification reaction; preferably, the method further includes extracting the DNA of the sample to be tested, and / or, the method is for non - diagnostic purposes.

7. The method according to claim 6, characterized in that, the method further includes the step of adding the non - specific double - stranded nucleic acid fluorescent chimeric dye to the reaction system before or after the amplification reaction occurs.

8. The method according to claim 6, characterized in that, the method further includes the step of detecting the fluorescence signal in the amplification reaction; optionally, it further includes the step of comparing the fluorescence signal detected in the amplification reaction with a standard curve.

9. The method according to claim 8, characterized in that, the standard curve is obtained by the following method: Dilute the VSV-G gene standard stock solution (1×10 10 copies / μL) by 10-fold serial dilution to 1×10 1 ~1×10 9 copies / μL for a total of 9 gradients to serve as the standard solution; Adding the primer pair according to claim 1 to a standard solution to carry out an amplification reaction, and obtaining the Ct value of the amplification reaction through fluorescence intensity; constructing a linear curve with the initial copy number of each concentration standard as the abscissa and the Ct value as the ordinate.

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