CN117737303A - Compositions, methods and kits for detecting replicating retrovirus RCR-GALV in cell therapy products - Google Patents

Abstract

The present invention relates to the field of molecular biological detection, and in particular to a detection composition, method and kit for replicative retrovirus RCR-GALV in cell therapy products. The detection composition includes primers and probes for detecting GALV as shown in the sequence list of SEQ ID NO: 1-3, and has the advantages of high sensitivity and high specificity. The invention also provides a detection kit for the replicative retrovirus RCR-GALV in cell therapy products, which can accurately and quickly detect the GALV content in the sample.

Description

Compositions, methods and kits for detecting replicating retrovirus RCR-GALV in cell therapy products

Technical Field

The invention relates to the field of molecular biological detection, in particular to a composition, a method and a kit for detecting replication type retrovirus RCR-GALV in a cell therapy product.

Background

Retroviruses (retroviruses), also known as retroviruses, belong to the class of RNA viruses whose genetic information is stored not on deoxyribonucleic acid (DNA) but on ribonucleic acid (RNA). The retrovirus genome is diploid, two identical single-stranded positive-strand RNAs are provided with Long Terminal Repeated Sequences (LTRs) at two ends, and stronger promoters and enhancers are contained in the genome, so that the genome plays an important role in transcriptional control of virus DNA. The viral core comprises a reverse transcriptase and an integrase. Unlike other RNA viruses, RNA from retroviruses does not self-replicate and, after entering a host cell, RNA synthesizes double-stranded DNA by reverse transcriptase, which is integrated by integrase into host cell chromosomal DNA to form provirus, establishing a life-long infection and being transmitted to daughter cells as the host cell divides.

Retroviruses currently used in cell therapy are designed as replication defective viral vectors, whose genomic RNA lacks the gag, pol and env sequences required for retroviral replication, which theoretically greatly reduces the risk of producing replication competent viruses, but these packaging elements are essential during the production of the retroviral vector and therefore have the potential to produce replication competent retroviruses by homologous recombination. Because of the potential pathogenicity of the presence of replicating retroviruses (RCR), stringent assays are more needed to rule out the presence of RCR in retroviral vector-based human cell therapy products.

For the determination of the titer of gibbon leukemia virus (GALV) in retroviruses, the conventional method is to determine by plaque assay, which aims at determining the viral plaque forming units (PFU, plaque forming unit) for the determination of the viral titer and for the calibration of the infectious capacity of the virus. The plaque method is relatively complex to operate and requires higher demands on the experimenters, so that the stability of experimental results is relatively poor.

Replication Competent Retrovirus (RCR) assay: according to the current state of the art, sensitive indicator cell culture methods are generally used for RCR detection of gamma-retroviral vectors. The RCR amplified cells and the sample to be tested are co-cultured to amplify RCR to the greatest extent, a proper amount of supernatant is taken to inoculate RCR indication cells for culture after subculturing for a certain passage times and time, cytopathic colonies are observed and counted or detection of RCR markers is carried out, and the conventional method only depends on cytopathic effect judgment results, so that the possibility of false positive exists.

Disclosure of Invention

The invention aims to provide a detection composition of replication type retrovirus RCR-GALV in cell therapy products, which has the advantages of high sensitivity and high specificity.

The invention also provides a detection kit for the replication type retrovirus RCR-GALV in the cell therapy product, and the content of the GALV in the sample can be accurately and rapidly detected by using the kit. The gamma-retrovirus vector used at present is a manually modified retrovirus vector, and the corresponding positive control viruses are generally determined according to the selected envelope genes during detection, so that the positive control viruses are GALV.

The technical scheme adopted for solving the technical problems is as follows:

a detection composition for replication competent retrovirus RCR-GALV in a cell therapy product, the detection composition comprising primers and probes for detecting GALV as follows:

Forwardprimer：AGCAGCAGCTGTTTTACTGGGGTTGGGA，SEQ ID NO：1，

reverse primer: GCGCTGACTGAGTCTTGGAGGGC, SEQ ID NO:2, probe: 5'-FAM-TCGCCATAGATGCTGACCTCCG-BHQ1-3', SEQ ID NO:3,

a kit for detecting a replication competent retrovirus RCR-GALV in a cell therapy product, the kit comprising the detection composition of the invention.

Preferably, the kit further comprises: GALV positive control virus; GALV-sensitive cells, such as HEK293 cells; GALV positive RNA reference.

Preferably, the kit is mainly composed of a reagent A and a reagent B:

the reagent A is qPCR reaction premix liquid and comprises a reagent 2 xOne Step RT-PCR Buffer III special for a probe method qPCR, taKaRa Ex Taq HS (5U/. Mu.L), primeScript RT Enzyme Mix II, ROX Reference Dye internal reference dye, and nuclease-free water, wherein the primer and the probe are used for detecting GALV;

reagent B was a positive control containing GALV positive RNA reference at a concentration of 2E9 copies/. Mu.L.

A method for detecting replication competent retrovirus RCR-GALV in a cell therapy product, the method comprising the steps of:

s1, amplifying and indicating culture of a sample to be detected, and extracting RNA of the sample to be detected;

s2, establishing a fluorescent quantitative PCR reaction system by adopting the detection composition;

s3, performing fluorescent quantitative PCR detection;

s4, analyzing and judging the result of the sample to be tested.

Preferably, the fluorescent quantitative PCR reaction system is 20. Mu.L, comprising 15. Mu.L of reagent A and 5. Mu.L of template; the template is RNA of a sample to be detected, a positive control reference substance or a negative control reference substance.

Preferably, the reaction conditions of the reaction system are: reverse transcription is carried out at 42 ℃ for 5m, and pre-denaturation is carried out at 95 ℃ for 3m; denaturation at 95℃for 5s, annealing at 55℃for 34s,5 cycles; denaturation at 95℃for 5s, annealing at 60℃for 34s,40 cycles.

Preferably, the detection result in S4 satisfies the following conditions at the same time, and the detection result is determined to be valid:

the negative control group had no CPE, and the GALV qPCR detection result was undetected;

the positive control group has CPE, and the Ct value of the GALV qPCR detection result is less than 35.

Preferably, in S4, the result judgment criteria are:

if the detection result of the sample to be detected is undetected and no CPE exists, judging that the batch of samples do not detect the replication competent retrovirus GALV;

if the sample to be tested has a Ct value of < 35 and has significant CPE, determining that the replication competent retrovirus GALV is detected in the batch of samples.

Compared with the prior art, the invention has the beneficial effects that:

1. the GALV titer determination method of the invention selects TCID ₅₀ Method, TCID ₅₀ The method reflects the virus titer by calculating the amount of virus required to cause 50% cytopathic or death (cytopathic effect, CPE), while the PFU method reflects the virus titer by calculating the number of plaques formed by the virus on a monolayer of cells. TCID (TCID) ₅₀ Represents a virus particle with infectious capacity (infectious viral particle), and a plaque also represents a virus particle with infectious capacity, thus TCID ₅₀ Proportional to infectious titer, PFU is also proportional to infectious titer;

compared with TCID ₅₀ The plaque method cannot be used to detect viruses that do not plaque a monolayer of cells. Meanwhile, the plaque method needs to cover a semi-solid agar layer in the operation process, and can influence the adsorption and infection of viruses and cells. TCID (TCID) ₅₀ The limit of detection is higher than that of the plaque method, i.e. TCID ₅₀ The method is capable of detecting lower concentrations of virus. This is because of TCID ₅₀ The method is a probabilistic statistics-based method, and the plaque method is a count-based method. TCID (TCID) ₅₀ The TCID can be calculated by observing CPE in a certain proportion of cells ₅₀ Whereas the plaque principle requires the formation of visible plaques on a monolayer of cells to calculate PFU;

2. the invention relates to a detection composition of gibbon leukemia virus GALV, wherein the 5 'end of a probe adopts a FAM fluorescence report group, and the 3' end adopts a BHQ1 fluorescence quenching group;

the target point of the primer probe design is positioned on the ENV, so that whether the infected virus is GALV virus can be detected specifically;

3. the detection kit for the gibbon leukemia virus GALV comprises a positive control virus reagent for detecting the GALV, wherein a detected sample and HEK293 cells are subjected to amplification in a co-culture mode, and RNA is extracted finely as a sample after the culture is indicated on PG-4 cellsThe product was subjected to fluorescent quantitative PCR detection to determine whether GALV was detected, with a limit of detection of 1TCID ₅₀ GALV；

The method for detecting the gibbon leukemia virus GALV is characterized in that the method is used for culturing the gibbon ape leukemia virus GALV on HEK293 cells for 21 days, the passage times are not less than 5 times, then the culture is subjected to three-freezing and three-thawing, and then the culture is indicated to be cultured on PG-4 cells for 7 days, so that RCR possibly existing in the culture can be amplified to the greatest extent, meanwhile, cell lysate supernatant is collected and passed on the PG-4 cells, cytopathic CPE is observed for preliminary judgment, and further real-time fluorescent quantitative PCR detection is carried out, so that the sensitivity of the detection method is improved, and GALV is detected specifically.

Drawings

FIG. 1 is a graph showing the results of PG-4 cell CPE;

FIG. 2 is a qPCR amplification curve;

FIG. 3 is a qPCR standard curve;

FIG. 4 is a flow chart of an RCR detection experiment;

FIG. 5 is 1TCID ₅₀ The initial viral load is subjected to an amplification curve after culture amplification by the detection method.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

The invention will now be further illustrated with reference to specific examples, which are given solely for the purpose of illustration and are not to be construed as limiting the invention. The test specimens and test procedures used in the following examples include those (if the specific conditions of the experiment are not specified in the examples, generally according to conventional conditions or according to the recommended conditions of the reagent company; the reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified).

Experimental materials used in the examples of the present invention:

one Step PrimeScriptTMRT-PCR Kit (Perfect Real Time) and ROX manufacturer TAKARA, and non-ribozyme water manufacturer Invitrogen;

HEK293 cells and PG-4 cells were from ATCC cell banks;

DMEM medium: purchased from Gibco, cat No.: 11995065;

McCoy's 5A medium: purchased from Gibco, cat No.: 16600-082

Fetal bovine serum: purchased from ExCell Bio escitalopram, cat: FND500;

RNA extraction kit: purchased from bi yun tian, cat No.: R0035M;

one Step PrimeScriptTMRT-PCR Kit (Perfect Real Time) was purchased from TaKaRa (containing ROX Reference Dye reference dye), cat: RR390A;

the nucleotidase water was purchased from Invitrogen, cat No.: 10977-015.

EXAMPLE 1GALV titer detection

A method for determining the titer of infection by GALV virus, comprising the steps of:

counting PG-4 cell suspension, and regulating cell suspension to proper concentration with complete culture medium containing 10% fetal calf serum to reach cell amount of 2×10 ⁵ And each mL.

One or more 96-well cell culture plates were taken, diluted cell suspension was added at Kong Nadi, 50. Mu.L per well, and the cell culture plates were incubated overnight at 37℃in a 5% CO2 incubator.

Taking out the needed GALV virus/test sample, and placing the test sample at room temperature (10-30 ℃) for re-melting for standby.

According to the gradient dilution method, virus dilution without serum is used for treating diseasesDiluting the toxin/test sample to 10 ^-1 -10 ^-6 The dilution is carried out by gently shaking or gently blowing by a pipette for later use. Such as: 100. Mu.L of virus/test sample was added to 900. Mu.L of test sample dilution, which was 10 ^-1 Diluting again to 10 ^-2 Followed by 10-fold gradient dilution to the desired dilution.

The medium in the 96-well plate was first all aspirated. Diluted virus/test dilutions of different gradients were then added to 96-well plates, 50 μl/well, 6 replicate wells per dilution. Negative Control (Negative Control) was added with 50. Mu.L of serum-free diluent. After 24h incubation, 100. Mu.L of complete medium with 10% serum was then supplemented. The added 6-well cell culture plate is subjected to 5% CO at 37+ -1deg.C ₂ Culturing for 7 days, and storing at-80deg.C.

Negative-positive wells were judged by cytopathic effect (CPE) at the end of the experiment and counted, cytopathic effects are shown in fig. 1.

The median infection (TCID 50) was calculated according to the Reed-Muench formula:

according to the Reed-Muench formula, to calculate the infection rate of the virus, the concepts of cumulative positive value, cumulative negative value and infection rate were introduced, i.e. the infected units (positive) at dilution 10-7 will also be infected at dilution 10-6. The cumulative value is calculated by adding the minimum value to the maximum value. The dilution point of 50% infection rate was defined by calculating the infection rate for each dilution. The infection rate was calculated as:

by confirming the dilution interval in which 50% infection rate is located, the Proportional Distance (PD) of the 50% infection rate dilution point to the upper and lower dilutions is calculated:

calculated to obtain

logTCID ₅₀ Log (higher than 50% infection rateThe first dilution) +the proportional distance × { -log (dilution factor) }

The infectious titer of the virus/test was finally determined according to the following formula:

where A is inoculum size in mL.

Example 2GALV primer probe design

The reason for selecting the target site of the primer design is as follows: the main elements of replication competent retrovirus are gag, pol, env (code VSV-G, GALV, etc.). The current research shows that env gene has smaller variation compared with gag and pol genes, and the envelope protein expressed by env gene determines the type of susceptible cells. Therefore, we selected the env sequence of GALV as the detection target. In addition, GALV is ssRNA virus, and the sequence included in the upstream primer and the downstream primer is synthesized into an RNA reference, so that the RNA nucleic acid sequence of the virus is simulated more truly, and a more accurate amplification result is obtained. The primer probe sequences are shown below:

Forwardprimer：AGCAGCTGTTTTACTGGGGTTGGGA，SEQ ID NO：1，

Reverse primer：GCTGACTGAGTCTTGGAGGGC，SEQ ID NO：2，

and (3) probe: 5'-FAM-TCGCCATAGATGCTGACCTCCG-BHQ1-3', SEQ ID NO:3.

selecting RNA reference to carry out screening test on primers, and carrying out screening test on the RNA reference from 2.000X10 ⁷ 10-fold gradient dilutions to 20.000 copies/. Mu.L were made for a total of 7 dilutions, with 5. Mu.L of nuclease-free water as negative control template. The qPCR reaction premix was prepared as shown in table 1 below.

TABLE 1 qPCR reaction System

The qPCR reaction procedure was set according to table 2 using an ABI7500PCR instrument, and signals were collected at 60 ℃.

TABLE 2qPCR reaction procedure

The results are shown in Table 3, and the standard curve R obtained by the primer probe ² =0.999, eff% = 101.175), all meet acceptable standards (R ² More than 0.99, 90% and less than or equal to 110% of Eff), the pair of primer probes is considered to meet detection requirements.

TABLE 3GALV primer probe linear range detection

UD：undetermined；NA：Notapplicable

The amplification curve obtained by the primer probe is shown in fig. 2, and the standard curve is shown in fig. 3.

EXAMPLE 3 precision detection

Precision refers to the degree of closeness between the results of multiple sample determinations of the same sample under prescribed conditions, and is generally expressed in terms of relative standard deviation (coefficient of variation, CV%). Further evaluating the precision of the detection method, selecting 2×10 ⁵ copies/μL、2×10 ⁴ COPIES/. Mu.L and 2X 10 ³ Samples of the standard of copies/. Mu.L were tested by three separate experiments each, each set with 3 replicates, to verify the reproducibility and intermediate precision of the method. The results of the precision of GALV qPCR assay are shown in Table 4.

TABLE 4 GALV qPCR assay precision

All results CV% values were calculated to satisfy CV% <25%, indicating that the method was accurate.

Example 4 detection limit detection

The detection limit refers to the lowest amount that can be detected. From the results of example 2, the detection limit of the present method was determined to be STD8 (100.000 copies/reaction). Selecting 2×10 ¹ At least 6 parallel samples were tested on the samples of the copies/. Mu.L standard, and three wells were set up for each experiment to verify that the limit samples of the method were detectable. The limit of detection of GALV qPCR is shown in table 5.

TABLE 5 GALV qPCR detection limit

Sample name	Complex 1Ct value	Compound well 2Ct value	Multiple well 3Ct value	Results
					DL1	32.825	29.764	31.200	Detection of
DL2	29.114	29.500	29.967	Detection of
					DL3	30.310	29.603	28.604	Detection of
DL4	28.956	29.232	32.847	Detection of
					DL5	31.472	31.650	29.648	Detection of
DL6	29.881	32.947	33.482	Detection of

EXAMPLE 5RCR-GALV detection method

A method for detecting GALV, one of replication-competent retroviruses, in cell therapy products, specifically comprises the following steps:

s1, amplifying and indicating culture of a sample to be detected, and extracting RNA of the sample to be detected

(1) HEK293 cells required for experiments were prepared one day in advance and inoculated 5X 10 ⁶ HEK293 cells were plated into T175 flasks. The following experimental groups were set for fine measurementCell infection inoculation experiment:

negative control group: infecting the cell sample with DMEM basal medium alone;

test article group: only adding a test sample to infect a cell sample, wherein the volume of the test sample is not more than 30mL;

positive control group: adding 1TCID ₅₀ Is a GALV virus-infected cell-like;

inhibition control group: adding test sample and 1TCID ₅₀ Is a GALV virus-infected cell-like;

after the above inoculum was added, the cells were placed in a 37℃incubator for 2 to 4 hours, and then the culture medium in all flasks was replaced with fresh complete medium.

(2) Placing the inoculated culture flask at 37deg.C and 5% CO ₂ Is cultured in an incubator of (a). The culture period is at least 5 times, the experiment groups are passaged according to a certain proportion, and HEK293 complete culture medium is supplemented after the passaging.

(3) When the culture was completed for 14 to 15 days, the preparation of PG-4 cells at the indicated stage was started, and the corresponding six well plates were plated on day 20.

(4) On day 21, cell suspensions of each experimental group were collected, triple-frozen (-80 ℃ C. Frozen for 1h or liquid nitrogen for 15 min) was performed (37 ℃ C. Water bath or metal bath was performed until the solution was thoroughly melted), viruses in the cells were completely released, the collected supernatant was inoculated onto PG-4 cells after centrifugation (4000 rpm,10 min), incubated for 2-4 hours, then the PG-4 cell culture medium was replaced, the culture was continued for 7 days, and the liquid replacement was performed once, if cytopathic effect occurred, the culture was stopped in advance, and the samples were frozen at-80 ℃ C. And treated together after the end of the experiment.

(5) The end-point products were observed for cytopathic effect (CPE) and their RNAs were extracted for subsequent testing.

S2, establishing a fluorescent quantitative PCR reaction system by adopting the detection composition

qPCR was performed using the above-mentioned extracted RNA as a detection template and the primer probe selected in example 2.

The primers and probes for detecting GALV are:

Forwardprimer：AGCAGCTGTTTTACTGGGGTTGGGA，SEQ ID NO：1，

reverse primer: GCTGACTGAGTCTTGGAGGGC, SEQ ID NO:2, probe: 5'-FAM-TCGCCATAGATGCTGACCTCCG-BHQ1-3', SEQ ID NO:3.

s3, performing fluorescent quantitative PCR detection

qPCR reaction premix configurations as shown in table 1, qPCR reaction procedure was set according to table 2, see example 2 for specific methods.

Determination of acceptable criteria for detection

The standard curve correlation coefficient (R ² ) Should not be less than 0.990; the amplification efficiency should be between 90% and 110%.

The NTC of each experiment of the detection method should be negative, and no obvious amplification curve exists.

The experiment is repeated repeatedly, the results have no obvious difference, and the results between different experimental batches are comparable and have good repeatability.

S4, analyzing and judging results of the sample to be tested

The negative control group had no CPE, and the GALV qPCR detection result was undetected;

the positive control group has CPE, and the Ct value of the GALV qPCR detection result is less than 35.

Among these, there are various types of test substances, such as EOPC (end cell), UPB (cell harvest liquid), and CAR-T cells. When testing for EOPC, fresh medium was added to HEK293 cells followed by the test sample. When UPB test samples are detected, HEK293 cells are directly co-cultured with the samples to avoid dilution. According to different types of test substances, the amount of the test substances used by the infected cells is calculated according to the following formula:

UPB detection volume= -initial transduced cell number MOI/(viral harvest drop size) ×ln (1-0.95)

EOPC/CAR-T cell detection volume= (1E 8 or 1% cell)/(cell concentration)

Positive virus GALV addition of 1TCID ₅₀ . Therefore, when the above three points are satisfied at the same time, the following determination is made on the detection result:

if the qPCR test of the test sample group is undetected and no CPE is detected, no replication competent retrovirus (GALV) is detected in the test sample, and the detection result is negative.

If the qPCR detection Ct value of the test sample group is less than 35 and the CPE is obvious, the test sample group detects the replication competent retrovirus (GALV) and the detection result is positive.

If the test subject applicability group qPCR detects Ct values < 35 and there is significant CPE, the test subject has no interference with the replication competent retrovirus (GALV) detection.

If the qPCR of the test sample applicability group is not detected and/or no CPE exists, the test sample has interference on the detection of the replication type lentivirus, and the applicability of the test sample needs to be reconfirmed after the influence of the test sample is eliminated by some methods, such as adjusting the inoculation proportion of the test sample.

The RCR-GALV test results are shown in Table 7.

TABLE 7RCR-GALV detection results

The detection method of the replication type retrovirus RCR-GALV in the cell therapy product implements an amplification culture stage of at least 5 passages for 21 days, so that the replication type retrovirus is amplified as much as possible, and the sensitivity of the method is improved; wherein the initial positive virus is controlled at 1TCID ₅₀ GALV virus, repeated stable detection of 3 bottles of positive control, small Ct value at the end point, very obvious positive result, FIG. 5 is 1TCID ₅₀ The positive control of (a) is an amplification curve of a qPCR experiment at the end point, can meet acceptable standards, and judges that the final detection limit of the method is 1TCID ₅₀ Sample.

Example 6 detection kit for replication competent retrovirus RCR-GALV in cell therapy product

The kit consists of 15 mu L of reagent A and 5 mu L of reagent B:

reagent A is qPCR reaction premix, specifically: 2 XOne Step RT-PCR Buffer III, taKaRa Ex Taq HS (5U/. Mu.L), primeScript RT Enzyme Mix II, ROX Reference Dye reference dye, and no ribozyme water, which are the primers and probes for detecting GALV;

the upstream and downstream primers and probes for detecting GALV are:

Forwardprimer：CCAACTCCATCACTAGGGGTTC，SEQ ID NO：1，

Reverse primer：TGCTCGTCAAGGTCGCTG，SEQ ID NO：2，

and (3) probe: 5'-FAM-ACCTTAATCACAATCTCGTAA-BHQ1-3', SEQ ID NO:3, a step of;

reagent B is a positive control, contains rcAAV8 positive plasmid standard substance, and has the concentration of 4 multiplied by 10 ⁷ cobies/. Mu.L; GALV positive virus with an infection titer of 3.16E+05TCID ₅₀ /mL. The quantification methods of GALV positive RNA reference and GALV positive virus are described in example 1.

In this specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, so that the same or similar parts between the embodiments are referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant points refer to the description of the method section.

The above detailed description of the compositions, methods and kits for detecting replication competent retrovirus RCR-GALV in a cell therapy product is provided. The principles and embodiments of the present invention have been described herein with reference to specific examples, the description of which is intended only to facilitate an understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims.

Claims (10)

1. A composition for detecting replication competent retrovirus RCR-GALV in a cell therapy product, characterized in that the composition comprises primers and probes as follows:

forward primer: AGCAGCAGCTGTTTTACTGGGGTTGGGA, as set forth in SEQ ID NO:1, and a nucleotide sequence shown in the specification;

reverse primer: GCGCTGACTGAGTCTTGGAGGGC, as set forth in SEQ ID NO:2, a nucleotide sequence shown in seq id no;

and (3) probe: 5'-TCGCCATAGATGCTGACCTCCG-3', as set forth in SEQ ID NO:3, and a nucleotide sequence shown in 3.

2. The composition for detecting replication competent retrovirus RCR-GALV in a cell therapy product according to claim 1, wherein: the fluorescence report group of the probe is FAM, and the quenching group is BHQ1.

3. A kit for detecting a replication competent retrovirus RCR-GALV in a cell therapy product, the kit comprising the detection composition of claim 1.

4. The test kit according to claim 3, further comprising: GALV positive control virus, GALV-sensitive cells and GALV positive RNA reference.

5. The detection kit according to claim 3, characterized in that it consists essentially of reagent a and reagent B:

reagent A is qPCR reaction premix, comprising a reagent special for probe method qPCR 2 xOne Step RT-PCR Buffer III, taKaRa Ex Taq HS (5U/. Mu.L), primeScript RT Enzyme Mix II, ROX Reference Dye reference dye, nuclease-free water, and the detection composition of replication type retrovirus RCR-GALV of claim 1;

reagent B was a positive control containing GALV positive RNA reference at a concentration of 2E9 copies/. Mu.L.

6. A method for detecting a replication competent retrovirus RCR-GALV in a cell therapy product of non-diagnostic or therapeutic interest, characterized in that the method comprises the steps of:

s1, amplifying and indicating culture of a sample to be detected, and extracting RNA of the sample to be detected;

s2, establishing a fluorescent quantitative PCR reaction system by adopting the detection composition of the replication competent retrovirus RCR-GALV of claim 1;

s3, performing fluorescent quantitative PCR detection;

s4, analyzing and judging the result of the sample to be tested.

7. The method according to claim 1, wherein: the fluorescent quantitative PCR reaction system is 20 mu L and comprises 15 mu L of reagent A and 5 mu L of template; the template is RNA of a sample to be detected, a positive control reference substance or a negative control reference substance.

8. The method according to claim 1, wherein: the reaction conditions of the reaction system are as follows: reverse transcription is carried out at 42 ℃ for 5m, and pre-denaturation is carried out at 95 ℃ for 3m; denaturation at 95℃for 5s, annealing at 55℃for 34s,5 cycles; denaturation at 95℃for 5s, annealing at 60℃for 34s,40 cycles.

9. The method according to claim 1, wherein: s4, the detection result meets the following conditions at the same time, and the detection result is judged to be valid:

the negative control group had no CPE, and the GALV qPCR detection result was undetected;

the positive control group has CPE, and the Ct value of the GALV qPCR detection result is less than 35.

10. The method according to claim 1, wherein: in S4, the result judgment criteria are:

if the detection result of the sample to be detected is undetected and no CPE exists, judging that the batch of samples do not detect the replication competent retrovirus GALV;

if the sample to be tested has a Ct value of < 35 and has significant CPE, determining that the replication competent retrovirus GALV is detected in the batch of samples.