CN113005223A - Primer for digital PCR detection aiming at RCR/RCL and detection method - Google Patents

Abstract

The invention discloses a primer for digital PCR detection aiming at RCR/RCL, which comprises the following primer groups: a VSVG4 primer set; a VSVG5 primer set; VSVG6 primer set. In the application, the inventor obviously improves the sensitivity of related RCL detection by screening related primer sequences and optimizing a reaction system, can establish a good technical basis for improving the stability of the treatment technical effect of the Car-T cell, and simultaneously provides good reference for the application of other virus detection technologies and cell treatment technologies.

Description

Primer for digital PCR detection aiming at RCR/RCL and detection method

Technical Field

The invention belongs to the technical field of molecular biology detection, and particularly relates to a digital PCR (dPCR) detection technology patent application for RCR/RCL.

Background

Among antitumor treatments, the Car-T cell therapy technology is a rapidly developing antitumor therapy technology in recent years due to its safety and wide applicability. However, in practice, there still remains a safety risk in the production of Car-T cell therapy products using Retroviruses or lentiviruses as vectors, mainly because the Retroviruses or lentiviruses as vectors may still produce specific Replication Competent viruses, i.e., Replication Competent Retroviruses (RCR) or Replication Competent Lentiviruses (RCL). Aiming at the risk, the FDA and the European Union have issued relevant documents and require to establish a sensitive and reliable RCR/RCL detection method so as to monitor the risk. In particular, for gene therapy products produced using gamma-retroviral and lentiviral vectors, it is necessary to separately control RCR/RCL at different stages of production and use, including the production of viral cell banks, end cells, viral vectors and CAR-T cells, and to monitor individual patients after CAR-T cell reinfusion to ensure that the patients are not infected by RCR/RCL.

In the prior art, the detection method of RCR/RCL mainly comprises a PCR method or QPCR method aiming at a specific gene (such as VSV-G gene, psi-gag gene or GALV gene) and a sensitive cell infection test method. In order to improve the sensitivity of the assay, the FDA recommends the use of a cell culture-based infectivity assay for RCR/RCL detection, the main process of which is: the possible RCR/RCL is amplified on sensitive cells first, and then detected by a sensitive, rapid detection method at the end of the culture. The QPCR method has higher sensitivity and specificity compared with the common PCR method, but the detection of the QPCR method is about 10 copies. However, the cell co-culture method requires about 6 weeks or more for obtaining results due to a long period, and thus cannot meet the requirement of rapid detection in practical detection.

The digital PCR technology, as a third-generation PCR technology, is a single-molecule amplification technology and also an absolute quantitative technology which does not depend on a standard product. Compared with the traditional fluorescence quantification, the method can realize single-molecule detection, and the sensitivity can be improved by more than 100 times; has the advantages of strong PCR amplification inhibition resistance and high detection accuracy. Like QPCR, the digital PCR technology can utilize TaqMan probes or SYBR Green dyes to carry out fluorescent signal detection, and finally utilizes an end-point signal detection method and Poisson distribution statistical calculation to realize absolute quantification of samples.

When the TaqMan probe is used for detection, the TaqMan probe design has the technical characteristic of high specificity, and the probe design core technology is that a specific site sequence of 18-30nt is designed at the middle position (the position closer to the upstream) of an upstream and downstream amplification primer, a fluorescent group is marked at the 5 'end of the probe, and a quenching group is marked at the 3' end of the probe. The probe does not emit light in a free state, and the TaqMan probe encountered is hydrolyzed due to the 5 '-3' exonuclease activity of the Taq enzyme in the PCR amplification process, so that fluorescence is emitted. And finally, absolute quantification of the sample is realized by using an end-point signal detection method and Poisson distribution statistical calculation.

In the conventional RCR/RCL detection, although FDA allows the detection of RCR/RCL residues in a Car-T product by using a QPCR technology, the lower detection limit of the QPCR technology is about 10copies, so that the safety requirement of RCR/RCL '0 detection' is obviously difficult to meet from the absolute safety aspect of application, and on the other hand, the problem of low primer probe specificity is also encountered in the QPCR detection process. Therefore, the technology of RCR/RCL detection requirement is combined with the emerging high-sensitivity digital PCR technology to develop a suitable RCR/RCL detection method, and the method still has very important technical significance.

Disclosure of Invention

Based on a digital PCR (dPCR) detection technology, the application aims to provide a plurality of groups of primer pairs for RCR/RCL detection, thereby laying a certain technical foundation for strict control of the RCR/RCL amount in related gene products.

The technical solution adopted in the present application is detailed as follows.

The primer for digital PCR detection aiming at RCR/RCL comprises the following primer groups:

VSVG4 primer set:

VSVG4-F：5’-attcaagcagacggttggatgt-3’，

VSVG4-R：5’-CCATACCAGCGGAAATCACA-3’；

VSVG 4-Probe: FAM-catgcttccaaatgggtcacta-MGB;

the specific amplification sequence (68 bp) is shown as SEQ ID No.1, and specifically comprises the following steps:

ATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTCACTACTTGTGATTTCCGCTGGTATGG；

VSVG5 primer set:

VSVG5-F：5’-caggtgtctggttcgagatg-3’，

VSVG5-R：5’-agatggagcagagatacttgacc-3’；

VSVG 5-Probe: HEX-ttctgggcattcagggaatctggctg-BHQ 1;

the specific amplification sequence (92 bp) is shown as SEQ ID No.2, and specifically comprises the following steps:

CAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAAGGGTCAAGTATCTCTGCTCCATCT；

VSVG6 primer set:

VSVG6-F：5’-ggacatggtatgttggactccg-3’，

VSVG6-R：5’-gcgtcttgaatgtgaggatgttc-3’；

VSVG 6-Probe: FAM-catcttagctcaaaggctcag-MGB;

the specific amplification sequence (77 bp) is shown as SEQ ID No.3, and specifically comprises the following steps:

GGACATGGTATGTTGGACTCCGATCTTCATCTTAGCTCAAAGGCTCAGGTGTTCGAACATCCTCACATTCAAGACGC。

and (3) preparing a detection kit by using the primer for digital PCR detection of the RCR/RCL.

The RCL detection method using the primer for the digital PCR detection of RCR/RCL comprises the following steps of specifically designing a 15-microliter reaction system:

template, 1. mu.l;

VSVG-F，10μM、0.3μl；

VSVG-R，10μM、0.3μl；

VSVG-probe, 10. mu.M, 0.3. mu.l;

JN solution（20x），0.75μl；

Prob master Mix（2x），7.5μl；

NF water，4.85μl；

the reaction procedure is as follows: 95 deg.C for 5 min; at 95 deg.C, 50s, 60 deg.C, 1min30s, 40 cycles; 5min at 70 ℃; storing at 4 ℃.

In the application, the inventor obviously improves the sensitivity of related RCL detection by screening related primer sequences and optimizing a reaction system, can establish a good technical basis for improving the stability of the treatment technical effect of the Car-T cell, and simultaneously provides good reference for the application of other virus detection technologies and cell treatment technologies.

Drawings

FIG. 1 shows the detection result of the primer set VSVG1 for NTC, and it can be seen that there is no signal;

FIG. 2 shows the detection result of the primer set VSVG1 for pVSV-G, and it can be seen that there is no signal;

FIG. 3 shows the detection result of the primer set VSVG2 for pVSV-G, and it can be seen that the positive signal and the background signal cannot be effectively distinguished although there is a signal, and thus, the primer set is not suitable for the digital PCR detection;

FIG. 4 shows the results of detection of the primer set VSVG4 for RCL samples; wherein the probes in the VSVG4 primer group are marked by FAM, in the figure, Repeat1 and Repeat2 are 2 times of the same sample, and Plasmid is Pvsv-G Plasmid; the results in the figure show that the VSVG4 primer probe can detect RCL copy number, but has poor repeatability and pollution in NTC (later experiments prove that plasmid pollutes RCL and NTC in the operation process, and the RCL detection value minus NTC value is exactly the RCL actual copy number);

FIG. 5 shows the results of detection after decontamination of the primer set for VSVG 4;

FIG. 6 shows the results of detection of the primer set for VSVG 5;

FIG. 7 shows the results of detection after the reaction system was optimized for the VSVG4 primer set;

FIG. 8 shows the results of detection after the reaction system was optimized for the VSVG5 primer set;

FIG. 9 shows the results of the assay after optimization of the reaction program for the VSVG4 primer set;

FIG. 10 shows the results of an optimized reaction procedure for the VSVG5 primer set;

FIG. 11 is the results of RCL sample testing for the primer sets VSVG4, VSVG5, and VSVG 6;

FIG. 12 is a line of correlation between actual and theoretical results for a VSVG5 primer set;

FIG. 13 is a sample lower limit of detection RCL for the VSVG5 primer set;

FIG. 14 is a line of the correlation between the actual measurement results and the theoretical results for the VSVG6 primer set;

FIG. 15 is a sample lower limit of detection RCL for the VSVG6 primer set;

FIG. 16 shows the results of RCL sample detection when VSVG5 and VSVG6 primer sets were used in common.

Detailed Description

The present application is further illustrated by the following examples. Before describing the specific embodiments, a brief description will be given of some experimental background cases in the following embodiments.

Related biological materials:

the C8166T cell line (T cell for short) was purchased from Ningbo boat Biotechnology Ltd for extracting genomic DNA of the C8166 cell line;

pVSV-G plasmid (length: 6507 bp; concentration: 100ng/ul, as positive control) was purchased from Hunan Fenghui Biotech Ltd;

primers and probes for RCR/RCL detection were designed and synthesized by Invitrogen corporation;

the main reagents are as follows:

DNeasy Blood & Tissue Kits (for extracting genomic DNA from human T cell lines), product of QIAGEN;

probmaster Mix (2 x) (digital PCR reaction Mix), JN solution (20 x) (for improving digital PCR reaction efficiency), digital PCR blocking oil (blocking each microwell, preventing cross contamination), JN Medsys products;

the main apparatus comprises:

the Clarity digital PCR system, JN Medsys, Singapore;

general PCR instruments TC-96/G/H (b) C, Bori science and technology, Hangzhou;

NanoDrop2000 (micro uv spectrophotometer), Thermo Fisher Scientific, usa;

shaker MIX-25P, MIULAB (Hangzhou Miao instruments, Inc.);

mimi centrifuge C1301-T-230V, Labnet, KOREA.

Example 1

Based on pVSV-G plasmid, the application aims to provide a digital PCR detection technology aiming at RCR/RCL, thereby laying a certain technical foundation for the safe application of related gene products.

Before practical application of digital PCR technology, relevant primers and probes need to be designed, so the design principles of relevant primers and probes are briefly introduced as follows. In the present application, the design principle of the dPCR primer is as follows:

the length of the amplification product is about 60-100 bp; the product cannot form a secondary structure; the length of the primer is generally 15-30 nt; the content of G and C is between 40 and 60 percent; the four bases are randomly distributed; the 5' end of the primer does not need to be modified (the RCL primer is not modified); primer 3' may not modify any group; the last 5 bases at the 3' end of the primer have at least one (at most 2-3) G or C.

The design principle of the dPCR probe (Taqman probe) is as follows:

the optimal length is 20nt (no more than 30 nt), the length is more than 30nt, but the distance between the fluorescent group and the quenching group is 18-25 nt; the dissolving temperature (Tm) value of the Taqman probe is required to be about 10 ℃ higher than that of a primer, and is usually between 65 ℃ and 70 ℃; continuous single base repeats, especially G, should be avoided as this may affect the secondary structure of the probe and reduce hybridization efficiency; in addition, the 5' end of the probe should not contain too much G, since the G base quenches the fluorescent signal; the G/C content of the probe is about 50% (ideally 30% -80%), and if the amplified sequence is rich in AT sequence, nucleic acid analogues such as LNA (locked nucleic acid) or MGB can be doped; the length of the MGB modified probe is about 15 nt; the 5 'end of the probe is as close as possible to the 3' end of the upstream primer, but it does not overlap to ensure rapid probe cleavage by the Taq enzyme.

Based on the principle, the inventors preliminarily designed and obtained a series of primer pairs and corresponding probes by using pVSV-G plasmid as an amplification object, as follows.

A primer group:

VSVG1-F：5’-agcattggggagtcagactc-3’，

VSVG1-R：5’-cattcagggaatctggctgc-3’；

VSVG 1-Probe: FAM-caggtgtctggttcgagatggctga-BHQ 1;

using the primers, the specific amplification sequence was as follows (85 bp):

AGCATTGGGGAGTCAGACTCCCATCAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATG

VSVG2 primer set:

VSVG2-F：5’-cggagagctatcatccctgg-3’，

VSVG2-R：5’-gagtctgactccccaatgct-3’；

VSVG 2-Probe: FAM-gaaaggagggcacagggttcagaagt-BHQ 1;

using the above primers, the specific amplification sequence was as follows (118 bp):

CGGAGAGCTATCATCCCTGGGAAAGGAGGGCACAGGGTTCAGAAGTAACTACTTTGCTTATGAAACTGGAGGCAAGGCCTGCAAAATGCAATACTGCAAGCATTGGGGAGTCAGACTC

VSVG3 primer set:

VSVG3-F：5’-tgctgctccaatcctctcaa-3’，

VSVG3-R：5’-tgggtccaatttccacgtct-3’；

VSVG 3-Probe: FAM-ctgtgggatgactgggc-MGB;

using the above primers, the specific amplification sequence was as follows (107 bp):

TGCTGCTCCAATCCTCTCAAGAATGGTCGGAATGATCAGTGGAACTACCACAGAAAGGGAACTGTGGGATGACTGGGCACCATATGAAGACGTGGAAATTGGACCCA

VSVG4 primer set:

VSVG4-F：5’-attcaagcagacggttggatgt-3’，

VSVG4-R：5’-CCATACCAGCGGAAATCACA-3’；

VSVG 4-Probe: FAM-catgcttccaaatgggtcacta-MGB;

using the primers, the specific amplification sequence was as follows (68 bp):

ATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTCACTACTTGTGATTTCCGCTGGTATGG

VSVG5 primer set:

VSVG5-F：5’-caggtgtctggttcgagatg-3’，

VSVG5-R：5’-agatggagcagagatacttgacc-3’；

VSVG 5-Probe: HEX-ttctgggcattcagggaatctggctg-BHQ 1;

using the above primers, the specific amplification sequence was as follows (92 bp):

CAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAAGGGTCAAGTATCTCTGCTCCATCT。

from the practical point of view of detection, it is obvious that further detection verification is required to prove the effectiveness, accuracy and practical lower limit of detection. For this reason, the inventors have carried out relevant practical verification, and the specific experimental procedures are summarized as follows.

(one) extracting DNA template and preparing different control samples

Extracting T cell line DNA as a template for PCR amplification, and taking the DNA as a Negative Control (NC) sample;

taking nuclease-free water (NF water) as a template for PCR amplification as a blank NTC negative control sample;

the mixture of the extracted T cell line DNA and pVSV-G plasmid was used as a template for PCR amplification (1X 10³And mixing the same volume of the pVSV-G of copies/ul and the genomic DNA of the T cells of 20 ng/ul) to serve as a Positive Control (PC) sample, so as to evaluate the detection performances such as the design accuracy, the sensitivity and the like of the related primers.

It should be noted that, in the DNA extraction, after the cultured T cell line is discarded from the culture medium, the cells are washed 3 times by adding PBS to a cell-containing culture dish, and then the cells are lysed and the T cell genomic DNA is purified according to the instruction of the DNA extraction kit of QIAGEN corporation.

(II) PCR amplification

And (2) respectively utilizing the different samples set in the step (I) as templates to carry out PCR amplification by utilizing the designed primer groups, wherein a 15-microliter amplification system is designed as follows:

template, 1. mu.l;

VSVG-F，10μM、0.6μl；

VSVG-R，10μM、0.6μl；

VSVG-probe, 10. mu.M, 0.5. mu.l;

JN solution（20x），0.75μl；

Prob master Mix（2x），7.5μl；

NF water，4.05μl；

reaction procedure: 95 deg.C for 5 min; at 95 deg.C, 50s, 58 deg.C, 1min30s, 40 cycles; 5min at 70 ℃; storing at 4 ℃.

PCR reactions were repeated 3 times for each primer set, and for each template sample. The experimental results of different primer sets are described in detail below.

The results of the experiment for the VSVG1 primer set are shown in FIGS. 1 and 2. As can be seen, none of the NTC and pVSV-G plasmids were detected by VSVG1, indicating that the VSVG1 primer pair is not suitable for digital PCR detection. (i.e., no signal was confirmed by dPCR for VSVG1, and RCL was not detected by VSVG 1)

The results of the experiment on the VSVG2 primer set are shown in FIG. 3. It can be seen that, the VSVG2 detects NTC no signal, and the pVSV-G has positive signal, but the positive signal and the background signal are not separated, and the positive signal and the negative signal of the pVSV-G plasmid detected by the VSVG2 are still not separated by optimizing the conditions of primer and probe concentration, annealing temperature and the like, so that the positive result is difficult to accurately judge. Indicating that the VSVG2 primer pair is not suitable for digital PCR detection. (i.e., background signal was too high for VSVG2 detection by dPCR and RCL was not effective for VSVG2 detection)

The experimental result of the primer group of VSVG3 is similar to that of the primer group of VSVG1, and the VSVG3 detects no signals of both NTC and pVSV-G plasmids, which indicates that the primer pair of VSVG3 is not suitable for digital PCR detection. (i.e., no signal was confirmed by dPCR for VSVG3, and RCL was not detected by VSVG 3)

The results of the experiment on the VSVG4 primer set are shown in FIG. 4. The result shows that the NTC result detected by the VSVG4 is 26.82 copies/ul; detecting two repetitions of the RCL sample, wherein the results are 60.99copies/ul and 55.46copies/ul respectively; the result of detecting the pVSV-G positive plasmid is 568.89 copies/ul. As the 15ul digital PCR reaction system is added with 1ul template, the actual detection result of the pVSV-G positive plasmid is 568.89copies/ul × 15ul ÷ 1ul =8.53 × 10³copies/ul, ratio theoretical 8.3X 10³copies/ul are slightly larger but remain substantially the same. However, as shown by the results of RCL sample detection by VSVG4, the repeatability of the results of the two times is poor, and a large amount of amplification is detected in NTC. Further later experimental verification shows that the main reason for the result is that the plasmid is diluted due to the ringThere is aerosol in the environment, contaminating the RCL sample and NTC.

After the relevant pollution factors are eliminated, further detection results show (as shown in figure 5), the result of the VSVG4 detecting NTC is 0.4copies/ul, namely, trace copies still exist in NTC; the positive plasmid is 379.10 copies/ul; the two repeated RCL samples are respectively 37.75copies/ul, 38.10copies/ul, 95% confidence intervals, and the CV value is less than 10%, which indicates that the RCL sample detected by VSVG4 has good repeatability. The positive plasmid was much smaller than the first 568.89copies/ul, and the analysis was thought to be mainly due to the fact that the plasmid was easily degraded when stored at an ultra-low concentration, and the value of the same plasmid used later was smaller and smaller, but the copy number of the RCL sample was substantially unchanged.

The results of the experiment on the VSVG5 primer set are shown in FIG. 6. The result shows that the NTC result detected by the VSVG5 is 0.59 copies/ul; detecting positive plasmid as 284.56 copies/ul; the two duplicate RCL samples were 31.01copies/ul, 29.53copies/ul, respectively, and the CV value was <10% at the 95% confidence interval, indicating that the VSVG5 detects RCL samples with good reproducibility.

When the comparison is carried out, the RCL value detected by the VSVG5 is smaller than that detected by the VSVG4, and the analysis is mainly that the FAM fluorescence is marked by the VSVG4, the HEX fluorescence is marked by the VSVG5, and the FAM is relatively stronger than the HEX signal. In general, it can be assumed that: both VSVG4 and VSVG5 can accurately detect RCL samples, but there is still a trace copy number detection error when detecting NTC samples.

Example 2

Based on the preliminary verification of example 1, in order to further optimize the relevant detection effect, the inventors performed further optimization experiments on VSVG4, VSVG5, and the two primer sets, and the relevant optimization process and results are briefly described below.

(I) the problem of trace copy number in NTC is solved from the perspective of a reaction system

In order to solve the problem of trace copy number in NTC when VSVG4 and VSVG5 primer groups are used for detection, based on the prior experience and the prior art, the inventor preliminarily considers that the concentration of primers and probes is properly reduced so as to prevent the trace copy number possibly generated by the combination of the primers and the probes as much as possible. Therefore, the inventors redesigned the reaction system and performed PCR verification for primer sets VSVG4 and VSVG 5. Specifically, the method comprises the following steps:

the 15. mu.l reaction system was designed as follows:

template, 1. mu.l;

VSVG-F，10μM、0.3μl；

VSVG-R，10μM、0.3μl；

VSVG-probe, 10. mu.M, 0.3. mu.l;

JN solution（20x），0.75μl；

Prob master Mix（2x），7.5μl；

NF water，4.85μl；

95 deg.C for 5 min; at 95 deg.C, 50s, 58 deg.C, 1min30s, 40 cycles; 5min at 70 ℃; storing at 4 ℃.

For specific results:

VSVG4 assay results (shown in fig. 7): NTC is 0.52copies/ul, and it can be seen that, compared with the preliminary verification, the copy number is also reduced, but the reduction effect is limited; the positive plasmid is 195.75 copies/ul; the two repeated RCL samples are respectively 40.32copies/ul, 41.28copies/ul, 95% confidence intervals, and the CV value is less than 10%, which indicates that the VSVG4 has good repeatability in detecting the RCL sample;

VSVG5 assay results (shown in fig. 8)): the NTC is 0.08copies/ul, which is obviously reduced by a lot compared with the preliminary verification in the embodiment 1; the positive plasmid is 188.75 copies/ul; the two duplicate RCL samples were 30.23copies/ul, 33.47copies/ul, respectively, and the CV value was <10% at the 95% confidence interval, indicating that the VSVG5 detects RCL samples with good reproducibility.

After further comparison, it can be seen that the RCL value detected by VSVG5 is smaller than that detected by VSVG4, and the analysis suggests that this is mainly because the VSVG4 is labeled with FAM fluorescence, and the VSVG5 is labeled with HEX fluorescence, and that FAM is relatively stronger than HEX signal.

In summary, both VSVG4 and VSVG5 can accurately detect RCL samples, but after the reaction system is further optimized by an optimization mode of reducing the concentrations of the primers and the probes, the NTC detected by VSVG5 is reduced, and the copy number in the NTC detected by VSVG4 is basically unchanged, which indicates that the detection result of VSVG5 should be caused by the high concentration of the primers and the probes, while VSVG4 is not caused by the excessively high concentration of the primers and the probes.

(II) solving the problem of trace copy number in NTC from the perspective of reaction conditions

Based on the above experimental results, in order to further solve the problem of the trace copy number in NTC, the inventors further adjusted the PCR reaction conditions to investigate the influence of the PCR reaction conditions on the final amplification result. The specific experimental conditions are briefly described below.

The specific reaction operation can be referred to the above, and the specific 15 μ l reaction system is designed as follows:

template, 1. mu.l;

VSVG-F，10μM、0.3μl；

VSVG-R，10μM、0.3μl；

VSVG-probe, 10. mu.M, 0.3. mu.l;

JN solution（20x），0.75μl；

Prob master Mix（2x），7.5μl；

NF water，4.85μl；

the reaction program was readjusted to: 95 deg.C for 5 min; at 95 deg.C, 50s, 60 deg.C, 1min30s, 40 cycles; 5min at 70 ℃; storing at 4 ℃. (namely, the original annealing temperature was adjusted to 58 ℃ C. to 60 ℃ C.)

For specific results:

VSVG4 assay results (shown in fig. 9): NTC is 0.00 copies/ul; the positive plasmid is 39.19 copies/ul; the two repeated RCL samples are respectively 37.75copies/ul, 40.13copies/ul, 95% confidence intervals, and the CV value is less than 10%, which indicates that the RCL sample detected by VSVG4 has good repeatability.

VSVG5 assay results (shown in fig. 10): NTC is 0.00 copies/ul; the positive plasmid is 43.07 copies/ul; the two duplicate RCL samples were 35.13copies/ul, 33.83copies/ul, respectively, and the CV value was <10% at the 95% confidence interval, indicating that the VSVG5 test RCL samples were well reproducible.

After further comparison, it was found that the RCL value detected by VSVG5 was still smaller than that detected by VSVG4, mainly because VSVG4 labeled FAM fluorescence and VSVG5 labeled HEX fluorescence, and that FAM was relatively stronger than HEX signal.

In summary, it is considered that both VSVG4 and VSVG5 can accurately detect RCL samples and no copy number in NTC by optimizing the reaction system and the reaction procedure.

(III) influence on accuracy of RCL detection sample by fluorescence detection type

As shown in the previous detection results, the results of detecting RCL samples by HEX-labeled probes are small. To determine that this effect is indeed due to the type of signal from the fluorescent label, the inventors performed further experimental verification, and the specific experimental profile is as follows.

It should be noted that, based on the above optimization experiment, the inventors further designed a VSVG6 primer set for the primer sequence for detection to perform auxiliary experiments and verification. The VSVG6 primer set:

VSVG6-F：5’-ggacatggtatgttggactccg-3’，

VSVG6-R：5’-gcgtcttgaatgtgaggatgttc-3’；

VSVG 6-Probe: FAM-catcttagctcaaaggctcag-MGB.

Using the above primers, the specific amplification sequence was as follows (77 bp):

GGACATGGTATGTTGGACTCCGATCTTCATCTTAGCTCAAAGGCTCAGGTGTTCGAACATCCTCACATTCAAGACGC。

during the experiment, RCL samples are detected by a VSVG6 primer group (marked FAM), a VSVG4 primer group (marked FAM) and a VSVG5 primer group (marked HEX) respectively; the specific reaction system and the reaction program refer to the optimized reaction system and the optimized reaction program.

For specific results (as shown in fig. 11):

NTC corresponding to three pairs of primer probes of VSVG4, VSVG5 and VSVG6 are all 0.00 copies/ul;

the RCL samples detected by VSVG4, VSVG5 and VSVG6 are 41.8copies/ul, 37.84copies/ul and 42.50copies/ul respectively; and the detection results of the FAM marked VSVG4 and the VSVG6 are consistent and are larger than that of the HEX marked VSVG5, which can indicate that the HEX probe has smaller results due to weaker signals.

In summary, the detection result of the HEX-labeled probe is smaller than that of the FAM-labeled probe, which is actually caused by the weak HEX signal, so that the dual probes can be used as a reference to each other to quantify RCL more accurately.

(IV) determining the lower detection limit of RCL sample in the dPCR detection

To determine the lower limit of RCL detection using dPCR detection with the primer sets described herein, the inventors further tested RCL samples after gradient dilution (3 replicates per gradient) using VSVG5 and VSVG6 primer sets, respectively.

Aiming at the primer group of VSVG5, the early experimental result shows that the RCL result detected by VSVG5 is 35 copies/ul. Based on this, the inventors diluted the template sample to 0.125copies/ul and designed 3 NTC replicate wells.

For the VSVG6 primer set, the early VSVG6 detected RCL with 40copies/ul, the template samples were diluted to 0.1875copies/ul, and 3 NTC duplicate wells were designed.

The specific reaction system and the reaction program can be operated after referring to the optimization.

For specific results:

the VSVG5 detects RCL samples with gradient dilution (the concrete results are shown in FIG. 12 and FIG. 13), the correlation coefficient R2 of the actual detection result and the theoretical result is 99.53%, the lower line of the detection of the RCL samples by the VSVG5 can reach 0.28copies/ul, the lower line of the detection of the RCL samples at 0.28copies/ul is 3 repeated results, and the CV value is less than 10% at the 95% confidence interval.

The VSVG6 detects RCL samples with gradient dilution (the concrete results are shown in FIG. 14 and FIG. 15), the correlation coefficient R2 of the actual detection result and the theoretical result is 99.94%, the lower line of the detection of the RCL samples by the VSVG6 can reach 0.325copies/ul, the lower line of the detection of the RCL samples at 0.325copies/ul is 3 repeated results, and the CV value is less than 10% at the 95% confidence interval.

(V) Dual Probe detection

The results of the foregoing experiments have demonstrated that both VSVG5 and VSVG6 can accurately and specifically detect RCL samples. On the basis, in order to prove whether the RCL can be accurately detected and how the detection result can be still detected when VSVG5 and VSVG6 are simultaneously applied in the same reaction system, the inventor carries out further experimental verification. Specifically, the method comprises the following steps:

with reference to the optimized reaction system, the RCL sample and the NTC blank were tested simultaneously in the same reaction system using VSVG5 and VSVG6, respectively, with 3 replicates of each experimental and NTC group.

The results of the experiment are shown in FIG. 16. Analysis can see that the results of simultaneous detection of RCL samples with VSVG5 and VSVG6 are the same as the results of detection of RCL samples with VSVG5 and VSVG6, respectively, with CV values less than 10% at the 95% confidence interval.

Therefore, the results of simultaneous detection of RCL by VSVG5 and VSVG6 and detection of RCL by VSVG5 and VSVG6 were the same for each RCL sample. Therefore, from the perspective of actual detection cost, it is obvious that independent primer sets can be used for detection, but from the perspective of accuracy, a double-primer set mode can be used to compare each other, so as to verify and ensure the accuracy of the detection result.

Sequence listing

<110> Hangzhou Jingbai Biotechnology Ltd

<120> primers for digital PCR detection for RCR/RCL and detection method

<130> none

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 68

<212> DNA

<213> pVSV-G plasmid (pVSV-G)

<400> 1

attcaagcag acggttggat gtgtcatgct tccaaatggg tcactacttg tgatttccgc 60

tggtatgg 68

<210> 2

<211> 92

<212> DNA

<213> pVSV-G plasmid (pVSV-G)

<400> 2

caggtgtctg gttcgagatg gctgataagg atctctttgc tgcagccaga ttccctgaat 60

gcccagaagg gtcaagtatc tctgctccat ct 92

<210> 3

<211> 77

<212> DNA

<213> pVSV-G plasmid (pVSV-G)

<400> 3

ggacatggta tgttggactc cgatcttcat cttagctcaa aggctcaggt gttcgaacat 60

cctcacattc aagacgc 77

Claims (5)

1. The primer for digital PCR detection aiming at RCR/RCL is characterized by comprising the following primer groups:

VSVG4 primer set:

VSVG4-F：5’-attcaagcagacggttggatgt-3’，

VSVG4-R：5’-CCATACCAGCGGAAATCACA-3’；

VSVG 4-Probe: FAM-catgcttccaaatgggtcacta-MGB;

the length of an amplification sequence using the VSVG4 primer group is 68bp, and the amplification sequence is shown as SEQ ID No.1 and specifically as follows:

ATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTCACTACTTGTGATTTCCGCTGGTATGG；

VSVG5 primer set:

VSVG5-F：5’-caggtgtctggttcgagatg-3’，

VSVG5-R：5’-agatggagcagagatacttgacc-3’；

VSVG 5-Probe: HEX-ttctgggcattcagggaatctggctg-BHQ 1;

the length of an amplification sequence using the VSVG5 primer group is 92bp, and the amplification sequence is shown as SEQ ID No.2 and specifically as follows:

CAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAAGGGTCAAGTATCTCTGCTCCATCT；

VSVG6 primer set:

VSVG6-F：5’-ggacatggtatgttggactccg-3’，

VSVG6-R：5’-gcgtcttgaatgtgaggatgttc-3’；

VSVG 6-Probe: FAM-catcttagctcaaaggctcag-MGB;

the length of an amplification sequence using the VSVG6 primer group is 77bp, and the amplification sequence is shown as SEQ ID No.3 and specifically as follows:

GGACATGGTATGTTGGACTCCGATCTTCATCTTAGCTCAAAGGCTCAGGTGTTCGAACATCCTCACATTCAAGACGC。

2. a detection kit prepared by using the primers for digital PCR detection of RCR/RCL according to claim 1.

3. The RCL detection method using the primers for digital PCR detection of RCR/RCL according to claim 1,

extracting pVSV-G plasmid genome as template, and PCR amplification.

4. The RCL detection method according to claim 3, wherein in the specific reaction, a 15. mu.l reaction system is designed as follows:

template, 1. mu.l;

VSVG-F，10μM、0.3μl；

VSVG-R，10μM、0.3μl；

VSVG-probe, 10. mu.M, 0.3. mu.l;

JN solution at 20X, 0.75. mu.l;

2x Prob master Mix, 7.5. mu.l;

NF water，4.85μl。

5. the RCL detection method of claim 3, wherein the specific PCR reaction procedure is: 95 deg.C for 5 min; at 95 deg.C, 50s, 60 deg.C, 1min30s, 40 cycles; 5min at 70 ℃; storing at 4 ℃.

Images