CN116622908B - Primer probe, kit and method for rapidly detecting wild adeno-associated virus and application - Google Patents

Primer probe, kit and method for rapidly detecting wild adeno-associated virus and application Download PDF

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CN116622908B
CN116622908B CN202310393586.1A CN202310393586A CN116622908B CN 116622908 B CN116622908 B CN 116622908B CN 202310393586 A CN202310393586 A CN 202310393586A CN 116622908 B CN116622908 B CN 116622908B
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郝瑶
许静
罗航
徐国东
王明珍
刘愈杰
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Canvest Wuhan Biotechnology Co ltd
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Abstract

The invention discloses a primer probe, a kit and a method for rapidly detecting wild adeno-associated virus and application thereof, and relates to the technical field of adeno-associated virus detection. The primer probe comprises two groups of primer probe combinations; the forward primer sequence of the first group of primer probe combination is shown as SEQ ID NO.1, the reverse primer sequence is shown as SEQ ID NO.2, and the probe sequence is shown as SEQ ID NO. 3; the forward primer sequence of the second group of primer probe combination is shown as SEQ ID NO.4, the reverse primer sequence is shown as SEQ ID NO.5, and the probe sequence is shown as SEQ ID NO. 6. The primer probe combination can detect 1-13 serotype AAV virus at one time and specifically, has high sensitivity, can reach 25 copies/ml for each AAV serotype, is 1-2 orders of magnitude higher than the conventional PCR, and is higher than SYBR green I qPCR method.

Description

Primer probe, kit and method for rapidly detecting wild adeno-associated virus and application
Technical Field
The invention relates to the technical field of adeno-associated virus detection, in particular to a primer probe, a kit and a method for wild adeno-associated virus and application thereof.
Background
Adeno-associated viruses (AAV) belong to the family of parvoviridae, dependovirus, and were first discovered in 1965 as contaminants in adenovirus preparations. Adeno-associated virus is a non-enveloped virus having an icosahedral structure with a viral particle diameter of 20-25nm and comprising an ssDNA genome of about 4.7 kb. The AAV genome has 145bp inverted terminal repeats (inverted terminal repeat, ITR) at both ends, and the coding region of AAV is located between the ITR sequences, and contains two open reading frames (open reading frames, ORF). The ORF at the 5' end encodes a sequence comprising 4 overlapping genes encoding nonstructural proteins (Rep) required for AAV replication and integration; the ORF at the 3' end encodes the 3 capsid proteins (Cap) of the virus, i.e.VP 1, VP2, VP3, determine the tissue tropism of the virus. ITRs contain cis-acting elements required for viral replication, integration, rescue and packaging, and have transcriptional promoter activity. AAV is a replication-defective virus, and is only subject to virulent infection under co-infection with helper virus (adenovirus or herpes virus). In the absence of helper virus, AAV viral genomes favor integration into the human genome chromosome 19 to establish latent infection.
To date, more than 100 serotypes of AAV have been isolated, with AAV1-AAV13 belonging to primate serotypes. AAV2, 3, 5 and 6 are found in human cells, whereas AAV1, 4 and 7-13 are derived from non-human primates. Except that AAV5 was isolated from human condyloma acuminatum, all primate-derived AAV was originally discovered as a contaminant of adenovirus preparations. Although there is no direct evidence at present that AAV is associated with human disease, as a pollution control for adenovirus production, the FDA has a clear regulation of quality control of AAV residues in the first draft of Guidance for Human Somatic Cell Therapy and Gene Therapy, suggesting AAV detection of master cell banks, master virus seed banks and adenovirus end products.
At present, the quality control is mainly carried out by amplifying the conserved sequences of common adeno-associated viruses by adopting common PCR in the quality control at home and abroad. The common PCR needs to be identified by gel electrophoresis, has complex operation and low sensitivity, and is inconvenient to accurately quantify. The universal primer for detecting wild adeno-associated virus disclosed in Chinese patent application CN114075610A detects AAV by SYBR green I qPCR method for the first time, the sensitivity can reach 100copies/reaction, but the standard group without adding plasmid can also peak at 35 ct. The TaqMan real-time fluorescent quantitative PCR technology has the characteristics of rapidness, specificity, sensitivity, accurate quantification and the like, and is widely applied to the detection of virus nucleic acid. At present, there is no TaqMan real-time fluorescence quantification method capable of rapidly, efficiently and quantitatively detecting 13 serotypes of wild type AAV (AAV 1-13).
Disclosure of Invention
In order to solve the defects that the common PCR is complicated in operation and low in sensitivity and is inconvenient to accurately quantify when detecting adeno-associated viruses, and realize rapid, efficient and quantitative detection of 13 serotypes of wild AAV1-13, the invention provides a primer probe, a kit and a method for detecting the wild adeno-associated viruses and application of the primer probe, the kit and the method, and adopts a TaqMan dual real-time fluorescence quantification technology, two groups of primer probes are designed for a Rep conserved sequence of AAV1-AAV13, so that not only can all adeno-associated viruses from primates be rapidly, accurately and efficiently detected, but also AAV5 with large sequence differences can be distinguished from other serotypes. To achieve the above object, the present invention is specifically achieved by the following technique.
The primer probe combination for rapidly detecting the wild adeno-associated virus comprises two groups of primer probe combinations; the nucleotide sequence of the forward primer of the primer probe combination of the first group is shown as SEQ ID NO.1, the nucleotide sequence of the reverse primer is shown as SEQ ID NO.2, and the nucleotide sequence of the probe is shown as SEQ ID NO. 3; the nucleotide sequence of the forward primer of the second group of primer probe combinations is shown as SEQ ID NO.4, the nucleotide sequence of the reverse primer is shown as SEQ ID NO.5, and the nucleotide sequence of the probe is shown as SEQ ID NO. 6;
different fluorescent labels are coupled to the 5 'ends of the probes of the two groups of primer probe combinations, and quenching groups are coupled to the 3' ends of the probes. In the probe sequence of the two primer probe combinations, the degenerate base V represents C, G or A, the degenerate base Y represents C, T, the degenerate base S represents G, C, and the degenerate base R represents A, G.
Preferably, the fluorescent label is a FAM, TET, NED, ROX, CY, CY5, VIC, JOE or HEX fluorescent label; the quenching group is TAMRA, NFQ, ECLIPSE, DABCYL, BHQ1 or BHQ2 quenching group.
More preferably, the 5 'end of the probe of the first group of primer probe combinations is coupled with FAM fluorescent markers, and the 3' end of the probe combinations is coupled with a quenching group of BHQ1; and the 5 'end of the probe of the second group of primer probe combination is coupled with HEX fluorescent label, and the 3' end of the probe is coupled with a quenching group of BHQ1.
The invention also provides application of the primer probe combination in preparing a product for rapidly detecting wild adeno-associated virus.
The invention also provides a kit for rapidly detecting the wild-type adeno-associated virus, which comprises the primer probe combination.
Preferably, the kit further comprises PCR reaction liquid, positive control inactivated virus, negative control, first positive standard plasmid, second positive standard plasmid and PCR grade water; the first positive standard plasmid and the second positive standard plasmid respectively contain the front 1440bp fragments of the Rep genes of AAV2 and AAV 5.
The quantitative reagent provided by the invention is stored at the temperature of-20 ℃ and repeated freezing and thawing are reduced as much as possible.
More preferably, the PCR reaction solution is Taq Pro HS Universal U +Probe Master Mix, which contains PCR buffer solution, dNTPs, hot start Taq polymerase and MgCl 2 The method comprises the steps of carrying out a first treatment on the surface of the The positive control inactivated virus is an AAV2 virus that has been amplified by adenovirus helper and has been inactivated.
The invention also provides a method for rapidly detecting wild-type adeno-associated virus without aiming at disease diagnosis and treatment, and the kit is used for qualitatively/quantitatively detecting AAV pollution in adenovirus gene therapy products or AAV pollution in other biological products, cultured cells, human tissues or blood samples.
Preferably, the above detection method includes the steps of:
s1, extracting DNA of a sample to be detected, eluting the DNA of the sample to be detected for a plurality of times by using PCR grade water; diluting the positive control inactivated virus 10000 times; taking a sterile PBS solution and a sample to be tested for synchronous operation in a negative control mode;
will be 1X 10 9 The first positive standard plasmid and the second positive standard plasmid of the copies/. Mu.l, respectively, were diluted (with PCR grade water) to 2X 10 8 The copies/. Mu.l, and mixing the two in equal volume; then sequentially diluting to 1×10 according to 10-fold gradient 1 COPIES/. Mu.l (i.e. 1X 10 respectively) 7 、1×10 6 、1×10 5 、1×10 4 、1×10 3 、1×10 2 And 1X 10 1 cobies/. Mu.l), plasmid standard solutions were obtained; taking each plasmid standard solution, drawing and obtaining a standard curve by taking the initial copy number as an abscissa and the Cq value as an ordinate;
s2, respectively carrying out qPCR detection on the DNA of the sample to be detected, the positive control and the negative control;
s3, judging a detection result according to an amplification curve and a Cq value generated by qPCR reaction;
if the Cq value of the fluorescence labeling channel corresponding to the first group of primer probe combinations of the sample to be detected is less than or equal to 35, an obvious amplification curve is provided, which indicates that AAV (AAV) pollution of other serotypes except AAV5 exists in the sample;
if the Cq value of the fluorescence labeling channel corresponding to the second group of primer probe combinations of the sample to be detected is less than or equal to 35, an obvious amplification curve is provided, which indicates that AAV5 pollution exists in the sample; if the Cq value of the sample to be tested is more than 35 and the obvious amplification curve exists, repeating the experiment;
if the quantitative determination is needed, the AAV content in the sample to be detected (namely the DNA of the sample to be detected) can be quantitatively determined according to a standard curve.
In the above detection method, the number of elution in step S1 is generally two.
More preferably, in step S2, the reaction system of qPCR detection includes: DNA of sample to be tested or standard curve template plasmid 5. Mu.l, 10. Mu.M primer probe Mix 2.4. Mu.l, H 2 O2.6. Mu.l, taq Pro HS Universal U +Probe Master Mix 10. Mu.l; the primer probe Mix is 0.5 mu l of each of four primers and 0.2 mu l of each of two probes; the positive control and the negative control are operated synchronously; amplification was performed using conventional qPCR reaction procedures.
The reaction program of the qPCR detection is as follows: (1) 2min at 37 ℃; (2) 95 ℃ for 5min; (3) the plate was read after a single cycle for 42 cycles at 95℃15s,56℃25s, and 72℃20 s.
Compared with the prior art, the invention has the following advantages:
1. the primer probe combination provided by the invention can detect AAV virus of 1-13 serotypes at one time and specifically, and the detected AAV serotypes are wide in coverage, so that all AAV serotypes possibly existing in primates are covered, and missed detection is avoided;
2. the primer probe combination provided by the invention has high detection sensitivity, and based on sequence consistency, the sensitivity of the kit to each AAV serotype can reach 25copies/reaction by using 4 template plasmids capable of representing 1-13 AAV serotypes, which is 1-2 orders of magnitude higher than that of the conventional PCR method and higher than that of SYBR green I qPCR method;
3. the primer probe combination provided by the invention has good specificity and strong durability; the method has the advantages that the method can not carry out nonspecific amplification with the genome DNA of the human virus Ad5 and the common cells CHO, 293 and Vero in industrial production, and has stronger and more stable specificity compared with the SYBR green I qPCR method; amplification of low template quantity AAV is not significantly interfered by the genomic DNA of Ad5, CHO, 293 and Vero;
4. the kit provided by the invention provides standard plasmids of AAV2 and AAV5, and can quantitatively detect AAV. When the standard curves of AAV2 and AAV5 are used for measuring the AAV1, AAV2, AAV4 and AAV5 content, the deviation between the measured concentration and the theoretical concentration is less than 30 percent, so that the AAV content in a sample can be accurately reflected;
5. the primer probe in the kit can distinguish AAV5 from AAV of other serotypes; the two primer probes used do not cross react with the other template, so AAV5 with large sequence differences can be distinguished from other serotypes.
Drawings
FIG. 1 is a map of the constructed pGEM-AAV-Rep plasmids of the different serotypes of example 1; in the figure, A is pGEM-AAV1-Rep plasmid map, B is pGEM-AAV2-Rep plasmid map, C is pGEM-AAV4-Rep plasmid map, and D is pGEM-AAV5-Rep plasmid map;
FIG. 2 is a preliminary verification of the amplification effect of each set of AAV primers of example 1; in the figure, A, B, C is the fluorescence quantitative amplification curve of primer combinations (1), (2) and (3) on pGEM-AAV2-Rep respectively; D. e is the fluorescence quantitative amplification curve of the primer combination (4) and (5) on pGEM-AAV5-Rep respectively. The concentration of the standard plasmid was 2X 10, respectively 6 copies/μl、2×10 5 copies/μl、2×10 4 copies/μl、2×10 3 copies/μl、2×10 2 copies/μl、2×10 1 copies/μl；
FIG. 3 is a diagram of the primer probe design region of AAV1-13 (except AAV 5) of example 1; in the figure, the red frame is the forward primer design region, the yellow frame is the reverse primer design region, and the green frame is the probe design region (designed by reverse complementary sequence);
FIG. 4 is an AAV single qPCR amplification curve versus standard curve of example 2; in the figure, A and B are respectively a fluorescent quantitative amplification curve and a standard curve of AAV 1; c and D are respectively a fluorescent quantitative amplification curve and a standard curve of AAV 2; e and F are respectively a fluorescent quantitative amplification curve and a standard curve of AAV 4; g and H are the fluorescent quantitative amplification curve and standard curve of AAV5, respectively. The concentration of the standard plasmid was 1X 10, respectively 7 copies/μl、1×10 6 copies/μl、1×10 5 copies/μl、1×10 4 copies/μl、1×10 3 copies/μl、1×10 2 copies/μl、1×10 1 copies/μl；
FIG. 5 is a cross-reaction validation of the primer probe of AAV combination (2) of example 2 with AAV 5;
FIG. 6 is a cross-reaction validation of primer probes of AAV combination (5) of example 2 with AAV of other serotypes;
FIG. 7 shows AAQ2+AAQV5 double qPCR amplification curve (A) and standard curve (B) of example 3; in the figure, the concentrations of the mixed standard plasmids were 1X 10, respectively 7 、1×10 6 、1×10 5 、1×10 4 、1×10 3 、1×10 2 And 1X 10 1 COPIES/. Mu.l. Blue underline indicates pGEM-AAV2-Rep amplification curve, green underline indicates pGEM-AAV5-Rep amplification curve;
FIG. 8 is a real viewThe dual qPCR system of example 4 AAV2 corresponds to accuracy verification of serotype plasmid samples; the positions of various samples of different dilutions of pGEM-AAV1-Rep, pGEM-AAV2-Rep and pGEM-AAV4-Rep on an AAV2 standard curve are shown in the figure, wherein 'omicron' represents various points of standard samples with different concentrations, and 'X' represents theoretical values of 1X 10 5 、1×10 4 And 1X 10 3 Actual position of each AAV plasmid sample of copies/. Mu.l on the standard curve;
FIG. 9 is an accuracy verification of AAV5 plasmid samples of the dual qPCR system of example 4; the position of each sample of pGEM-AAV5-Rep at different dilutions on the AAV5 standard curve is shown, wherein "omicron" indicates each point of the standard at different concentrations and "×" indicates a theoretical value of 1X 10 5 、1×10 4 And 1X 10 3 Actual position of each AAV plasmid sample of copies/. Mu.l on the AAV5 standard curve;
FIG. 10 is AAV dual fluorescence qPCR system specificity verification of example 5;
FIG. 11 is the effect of sample matrices of example 5 on AAV2 amplification curves;
FIG. 12 is the effect of sample matrices of example 5 on AAV5 amplification curves.
Detailed Description
The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are only some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.
The TaqMan PCR technology is one of real-time fluorescent PCR, and compared with the traditional PCR, the TaqMan PCR technology is provided with a probe with two ends respectively marked with fluorescent marks and quenching groups in a reaction system. When the probe structure is complete, the energy of fluorescence emitted by the fluorescent label is transferred to the quenching group, and the quenching effect is presented. If the target sequence exists in the amplification process, the probe molecules are gradually hydrolyzed and cut off, the fluorescent reporter group and the quenching group are dissociated from each other, the fluorescence resonance energy transfer effect between the fluorescent reporter group and the quenching group is blocked, and the fluorescent reporter group emits a fluorescence signal. As the amplification proceeds, the fluorescent signal exhibits a linear increase with the amplification of the fragment of interest.
In the two groups of primer probe combinations provided by the invention, the 5' ends of the probes are respectively coupled with different fluorescent markers so as to distinguish AAV5 from other 12AAV serotypes; the 3' ends are coupled with quenching groups. Thus, based on the commonly used fluorescent label and the type of quencher, the fluorescent label may be selected from FAM, TET, NED, ROX, CY, CY5, VIC, JOE or HEX fluorescent labels; the quenching group may be selected from TAMRA, NFQ, ECLIPSE, DABCYL, BHQ or BHQ2. For convenience of verification, in the following example 1, when designing the primers and probes, the 5 'end of the probes of the first primer probe set is coupled with FAM fluorescent label, and the 3' end of the probes is coupled with a quenching group of BHQ1; and the HEX fluorescent label is coupled to the 5 'end of the probe of the second group of primer probe combination, and the quenching group coupled to the 3' end is BHQ1.
The DNA extraction kit used in the present invention may be a commercial kit, for example: taKaRa MiniBEST Viral RNA/DNA Extraction Kit.
The primers or nucleotide sequences used in the present invention, unless otherwise specified, were synthesized by the division of biological engineering (Shanghai); the conventional reagents (such as qPCR reaction buffer, negative control, etc.) are all commercial products of Nanjinouzan biotechnology Co., ltd; the positive control inactivated virus was prepared by the company of Wohan's Biotechnology Co., ltd. Unless otherwise indicated, all technical schemes used in the following examples are conventional molecular biology techniques.
Example 1: adeno-associated virus primer, probe design, standard plasmid construction and primer probe effect preliminary verification
1. Primer and probe design
The gene sequence of AAV1-13 is obtained from NCBI search, after DNAMAN comparison, a relatively conserved Rep gene design primer probe is selected, and the GeneBank numbers corresponding to each serotype of AAV are respectively: AAV1 (AF 063497), AAV2 (AF 043303), AAV3 (U48704), AAV4 (U89790), AAV5 (AF 085716), AAV6 (AF 028704), AAV7 (AF 513851), AAV8 (AF 513852), AAV9 (MB 418532), AAV10 (AY 631965), AAV11 (AY 631966), AAV12 (DQ 813647), and AAV13 (EU 285562). The AAV has more point mutations among serotypes, and the AAV5 has relatively large sequence difference from AAV of other serotypes through comparison, so that the AAV is separated to design a primer probe. 3 groups of primer probes are designed for the Rep genes of other AAV except AAV5, 2 groups of primer probes are designed for the Rep genes of AAV5, and the probes are coupled with different fluorescent markers, so that AAV5 with larger sequence difference can be distinguished from other serotypes. Primer probe sequences and corresponding detectable AAV serotypes are shown in table 1.
TABLE 1AAV-Rep primers and probes
2. Standard plasmid construction
According to sequence differences of AAV at the set primer probe sites, AAV1 (AF 063497), AAV2 (AF 043303), AAV4 (U89790) and AAV5 (AF 085716) are selected as target serotypes, the front 1440bp of the Rep gene CDS region is artificially synthesized, and transferred into pGEM-T easy plasmid to construct pGEM-AAV1-Rep (figure 1-A), pGEM-AAV2-Rep (figure 1-B), pGEM-AAV4-Rep (figure 1-C) and pGEM-AAV5-Rep (figure 1-D) standard plasmids.
3. Primer probe effect preliminary verification
Preliminary verification of the amplification effect of primer combinations (1) (2) (3) was performed using pGEM-AAV2-Rep, and preliminary verification of the amplification effect of primer combinations (4) (5) was performed using pGEM-AAV5-Rep plasmid (FIG. 2). And selecting a combination (2) and a combination (5) with better amplification effect for subsequent further verification.
The design area of the primer probe of the combination (2) is shown in figure 3, and the AAV of other serotypes except AAV5 is amplified, and the amplification length is 98bp; the amplified sequences of combination (5) are:
gtcattgttcgcgtcccatttgacgtggaggaacatctgcctggaatttctgacagctttgtggactg ggtaactggtc aaatttgggagctgcctccagagtcagathe Rep gene of AAV5 is amplified, and the amplification length is 107bp.
Example 2: AAV single qPCR validation
1. Establishment of single qPCR reaction system and program
AAV single qPCR reaction system is divided into a group A and a group B, and is 5 μl of sample DNA to be detected or 10 μM primer probe Mix 2.4 μl (0.5 μl of each of four primers, 0.2 μl of each of two probes) and H 2 O 2.6μl、Taq Pro HS Universal U+Probe Master Mix 10μl。
Specific group a: 2X Taq Pro HS Universal U +Probe Master Mix 10. Mu.l, 10. Mu.M FP 20.5. Mu.l, 10. Mu.M RP2 0.5. Mu.l, 10. Mu.M Probe2 0.20. Mu.l, sample template 5. Mu.l, H 2 O 3.8μl。
Specific group b: 2X Taq Pro HS Universal U +Probe Master Mix 10. Mu.l, 10. Mu.M FP 50.5. Mu.l, 10. Mu.M RP5 0.5. Mu.l, 10. Mu.M Probe5 0.20. Mu.l, sample template 5. Mu.l, H 2 O3.8 μl. Group a can detect AAV viruses of serotypes other than AAV5 and group b can be used to detect AAV5 viruses.
The reaction procedure is: (1) 37 ℃ for 2min; (2) 95 ℃ for 5min; (3) the plate was read after a single cycle for 42 cycles at 95℃15s,56℃25s, and 72℃20 s.
2. Single qPCR standard curve and amplification efficiency validation
Based on the size of each standard plasmid and the measured concentration, the standard plasmid was converted to copy number (copies/. Mu.l) from 1X 10 7 The copies/. Mu.l were serially diluted 10-fold to 1X 10 1 The copies/. Mu.l was used as each point of the standard curve, while the non-nucleic acid water was used as a negative control. Amplifying by adopting the reaction system and the reaction program, wherein the group a reaction system detects and detects the amplification efficiency of pGEM-AAV1-Rep, pGEM-AAV2-Rep and pGEM-AAV 4-Rep; and b, detecting the amplification efficiency of pGEM-AAV5-Rep by the reaction system of the group b. The amplification curves and standard curves of the respective template plasmids are shown in FIG. 4, and the amplification efficiencies are summarized in Table 2.
TABLE 2AAV Standard plasmid amplification efficiency
AVV template Efficiency of R2 Slope of Dilution range
pGEM-AAV1-Rep 99.50％ 0.999 -3.333 1×10 7 -1×10 1 copies/μl
pGEM-AAV2-Rep 99.50％ 0.999 -3.333 1×10 7 -1×10 1 copies/μl
pGEM-AAV4-Rep 98.60％ 0.999 -3.355 1×10 7 -1×10 1 copies/μl
pGEM-AAV5-Rep 99.20％ 0.999 -3.341 1×10 7 -1×10 1 copies/μl
The results of the single qPCR show that the amplification efficiencies of the standard yeast plasmids AAV1, AAV2, AAV4 and AAV5 are ≡90%, and the NTC has no amplification curve. Compared with the sequence consistency of AAV1, AAV2, AAV4 and AAV5 and other primate AAV serotypes at the primer probe binding site, the primer can comprehensively cover AAV1-13 and cannot cause the omission of the AAV serotypes of a certain primate.
3. Single qPCR sensitivity detection
The sensitivity of AAV1, AAV2 and AAV4 is detected by adopting the reaction system of the group a, and the sensitivity of AAV5 is detected by adopting the reaction system of the group b. pGEM-AAV1-Rep, pGEM-AAV2-Rep, pGEM-AAV4-Rep, pGEM-AAV5-Rep Standard plasmid was transformed from 1X 10 7 The copies/. Mu.l were diluted in a 10-fold gradient to 1X 10 in succession 6 、1×10 5 、1×10 4 、1×10 3 、1×10 2 、1×10 1 And 1X 10 0 The copies/. Mu.l was additionally diluted to 5X 10 0 cobies/. Mu.l; wherein 1×10 1 、5×10 0 And 1X 10 0 10 wells were made per μl, and experiments were independently repeated 2 times for a total of 20 wells.
Each standard plasmid 1X 10 1 、5×10 0 And 1X 10 0 The results of the Cq statistics of copies/. Mu.l are shown in tables 3, 4, 5 and 6, respectively.
TABLE 3 Single qPCR AAV1 sensitivity statistics
TABLE 4 Single qPCR AAV2 sensitivity statistics
TABLE 5 Single qPCR AAV4 sensitivity statistics
TABLE 6 Single qPCR AAV5 sensitivity statistics
4. Cross reaction verification of single qPCR primer probe and template plasmid
At 2X 10 5 Detecting the amplification condition of pGEM-AAV5-Rep by using a primer probe system of the group a by taking copies/μl of pGEM-AAV5-Rep as a template; at 2X 10 5 The amplification conditions of pGEM-AAV1-Rep, pGEM-AAV2-Rep and pGEM-AAV4-Rep are detected by using the primer probe system of the group b by taking copies/μl of pGEM-AAV1-Rep, pGEM-AAV2-Rep and pGEM-AAV4-Rep as templates.
The verification result shows that the primer probe system of group a has good amplification curve on pGEM-AAV2-Rep template and 2X 10 5 The copies/. Mu.l pGEM-AAV5-Rep no-amplification curve (shown in FIG. 5); the primer probe system of the group b has good amplification curve on pGEM-AAV5-Rep template and 2 multiplied by 10 5 The amplification-free curves of the copies/. Mu.l pGEM-AAV1-Rep, pGEM-AAV2-Rep and pGEM-AAV4-Rep (shown in FIG. 6) show that the primer probe systems of the a group and the b group have no cross reaction with the opposite template.
Example 3: AAV double qPCR amplification curve and sensitivity verification
1. Establishment of double qPCR reaction system and program
The reaction system of the double qPCR is as follows: 2× Taq Pro HS Universal U +Probe Master Mix 10. Mu.l, 10. Mu.M FP2 0.5. Mu.l, 10. Mu.M RP2 0.5. Mu.l, 10. Mu.M Probe2 0.20. Mu.l, 10. Mu.M FP5 0.5. Mu.l, 10. Mu.M RP5 0.5. Mu.l, 10. Mu.M Probe5 0.20. Mu.l, sample template 5. Mu.l, H 2 O 2.6μl。
The reaction procedure for double qPCR was identical to that described in step 1 of example 2.
2. Double qPCR standard curve and amplification efficiency verification
Will be 2X 10 8 Equal volume mixing of copies/. Mu.l pGEM-AAV2-Rep and pGEM-AAV5-Rep resulted in a final plasmid concentration of 1X 10 after mixing 8 COPIES/. Mu.l. Sequentially 10 times of gradient diluted to 1×10 1 COPIES/. Mu.l, 1X 10 7 -1×10 1 The copies/. Mu.l plasmid standard solution was used as the standard curve point and water was used as the negative control. And (3) performing amplification by adopting the double qPCR reaction system and the reaction program. Experiments were independently repeated three times.
The amplification curves and standard curves of AAV2 and AAV5 double qPCR of HEX channels of FAM channel are shown in figure 7, the amplification efficiencies of AAV2 and AAV5 are close to 100%, the amplification efficiencies are basically consistent with that of single qPCR, NTC has no amplification curve, and the mixture of the primer and the probe can not interfere the amplification of each template. Similar results are obtained by repeated experiments at different times, which shows that the detection method has good stability and higher repeatability.
3. Dual qPCR sensitivity verification
The plasmid mixture of pGEM-AAV2-Rep and pGEM-AAV5-Rep was used to generate a plasmid sequence of 1X 10 7 The copies/. Mu.l was diluted to 1X 10 in a 10-fold gradient 6 、1×10 5 、1×10 4 、1×10 3 、1×10 2 、1×10 1 、1×10 0 The copies/. Mu.l was additionally diluted to 5X 10 0 cobies/. Mu.l; wherein 1×10 1 、5×10 0 And 1X 10 0 10 multiplex wells were made for each of the copies/. Mu.l, 2 multiplex wells were made for the other concentrations, and the amplification at each concentration was detected using the double qPCR reaction system described above. The above experiment was independently repeated 2 times.
And judging the detection result according to an amplification curve and a Cq value generated by the qPCR reaction. The BIO-RAD CFX96 instrument Cq Determination Mode can select the Regression mode, or select Single Threshold mode, manually drag the threshold line to the positive control linear amplification start position. AAV2 and AAV5 Standard plasmids 1×10 1 、5×10 0 And 1X 10 0 The results of the Cq statistics for copies/. Mu.l are shown in tables 7 and 8, respectively.
TABLE 7 double qPCR AAV2 sensitivity statistics
Table 8 dual qPCR AAV5 sensitivity statistics
AAV2 and AAV5 template plasmids were detected at 1X 10 1 、5×10 0 The copies/μl can be stably detected, and the Cq average value is less than or equal to 35; 1X 10 0 Undetected conditions were found in each of the copies/. Mu.l. This result is consistent with the single qPCR sensitivity result, so the detection sensitivity of the method was determined to be 5X 10 0 copies/. Mu.l, 25 copies/reaction.
Example 4: AAV dual qPCR repeatability and accuracy detection
The standard plasmids pGEM-AAV1-Rep, pGEM-AAV2-Rep, pGEM-AAV4-Rep and pGEM-AAV5-Rep were diluted to 1X 10, respectively 5 、1×10 4 、1×10 3 The actual measurement of each AAV plasmid was measured using the double fluorescent quantitative reaction system of example 3, with copies/. Mu.l (theory), and 4 wells were made for each AAV sample. Measuring the variation coefficient between samples corresponding to each concentration, so as to evaluate the repeatability of the experiment; and measuring the relative deviation between the actual value and the theoretical value of each concentration sample, thereby evaluating the accuracy of the experiment.
The positions of the plasmid samples of each dilution of AAV1, AAV2 and AAV4 on the AAV2 standard curve are shown in FIG. 8, and the positions of the plasmid samples of each dilution of AAV5 on the AAV5 standard curve are shown in FIG. 9. The results in the graph show that 1X 10 5 、1×10 4 、1×10 3 Actual position of each plasmid sample of copies/. Mu.l was 1X 10 on the corresponding standard curve 5 、1×10 4 、1×10 3 The points of the copies/. Mu.l are more consistent. Tables 9, 10, 11, and 12 below show the results of the repeatability and accuracy verification of AAV1, AAV2, AAV4, and AAV5 plasmid samples, respectively.
TABLE 9AAV1 plasmid sample repeatability and accuracy statistics
TABLE 10AAV2 plasmid sample repeatability and accuracy statistics
TABLE 11AAV4 plasmid sample repeatability and accuracy statistics
TABLE 12AAV5 plasmid sample repeatability and accuracy statistics
Proved by verification, CV values in plasmid sample groups with various concentrations of AAV1, AAV2, AAV4 and AAV5 are all less than 10%, which shows that the repeatability is good. The relative deviation between the measured value and the theoretical value of each sample is less than 30%, and the accuracy is qualified. By comparing the sequence consistency of AAV1, AAV2, AAV4 and AAV5 with other primate AAV serotypes at primer probe binding sites, the system can accurately quantify 13 primate AAV serotypes of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and AAV 13.
Example 5: AAV dual qPCR specificity and matrix durability validation
1. Specificity verification
At 5X 10 5 The samples of the genome DNA of the cobies/. Mu.l adenovirus pBHG/DC316-mock (certain non-replicative adenovirus), AD-D55 (certain oncolytic adenovirus) and wtAd5 (wild-type adenovirus) and the 20 ng/. Mu.l genome DNA of 293, CHO and Vero were verified that the AAV double qPCR system had non-specific amplification for each of the above samples.
The experimental results are shown in FIG. 10, and the AAV double qPCR primer probe system has no amplification curve for each of the above genomic DNA and negative control (RNase-free water), indicating that the established method has good specificity.
2. Matrix durability verification
50copies/reaction pGEM-AAV2-Rep and pGEM-AAV5-Rep mixed plasmids are added into an AAV double qPCR reaction system, and 100ng/reaction CHO, 293 and Vero cell genome DNA and 5×10 are respectively added 9 Ad5 genome of copies/reaction, simultaneously adding H 2 O negative control, validating the effect of cell genome on low concentration AAV amplification curve.
As shown in FIGS. 11 and 12, the CHO, 293, vero cell genomes and 5X 10 of 100ng/reaction 8 The Ad5 genome of copies/reaction did not affect the normal detection of AAV2 and AAV5 templates of 50 copies/reaction.
The result shows that the qPCR detection method provided by the invention has good specificity and sensitivity to AAV, has good anti-interference performance to adenovirus and cell genome, can be used for AAV detection in adenovirus gene therapy products and vaccines, and can also be used for AAV detection in samples of other biological products, cultured cells, human tissues, blood and the like.
Example 6: positive virus detection
AAV2 was amplified using wild-type Ad5 as a helper virus. 200. Mu.l of the virus sample was extracted using TaKaRa MiniBEST Viral RNA/DNA Extraction Kit and the sample DNA was eluted twice with 50. Mu.l of nuclease-free water. And detecting the extracted viral nucleic acid by adopting an AAV double fluorescence quantitative system. In the detection result, only AAV2 represented by FAM channel has a specific amplification curve, and AAV5 represented by HEX channel has no amplification curve. The statistical results of the three independent replicates are shown in table 11 below.
TABLE 11AAV2 viral fluid genome titre assay
Measurement 1 Measurement 2 Measurement 3 Mean value of SD RSD
1.17E+08 9.60E+07 1.08E+08 1.07E+08 1.03E+07 9.69％
The genome titer of AAV2 virus amplified in this laboratory was (1.07 E+08) copies/. Mu.l (i.e., (1.07 E+11) copies/ml). This result also demonstrates that the method can specifically recognize and distinguish AAV viruses, while demonstrating the effectiveness of the detection system.
In summary, the two primer probe combinations in the kit provided by the invention can detect AAV virus of 1-13 serotypes at one time, cover all AAV serotypes possibly existing in primates, and effectively avoid missed detection. The detection sensitivity of the kit is high, and the sensitivity of each AAV serotype can reach 5 multiplied by 10 0 The copies/. Mu.l (25 copies/reaction) and the AAV virus can be quantitatively detected by combining with the standard plasmid in the kit. The primer probe in the kit can not carry out nonspecific amplification with human virus Ad5 and genomic DNA of common cells CHO, 293 and Vero in industrial production, and meanwhile, the amplification of low-template AAV can not be obviously interfered by the genomic DNA of Ad5, CHO, 293 and Vero. The primer probe in the kit can be used for combining AAV5 with larger sequence differenceAAV of other serotypes are differentiated. The kit is mainly used for qualitatively or quantitatively detecting AAV pollution in adenovirus gene therapy products or vaccines, and can also be used for detecting whether AAV pollution exists in other biological products, cultured cells, human tissues and blood samples.
The above detailed description describes in detail the practice of the invention, but the invention is not limited to the specific details of the above embodiments. Many simple modifications and variations of the technical solution of the present invention are possible within the scope of the claims and technical idea of the present invention, which simple modifications are all within the scope of the present invention.

Claims (10)

1. The primer probe combination for rapidly detecting the wild adeno-associated virus is characterized by comprising two groups of primer probe combinations; the nucleotide sequence of the forward primer of the primer probe combination of the first group is shown as SEQ ID NO.1, the nucleotide sequence of the reverse primer is shown as SEQ ID NO.2, and the nucleotide sequence of the probe is shown as SEQ ID NO. 3; the nucleotide sequence of the forward primer of the second group of primer probe combinations is shown as SEQ ID NO.4, the nucleotide sequence of the reverse primer is shown as SEQ ID NO.5, and the nucleotide sequence of the probe is shown as SEQ ID NO. 6;
the 5 'ends of the probes of the two groups of primer probe combinations are coupled with different fluorescent labels, and the 3' ends of the probes of the two groups of primer probe combinations are coupled with quenching groups; the degenerate base V represents base C, G or A, the degenerate base Y represents base C, T, the degenerate base S represents base G, C, and the degenerate base R represents base A, G.
2. The primer probe combination of claim 1, wherein the fluorescent label is FAM, TET, NED, ROX, CY3, CY5, VIC, JOE or HEX fluorescent label; the quenching group is TAMRA, NFQ, ECLIPSE, DABCYL, BHQ1 or BHQ2 quenching group.
3. The primer probe combination according to claim 2, wherein the probes of the first set of the primer probe combination are coupled with FAM fluorescent markers at the 5 'end and a quenching group coupled at the 3' end is BHQ1; and the 5 'end of the probe of the second group of primer probe combination is coupled with HEX fluorescent label, and the 3' end of the probe is coupled with a quenching group of BHQ1.
4. Use of the primer probe combination of any one of claims 1-3 in the preparation of a PCR detection reagent, kit for rapid detection of wild-type adeno-associated virus.
5. A kit for rapid detection of wild-type adeno-associated virus comprising a primer probe combination according to any one of claims 1-3.
6. The kit of claim 5, further comprising a PCR reaction solution, a positive control inactivated virus, a negative control, a first positive standard plasmid, a second positive standard plasmid, PCR grade water; the first positive standard plasmid and the second positive standard plasmid respectively contain the front 1440bp fragments of the Rep genes of AAV2 and AAV 5.
7. The kit according to claim 6, wherein the PCR reaction solution is Taq Pro HS Universal U +Probe Master Mix, and comprises PCR buffer solution, dNTPs, hot start Taq polymerase and MgCl 2 The method comprises the steps of carrying out a first treatment on the surface of the The positive control inactivated virus is an AAV2 virus that has been amplified by adenovirus helper and has been inactivated.
8. A method for rapid detection of wild-type adeno-associated virus not aimed at disease diagnosis and treatment, characterized in that the kit according to any one of claims 5-7 is used for qualitative and/or quantitative detection of AAV contamination in adenovirus gene therapy products or for qualitative and/or quantitative detection of AAV contamination in other biological products, cultured cells, human tissue or blood samples.
9. The method according to claim 8, comprising the steps of:
s1, extracting DNA of a sample to be detected, eluting the DNA of the sample to be detected for a plurality of times by using PCR grade water; diluting the positive control inactivated virus 10000 times; taking a sterile PBS solution and a sample to be tested for synchronous operation in a negative control mode;
will be 1X 10 9 The first positive standard plasmid and the second positive standard plasmid of the copies/. Mu.l were diluted to 2X 10 8 The copies/. Mu.l, and mixing the two in equal volume; then sequentially diluting to 1×10 according to 10-fold gradient 1 The copies/. Mu.l, plasmid standard solution was obtained; taking each plasmid standard solution, drawing and obtaining a standard curve by taking the initial copy number as an abscissa and the Cq value as an ordinate;
s2, respectively carrying out qPCR detection on the DNA of the sample to be detected, the positive control and the negative control;
s3, judging a detection result according to an amplification curve and a Cq value generated by qPCR reaction;
if the Cq value of the fluorescence labeling channel corresponding to the first group of primer probe combinations of the sample to be detected is less than or equal to 35, an obvious amplification curve is provided, which indicates that AAV (AAV) pollution of other serotypes except AAV5 exists in the sample;
if the Cq value of the fluorescence labeling channel corresponding to the second group of primer probe combinations of the sample to be detected is less than or equal to 35, an obvious amplification curve is provided, which indicates that AAV5 pollution exists in the sample;
if the Cq value of the sample to be tested is more than 35 and the obvious amplification curve exists, repeating the experiment;
and meanwhile, quantitatively determining the AAV content in the sample to be tested according to a standard curve.
10. The method according to claim 9, wherein in step S2, the reaction system of qPCR detection comprises: DNA of sample to be detected or standard curve template plasmid 5 μl, 10 μM primer probe combination 2.4 μl, H 2 O2.6. Mu.l, taq Pro HS Universal U +Probe Master Mix 10. Mu.l; the primer probe combination is that four primers are 0.5 mu l each and two probes are 0.2 mu l each; the positive control and the negative control were operated synchronously.
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