rc aav5 detection probe and primer combinations and uses thereof

Abstract

The present application provides a primer and probe combination for detecting rcAAV5 and use thereof. When detection is performed by the primer and probe combination of the present application, the contamination rate of rcAAV5 in rAAV5 can be detected quickly, sensitively, accurately and at low cost.

Description

Technical Field

[0001] This application relates to the field of virus detection, and more specifically to the detection of replicating recombinant adeno-associated virus in recombinant adeno-associated virus. Background Technology

[0002] Adeno-associated virus (AAV) is a non-enveloped linear DNA virus belonging to the Parvoviridae family. It can only replicate in the presence of a helper virus (usually adenovirus). AAV can infect various types of human cells and is therefore classified into multiple serotypes. A typical AAV2 genome is approximately 4800 bp, consisting of two inverted terminal repeats (ITRs, 145 bp) and two open reading frames (ORFs), rep and cap. The ITR plays a crucial role in viral replication and packaging and is essential for the synthesis of complementary DNA strands. The cap gene encodes the viral capsid protein, and the rep gene participates in viral replication and integration. It has been reported that 13 different serotypes of AAV exist in primates (AAV1-AAV13), of which AAV2, AAV3, and AAV9 originate from humans. AAV2 was the first virus to be cloned and is still the most thoroughly studied and widely used virus to date. Compared to AAV2, AAV5 is far removed from it in evolution, and there are few homologous sequences between the two, resulting in less research. However, AAV5 has a wide range of applications, capable of infecting important human organs and tissues such as the eyes, central nervous system, pancreas, and lungs.

[0003] Recombination adeno-associated virus (rAAV) is a gene vector modified from non-pathogenic wild-type AAV. The rAAV genome contains the deletion of all AAV protein-coding sequences and the addition of therapeutic gene expression cassettes. The sole viral source sequence is the ITR, which is essential for guiding genome replication and packaging during vector production. Due to its advantages such as variability, extremely low immunogenicity, high safety, broad host cell range (infecting both dividing and non-dividing cells), strong dispersibility, and long in vivo gene expression time, rAAV viral vectors are considered one of the most promising vectors for gene research and gene therapy.

[0004] However, during the production of rAAV vectors, the plasmids used for transfection or the host cell DNA and rAAV may undergo a large number of non-homologous recombinations due to physical proximity, resulting in the formation of a replication-competent AAV (rcAAV).

[0005] Although rcAAV particles are not associated with any known human diseases, as contaminants, they may affect rAAV gene expression and are an uncontrolled variable in many AAV gene transfer studies. Recent animal studies have shown that expression of the cap gene in vivo can trigger a severe immune response, while rcAAV packaged with other DNA impurities poses a potential risk of tumorigenicity or the introduction of antibiotic resistance. The rcAAV contamination rate in rAAV produced using conventional processes can be as high as 10%, and even with optimized processes, the contamination rate can reach 0.4%–1%. Therefore, determining the rcAAV contamination rate in rAAV is essential.

[0006] Because rcAAV in rAAV products poses a significant hazard, although the contamination level is relatively low, a highly sensitive method must be designed for its detection and quantitative analysis. There are two existing detection methods: one utilizes cell culture combined with qPCR, and the other is a rapid qPCR detection method. The former is currently the most commonly used, but it also has unavoidable drawbacks: First, its sensitivity depends on the infection efficiency of rcAAV on target cells. Compared to AAV type 2, many other AAV serotypes (1, 3, 5, 6, 7) cannot effectively infect target cells during culture (e.g., HECK293 / 293T cells), leading to reduced detection sensitivity and lower than the actual value. Second, this method is time-consuming and costly. The rapid qPCR detection method (direct detection method) can precisely compensate for the shortcomings of the cell culture method. It can detect rcAAV in a short time, but because the detection fragment is too short, it cannot guarantee that the sample contains the complete sequence required for rcAAV replication. Therefore, the detected rcAAV may not necessarily have replication ability, and the detection value is generally higher than the actual value.

[0007] However, there is still a need in the field for a low-cost method that can quickly, sensitively, and accurately detect the rcAAV5 contamination rate in rAAV5. Summary of the Invention

[0008] This invention is based on the following discovery: The inventors discovered for the first time that primer and probe sequences designed for the ITR-Rep (ITR, or inverted terminal repeat sequence), the target gene of type 5 rcAAV (rcAAV5), have higher accuracy than those for detecting single genes. These sequences can be used in both cell culture and rapid detection methods, and are particularly suitable for qPCR rapid detection, achieving mutual verification between the two methods. For example, when detecting rcAAV5 in rAAV5 products, qPCR can be used first. If the quality control standards are met, cell culture testing is unnecessary; if the standards are exceeded, cell culture testing is required. Furthermore, when testing samples, it is preferable to first treat the sample with DNase I to remove free nucleic acids not encapsulated by the viral capsid, which further improves detection accuracy.

[0009] In the first aspect, a primer and probe combination for detecting rcAAV5 is provided, including amplification primer sequences of the target sequences shown in SEQ ID No. 2 and 3, and probe sequences of the target sequences shown in SEQ ID No. 4.

[0010] In some embodiments, the primer and probe combination further includes amplification primer sequences of the internal reference sequences shown in SEQ ID No. 6 and 7, and probe sequences of the internal reference sequences shown in SEQ ID No. 8.

[0011] In this paper, the primer and probe combination is used to detect the contaminant rcAAV5 in rAAV5. The probe sequence has a fluorescent group and a quencher group attached to both ends, for example, a quencher group at the 3' end and a fluorescent group at the 5' end. The fluorescent groups of the target gene and internal reference gene can be FAM, VIC, TAMRA, CY5, etc.; the quencher group of the target gene can be MGB-NFQ; and the quencher group of the internal reference gene can be BHQ1, BHQ2, BHQ3, etc.

[0012] In some implementations, SEQ ID No. 4 has FAM connected to its 5' end and MGB-NFQ connected to its 3' end; SEQ ID No. 8 has CY5 connected to its 5' end and BHQ3 connected to its 3' end.

[0013] In a second aspect, a kit is provided that includes the primer and probe combination described in the first aspect.

[0014] In some embodiments, the kit further includes a quantitative reference, which may include, for example, the sequence shown in SEQ ID NO. 17. More specifically, the quantitative reference may be a linearized plasmid containing the sequence shown in SEQ ID NO. 17.

[0015] In some embodiments, the kit further includes qPCR polymerase, DNA dilution buffer (e.g., TE buffer), and quantitative reference. In the kit, the internal control and primers / probes for the target gene may be present in premixed form.

[0016] Furthermore, the use of the primer and probe combination of the first aspect in the preparation of a kit for detecting the contamination rate of rcAAV5 in rAAV5 is also provided.

[0017] As is known in the art, broadly speaking, rcAAV includes rcAAV that has undergone non-homologous recombination and wild-type AAV (wtAAV). Therefore, the detection of rcAAV5 in this paper also includes the detection of wtAAV5.

[0018] Furthermore, a method for detecting the contamination rate of rcAAV5 in rAAV5 using the primer and probe combination of the first aspect or the kit of the second aspect is also provided, including the following steps:

[0019] 1) Optionally, treat the sample with DNaseI to remove free nucleic acids that are not encapsulated by the viral capsid;

[0020] 2) Extract total DNA from the sample to be tested. For example, for cell samples, the cells need to be lysed first, centrifuged, and the supernatant is collected before DNA extraction.

[0021] 3) Using total DNA as a template, perform qPCR using the primers and probes described above; and

[0022] 4) Based on the amplification results, determine whether rcAAV5 exists in the sample to be tested, and quantify its content.

[0023] In some implementations, the sample is a cell bank, stock solution, or final product involved in the production of recombinant adeno-associated virus, or a cell sample for detecting rcAAV5 contamination rate based on cell culture methods.

[0024] In some implementations, the quantification includes the step of preparing a standard curve using a quantification reference.

[0025] In some implementations, the qPCR reaction conditions are: 95°C for 10 min; 95°C for 15 s, 60°C for 30 s, 72°C for 30 s, for a total of 40 cycles.

[0026] The kit of this invention has low detection sensitivity and a limit of quantitation of 2 copies / μL (1.90 particles quantitatively). -7The kit (using linearized plasmid quantitative reference) exhibits significantly higher detection sensitivity than the limits of quantitation (LOQ) of Rep and Cap sequences commonly used in cell culture methods. The quantitative reference in this kit contains both the target gene and the internal control gene, with separate standard curves for each gene. Therefore, the ratio of rcAAV5 to rAAV5 can be easily calculated to confirm whether it meets the standard of <1 rcAAV5 / 10n. 8 The industry standard for rAAV5VG (Vector Genome) is... Furthermore, the significance of using inverted terminal repeat (ITR) sequences as internal control genes lies in two aspects: firstly, ensuring the effectiveness of nucleic acid extraction, allowing direct determination of the vector genome copy number in rAAV5 viral samples extracted and recovered using kits or other methods; secondly, the vector concentration of rAAV5 determined by ITR plays a crucial role in determining whether a sample meets the standards, generally considered to be <1 rAAV / 10... 8 rAAV VG, therefore, this detection serves as the denominator in the testing standards. This test kit can effectively be used as a quality control method for rAAV5 contamination rate in the production process of gene therapy products.

[0027] In addition, the kit of the present invention has the advantages of high accuracy, specificity, reproducibility, stability during repeated freeze-thaw cycles, and compatibility with a variety of qPCR instruments. Attached Figure Description

[0028] The embodiments will now be described in conjunction with the accompanying drawings, thereby making the above and other aspects and advantages of the present invention apparent and readily understood.

[0029] Figure 1 Comparison of rcAA5 and rAAV5 genome maps.

[0030] Figure 2 : Prediction results of ITR secondary structure of AAV5.

[0031] Figure 3 : Amplification curve of the target gene.

[0032] Figure 4 Standard curve of target gene.

[0033] Figure 5 Amplification curve of the internal reference gene.

[0034] Figure 6 Standard curve of internal reference gene.

[0035] Figure 7 : Amplification curves specific to the target gene.

[0036] Figure 8 : Amplification curves specific to the internal reference gene. Detailed Implementation

[0037] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are merely illustrative and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature or the product / instrument instruction manual. All reagents or instruments (including those without a specified manufacturer) are commercially available.

[0038] Example 1. Design and synthesis of related sequences

[0039] In a broad sense, rcAAV5 actually includes rcAAV5 that has undergone non-homologous recombination and wild-type AAV5 (wtAAV5). By comparing the genome maps of rcAAV5 and rAAV5, it can be seen that the ITR-Rep linker sequence, Rep, and Cap sequences are unique to rcAAV5 and not found in the rAAV5 genome.

[0040] The inventors selected the ITR-Rep linker sequence as the target gene for this kit. Analysis of the AAV5 whole-genome sequence in NCBI determined the location of the AAV5 inverted terminal repeat (ITR) to be 1-161 nt, and the Rep sequence to be 359-2191 nt. Next, the 1-161 nt ITR sequence was used to predict the D sequence location of the AAV5 ITR using the secondary structure prediction website https: / / www.vectorbuilder.cn / tool / dna-secondary-structure.html (see...). Figure 2 The target gene primer is 145-161 nt, and a 428-445 nt position on the rep gene is selected as the downstream primer. Correspondingly, the probe position is determined to be 402-420 nt. The primers and probes for the internal reference gene in this invention use industry-recognized sequences to quantify the vector genomic concentrations of human rAAV and rcAAV.

[0041] Specifically, the designed and synthesized primer and probe sequences are shown in Table 1 below.

[0042] Table 1: Primers, probes, and amplification sequences

[0043]

[0044]

[0045] The target sequence is the ITR-Rep linker sequence (ITR-Rep) (NCBI accession number NC_006152.1: bits 145-445), and the internal parameter sequence is the inverted terminal repeat sequence (ITR) (NCBI accession number NC_006152.1: bits 93-161).

[0046] In addition, the inventors selected partial sequences of ITR, Rep, and Cap for gene synthesis, and then ligated the synthesized sequences into the plasmid vector pUC57-Kan-mini, obtaining a sequence SEQ ID NO.17. In this sequence, 66-2549nt is synthetic, and the remaining portion comes from the aforementioned plasmid vector. This sequence was used as the quantitative reference sequence for this invention. The specific sequence is as follows:

[0047] The sequence of the rcAAV5 quantitative reference material is SEQ ID NO.17.

[0048]

[0049] The obtained plasmid reference standard was linearized using the restriction endonuclease PVUI (NEB). The digestion products were then cleaned and recovered, and their concentration (μg / mL) was determined. Finally, the copy number was calculated using the following formula:

[0050]

[0051] The plasmid was 4.308 kb in size, and the linearized quantitative reference standard was finally diluted to 2 × 10⁻⁶ using DNA dilution buffer (TE buffer). 8 Prepare a quantitative reference solution of rcAAV5 by copying / μL and store at -20℃ for later use.

[0052] Example 2. Detection Instruments and Detection System

[0053] The detection instrument was a SHENTEK-96S real-time PCR instrument (Huzhou Shenke).

[0054] The reference stock solution was serially diluted 10-fold using DNA dilution buffer (TE buffer) to prepare 2×10⁻⁶ solutions. 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 Seven concentrations were prepared (ST1 to ST7) at 2 copies / μL and 2 copies / μL. Template-free negative controls (NTCs) were prepared using DNA dilution buffer instead of the reference standard.

[0055] Testing system:

[0056] First, primers and probes targeting the target gene, internal reference gene, rep, and cap were prepared into mixtures using DNA dilution buffer, with each primer and probe having a concentration of 200 nM.

[0057] The qPCR reaction system consisted of 30 μL, containing 8 μL of 2×qPCR SHENmix (Huzhou Shenke) + 2 μL of primer and probe mixture + 20 μL of rcAAV5 quantitative reference. Simultaneously, 20 μL of DNA dilution buffer was used to replace the reference to form a template-free control (NTC) system; and 20 μL of nucleotide solution of the test sample was used to prepare the test sample system.

[0058] qPCR reaction process:

[0059] 95℃ for 10 min; 95℃ for 15 s, 60℃ for 30 s, 72℃ for 30 s, 40 cycles.

[0060] Example 3. Standard curve verification of probe and primer combinations

[0061] Using the above-described qPCR reaction system and process, the performance of the primers and probes was verified through qPCR experiments.

[0062] The qPCR standard curve used 2×10 6 Copy / μL to 2×10 -1 Reference samples at 8 concentrations per copy / μL. NTC is a template-free negative control (DNA dilution).

[0063] Experimental results are as follows Figures 3-6 As shown. Among them Figure 3 and 5 These are the amplification curves of the target gene and the internal reference gene, respectively. Figure 4 and 6 These are the standard curves for the target gene and the internal reference gene, respectively. Figure 3 and Figure 5 It can be seen that the amplification curves of both the target gene and the internal reference gene show a clear exponential growth phase. Figure 4 and Figure 6 It can be seen that when the concentration of the reference sample is 2×10 6 Copy / μL to 2×10 -1 At a concentration of copies / μL, the amplification efficiency of the target gene was E = 96.2%, and the correlation coefficient (R) was [missing value]. 2 =1.000; the amplification efficiency of the internal reference gene E = 100.5%, and the correlation coefficient (R) 2 The value was 1.000. This indicates that the primer and probe sequences meet the requirements of the qPCR method in Chapter 3407 of the 2020 edition of the Chinese Pharmacopoeia for the standard curve parameters of the detection system.

[0064] Example 4. Validation of the linear detection range of the probe and primer combination

[0065] The inventors verified the linear range of the above primers and probes through qPCR experiments. The qPCR detection samples included eight concentrations of linearization quantitative reference standard, ranging from 2×10⁶ copies / μL to 2×10⁻¹ copies / μL. NTC was used as a template-free negative control (DNA dilution), and three replicates were set for each concentration.

[0066] The mean is calculated using the AVERAGE function; the standard deviation (SD) is calculated using the STDEV function; the coefficient of variation (CV) is calculated as SD / mean × 100%; the relative deviation is calculated as the absolute value of (actual concentration - theoretical concentration) / theoretical concentration × 100%.

[0067] Table 2: Detection Range

[0068]

[0069]

[0070] Experimental results show that the R-values ​​of the standard curves for the ITR-Rep target sequence and the internal reference sequence are significantly different. 2 All values ​​were greater than 0.990, and the amplification efficiencies were all within the range of 90% ≤ E ≤ 110%. These parameters all meet the pharmacopoeia requirements for standard curve parameters. At a reference concentration of 2 × 10⁻⁶... 6 Within the range of 2 copies / μL to 2 copies / μL, the relative deviation between the measured and theoretical concentrations at each point was <20% (the relative deviation at all points except the lowest and highest concentrations was <15%), and the coefficient of variation (CV) at each concentration point was <20% (the coefficient of variation at all points except the lowest and highest concentrations was <15%). Therefore, 2×10 6 The range of copies / μL to 2 copies / μL can be considered as the linear range for detection.

[0071] Example 5. Specificity verification of probe and primer combinations

[0072] Since rAAV is generally produced from sf9, 293, or 293T cells, even after purification processes such as digestion, residual DNA from these engineered cells may still be present in the rAAV test sample. Furthermore, because the quantitative reference standard in this invention's kit is produced from *E. coli*, it inevitably contains a small amount of *E. coli* DNA. Based on these facts, it is essential that the primer and probe combination for the target gene in this invention's kit specifically detects rcAAV but not residual DNA from engineered cells and *E. coli*, and that the primer and probe combination for the internal reference gene specifically detects both rcAAV and rAAV but not residual DNA from engineered cells and *E. coli*.

[0073] The inventors used the genomic DNA of several common engineered cells and Escherichia coli as templates (see Table 4) and verified the detection specificity of primer and probe combinations for target genes and internal reference genes through qPCR experiments.

[0074] Standard curve: Reference DNA diluted to a concentration of 2 × 10⁻⁶ 6 Seven concentrations were available, ranging from 2 copies / μL to 2 copies / μL. NTC was used as a template-free negative control (DNA dilution).

[0075] Specific detection: Genomic DNA from CHO cells, Vero cells, 293T cells, HEK293 cells, MDCK cells, E. coli, and sf9 cells was diluted to a concentration of 300 pg / μL and used as a template for specific detection.

[0076] In the specific qPCR detection system, 20 μL (300 pg / μL) of genomic DNA from engineered cells or E. coli was used. Three replicates were set up.

[0077] Table 3: Specific Detection Results

[0078]

[0079] The experimental results are shown in Table 3 and Figure 7 As shown in the figure. The experimental results showed that CT values ​​were not detected, and the detection values ​​were all <1.000E-5. The amplification curves were horizontal, indicating that the interfering DNA did not affect the detection results of the target gene and the internal reference gene, proving that the primer and probe combination of this kit has excellent specificity.

[0080] Example 6. Limit of Quantitation for Probe and Primer Combinations

[0081] Since the limit of quantitation (LOQ) is a crucial indicator for evaluating kit performance, this invention uses the LOQ of the rep and cap genes, commonly used in cell culture methods for detecting rcAAV, as a control group to assess the quantitative detection performance of the target genes. Because the difference between replicates begins to increase when the CT value exceeds 30, this invention selects several concentration points with CT values ​​around 30 based on the standard curve: 8 or 4 and 2 or 1 (copy / μL) as the expected LOQ of this kit. Each concentration point is tested 10 times, and the coefficient of variation (CV) and relative deviation are calculated. A CV and relative deviation of no more than 20% are considered acceptable.

[0082] The mean is calculated using the AVERAGE function based on the average concentration of 10 replicates; SD is calculated using STDEV; the coefficient of variation (CV) is calculated as SD / mean; and the relative deviation is calculated as the absolute value of (actual concentration - theoretical concentration) / theoretical concentration.

[0083] The standard curve for qPCR uses a concentration of 2×10⁻⁶. 6 Reference samples were available at eight concentrations, ranging from 2 copies / μL to 2 copies / μL. NTC was used as a template-free negative control (DNA dilution).

[0084] Table 4: Results of Limit of Quantitation Detection

[0085]

[0086]

[0087] The experimental results are shown in Table 4. Based on the validation criteria of a coefficient of variation (CV) and a relative deviation of no more than 20%, it can be seen that the limits of quantitation (LOQs) for the rep and cap genes, commonly used in cell culture methods for detecting rcAAV, are 8 copies / μL and 4 copies / μL, respectively. In contrast, the LQs for both the target gene and the internal control gene in this kit are 2 copies / μL. Therefore, the target gene selected in this kit has higher detection sensitivity than the rep and cap genes commonly used in cell culture methods for detecting rcAAV.

[0088] Example 7. Validation of the repeatability of primer and probe detection combinations

[0089] Repeatability of the test is another important indicator for evaluating the performance of the kit. Based on the linear range of the kit, a high concentration of 2×10⁻⁶ was selected. 5 Ten repeated tests were performed at a low concentration of 20 (copies / μL) and a low concentration of 20 (copies / μL), and the coefficient of variation (CV) was calculated for each concentration point. The concentration with the closest CV and not greater than 20% and the relative deviation not greater than 20% was considered acceptable.

[0090] The standard curve for qPCR uses a linearized quantitative reference concentration of 2 × 10⁻⁶. 6 Seven concentrations were available, ranging from 2 copies / μL to 2 copies / μL. NTC was used as a template-free negative control (DNA dilution).

[0091] The mean is calculated using the AVERAGE function; the standard deviation (SD) is calculated using the STDEV function; the coefficient of variation (CV) is calculated as SD / mean × 100%; the relative deviation is calculated as the absolute value of (actual concentration - theoretical concentration) / theoretical concentration × 100%.

[0092] Table 5: Repeatability Test Results

[0093]

[0094]

[0095] Experimental results show that the parameters of the standard curves for both the target gene and the internal reference gene satisfy R0. 2 The kit meets the requirements of >0.990, 90%≤E≤110%, and the CV of each concentration is less than 20% and the relative deviation is less than 20%, so the repeatability of the test is qualified.

[0096] Example 8. Blank limit verification of primers and probes

[0097] The limit of blank (LoB) is also an important indicator for kit evaluation. The limit of blank (LoB) is a term used in the Clinical and Laboratory Standards Institute (CLSI) EP17-A document; it refers to the maximum detectable result that can be observed in a blank sample under specified probabilities. Ideally, the result for every blank sample should be 0, but due to random error, blank sample detectable values ​​will be distributed within a low range. The inventors used DNA dilution as a template, repeated the test eight times, and performed three independent experiments to determine the kit's limit of blank.

[0098] Table 6: Results of Blank Limit Detection of Reagent Kit

[0099]

[0100]

[0101] The experimental results are shown in Table 6. The blank limits for both the internal reference gene and the target gene were not detected, so the blank limit was considered to be 0.

[0102] Example 9. Further detection of primer and probe combinations

[0103] In this embodiment, the inventors tested the rcAAV5 contamination rate in actual rAAV5 samples. The actual samples were produced using a three-plasmid system, with the specific production process as follows: A transfer plasmid pAAV carrying the GFP gene and type 5 ITR sequence, a type 5 Rep and Cap expression plasmid pHelper, and an auxiliary plasmid pAV encoding adenovirus genes (E2, E2A, and VA) that mediate adeno-associated virus replication were co-transfected into HEK293T packaging cells. After a brief incubation, cell lysates were collected, centrifuged, and the supernatant was used to obtain viral particles. The viral particles were concentrated with PEG, and excess plasmids were digested with DNase to obtain the test sample.

[0104] The sample was treated with DNase I (NEB) to remove free nucleic acids that were not encapsulated by the viral capsid.

[0105] Nucleic acid was extracted from the test samples using the QIAGEN AllPrep PowerViral DNA / RNA Kit (the total volume of the test samples after elution was 50 μL), and the nucleic acid concentration was determined using a spectrophotometer after extraction.

[0106] Take 20 μL of the original sample solution to detect the target gene, and set up 2 replicates. Serially dilute the remaining sample solution to 1 / 10, 1 / 100, and 1 / 1000 of the original solution to detect the internal control gene. Set up 2 replicates for each concentration.

[0107] Reaction system: 8 μL 2×qPCR SHENmixer + 2 μL primer and probe mixture + 20 μL nucleotide solution of the sample to be tested, to detect the target gene and internal reference gene in the previous step respectively.

[0108] The standard curve for qPCR uses 2×10 6 Eight reference concentrations were provided, ranging from 2 copies / μL to 2 copies / μL. NTC was used as a template-free negative control (DNA dilution). Each concentration was tested in triplicate.

[0109] Data Analysis: Set the threshold to 0.06 and the baseline to automatic. The analysis software (SHENTEK PCR Analysis System) will calculate the concentration at each detection point of the sample based on the standard curve.

[0110] The actual concentration of the sample to be tested can be calculated by dilution factor. Since the actual rAAV5 and rcAAV5 each contain an ITR gene at the 3' end and 5' end, the actual concentration of the internal reference gene should be half of the measured concentration.

[0111] The contamination rate of rcAAV5 in rAAV5 is calculated by dividing the copy number of the target gene by (the measured copy number of the internal reference gene × 1 / 2 - the copy number of the target gene).

[0112] To verify the authenticity of the detected target genes, the qPCR products need to be sent to a sequencing company for sequencing. The authenticity of the internal reference genes and target genes in the actual samples is determined by comparing the detected target genes and internal reference genes with the theoretical sequences.

[0113] Table 7: Detection of rcAAV5 contamination rate in rAAV5 using the kit

[0114]

[0115]

[0116] Table 7 shows that the concentration of rcAAV5 in the actual sample is 980 copies / μL, i.e., the total amount of rcAAV5 = 980 copies / μL × 50μL = 4.9 × 10⁻⁶. 4 copy.

[0117] The concentration of rAAV5 in the actual sample is approximately (9.17E+06+1.10E+07+1.03E+07)÷3=1.02×10 7 Copy / μL, i.e., total rAAV5 = 1.02 × 10⁻⁶ 7 Copy / μL×50μL×1 / 2-4.9×10 4 Copy = 5.1 × 10 8 Copy ≈ 2.55 x 108 copy.

[0118] The actual contamination rate of rcAAV5 in the rAAV5 sample was 4.9 × 10⁻⁶. 4 Copy rcAAV5 / 2.55x10 8 Copy rAAV5 = 1 rcAAV5 / 5.20 x 10 4 rAAV5 VG.

[0119] Meanwhile, the sequencing results show that the sequences of the target gene and internal reference gene amplified in the actual sample are completely matched with the theoretical sequences, indicating that the primer and probe design of this kit is correct and also demonstrating the accuracy of the results.

[0120] Example 10. The repeated freeze-thaw stability of the kit of the present invention

[0121] In practice, the kit inevitably undergoes repeated freeze-thaw cycles. Therefore, the stability of the kit under these conditions is another indicator for evaluating its performance. The inventors conducted performance tests on kits subjected to 1, 3, and 5 freeze-thaw cycles. The specific performance indicator was whether the limit of quantitation (LOQ) for both the target gene and the internal reference gene was 2 copies / μL. The criteria for acceptance were that both the coefficient of variation (CV) and the relative deviation were no greater than 20%.

[0122] qPCR standard curve range: linearized quantitative reference concentration of 2×10⁻⁶ 6 Reference samples were available at eight concentrations, ranging from 2 copies / μL to 2 copies / μL. NTC was used as a template-free negative control (DNA dilution).

[0123] The mean is calculated using the AVERAGE function based on the average concentration of 10 replicates. SD is the standard deviation calculated using STDEV. The coefficient of variation (CV) is calculated as SD / mean. The relative deviation is calculated as the absolute value of (actual concentration - theoretical concentration) / theoretical concentration.

[0124] The Grubbs test was chosen as the method for identifying outliers. It has the best power to identify outliers when the number of outliers detected is limited to no more than one. The specific test method is as follows: First, arrange the sample measurements from smallest to largest. If the maximum value is suspected to be an outlier, use the upper-side test; if the minimum value is suspected to be an outlier, use the lower-side test; if it is uncertain whether the maximum or minimum value might be an outlier, use the two-side test.

[0125] Table 8: Test results of the reagent kit's stability under repeated freeze-thaw cycles

[0126]

[0127] As shown in Table 8, the coefficient of variation (CV) and relative deviation of the limit of quantitation (2 copies / μL) of the target gene and internal reference gene of the kit were not greater than 20% after repeated freeze-thaw cycles of 1, 3 and 5 times. Therefore, it is believed that the detection performance of the kit in this paper remains stable after repeated freeze-thaw cycles of 5 times.

[0128] Example 11. Instrument suitability of the reagent kit of the present invention

[0129] To demonstrate that the kit is applicable not only to the SHENTEK-96S real-time PCR instrument but also to other models, the inventors conducted performance tests on three commonly used PCR instruments: the CFX96 (Bio-Rad), the PRISM 7500 Real-Time PCR System (ABI), and the LightCycler 480 II (Roche). The main performance indicators were that the coefficient of variation (CV) and relative deviation of the kit's limit of quantitation were both no greater than 20%. Based on the limit of quantitation of 2 copies / μL on the SHENTEK-96S instrument, the limits for other instruments were set at 2 copies / μL and 1 copy / μL, respectively.

[0130] The standard curve for qPCR uses 2×10 6 Reference samples were available at eight concentrations, ranging from 2 copies / μL to 2 copies / μL. NTC was used as a template-free negative control (DNA dilution).

[0131] The mean is calculated using the AVERAGE function based on the average concentration of 10 replicates. SD is the standard deviation calculated using STDEV. The coefficient of variation (CV) is calculated as SD / mean. The relative deviation is calculated as the absolute value of (actual concentration - theoretical concentration) / theoretical concentration.

[0132] The Grubbs test was chosen as the method for identifying outliers. It has the best power to identify outliers when the number of outliers detected is limited to no more than one. The specific test method is as follows: First, arrange the sample measurements from smallest to largest. If the maximum value is suspected to be an outlier, use the upper-side test; if the minimum value is suspected to be an outlier, use the lower-side test; if it is uncertain whether the maximum or minimum value might be an outlier, use the two-side test.

[0133] Table 9: Test Results of Instrument Suitability of Reagent Kit

[0134]

[0135]

[0136] As shown in Table 9, the limit of quantitation (LOQ) of the kit of the present invention is 2 copies / μL on three real-time PCR instruments: CFX96 (Bio-Rad), PRISM 7500 Real-Time PCR System (ABI), and LightCycler 480 II (Roche). This is consistent with the LOQ of the SHENTEK-96S real-time PCR instrument, indicating that the kit of the present invention has excellent instrument compatibility.

[0137] Although the invention has been disclosed with reference to certain embodiments, it will be apparent that modifications and variations can be made without departing from the spirit and scope of the invention as disclosed herein and as set forth in the appended claims. Furthermore, it should be understood that while all embodiments disclosed illustrate implementations of the invention, they are provided only as non-limiting examples and should not be construed as limiting the various aspects of the invention thus illustrated. The invention is intended to have the full scope defined by the language of this disclosure, the following claims, and any equivalents thereof. Therefore, the drawings and detailed descriptions should be considered illustrative rather than restrictive.

Claims

1. A primer and probe combination for detecting rcAAV5, comprising amplification primers and probes targeting a target sequence, wherein the sequences of the amplification primers are SEQ ID No. 2 and SEQ ID No. 3, and the sequence of the probe is SEQ ID No.

4.

2. The primer and probe combination according to claim 1, further comprising amplification primers and probes targeting an internal reference sequence, wherein the sequences of the amplification primers are SEQ ID No. 6 and SEQ ID No. 7, and the sequence of the probe is SEQ ID No.

8.

3. The primer and probe combination according to claim 1 or 2, wherein the probe sequence is connected to a fluorescent group and a quenching group at both ends, respectively.

4. The primer and probe combination according to claim 3, wherein the fluorescent group is FAM, VIC, TAMRA or CY5, and the quenching group is MGB-NFQ, BHQ1, BHQ2 or BHQ3.

5. The primer and probe combination according to claim 3 or 4, wherein SEQ ID No. 4 has FAM attached to the 5' end and MGB-NFQ attached to the 3' end; SEQ ID No. 8 has CY5 attached to the 5' end and BHQ3 attached to the 3' end.

6. The primer and probe combination according to any one of claims 1-5, used to detect the contamination rate of rcAAV5 in rAAV5.

7. A kit comprising the primer and probe combination as described in any one of claims 1-6.

8. The kit of claim 7, further comprising a quantitative reference comprising the linearized plasmid shown in SEQ ID NO.

17.

9. Use of the primer and probe combination of any one of claims 1-6 in the preparation of a kit for detecting the contamination rate of rcAAV5 in rAAV5.

10. A method for detecting the contamination rate of rcAAV5 in rAAV5 of a test sample using the primer and probe combination of any one of claims 1-6 or the kit of any one of claims 7-8, comprising the steps of: 1) Treat the sample with DNaseI to remove free nucleic acids that are not encapsulated by the viral capsid; 2) Extract total DNA from the sample processed in step 1); 3) Using the total DNA extracted in step 2) as a template, perform a qPCR reaction using the primers and probes described above; and 4) Based on the amplification results, determine whether rcAAV5 exists in the sample to be tested, and quantify its content.

11. The method according to claim 10, wherein the sample is a cell bank, stock solution, or final product used in the production of rAAV5, or a cell sample for detecting rcAAV5 based on cell culture methods.

12. The method according to claim 11, wherein, The contamination rate of rcAAV5 in rAAV5 was calculated using the formula: "copy number of target gene ÷ (copy number of internal reference gene × 1 / 2 - copy number of target gene)".

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