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CN-116121447-B - RcAAV5 detection probe and primer combination and use thereof

CN116121447BCN 116121447 BCN116121447 BCN 116121447BCN-116121447-B

Abstract

The invention provides a primer and probe combination for detecting rcAAV and application thereof. When the primer and probe combination is used for detection, the pollution rate of rcAAV in rAAV5 can be rapidly, sensitively, accurately and at low cost.

Inventors

  • ZHANG QIMENG
  • YANG ZHIXING
  • ZONG WEIYING
  • LU SHUANGHONG

Assignees

  • 湖州申科生物技术股份有限公司

Dates

Publication Date
20260508
Application Date
20220812

Claims (12)

  1. 1. And detecting rcAAV by using a primer and probe combination, wherein the primer and probe combination comprises an amplification primer and a probe aiming at a target sequence, the sequences of the amplification primers are SEQ ID No.2 and SEQ ID No. 3, and the sequences of the probes are SEQ ID No.4.
  2. 2. The primer and probe combination of claim 1, further comprising an amplification primer and probe for an internal reference sequence, wherein the amplification primer has the sequences of SEQ ID No. 6 and SEQ ID No. 7 and the probe has the sequence of SEQ ID No.8.
  3. 3. The primer and probe combination according to claim 1 or 2, wherein both ends of the probe sequence are respectively attached with a fluorescent group and a quenching group.
  4. 4. The primer and probe combination of claim 3, wherein the fluorescent group is FAM, VIC, TAMRA or CY5 and the quenching group is MGB-NFQ, BHQ1, BHQ2, or BHQ3.
  5. 5. The primer and probe combination according to claim 3 or 4, wherein SEQ ID No.4 is linked at the 5 'end to FAM and at the 3' end to MGB-NFQ, and SEQ ID No.8 is linked at the 5 'end to CY5 and at the 3' end to BHQ3.
  6. 6. The primer and probe combination of any one of claims 1-5 for use in detecting a contamination rate of rcAAV in rAAV 5.
  7. 7. A kit comprising the primer and probe combination of any one of claims 1-6.
  8. 8. The kit of claim 7, further comprising a quantitative reference comprising a linearized plasmid set forth in SEQ ID No. 17.
  9. 9. Use of a primer and probe combination according to any one of claims 1-6 in the manufacture of a kit for detecting the contamination rate of rcAAV in rAAV 5.
  10. 10. A method for detecting the contamination rate of rcAAV in rAAV5 of a sample to be tested 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) Treating the test sample with DNaseI to remove free nucleic acids not encapsulated by the viral capsid; 2) Extracting total DNA in the sample treated in the step 1); 3) Performing qPCR reaction using the total DNA extracted in step 2) as a template and using the primers and probes, and 4) And judging whether rcAAV exists in the sample to be detected according to the amplification result, and quantifying the content of rcAAV.
  11. 11. The method of claim 10, wherein the sample is a cell bank, stock solution, or end product of a process for producing rAAV5, or a cell sample of rcAAV detected based on a cell culture method.
  12. 12. The method of claim 11, wherein the contamination rate of rcAAV in rAAV5 is calculated according to the formula "copy number of target gene ≡ (measured copy number of reference gene × 1/2-target gene copy number)".

Description

RcAAV5 detection probe and primer combination and use thereof Technical Field The application relates to the field of virus detection, in particular to detection of replication type recombinant adeno-associated virus in recombinant adeno-associated virus. Background Adeno-associated virus (AAV) belongs to the parvoviridae family of non-enveloped linear DNA viruses, and viral replication is achieved only in the presence of helper virus (typically adenovirus). AAV is capable of infecting multiple types of human cells and is therefore classified into multiple serotypes. A typical AAV2 genome is about 4800bp and consists of two inverted terminal repeats (INVERTED TERMINAL REPEAT or ITRs, 145 bp) and two Open Reading Frame (ORF) rep and cap genes. ITR plays a decisive role in viral replication and packaging and is necessary for the synthesis of complementary DNA strands. The cap gene encodes viral capsid proteins and the rep gene is involved in viral replication and integration. AAV of 13 different serotypes (i.e., AAV1-AAV 13) are reported in primates, wherein AAV2, AAV3, AAV9 are derived from humans themselves, AAV2 being the earliest cloned virus and the most thoroughly studied and widely used virus to date. AAV5 is far from AAV2 in evolution, and has few homologous sequences between the AAV5 and AAV, and has few researches, but AAV5 has wide application range, and can infect important organs of human body such as eyes, central nervous system, pancreas, lung and the like. Recombinant adeno-associated virus (rAAV) is a genetic vector engineered on the basis of nonpathogenic wild-type AAV, the genome of the rAAV package has deleted all AAV protein coding sequences and a therapeutic gene expression cassette has been added, the only viral-derived sequences being ITRs, which are necessary to direct genome replication and packaging during vector production. The rAAV virus vector has the advantages of diversity, extremely low immunogenicity, high safety, wide host cell range (both dividing cells and non-dividing cells have infectivity), strong diffusion capability, long in-vivo gene expression time and the like, so the rAAV virus vector is regarded as one of the most promising gene research and gene therapy vectors. However, in the production of rAAV vectors, large amounts of rAAV vector genome, rep and cap genes, and ITR sequences undergo non-homologous recombination due to physical proximity between the transfection plasmid or host cell DNA and the rAAV, thereby forming replication competent AAV (Replication competent AAV, rcAAV). Although rcAAV particles are not associated with any known human disease, rcAAV particles as a contaminant may affect rAAV gene expression and are an uncontrolled variable in many AAV gene transfer studies. Recent animal experiments have shown that expression of cap gene in vivo can elicit a severe immune response, while rcAAV, packed with other DNA impurities, carries the potential risk of tumorigenicity or introduction of antibiotic resistance. In rAAV produced by the traditional process, the rcAAV pollution rate can reach 10%, and even if the optimized process is adopted, the rcAAV pollution rate can reach 0.4% -1%, so that the determination of rcAAV pollution rate in rAAV is very necessary. Because rcAAV in the rAAV preparation is not harmful, but the pollution amount is relatively low, a high-sensitivity method must be designed to detect and quantitatively analyze the rAAV preparation. The existing detection methods are two, one is a method of combining a cell culture method with qPCR, and the other is a qPCR rapid detection method. The former method is most commonly used at present, but has unavoidable drawbacks in that firstly, its sensitivity depends on the infection efficiency of rcAAV on target cells, many other AAV serotypes (1, 3, 5, 6, 7) cannot effectively infect target cells (e.g. HECK293/293T cells) during the culture process compared with AAV type 2, resulting in reduced detection sensitivity, lower detection value than practical value, and secondly, the method takes longer time and has higher input cost. The qPCR rapid detection method (direct detection method) can exactly make up the defect of a cell culture method, and can detect rcAAV in a short time, but the detection fragment is too short to ensure that a detected object has a complete sequence required by rcAAV replication, so that the detected rcAAV does not have replication capacity necessarily, and the detection value is generally higher than an actual value. There remains a need in the art for low cost methods that can rapidly, sensitively, and accurately detect rcAAV contamination rates in rAAV 5. Disclosure of Invention The invention is completed based on the finding that the inventors found for the first time that the primer and probe sequence designed for the target gene ITR-Rep (ITR, inverted terminal repeat sequence) of type-5 rcAAV (rcAAV) is more accurate than that of single gene detection, can be used