Search

CN-122012812-A - Probe primer combination for detecting AdV wild type virus residues as well as method and application thereof

CN122012812ACN 122012812 ACN122012812 ACN 122012812ACN-122012812-A

Abstract

The invention belongs to the technical field of biological detection, and relates to a probe primer combination for detecting AdV wild type virus residues, a method and application thereof. According to the invention, two sets of primer probe combinations respectively targeting the E1A-CR2 and E3 regions of wild adenovirus are designed, and double-target synchronous detection and signal distinction are realized by using dPCR. By calculating the concentration of the linkage molecules (linkage) of the two targets on the same DNA chain, false positive results caused by host cell DNA residues, incomplete E3 region knockout or single region non-specific amplification can be effectively eliminated, so that the specific recognition of wild adenovirus is realized. The method is convenient and quick, can finish the detection of the AdV wild toxin residues in a short time, does not need expensive instruments and equipment, has high sensitivity and good specificity, improves the sensitivity by 2 orders of magnitude compared with a PCR method (about 1/10 7 ), and can improve the sensitivity to more than 0.5/10 9 .

Inventors

  • PAN JUNYI
  • CHEN HUILING
  • HUANG LIULIU
  • WANG SHUANGTING
  • ZHANG HAIYANG
  • WANG BIN
  • LI HONGLING

Assignees

  • 和元智造(上海)基因技术有限公司

Dates

Publication Date
20260512
Application Date
20260121

Claims (10)

  1. 1.A method for detecting adenovirus wild-type viral residues comprising the steps of: S1, the sample to be tested comprises modified adenovirus and/or wild adenovirus, wherein the modification at least comprises deletion or modification of CR2 region of early 1 (E1) A gene and/or early 3 (E3) gene; s2, performing double amplification detection on the sample to be detected by using a digital PCR (dPCR) reaction system, wherein the dPCR reaction system comprises: (1) A first set of primers and probes that target the E1A-CR2 region of a wild-type adenovirus, wherein the 3 'end of the reverse primer and/or the 5' end of the probe is located within the CR2 region or is designed across the boundary of the CR2 region such that an adenovirus engineered with the CR2 region cannot be effectively amplified or detected; (2) The second group of primers and probes target the E3 gene region of the wild adenovirus, so that the adenovirus subjected to E3 gene deletion or modification cannot be effectively amplified or detected; S3, performing dPCR amplification and detection, obtaining fluorescent signals corresponding to the E1A-CR2 target and the E3 target in each droplet, and counting the number of double-positive droplets, E1A-CR2 positive droplets, E3 positive droplets and double-negative droplets; S4, calculating the concentration of a linkage molecule (linkage) existing on the same adenovirus DNA chain in the E1A-CR2 region and the E3 region in the sample to be detected according to the number of the microdroplets counted in the step S3 based on the Poisson distribution principle; S5, judging whether wild adenovirus residues exist in the sample to be detected according to the calculated concentration of the linkage molecules, wherein the wild adenovirus residues are judged to be positive when the concentration of the linkage molecules is larger than 0, and the wild adenovirus residues are judged to be negative when the concentration of the linkage molecules is smaller than or equal to 0.
  2. 2. The method of claim 1, wherein in S2, the first set of probes and the second set of probes carry distinguishable reporter labels.
  3. 3. The method of claim 2, wherein in S2, the first set of probes is labeled FAM and the second set of probes is labeled HEX.
  4. 4. The method according to claim 1, wherein in S1, the sample to be tested is not subjected to pretreatment before dPCR detection.
  5. 5. The method of claim 1, wherein the first set of primers and probes have the sequences shown in SEQ ID NOS.1-3 and the second set of primers and probes have the sequences shown in SEQ ID NOS.4-6.
  6. 6. The method for detecting adenovirus wild-type viral residues according to claim 1, wherein in S4 the concentration of said linked molecules (linkage concentration) is calculated based on the following formula or equivalent algorithm: poisson distribution statistics: ,( , , Three variables); When k= at the time of 0, the temperature of the liquid, )= )= ; Conc=CPD/V drop droplet volume=copies/nL; )= =conc.×v drop droplet volumes Assuming a is a double positive droplet, b and c are single positive droplets, d is a negative droplet, x is an unlinked double positive droplet, since = = X=can be obtained by simple calculation ; = = ; It can be deduced that: = ) According to the distribution conditions of different marked droplets, the concentration of the linkage can be calculated according to the volume of the droplets; Where k is the number of positives contained in the droplet, λ is the average number of molecules per droplet, N negative is the number of double negative droplets, and N total is the total number of valid droplets.
  7. 7. The method of claim 1, wherein in S3, the dPCR is a digital micro-PCR (dPCR).
  8. 8. A probe primer combination for detecting adenovirus wild-type virus residue, comprising a first set of primers and probes, a second set of primers and probes; The first group of primers and probes target the E1A-CR2 region of the wild adenovirus, wherein the 3 'end of the reverse primer and/or the 5' end of the probe are positioned in the CR2 region or are designed across the boundary of the CR2 region, so that the adenovirus modified by the CR2 region cannot be effectively amplified or detected; The second group of primers and probes target the E3 gene region of the wild adenovirus, so that the adenovirus subjected to E3 gene deletion or modification cannot be effectively amplified or detected, and the sequences of the second group of primers and probes are shown as SEQ ID NO. 4-6.
  9. 9. A kit for detecting adenovirus wild-type viral residues comprising: (a) The first set of primers and probes of claim 8, and the second set of primers and probes; (b) A reaction premix for performing digital PCR; (c) An oil phase reagent for generating droplets; (d) Wild adenovirus standard or positive quality control.
  10. 10. Use of the method of any one of claims 1-7, the probe-primer combination of claim 8 or the kit of claim 9 for the preparation of a detection formulation for detecting wild-type viral residues in an adenovirus vector preparation.

Description

Probe primer combination for detecting AdV wild type virus residues as well as method and application thereof Technical Field The invention belongs to the technical field of biological detection, and relates to a probe primer combination for detecting AdV wild type virus residues, a method and application thereof. Background Adenovirus (Adenovirus, adV) is a viral vector that is widely used in gene therapy and vaccine development. Its natural advantage is the ability to efficiently infect multiple cell types and induce strong innate and adaptive immune responses simultaneously in a mammalian host. As technology advances, adV has transformed from a tool for gene replacement therapy into a true vaccine delivery vehicle. In order to ensure the safety of clinical application, adV vectors are often genetically modified to reduce their replication capacity or to expand the loading capacity of exogenous genes. At present, the modification of the AdV can be realized by manipulating the early 1 (E1) region or part thereof, so that the AdV-based vector has replicative capacity or does not have replicative capacity, and common modification sites comprise E1A-CR2 and E1B-55K, E B-19K. The E1A-CR2 region can combine with retinoblastoma mutant gene to promote cell replication into S phase, and provide conditions for virus replication, so that common AdV viruses generally modify E1A-CR2 to increase safety. Furthermore, since the E3 gene is not essential for viral replication, the capacity of transgene insertion is enlarged by deleting the early 3 (E3) gene. During production, there is a risk of producing replicable wild-type AdV (RCA) due to homologous recombination or other genetic instability. Such wild-type viral residues may pose a safety risk to the subject, so the strict detection of AdV wild-type viral residues in the final product is a key element in quality control. Due to the lysis of cells by AdV, adV cannot be detected by classical culture methods, which are not suitable for efficient, direct detection. Currently, the industry mainstream relies on molecular biological methods, in particular Polymerase Chain Reaction (PCR) techniques. The PCR method utilizes the transformation characteristic of AdV, in order to avoid false positive caused by homologous recombination, a Long-fragment amplification primer is usually designed, electrophoresis detection is carried out after Long Range PCR amplification, the possibility is provided for detecting the wild virus residue of AdV, and the strategy design principle of combining Long Range PCR with agarose gel electrophoresis is that a Long-fragment amplification primer (usually the amplification length is more than 7 kb) which spans the deleted fragment is designed aiming at a genome region (such as a complete E1A-CR2 region or E3 region) which is special to wild type AdV and is transformed or deleted in a recombinant vector. Theoretically, only wild-type viral DNA containing the complete target sequence can be amplified efficiently, so that specific bands are observed by electrophoresis, whereas recombinant vectors lacking this region cannot produce efficient amplification. However, long Range PCR detection methods suffer from the disadvantages of being Long in time, relatively low in sensitivity, and causing false positives due to non-specific amplification: the sensitivity is low, and the amplification efficiency of Long Range PCR is low due to the Long amplification fragment. Furthermore, to ensure specificity, the reaction conditions are often stringent, which further limits the sensitivity. Typical detection limits of the existing methods are about 1 wild-type viral genome/10≡7 vector genome, and it is difficult to meet the detection requirements of extremely high purity products. Time consuming and low throughput Long Range PCR requires a longer extension time to ensure complete synthesis of Long fragments, with a single PCR cycle time much longer than conventional PCR. From nucleic acid extraction, long fragment PCR amplification to final agarose gel electrophoresis analysis, the whole process usually takes a whole day or even longer, and rapid detection cannot be achieved. Non-specific amplification long fragment PCR is easier to generate non-specific amplification products and primer dimers, resulting in multiple bands in the electropherogram, interfering with interpretation and possibly even misjudging as a positive signal. Disclosure of Invention In order to solve the problems of long time consumption, low sensitivity, poor specificity and false positives of the conventional PCR method, the invention develops an AdV wild toxin residue detection method capable of overcoming the defects. According to 2 different transformation areas of the AdV, 2 pairs of differently marked primers are designed in the corresponding areas of the wild type AdV, double dPCR detection is carried out, amplification conditions of the corresponding areas of the wild type AdV are detect