CN-114875181-B - Quality control product for human papilloma virus molecular detection and preparation method thereof

Abstract

The invention discloses a quality control product for human papilloma virus molecular detection and a preparation method thereof, belonging to the technical field of biological products. After 25 types of high-risk type (HPV16、HPV18、HPV26、HPV31、HPV33、HPV35、HPV39、HPV45、HPV51、HPV52、HPV53、HPV56、HPV58、HPV59、HPV66、HPV68、HPV73、HPV82), low-risk types (HPV 6, HPV11, HPV42, HPV43, HPV44, HPV81 and HPV 83) are synthesized, the HPV whole genome is connected into a pcDNA3.1 (+) framework vector system, and the HPV whole genome is respectively integrated into a human ovarian cancer cell line ovcar genome to obtain 25 human ovarian cancer cell lines with stably integrated HPV whole genome. Can be used for preparing novel quality control products or kits for various simulated clinical samples such as genome DNA, cell precipitation, cell suspension and the like, and is suitable for various detection platforms. The quality control product is a humanized cell line quality control product with 25 HPV typing full-length genomes stably integrated, can more accurately measure the viral load and control the whole HPV detection process.

Inventors

• LI JINGHUA
• CHENG YING
• HOU XIAOMEI
• LIU CHUXIN
• LIU QINGBO
• LIN XINXIN
• Request for anonymity
• Request for anonymity
• Request for anonymity

Assignees

• 菁良基因科技(深圳)有限公司

Dates

Publication Date

20260508

Application Date

20220629

Claims (1)

1. 1. The preparation method of the human papilloma virus molecular detection quality control product is characterized by comprising the following steps: (1) Synthesizing an HPV genome sequence, and adding enzyme cutting sites at two ends of the HPV genome sequence, wherein the enzyme cutting site at the 5 'end is NheI, and the enzyme cutting site at the 3' end is PmeI; (2) The HPV genome sequence is connected into pcDNA3.1 (+) framework vector through cleavage sites NheI and PmeI to obtain HPV typing plasmid, the HPV typing plasmid is transfected into a human ovarian cancer cell line ovcar, and a transfection system is configured as follows: jetPRIME® buffer:75 μL, HPV typing plasmid 1 mug, jetPRIME® reagent:3.6 μL, Mixing the above prepared system, standing at room temperature for 10min, adding the mixed system into 12-well plate inoculated with ovcar cell line in advance, incubating in incubator for 4-6 hr, and changing normal culture medium; (3) After the screening, the rest cells are subjected to single cell cloning, cloning holes are marked when the single cell cloning is formed, trypsin is added to digest the cells, and the digested cells are divided into two parts, wherein one part of the cells are used as an amplification template, and the single clone is amplified by using a primer and the sanger sequencing to verify whether the whole genome of HPV is successfully integrated; (4) Selecting sanger sequencing to verify single cell clone with HPV whole genome successfully integrated, amplifying and culturing the clone, extracting gDNA, detecting the copy number of HPV in the selected clone by adopting digital PCR, and preparing gradient quality control products with different copy numbers by taking ddPCR identification copy number as a reference, wherein the quality control products are in the form of cell suspension, cell precipitation or gDNA.

Description

Quality control product for human papilloma virus molecular detection and preparation method thereof Technical Field The invention belongs to the technical field of biological products, and particularly relates to a quality control product for human papilloma virus molecular detection and a preparation method thereof. Background Human papilloma virus (Human Papillomavirus, HPV) belongs to the family papillomaviridae, is a circular double-stranded DNA virus without envelope coating, consisting of a DNA core and a protein capsid consisting of a major capsid protein (L1) and a minor capsid protein (L2). The genome is divided into 3 functional regions, namely an early transcription region (E region, encoding early proteins such as E1, E2, E4, E5, E6, E7 and the like, involved in the functions of viral replication, transcription, translational regulation, transformation and the like), a late transcription region (L region, encoding capsid proteins L1 and L2) and a non-transcription region (long control region, LCR, containing the replication origin of dnas and regulatory elements required for expression, regulating viral transcription and replication), at about 8000 base pairs (bp). Over 200 HPV have been reported to infect humans by direct or indirect contact with contaminating items or sexually transmitted. The high-risk types (HPV16、HPV18、HPV26、HPV31、HPV33、HPV35、HPV39、HPV45、HPV51、HPV52、HPV53、HPV56、HPV58、HPV59、HPV66、HPV68、HPV73、HPV82), and low-risk types (HPV 6, HPV11, HPV42, HPV43, HPV44, HPV81, HPV 83) are classified into other subtypes according to the post-infection hazard of HPV. Epidemiological studies have shown that persistent infection of high-risk HPV in normal cervical epithelial cells of humans is one of the major causes of cervical cancer, rectal cancer, and low-risk HPV is associated with common warts, flat warts, plantar warts, and the like. HPV early detection, particularly accurate typing, is critical for early detection, prevention, early warning and treatment of cervical cancer. The clinical detection of HPV is mainly based on virus nucleic acid detection, and specific methods include a PCR-reverse dot hybridization method, a PCR-fluorescent probe method, a PC R-diversion hybridization method, a hybridization capture-chemiluminescence method and the like. The detection reagent has a plurality of types, has different principles and characteristics, and has different performance indexes such as analysis sensitivity, specificity and the like. In order to improve reliability and consistency of detection results of laboratories and different kits, inter-laboratory quality evaluation should be performed, comparison is performed on results of laboratory HPV detection or genotyping, detection capabilities of different platforms and laboratories are monitored, and accuracy of result reporting is improved. The calibrator (object) and the quality control (object) are main tools for realizing the accurate and consistent in-vitro diagnosis, clinical detection and supervision and inspection results, and are also physical metering standards for ensuring the quantity value transmission. The HPV international standard substance is obtained by diluting recombinant HPV genome plasmid by using HPV negative genome DNA extracted from human cervical cell line. Meanwhile, HPV quality control products in the market at present are all of plasmid sources, and part of HPV quality control products are obtained by adding plasmids into a humanized genome for dilution. Such standards cannot monitor sample processing procedures, and have large matrix differences with clinical samples, single in form. Secondly, in clinical detection, HPV viral load is an important reference index for evaluating cervical lesion extent, and further reflects the true viral infection extent. (HPV viral load: copy number of HPV in unit-infected epithelial cells). Studies have shown that different genotypes and viral loads of HPV are important predictors of CIN2+ and CIN3+. Patients with higher risk of cin2+ and cin3+ are identified based on HPV genotype and viral load, which is important for the formulation of personalized triage and follow-up strategies. The quality control prepared in the form of plasmid does not have the ability to detect viral load. Disclosure of Invention 1. Problems to be solved Aiming at the limitations of the standard substance/quality control substance in the practical use scene, the invention provides a novel quality control substance which is a humanized cell line quality control substance with 25 HPV typing full-length genomes stably integrated, can more accurately measure virus load and control the whole HPV detection process, and meanwhile, the quality control substance and the kit prepared by HPV typing can be prepared into various forms such as gDNA, cell precipitation, cell suspension and the like, and are suitable for various detection platforms. 2. Technical proposal In order to solve the problems, the inventio