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Combined primer for amplification and typing of human papilloma virogenes and application of combined primer

A virus gene and papilloma technology, applied in the field of gene detection, can solve the problems of high cost, limited number of tests, and inapplicability of cervical cancer screening, and achieve the effect of cost saving and easy access

Inactive Publication Date: 2015-04-29
南京普东兴生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the combination of these methods is not only costly, but also time-consuming and labor-intensive, and the number of single tests is limited, so it is not suitable for large-scale cervical cancer screening, and is generally only suitable for HPV detection and cervical cancer screening in economically developed areas

Method used

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  • Combined primer for amplification and typing of human papilloma virogenes and application of combined primer
  • Combined primer for amplification and typing of human papilloma virogenes and application of combined primer
  • Combined primer for amplification and typing of human papilloma virogenes and application of combined primer

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Embodiment 1

[0054] Embodiment 1: PCR amplification primer design

[0055] The present invention adopts the method of amplicon to amplify the L1 region, and the primers are designed as follows: figure 1 :

[0056] The design mainly follows:

[0057] The combined upstream primer is: 5'P2 adapter + barcode adapter + upstream primer 3'; the combined downstream primer is: 5'P1 adapter + downstream primer 3'; the total amplification length is about 200bp. Use software for comparison to ensure that the annealing temperature difference between the primer pairs is not large, preferably within 3 degrees. The absolute value of ΔG of the primer-dimer formed between the primer pair and the primer pair is less than 5.

[0058] The length of the primer is about 50bp, so when synthesizing the primer, a higher purification standard should be selected.

[0059] Table 1: Barcode sequence

[0060]

[0061] Note: Bc=barcode

[0062] Table 2: P1, P2 sequences

[0063]

[0064] Table 3: Primers wit...

Embodiment 2

[0072] Embodiment 2 PCR amplification

[0073] Each primer was mixed to a final concentration of 2 μM, and the amplification reaction was performed according to the following Takara reaction system and amplification procedure.

[0074]

[0075]

[0076] The PCR reaction conditions are: denaturation at 94°C for 30 seconds; denaturation at 94°C for 30 seconds, annealing at 58°C for 15 seconds, extension at 72°C for 1 minute, and a total of 35 cycles of amplification; finally, full extension at 72°C for 10 minutes;

Embodiment 3

[0077] Embodiment 3: the recovery of target fragment

[0078] Recover the target gene: prepare 2% agarose gel, put it in the electrophoresis tank, add TAE working solution, and submerge the gel block. Mix 10 μL of PCR product with 2 μL of 6×Loading buffer and add to the loading well. The voltage was adjusted to 90V, and the electrophoresis stopped when the front of the dye reached 3 / 4 of the gel plate. Rapidly excise the band of interest under UV light. According to the operation instructions of Tiangen Agarose Gel Recovery Kit (DP209-03), the target fragment was recovered, and the target gene was dissolved with 30 μL ddH2O, and the recovered product was the library.

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Abstract

The invention discloses a combined primer for the amplification and typing of human papilloma virogenes. The combined primer is divided into a combined forward primer and a combined reverse primer; the combined forward primer is composed of three parts, namely a joint part P2 sequence, a tag sequence and a forward primer sequence in order; the combined reverse primer is composed of a reverse primer sequence and a joint part P1 sequence in order. The invention also discloses a kit for the amplification and typing of the human papilloma virogenes. The invention also discloses the applications of the combined primer and the kit. The combined primer has real flux, and is low in cost and capable of accurately typing HPV. The type of HPV can be accurately determined by using a plurality of primers (6 forward primers and 5 reverse primers) for amplification and comparing the sequence information of the amplification product with an HPV database; as a result, a basis is provided for clinical diagnosis and treatment scheme selection.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to combined primers for the amplification and typing of Human Papilloma Virus (HPV) genes and applications thereof, in particular to HPV typing detection based on high-throughput sequencing and multiplex PCR technology new method. Background technique [0002] Human papillomavirus (HPV) is a small, non-enveloped, double-stranded circular DNA virus that infects humans mainly through direct or indirect contact with contaminated items or sexual transmission. HPV has host specificity and tissue specificity, and can only infect human skin and mucosal epithelial cells, causing a variety of papillomas or warts on human skin and hyperplastic lesions of the mucosal genital tract epithelium. At present, both the World Health Organization and the International Cancer Research Center have confirmed that persistent HPV infection is the main cause of cervical cancer. [0003] The cancer report sho...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/708C12Q1/6806C12Q1/6869
Inventor 叶珊戴琳超陆祖宏
Owner 南京普东兴生物科技有限公司
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