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Primer, probe and kit for fluorescence PCR (Polymerase Chain Reaction) detection of 18 high-risk human papilloma viruses

A technology for human papillomavirus and detection kits, applied in the directions of fluorescence/phosphorescence, biochemical equipment and methods, determination/inspection of microorganisms, etc.

Active Publication Date: 2014-04-30
英科新创(苏州)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

[0010] Therefore, there are many HPV products detected by the existing real-time fluorescent PCR technology, and the genotypes detected by most products are relatively small, but no matter how many genotypes are detected by the product, and whether the detection uses a specific type of probe, the existing products are a set of primers Probes can only be provided in one kit

Method used

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  • Primer, probe and kit for fluorescence PCR (Polymerase Chain Reaction) detection of 18 high-risk human papilloma viruses
  • Primer, probe and kit for fluorescence PCR (Polymerase Chain Reaction) detection of 18 high-risk human papilloma viruses
  • Primer, probe and kit for fluorescence PCR (Polymerase Chain Reaction) detection of 18 high-risk human papilloma viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Extraction of DNA

[0069] The HPV virus DNA is extracted by the magnetic bead extraction method (it can also be extracted by a fully automatic nucleic acid extractor). Its working principle and steps mainly include cell lysis, binding of nucleic acid and magnetic beads, washing the complex of magnetic beads and nucleic acid and eluting nucleic acid. The process of cell lysis is to destroy the cell structure and release the nucleic acid from the cell; the process of combining nucleic acid and magnetic beads is the process of purifying nucleic acid to remove impurities such as proteins, polysaccharides and other biological macromolecules. Washing the complex of magnetic beads and nucleic acid is to further purify nucleic acid and remove residues and trace salt ions on the surface of the complex. The elution of nucleic acid is to separate the nucleic acid from the magnetic beads to obtain the required high-purity nucleic acid.

Embodiment 2

[0070] Example 2: Primer Design and Synthesis

[0071] The DNA sequences of the L1 regions of each subtype of human papillomavirus were called out from GeneBank, and then the sequences of the L1 regions of each subtype were compared by DNAStar software, and primer sequences were designed and synthesized in the relatively conserved regions of the high-risk types. The primer sequences designed in the present invention have:

[0072] Primer 1 for amplifying human papillomavirus, its base sequence is shown in SEQ ID NO:1;

[0073] Primer 2 for amplifying human papillomavirus, its base sequence is shown in SEQ ID NO:2;

[0074] Primer 3 for amplifying human papillomavirus, its base sequence is shown in SEQ ID NO:3;

[0075] Primer 4 for amplifying human papillomavirus, its base sequence is shown in SEQ ID NO:4;

[0076] Primer 5 for amplifying human papillomavirus, its base sequence is shown in SEQ ID NO:5;

[0077] Primer 6 for amplifying human papillomavirus, its base sequenc...

Embodiment 3

[0081] Example 3: Probe Design and Synthesis

[0082] The DNA sequences of the L1 regions of each subtype of human papillomavirus were retrieved from GeneBank, and then the sequences of the L1 regions of each subtype were compared by DNAStar software. Molecular beacon probes for each high-risk type were synthesized. A total of 18 probes were designed, and the probe sequences are as follows:

[0083] Specific detection of human papillomavirus type 16 molecular beacon probe, the base sequence of which is shown in SEQ ID NO:7;

[0084] Specific detection of human papillomavirus type 18 molecular beacon probe, the base sequence of which is shown in SEQ ID NO:8;

[0085] Specific detection of human papillomavirus type 26 molecular beacon probe, the base sequence of which is shown in SEQ ID NO:9;

[0086] Specific detection of human papillomavirus type 31 molecular beacon probe, the base sequence of which is shown in SEQ ID NO:10;

[0087] Specific detection of human papillomavi...

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Abstract

The invention discloses a primer, a probe and a kit for fluorescence PCR (Polymerase Chain Reaction) detection of 18 types of high-risk human papilloma viruses. Different typing kits are detected by adding different specificity probes; the DNAs (Deoxyribose Nucleic Acids)) of 18 types of common high-risk human papilloma viruses internationally recognized and closely related to the cervical cancer can be detected once; and typing detection is carried out on HPV (Human Papilloma Virus)16 and 18. The application provides 6 universal primers and 18 specific molecular beacon probes. The DNAs of the 18 types of common high-risk human papilloma viruses can be amplified by the 6 universal primers; and meanwhile, the 18 probes are the specific molecular beacon probes designed by aiming at the 18 high-risk types; different types of probes are added according to the detection need and combined as the kits aiming at the detection need of the different high-risk types; and at the same time, the added probes are marked by different report genes, so that the purpose of carrying out typing detection on the high-risk HPV is achieved.

Description

technical field [0001] The invention relates to the field of kits for detecting human papillomaviruses, in particular to primers, probes and kits for fluorescent PCR detection of 18 high-risk types of human papillomaviruses. Background technique [0002] Human papillomavirus (Hμman papillomvirμs, HPV) is an epitheliophilic virus with a high degree of specificity, and humans are its only host. At present, more than 120 types of HPV have been identified, and about 30 types are directed to infect the stratified squamous epithelium of human genital tract skin and mucous membranes, resulting in genital warts and cervical lesions, and may even cause cervical cancer. Different subtypes of HPV infection lead to different lesions. According to the degree of lesions, HPV subtypes are divided into high-risk types and low-risk types. Low-risk types are related to sexually transmitted warts or genital warts, and generally do not induce cancer. The common types are 6, 11, 42, 43, and 44;...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 江春琴高清云
Owner 英科新创(苏州)生物科技有限公司
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