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General primers and process for detecting diverse genotypes of human papillomavirus by PCR

A technology for human papilloma and universal primers, applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of HPV lack of sensitivity, limited use, and primer set defects, etc.

Inactive Publication Date: 2006-01-04
CHA WILYINS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] However, these primer sets that are widely used worldwide are also defective because they can only detect limited HPV types compared with different HPV types that are classified into more than 120 types, and lack sensitivity to HPV types.
Therefore, although they have other values ​​and uses, the use of these universal primer sets in the clinical field is limited

Method used

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  • General primers and process for detecting diverse genotypes of human papillomavirus by PCR
  • General primers and process for detecting diverse genotypes of human papillomavirus by PCR
  • General primers and process for detecting diverse genotypes of human papillomavirus by PCR

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Example 1: PCR primer design and synthesis

[0050] (1) PCR primer design

[0051] According to the purpose of the present invention, a nucleotide sequence that can complementarily bind to different types of HPV and thereby amplify all HPV type DNA is designed as a universal primer for PCR.

[0052] First, export HPV 1a, 2a, 3, 4, 5, 6b, 7, 8, 9, 10, 11, 12, 13, 15, etc. from the NCBI (National Center for Biotechnology Information) and the National HPV database of Los Alamos. 16, 16r, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35h, 36, 37, 38, 39, 40, 41, 42, 44, 45, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 65, 66, 67, 68, Full-length nucleic acid sequences of 72 HPV types 70, 72, 73, 74, 75, 76, 77 and 80.

[0053] Using Clustal W computer program (DNSSTAR, MegAlign TM 5. DNASTAR Inc.) conducts DNA sequence homology research on the derived DNA sequence. This program screens representative base sequences in the group thr...

Embodiment 2

[0065] Example 2: PCR primer design and synthesis

[0066] Except that the designed universal PCR primers are set to about 24±3 bp and about 33±2 bp nucleotide sequences, and their amplified products are about 170-550 bp, Example 2 is completed under the same conditions and steps as Example 1.

[0067] Under the above conditions, the final 8 pairs of universal primers were selected. For ease of description in the following, each of the 8 pairs of combination primers according to the embodiment of the present invention is referred to as AlbioGP3, AlbioGP4, AlbioGP5, AlbioGP6, AlbioGP7, AlbioGP8, AlbioGP9, AlbioGP10, and AlbioGP11, respectively. Table 3 shows the nucleic acid sequence, SEQ ID No, the type of amplified HPV, the size of the amplified product, and the actual amplification result of the amplified region of the determined primer pair. And Table 4 shows the actual amplification product size of representative cervical cancer-related HPV types by using AlbioGP5, AlbioGP6, ...

Embodiment 3

[0075] Example 3: PCR amplification using universal primers

[0076] The 3 pairs of primers (AlbioGP1, AlbioGP2 and AlbioGP3) synthesized in Example 1 were used to amplify the target HPV 16 infected Caski cell line (ATCC CRL-1550), HPV 18 infected HeLa cell line (ATCC CCL-2) and Non-HPV infected K-562 cell line (KCLB-10243, Korean cell line bank).

[0077] Culture each of Caski, HeLa, and K-562 cells according to the manufacturer’s instructions, then add 0.25% trypsin solution to Caski and HeLa cells, and centrifuge the cultured cell lines from the culture flask. Wash each cultured Caski, HeLa and K-562 cell lines once with Dulbecco’s phosphate buffered saline (Gibco Inc.), and check the number of cells under a microscope. Then the 2×10 from the cultured cell line 4 The cells were suspended in 230 μL (microliter) of D.W and heated at 100°C (Celsius) for 15 minutes to extract cellular DNA. The extracted cellular DNA was cooled at room temperature and centrifuged at 12,000 rpm for ...

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Abstract

The presence of diverse human Papillomavirus genotypes in a sample can be detected by 1 cycle of polymerase chain reaction using primers. The primers used in the method are general primers which can bind complementarily to specific nucleic acid sequences of all HPV genotypes related to cervical carcinoma and its pre-stage lesions and can be used to amplify cervical-neoplasia related HPV genotypes including oncogenic high-risk groups and low-risk groups.

Description

Technical field [0001] The present invention relates to PCR primers for detecting and / or diagnosing the presence or absence of human papillomavirus (HPV) in a sample and a method for detecting and / or analyzing the presence or absence of HPV in a sample using the primers, more particularly , Relates to PCR primers containing oligonucleotides that complementarily bind to different types of HPV nucleic acid to obtain amplification products, and use the primers to detect and / or analyze the presence or absence of different types of HPV in a sample The method of existence. technical background [0002] It has been found that human papillomavirus (HPV) involves multiple tumor types including cervical cancer and nearly 120 different HPV genotypes. HPV is a circular double-stranded DNA virus with a genome size of approximately 8kb. After infection of the host cell, the E1 to E7 genes are related to the early steps in the HPV genome, while the L1 and L2 genes are related to the late steps....

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/708C12Q2531/113
Inventor 李相和金渊洙柳康烈金丞兆车光烈高正在
Owner CHA WILYINS
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