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Human papillomavirus HPV DNA fragment, specific primer and application thereof

A human papillomavirus-specific technology, applied in the field of detection and/or typing of human papillomavirus DNA, can solve problems such as inability to do typing

Inactive Publication Date: 2010-10-06
白向阳 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The hybrid capture second-generation (HC-II) detection technology launched by Qiagen in the United States is the only FDA-approved technology for detecting HPV DNA that can be used clinically. It is currently the most popular HPV molecular biotechnology detection kit, but this The kit can only detect 13 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) at the same time, and cannot perform specific typing

Method used

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  • Human papillomavirus HPV DNA fragment, specific primer and application thereof
  • Human papillomavirus HPV DNA fragment, specific primer and application thereof
  • Human papillomavirus HPV DNA fragment, specific primer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 : Selection of 31 HPV gene DNA fragments

[0079] All the genomes of 31 kinds of HPV viruses published on GenBank were compared, compared for homology, and the 31 kinds of HPV gene DNA fragments provided by the present invention were selected.

[0080]The gene accession numbers of the 31 HPV viruses are as follows (see brackets): HPV6[X00203], HPV 11[M14119], HPV 16[K02718], HPV 18[X05015], HPV 26[X74472], HPV 31[ J04353], HPV 33[M12732], HPV 35[X74477], HPV 39[M62849], HPV 40[X74478], HPV 42[M73236], HPV 43[AJ620205], HPV 44[U31788], HPV 45[X74479] , HPV 51[M62877], HPV 52[X74481], HPV 53[X74482], HPV 54[U37488], HPV 55[U31791], HPV 56[X74483], HPV 58[D90400], HPV 59[X77858], HPV 61[U31793], HPV 66[U31794], HPV 68[X67161], HPV 70[U21941], HPV 72[X94164], HPV 73[X94165], HPV 81[AJ620209], HPV 82[AB027021], and HPV 83[ AF151983].

[0081] According to the result of homology comparison, use MEGA4.0 software to make the phylogenetic tree model of each subtyp...

Embodiment 2

[0083] Example 2 : Design multiple pairs of different primers for 31 HPV gene DNA fragments

[0084] Before designing primers, analyze the properties of the target sequence to be tested, and select highly conserved regions with uniform base distribution for primer design. The length of oligonucleotide primers is 15-30bp. The Tm value of the primers is generally controlled at 55-60°C, and the Tm values ​​of the upstream and downstream primers should be as consistent as possible, generally not exceeding 2°C. The proportion of (G+C) in effective primers is generally 40-60%.

[0085] The present invention uses primer software to design and select according to the score of each primer. For each fragment, 10 pairs of primers with the highest scores were selected for testing, and the primers that could not amplify the target band were deleted and re-selected.

[0086] The specific primers of all 31 kinds of human papillomavirus DNA fragments described in Example 1 designed accor...

Embodiment 3

[0118] Example 3 : Amplify 31 HPV DNA templates with 31 pairs of single primers

[0119] Using the 31 pairs of specific primers designed according to Example 2, the corresponding 31 HPV DNA templates were individually amplified by PCR. The primer pairs used are SEQ ID NO: 32-33, SEQ ID NO: 40-41, SEQ ID NO: 48-49, SEQ ID NO: 56-57, SEQ ID NO: 64-65, SEQ ID NO: 72-73 , SEQ ID NO: 80-81, SEQ ID NO: 88-89, SEQ ID NO: 96-97, SEQ ID NO: 104-105, SEQ ID NO: 112-113, SEQ ID NO: 120-121, SEQ ID NO: 120-121, SEQ ID NO: ID NO: 128-129, SEQ ID NO: 136-137, SEQ ID NO: 144-145, SEQ ID NO: 148-149, SEQ ID NO: 156-157, SEQ ID NO: 164-165, SEQ ID NO: 172-173, SEQ ID NO: 180-181, SEQ ID NO: 188-189, SEQ ID NO: 196-197, SEQ ID NO: 204-205, SEQ ID NO: 212-213, SEQ ID NO: 220- 221. SEQ ID NO: 228-229, SEQ ID NO: 236-237, SEQ ID NO: 244-245, SEQ ID NO: 252-253, SEQ ID NO: 262-261 and SEQ ID NO: 268-269.

[0120] 1) Single primer PCR amplification:

[0121] a) Prepare a 25 μL reaction system ...

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Abstract

The invention provides a human papillomavirus HPV DNA fragment, a mix primer for human papillomavirus HPV DNA detection and / or parting and application thereof. The mixed primer can specifically amplify to obtain one or a plurality of DNA sequences from HPV6, HPV 11, HPV 16, HPV 18, HPV 26, HPV 31, HPV 33, HPV 35, HPV 39, HPV 40, HPV 42, HPV 43, HPV 44, HPV45, HPV 51, HPV 52, HPV 53, HPV 54, HPV 55, HPV 56, HPV 58, HPV 59, HPV 61, HPV 66, HPV 68, HPV 70, HPV 72, HPV 73, HPV 81, HPV 82 and HPV 83, can be used for the human papillomavirus HPV DNA detection and / or parting and prepared into a PCR kit. The invention can detect common types of HPV at a time, greatly improve the HPV detection efficiency, overcome the defect of missed detection of latent infection by a serology and IHC detection method, realize early diagnosis of HPV diseases, and is suitable for clinical HPV gene detection and parting and guides prevention and cure of cervical carcinoma.

Description

technical field [0001] The invention belongs to biotechnology and relates to the detection and / or typing of human papillomavirus DNA. Background technique [0002] Human papilloma virus (HPV) is a small DNA double-stranded virus. More than 100 HPV DNAs have been isolated, and more than 40 of them are related to cervical infection and lesions. According to the pathogenicity of HPV, it is divided into high-risk type and low-risk type. The low-risk type mainly causes exophytic condyloma lesions, flat condyloma lesions and low-grade cervical intraepithelial lesions in the genital tract, perianal skin and lower vagina. Neoplastic change (CIN I); the high-risk type mainly leads to the occurrence of CIN II and III grade lesions and cervical cancer. Persistent high-risk HPV infection is an inevitable process for CIN to develop into cervical cancer. The time interval from infection with high-risk HPV to development of cervical cancer is 15 years. Therefore, cervical cancer is a spec...

Claims

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Application Information

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IPC IPC(8): C12N15/37C12N15/11C12Q1/70C12Q1/68C12R1/93
Inventor 白向阳赵珊珊
Owner 白向阳
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