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Compositions and methods for detecting human papillomavirus

A technology for papilloma and composition, which is applied in the directions of biochemical equipment and methods, recombinant DNA technology, and microbial determination/examination, and can solve problems such as urethral swab pain

Active Publication Date: 2020-11-13
HANGZHOU NEW HORIZON HEALTH TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, fewer HPV tests are available in men in current clinical practice, and the commonly used sampling method for men (ie, urethral swab) is painful

Method used

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  • Compositions and methods for detecting human papillomavirus
  • Compositions and methods for detecting human papillomavirus
  • Compositions and methods for detecting human papillomavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0323] Example 1: Design and testing of primers / probes

[0324] Snijders et al. (J. Gen. Virol., 71 (1990), 173-181) and Surentheran et al. (J. Clin. Path., 51 (1998), 606-610) describe a method for detecting HPV L1 gene DNA PCR method. A disadvantage of both methods is that only a limited number of HPV types can be detected. For example, the primers described by Snijder et al. can only detect some HPV types, such as HPV30, HPV39, HPV51, but the sensitivity is greatly reduced. Furthermore, certain HPV types, such as HPV18, resulted in the formation of additional bands when using the primers described by Snijder et al. Therefore, existing assays can only detect a limited spectrum of HPV subtypes, and some rare HPV subtypes cannot be adequately detected. The compositions and methods provided herein can be used to detect the genotypes of up to 14 high-risk HPV subtypes and / or two low-risk HPV subtypes in one or two test tubes.

[0325] Design primers and probes

[0326] Mult...

Embodiment 2

[0334] Embodiment 2: high-risk HPV detection kit

[0335] As a non-limiting example, a typical kit may include the following parts:

[0336] (1) High-risk HPV qPCR mixture: 1×buffer (20mM Tris-HCl, 50mM KCl, pH8.4), 0.15-0.3mM dNTP, 2-4mM MgCl2, 0.2-1.2μM primers / probes (14 high-risk type), 0.1~1mg / ml BSA, 0.2%~2% (V / V) formamide, 0.2mM~2mM spermidine, 10mM~30mM tetramethylammonium chloride, 0.01mM~0.1mM DTT, 0.2% ~2% 2-pyrrolidone and H2O

[0337] (2) Taq enzyme: 1~6U / μl Taq enzyme

[0338] (3) Positive control: high-risk HPV16, HPV18, and HPV45 L1 gene plasmids (10 per template 3 copies / ml), and high-risk HPV DNA-negative urine

[0339] (4) Negative control: high-risk HPV DNA negative urine

Embodiment 3

[0340] Example 3: HPV detection kits for the detection of 14 high-risk subtypes and 2 low-risk subtypes

[0341] As a non-limiting example, a typical kit to detect 14 high-risk subtypes and two low-risk subtypes may include the following parts:

[0342] (1) HPV qPCR reaction solution I: 1×buffer (20mM Tris-HCl, 50mM KCl, pH 8.4), 0.2-0.3mM dNTP, 2-3mM MgCl2, 0.2-0.8μM primers and probes (HPV16, HPV18, HPV35, HPV39, HPV68, HPV59, HPV56, HPV66, HPV51), 0.1~1mg / ml BSA, 0.2%~2% (V / V) formamide, 0.2mM~2mM spermidine, 10mM~30mM tetramethyl chloride Ammonium, 0.01mM ~ 0.1mM DTT, 0.2% ~ 2% 2-pyrrolidone and H2O;

[0343] (2) HPV qPCR reaction solution II: 1×buffer (20mM Tris-HCl, 50mM KCl, pH 8.4), 0.2-0.3mM dNTP, 2-3mM MgCl2, 0.2-0.8μM primers and probes (HPV6, HPV11, HPV33 , HPV58, HPV31, HPV45, HPV52, β-actin), 0.1~1mg / ml BSA, 0.2%~2% (V / V) formamide, 0.2mM~2mM spermidine, 10mM~30mM tetramethyl chloride ammonium chloride, 0.01mM ~ 0.1mM DTT, 0.2% ~ 2% 2-pyrrolidone and H2O;

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Abstract

Provided are compositions and methods for detecting and / or identifying the genotype of a human papillomavirus (HPV) in a biological sample obtained from a subject in need thereof. Particularly, provided are primers, probes and kits for simultaneously detecting multiple HPV genotypes in a single-tube PCR reaction.

Description

[0001] cross reference [0002] This application claims priority to PCT application PCT / CN2019 / 070277, filed January 3, 2019, the entirety of which is incorporated herein by reference. [0003] Field of Invention [0004] The present invention relates to compositions and methods for detecting and / or genotyping human papillomavirus (HPV). [0005] Background of the Invention [0006] Human papillomavirus (HPV) is a virus of the genus Papillomavirus in the family Papillomaviridae. It is host-specific and tissue-specific and typically infects human skin and mucosal cells. It is a common pathogen that is spread primarily through sexual activity. [0007] The HPV genome is a double-stranded circular DNA. The genome can be divided into three regions, namely early region (E region), late region (L region) and non-coding region (NCR). The E region can be further divided into seven open reading frames (E1-E7), which mainly encode proteins involved in viral replication, transcriptio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/708C12Q1/686C12Q2600/112C12Q2600/158C12Q2600/16C12Q2563/107C12Q2537/143C12Q2545/101C12Q2600/156
Inventor 吴扬刘刚吕宁陈一友
Owner HANGZHOU NEW HORIZON HEALTH TECH CO LTD
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