Method for detecting HPV (human papillomavirus) and kit

A technology for detecting probes and nucleotide sequences, which is applied in the detection of human papillomavirus infection, molecular biology and virus detection, and can solve the problems of high cost, lack of detection methods for multiple HPV types, low efficiency, etc. question

Inactive Publication Date: 2017-10-20
GENMAG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are also disadvantages of relatively low efficiency and high cost.
In addition, the invention does not provide a method for detecting multiple HPV types

Method used

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  • Method for detecting HPV (human papillomavirus) and kit
  • Method for detecting HPV (human papillomavirus) and kit
  • Method for detecting HPV (human papillomavirus) and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0182] Primer pairs and probe-specific amplification and recognition of 14 HPV genotype genes

[0183] The following 14 HPV types were designed and synthesized (HPV16, HPV18, HPV31, HPV35, HPV45, HPV52, HPV56, HPV59, HPV66, HPV68, HPV33, HPV39, HPV51 or HPV58) specific primer pairs and probe sets. The standard plasmids containing artificially synthesized E6E7 gene sequence fragments corresponding to HPV types were used as templates to test the specific amplification effect of each HPV type primer pair and probe set pair corresponding to HPV types.

[0184] Table 1.14 Specific primer pairs and probe sets for HPV type genes:

[0185]

[0186]

[0187] illustrate:

[0188] HPV16 type:

[0189] Upstream primer 16E-S1: nucleotide sequence such as SEQ ID NO: 1,

[0190] Downstream primer 16E-A1: nucleotide sequence such as SEQ ID NO: 2,

[0191] Taqman fluorescent probe 16E-P1: nucleotide sequence such as SEQ ID NO: 3;

[0192] HPV18 type:

[0193] Upstream primer 18E-S...

Embodiment 2

[0269] Sensitivity test of primer pairs and probes for 14 HPV genotype genes

[0270] Test the sensitivity of the 14 HPV type-specific primer pairs and probe sets in Table 1 of Example 1 to determine the minimum detection template concentration. A 10-fold serial dilution of template DNA with a known concentration was performed, and the sensitivity of the primers and probes was determined by the least effective amplified template sample.

[0271] Use the Roche lightCycler 480II fluorescence quantitative PCR instrument to carry out amplification and fluorescence detection with the fluorescent PCR reaction condition of embodiment 1, carry out 10 times of gradient dilution with the standard plasmid of the corresponding HPV type gene fragment of known concentration and be template carry out testing. The result is as follows:

[0272] HPV16 type: 3.547×10 -8 ng / ul(4copy / ul)

[0273] HPV18 type: 3.133×10 -8 ng / ul(4copy / ul)

[0274] HPV31 type: 1.78×10 -7 ng / ul(20copy / ul)

[0...

Embodiment 3

[0288] Effectiveness and Specificity of HPV Screening Kits

[0289] Utilize the method of the present invention to carry out HPV screening to biological sample, wherein said biological sample is respectively amplified with the combination of specific primer pair and probe of following two groups of HPV type genes:

[0290] Group (1): Type 16 and Type 18 specific primer pairs and probes;

[0291] Panel (2): Type 45, Type 31, Type 39, Type 51, Type 52, Type 56, Type 58, Type 59, Type 33, Type 35, Type 66, Type 68 specific primer pairs and probes; and β - Actin specific primer pair and probe, its sequence is as follows:

[0292] Upstream primer Act517-S1: nucleotide sequence such as SEQ ID NO: 43,

[0293] Downstream primer Act517-A1: nucleotide sequence such as SEQ ID NO: 44,

[0294] Taqman fluorescent probe Act517-P1: nucleotide sequence such as SEQ ID NO:45.

[0295] Primers / Probes

Sequence (5'to3')

Upstream primer Act517-S1:

TCACCCACACTGTGCCCATCTA...

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Abstract

The invention provides a method for detecting HPV (human papillomavirus) nucleic acid in a biological sample. The method comprises the following steps of (i) respectively using 14 types of HPV specific primer pairs and primers in a probe group to amplify nucleic acid segments in a sample; (ii) using the amplified segments in step (i) to respectively touch the 14 types of HPV gene specific primer pairs and the probes in the probe group; (iii) detecting signals released by the probes, and judging the existence of the HPV in the biological sample and the type of the HPV. The invention also provides a kit using the method to detect the HPV nucleic acid in the biological sample.

Description

field of invention [0001] The invention relates to the fields of molecular biology and virus detection. In particular, the invention relates to the field of detection of human papillomavirus (HPV) infection. Background of the invention [0002] HPV is a virus closely related to cervical cancer. More than 100 HPV types have been discovered in the art. HPV typically infects the skin (cutaneous type) or the mucous membranes of the respiratory and anogenital tracts (mucosal type). More than 40 HPV types are known to infect the cervix. Based on the induced benign, premalignant, or malignant lesions, HPVs are classified into low-risk (e.g., HPV types 6, 11, 42, 43, and 44) ​​and high-risk types (e.g., types 16, 18, 31, 33, and 45). High-risk types account for more than 99 percent of all invasive cervical cancers. Therefore, the detection and identification of HPV types is very important. The high-risk type is defined as always found in high-grade SIL (squamous intraepitheli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/708C12Q2600/166C12Q2561/101C12Q2545/101C12Q2545/113
Inventor 赵海峰
Owner GENMAG BIOTECH
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