Kit for detecting HPV (Human Papillomavirus)

A kit and sequence technology, applied in the field of bioengineering, can solve the problems of low specificity and sensitivity, and achieve the effects of improving sensitivity and specificity, shortening reaction time, and improving detection efficiency

Inactive Publication Date: 2018-12-21
SHENZHEN SINGUWAY BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a kit for detecting HPV, aiming to solve the technical problems of low specificity and low sensitivity when the existing RPA method detects HPV

Method used

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  • Kit for detecting HPV (Human Papillomavirus)
  • Kit for detecting HPV (Human Papillomavirus)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 is used to detect the test kit of HPV

[0046] A kit for detecting HPV based on a recombinant high-sensitivity amplification method, comprising:

[0047] A first primer pair (SEQ ID NO.1 and SEQ ID NO.2) for amplifying HPV16 and a second primer pair (SEQ ID NO.3 and SEQ ID NO.4) for amplifying HPV18; Uvs X (SEQ ID NO.5), Uvs Y (SEQ ID NO.6), SSB (SEQ ID NO.7) and DNA polymerase (SEQ ID NO.8); and amplification buffer: 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 50mM KCl, 2mM MgSO 4 and 0.1% 20 (pH=8.8).

Embodiment 2D

[0048] Example 2 DNA extraction (extraction method)

[0049] The extraction of DNA in the detection sample includes the following steps:

[0050] 1. Add 1ml of extraction buffer (100mM Tris-HCl, pH=8.0; 50mM EDTA, pH=8.0; 500mM NaCl, +0.07% mercaptoethanol) to cervical tissue (Cervical tissue) cells, and mix thoroughly.

[0051] 2. Add 130ul 10% SDS and a small amount of proteolytic enzyme K, shake it upside down several times, and place it in a water bath at 65°C for 1 hour.

[0052] 3. Add 300ul 5M potassium acetate (potassium acetate), mix thoroughly, and then place on ice for 30 minutes to facilitate protein precipitation.

[0053] 4. Centrifuge at 12000 rpm for 30 minutes, take out the supernatant and transfer to a new centrifuge tube (eppendorf tube).

[0054] 5. Add twice the volume of isopropanol and centrifuge at 12000rpm for 30 minutes.

[0055] 6. Pour off the supernatant, add 70% ethanol, and centrifuge at 12000 rpm for 30 minutes.

[0056] 7. Pour off the supern...

Embodiment 3

[0059] Embodiment 3HPV detects

[0060] The kit in Example 1 and the extracted DNA sample in Example 2 were added to the micro test tube (final volume: 50ul) according to the proportions in Table 1 below, and shaken homogeneously in an environment of 37-40°C React at 300rpm for 10-20 minutes.

[0061] Table 1

[0062]

[0063] After the reaction is finished, the reaction product is electrophoresed in 3-4% agaric gum, and the size of the DNA product is judged (adding a tracking dye, performing contrast electrophoresis with the control group, and putting it into a DNA marker for comparison). The power plug (note the position of the positive and negative poles) is connected to the power supply. During the electrophoresis process, the negatively charged DNA molecules will move from the negative pole to the positive pole, and run the gel in 100vol for about 10 minutes. Put the agaric gum on the strong UV transmission light source plate with gloves on, and then take a photo of ...

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Abstract

The invention belongs to the field of bioengineering, and specifically relates to a kit for detecting HPV (Human Papillomavirus). The kit is prepared from a primer for amplifying the HPV as well as Uvs X, Uvs Y, SSB and DNA polymerases. In the kit for detecting the HPV, provided by the invention, a T4-series enzyme combination of Uvs X and Uvs Y is selective for replacing existing recombinase. Theenzyme system not only is capable of further increasing the sensitivity and the specificity of HPV detection, but also can be more diversified in selecting primers for amplifying the HPV, and 100 to200 bp products can be amplified under a shorter primer condition, so that the reaction time can be shortened, the interference of a background signal can be reduced, the detection efficiency can be finally increased, and the detection cost can be remarkably reduced.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a kit for detecting HPV. Background technique [0002] Human papillomavirus (Human Papillomavirus, HPV) is a microorganism smaller than bacteria, and it is a kind of viral virus. Among the currently known more than 200 subtypes of HPV, about 40 subtypes can invade the female reproductive tract, and at least 15 subtypes are related to invasive cervical cancer, called oncogenic or high-risk types, of which type 16 The most common, followed by type 18, the two together accounted for 70% of cervical cancer. Human papillomaviruses are highly host-specific and specifically infect human skin and mucous membranes. Infection with HPV types 16 and 18 is associated with the development of cervical, penile, perineal, and anal cancers. [0003] At present, there are many HPV detection methods on the market, including hybridization capture method, fluorescence in situ hybridization method, chi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844
CPCC12Q1/6844C12Q1/708C12Q2600/166C12Q2522/101C12Q2521/507C12Q2537/1376C12Q2531/119
Inventor 黄雅淑翁义杰
Owner SHENZHEN SINGUWAY BIOTECH CO LTD
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