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Gene chip and kit for detecting human papillomavirus (HPV)

A technology of human papillomavirus and gene chip, which is applied in the field of life science and biology, can solve the problems of common clinicians who are easy to misdiagnose, unfavorable clinical follow-up examination and treatment, and unable to identify HPV virus subtypes, etc., and achieve high sensitivity and operation simple effect

Active Publication Date: 2012-02-08
济南艾迪康医学检验中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method can only detect whether there is HPV infection in the sample, and cannot identify the specific HPV virus subtype, which is not conducive to clinical follow-up inspection and treatment.
[0007] The integration of high-risk HPV DNA into the host genome will cause host cells to produce a series of irreversible chain reactions including functional defects of tumor suppressor genes, activation of oncogenes, genome instability and cell immortalization, which is one of the important mechanisms of cervical carcinogenesis. Its warning value is higher than that of the current general HPV-DNA infection detection, but the probes used in the current commercially available kits lack the detection of HPV integration. In addition, the existing HPV detection membrane strips are not clearly divided to detect HPV , it is easy for ordinary clinicians to misdiagnose. The present invention detects and improves the above two clinically important indicators, making the detection of clinical HPV infection richer, more accurate, more practical and more valuable

Method used

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  • Gene chip and kit for detecting human papillomavirus (HPV)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1: the preparation of gene chip

[0074] According to the research results of HPV infection worldwide and the actual situation of HPV infection in China, 27 HPV subtypes were selected, including 22 high-risk and medium-risk HPV types, 5 low-risk HPV types and 2 integrated types (16 / 18 type). For each HPV subtype, specific probes (SEQID No.9-37) with amino groups at the 3' end were designed and synthesized.

[0075] Cut the membrane strips as required; soak in the freshly prepared EDAC solution for 30 minutes; wash the membrane with distilled water for 3 times, 2 minutes each time, dry on the filter paper, and fix the membrane on the filter paper; Scratch the probe 3 times; place the spotted film at room temperature for 20 minutes to dry; soak the film strip with 0.5N NaOH for 5 minutes; discard the NaOH, wash with distilled water 3 times, 2 minutes each time; after fully drying, store in a dry bottle spare.

Embodiment 2

[0076] Embodiment 2: sample preparation

[0077] Preparation and purification of DNA from clinical samples by boiling.

[0078] The PCR reaction system is 20ul, and the specific formula is as follows:

[0079] 2X PCR Mix 10μl

[0080] Plus solution 2μl

[0081] SeqNo.1-2 (100uM) 0.03μl

[0082] SeqNo.2-4 (100uM) 0.03μl

[0083] SeqNo.5-6(100uM) 0.03μ1

[0084] Deionized water 5.91μl

[0085] Template DNA 2ul

[0086] A negative control was set up for each experiment, that is, 2ul sterile water was used as a template.

[0087] Amplify according to the following conditions:

[0088] Stage1: 94°C 3min

[0089] Stage2: 94°C 20s

[0090] 51℃ 1min

[0091] 72°C 30s

[0092] 40cycles

[0093] Stage3: 72°C 2min

Embodiment 3

[0094] Example 3: HPV DNA detection

[0095] The DNA sample amplified in Example 2 was hybridized with the gene chip prepared in Example 1, and finally judged according to the color development, and the position with color development indicated the corresponding HPV subtype.

[0096] 1. Hybridization

[0097] Take a 5ml centrifuge tube, mark the patient number, put the membrane strip also marked with the patient number, add 3ml hybridization solution (0.3mol / L NaCl, 10mmol / L NaH 2 PO 4 , 2mmol / L EDTA, 0.1% SDS, pH 7.4) and PCR products, in a boiling water bath for 10 minutes; hybridization at 51°C for 1 hour.

[0098] Take a 50ml centrifuge tube, add 40ml B solution to preheat to 51°C.

[0099] Control: take 10ul PC and add it to the hybridization tube of the negative control for hybridization, the method is the same as above.

[0100] 2. Membrane washing

[0101] Take out the membrane strip and move it to a place containing preheated membrane washing solution (75mmol / L N...

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Abstract

The invention discloses a gene chip for detecting human papillomavirus (HPV), comprising a solid carrier and a human papillomavirus detecting probes fixed on the solid carrier. The detecting probes comprise nucleotide sequences in SEQ ID No.9-37, and the 27 types of HVP subtype specific probes in the invention correspondingly comprise 22 kinds of high-risk and mediate-risk HPV, and 5 kinds of low-risk HPV and two kinds of high-risk HPV integration (HPV16 / HPV18). The invention also discloses a detecting kit for detecting various subtypes of HPV in a sample with high speed and high flux, and the kit comprises the gene chip, a primer sequence for detection, a human GAPDH gene, special HPV lysing solution, PCR reaction liquid and a color reagent, wherein the detecting probe fixed on the gene chip has the nucleotide sequence in the SEQ ID No.9-37; the primer sequence for detection is used for amplifying the DNA sequence of the HPV in a clinical sample, and the human GAPDH gene is used for amplifying the clinical sample. The HPV detection has important significance for the clinical diagnosis of HPV as well as the early prevention, the diagnosis, the treatment and the postoperation tracking of cervical cancer.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, in particular to a gene chip containing human papillomavirus (HPV) subtype probes, and also relates to a kit for HPV detection and typing. Background technique [0002] About 510,000 women worldwide are diagnosed with cervical cancer every year, which is the second most common gynecological malignancy after breast cancer, and its incidence is now showing a younger trend. In China, about 135,000 new cases of cervical cancer are added each year, accounting for 1 / 3 of the global incidence, and about 80,000 people die from the disease. Studies have found that more than 90% of cervical cancer patients are associated with human papillomavirus (human papillomaviruses, HPV) infection. The occurrence of cervical cancer and its precancerous lesions is the result of the synergy of many factors. High-risk HPV infection is the initiating factor for the evolution of cervical cancer, and the integ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 郭兴中石慧董正伟方国伟郭尧
Owner 济南艾迪康医学检验中心有限公司
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