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Human papillomavirus gene parting detecting reagent case and method for preparing gene chip thereof

A technology of papilloma virus and detection kit, which is applied in the direction of biochemical equipment and methods, and the determination/testing of microorganisms, can solve the problems of less subtypes covered, omissions and lack of detection, etc., to shorten the detection time, Easy to produce and operate, good elastic effect

Active Publication Date: 2008-05-14
潮州凯普生物化学有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the probe used in this method is RNA, which may affect the stability of the kit
The most important thing is that this method can only detect whether there is HPV virus infection in the sample, and cannot identify the specific HPV virus subtype, which is not conducive to clinical follow-up detection and treatment
[0006] Moreover, most of the probes used in the gene kits sold on the market lack the common subtypes (53 / 66 / CP8304) of the Chinese population, and cover a small number of subtypes, which are not suitable for the situation of Chinese people, and often easily lead to omissions in detection

Method used

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  • Human papillomavirus gene parting detecting reagent case and method for preparing gene chip thereof
  • Human papillomavirus gene parting detecting reagent case and method for preparing gene chip thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1 Preparation of gene chip

[0089] According to the research results of HPV infection worldwide and the actual situation of HPV infection in China, 21 subtypes of HPV were selected, including 13 high-risk subtypes (16 / 18 / 31 / 33 / 35 / 39 / 45 / 51 / 52 / 56 / 58 / 59 / 68), five low-risk subtypes (6 / 11 / 42 / 43 / 44) and three common subtypes in the Chinese population (53 / 66 / CP8304). For each HPV subtype, design and synthesize specific gene probes with amine groups at the 5' end, and prepare IC and Bio for HPV detection.

[0090] The production steps of the DNA gene chip are as follows: the prepared probes, IC and Bio were first dissolved in ultrapure water to make a mother solution with a concentration of 200uM, and then dissolved in 0.5M Na 2 CO 3 and 0.5M NaHCO 3 solution to a final concentration of 2uM.

[0091] To treat the nylon membrane, first soak it in 0.1M HCl solution for 30 seconds, then put the membrane that has removed the residual solution into 20% EDAC solution an...

Embodiment 2

[0093] Example 2 sample preparation

Embodiment 2-1

[0094] The preparation of embodiment 2-1 positive control group sample

[0095] The HPV18 fragment inserted into the pMD-T vector was amplified using MY09 / MY11 primers. The PCR reaction system is 25ul, including 10x buffer, 2.5ul; 25mM MgCl 2 , 6.0ul; 10mM d(A / C / G)TP, 0.5ul; 100uM biotinylated primer MY09, 0.25ul; 100uM biotinylated primer MY11, 0.25ul; internal control DNA, 1.0ul; ddH 2 O, 12.4ul; 100mM dUTP, 0.1ul; UNGase (1U / ul), 0.25ul; TaqDNA polymerase, 0.25ul; membrane DNA, 1.0ul; a total of 25ul. The PCR reaction program was as follows: first denaturation at 95°C for 9 minutes, then 95°C for 20 seconds; 55°C for 30 seconds; 72°C for 30 seconds; 40 cycles, and finally 72°C for 5 minutes.

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Abstract

The invention discloses a probe with a nucleotide sequence complementary to human papilloma virus HPV DNA, a genotyping detection kit containing the probe and a gene chip preparation method thereof. The probe is selected from SEQ ID Nos: 1-21 and sequences complementary to SEQ ID Nos: 1-21. The kit includes a gene chip with at least one of the above-mentioned probes, a DNA sequence marked with biotin spots, a DNA sequence coding a part of β-globulin, and various primers selected from MY09 / MY11. The gene chip of the kit is prepared through the steps of preparing DNA probes, fixing the probes, using sodium hydroxide to stop the reaction, and the like. The kit of the invention can quickly and accurately detect HPV infection and identify specific subtypes, is more suitable for Chinese people, and has great significance for early diagnosis, prevention, treatment and follow-up of cervical cancer.

Description

technical field [0001] The invention relates to a probe with a nucleotide sequence complementary to human papillomavirus HPV DNA, a genotyping detection kit for detecting specific subtypes of HPV containing the probe and a gene chip preparation method thereof. Background technique [0002] Cervical cancer is one of the most common malignancies in women. There are about 500,000 new cases of cervical cancer in the world every year, about 380,000 in Asia, and about 150,000 in China. About 230,000 women die of cervical cancer in the world every year, about 190,000 in Asia, and about 80,000 in China. Cervical cancer is the second most common malignant tumor in women. In particular, human papillomavirus infection has led to an increasing incidence of cervical cancer and precancerous lesions and tends to be younger, which seriously threatens the physical and mental health of women. Therefore, the prevention, diagnosis and treatment of cervical cancer is an urgent problem to be sol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 谢龙旭
Owner 潮州凯普生物化学有限公司
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