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
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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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