Kit for early screening and detecting carcinoma of urinary bladder by demethylation fluorescent quantitation PCR method

Abstract

The invention belongs to the field of molecular biology, and relates to a kit for early screening and detecting carcinoma of urinary bladder by a demethylation fluorescent quantitation PCR method. Thekit comprises primers and probes of the abovementioned two target genes p16 and RASSF1A, a forward primer and a probe of GAPDH, PCR Master Mix, a cracking solution, protease K, a washing solution, ahydrosulphite solution and ddH2O. According to the kit, two target genes which are p16 and RASSF1A are combined to detect the carcinoma of urinary bladder; the detection sensitivity is high; and the specificity is high.

Description

technical field

[0001] The invention belongs to the field of molecular biology, and relates to a kit for early screening and detection of bladder cancer by double methylation fluorescence quantitative PCR method. Background technique

[0002] P16 gene, also called MTS (multiple tumor suppressor 1) gene, is a new anti-cancer gene discovered by Kamb and others in Cold Spring Laboratory in the United States in 1994. It is a basic gene in the cell cycle, directly involved in the regulation of cell cycle, and negatively regulates cell Proliferation and division. Homozygous deletion and mutation are found in 50% of human tumor cell lines. It is believed that P16 is a new anti-cancer gene that is more important than P53. The product encoded by the P16 gene is a 16KD protein, that is, the P16 protein, which is located in the nucleus. The P16 protein is an inhibitor of CDK4, one of the key enzymes of the cell division cycle (Cell Division Cycle). Once its expression fails, it will l...

Examples

Embodiment 1

[0037] Example 1 Design of primers and probes for detecting the methylation level of bladder cancer urine precipitate cells

[0038]According to the human whole genome sequence p16 and RASSF1A gene sequence published by National Center for Biotechnology Information (NCBI), the present invention designs primers, the primers are synthesized by TAKARA (Treasure Bioengineering Co., Ltd.), and the probes are synthesized by LifeTechnologies.

[0039] (1) Detect the nucleic acid sequence of p16 gene fragment methylation (FAM fluorescence channel)

[0040] The forward primer sequence is SEQ ID NO.1 (5'-AGCGGGCGGCGGGGAGTAGTA-3'),

[0041] The reverse primer sequence is SEQ ID NO.2 (5'-AGTCGGCGGCGGGGAGTAGTATG-3'),

[0042] The fluorescent probe sequence is SEQ ID NO.3 (5'-FAM-GATTGGTTGGTTACGGTCGCGGTT-MGB-3');

[0043] The 5' end of the fluorescent probe SEQ ID NO: A3 is labeled with FAM (fluorescence reporter group), and the 3' end is labeled with MGB (fluorescence quencher group).

...

Embodiment 2

[0048] Embodiment 2 A kind of test kit

[0049] The kit includes primers and probes for the above two target genes of p16 and RASSF1A; in addition, it also includes forward primers and probes for detecting GAPDH, PCR Master Mix, lysate, proteinase K, rinse solution, bisulfite solution and ddH 2 O.

[0050] The forward primer sequence of p16 is shown in SEQ ID NO.1, the reverse primer sequence is shown in SEQ ID NO.2; the fluorescent probe sequence is SEQ ID NO.3, the 5' end of the probe is labeled with FAM, 3 ' end labeled MGB.

[0051] The forward primer sequence of RASSF1A is shown in SEQ ID NO.4, the reverse primer sequence is shown in SEQ ID NO.5; the fluorescent probe sequence is SEQ ID NO.6, the 5' end of the probe is labeled with FAM, 3 ' end labeled MGB.

[0052] Forward primers and probes for detecting GAPDH can be primers and probes known in the art.

Embodiment 3

[0053] The using method of kit described in embodiment 3

[0054] (1) Extraction of genomic DNA from urine precipitated cells: take the patient's urine precipitated cells, use lysate, proteinase K, rinse solution and ddH 2 O is extracted to obtain a genomic DNA solution;

[0055] The specific steps are:

[0056] Aspirate 200 μL of urine sample, put it into a 1.5mL centrifuge tube, add 1mL Lysis Solution 1, then continue to vortex and shake for 1min, and then place the 1.5mL centrifuge tube in a 70°C water bath for 5min.

[0057] Take the 1.5mL centrifuge tube out of the water bath, centrifuge at 12,000rpm for 5min, and slowly pipette 300μL of the supernatant into a new 1.5mL centrifuge tube.

[0058] Add 200 μL Lysis Solution 2 and 20 μL Proteinase K to a new 1.5 mL centrifuge tube in sequence, and vortex to mix.

[0059] Place a new 1.5mL centrifuge tube in a 70°C water bath for 10min.

[0060] Add 200 µL of isopropanol and vortex to mix.

[0061] Put all the solution ob...

technical field

[0001] The invention belongs to the field of molecular biology, and relates to a kit for early screening and detection of bladder cancer by double methylation fluorescence quantitative PCR method. Background technique

[0002] P16 gene, also called MTS (multiple tumor suppressor 1) gene, is a new anti-cancer gene discovered by Kamb and others in Cold Spring Laboratory in the United States in 1994. It is a basic gene in the cell cycle, directly involved in the regulation of cell cycle, and negatively regulates cell Proliferation and division. Homozygous deletion and mutation are found in 50% of human tumor cell lines. It is believed that P16 is a new anti-cancer gene that is more important than P53. The product encoded by the P16 gene is a 16KD protein, that is, the P16 protein, which is located in the nucleus. The P16 protein is an inhibitor of CDK4, one of the key enzymes of the cell division cycle (Cell Division Cycle). Once its expression fails, it will l...

Examples

Embodiment 1

[0037] Example 1 Design of primers and probes for detecting the methylation level of bladder cancer urine precipitate cells

[0038]According to the human whole genome sequence p16 and RASSF1A gene sequence published by National Center for Biotechnology Information (NCBI), the present invention designs primers, the primers are synthesized by TAKARA (Treasure Bioengineering Co., Ltd.), and the probes are synthesized by LifeTechnologies.

[0039] (1) Detect the nucleic acid sequence of p16 gene fragment methylation (FAM fluorescence channel)

[0040] The forward primer sequence is SEQ ID NO.1 (5'-AGCGGGCGGCGGGGAGTAGTA-3'),

[0041] The reverse primer sequence is SEQ ID NO.2 (5'-AGTCGGCGGCGGGGAGTAGTATG-3'),

[0042] The fluorescent probe sequence is SEQ ID NO.3 (5'-FAM-GATTGGTTGGTTACGGTCGCGGTT-MGB-3');

[0043] The 5' end of the fluorescent probe SEQ ID NO: A3 is labeled with FAM (fluorescence reporter group), and the 3' end is labeled with MGB (fluorescence quencher group).

...

Embodiment 2

[0048] Embodiment 2 A kind of test kit

[0049] The kit includes primers and probes for the above two target genes of p16 and RASSF1A; in addition, it also includes forward primers and probes for detecting GAPDH, PCR Master Mix, lysate, proteinase K, rinse solution, bisulfite solution and ddH 2 O.

[0050] The forward primer sequence of p16 is shown in SEQ ID NO.1, the reverse primer sequence is shown in SEQ ID NO.2; the fluorescent probe sequence is SEQ ID NO.3, the 5' end of the probe is labeled with FAM, 3 ' end labeled MGB.

[0051] The forward primer sequence of RASSF1A is shown in SEQ ID NO.4, the reverse primer sequence is shown in SEQ ID NO.5; the fluorescent probe sequence is SEQ ID NO.6, the 5' end of the probe is labeled with FAM, 3 ' end labeled MGB.

[0052] Forward primers and probes for detecting GAPDH can be primers and probes known in the art.

Embodiment 3

[0053] The using method of kit described in embodiment 3

[0054] (1) Extraction of genomic DNA from urine precipitated cells: take the patient's urine precipitated cells, use lysate, proteinase K, rinse solution and ddH 2 O is extracted to obtain a genomic DNA solution;

[0055] The specific steps are:

[0056] Aspirate 200 μL of urine sample, put it into a 1.5mL centrifuge tube, add 1mL Lysis Solution 1, then continue to vortex and shake for 1min, and then place the 1.5mL centrifuge tube in a 70°C water bath for 5min.

[0057] Take the 1.5mL centrifuge tube out of the water bath, centrifuge at 12,000rpm for 5min, and slowly pipette 300μL of the supernatant into a new 1.5mL centrifuge tube.

[0058] Add 200 μL Lysis Solution 2 and 20 μL Proteinase K to a new 1.5 mL centrifuge tube in sequence, and vortex to mix.

[0059] Place a new 1.5mL centrifuge tube in a 70°C water bath for 10min.

[0060] Add 200 µL of isopropanol and vortex to mix.

[0061] Put all the solution ob...

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