CN101139629A - Reagent case for lung carcinoma susceptibility detection through PARP1 gene - Google Patents

Abstract

The invention discloses a reagent box for detecting lung cancer susceptibility. The reagent box comprises a pair of specificity primers for detecting 161st SNP site in SEQ ID NO:1 of PARP1 gene, a specificity fluorescent probe and an ordinary assembly for fluorescent quantitative PCR detection. The reagent box predicts the susceptibility of individuals on lung cancer by detecting and analyzing the type of carried gene at SNP site of individual PARP1 gene.

Description

A kind of test kit by PARP1 gene test lung cancer susceptibility

Technical field

The present invention relates to the test kit that a kind of disease susceptibility detects usefulness, especially a kind of test kit of detection of lung cancer susceptibility, by detecting poly ADP ribose transferring enzyme family member 1 (poly (ADP-ribose) polymerase family, member 1, PARP1 is abbreviated as ADPRT again) single nucleotide polymorphism (SNP) site predict individual susceptibility to lung cancer.

Background technology

Primary bronchogenic carcinoma of lung is called for short lung cancer, is one of modal malignant tumour in our times various places, is the disease of a kind of serious threat human health and life.Tumour cell comes from tunica mucosa bronchiorum or body of gland, and regional lymphoglandula and hematogenous metastasis are often arranged, and respiratory symptoms such as irritable cough, sputum mixed with blood are often arranged in early days, and disease progression speed is relevant with the biological nature of cell.

Since half a century, the sickness rate of countries in the world lung cancer and case fatality rate all have and obviously increase trend.The World Health Organization's year 2000 report: totally 706.5 ten thousand people of malignant tumour are died from the whole world in 1997, account for 12.6% of death toll, and wherein lung cancer accounts for 19% of malignant tumour death, occupies first of the malignant tumour cause of the death.China sample survey in 1990～1992 years shows, in the tumor mortality of urban population, lung cancer rises to the 1st by original the 4th, and rising the fastest on the rural area also is lung cancer.The lung cancer morbidity rate of Yunnan tin ore is to be to rise to 219.10/10 ten thousand in the period of 197.87/10 ten thousand, 1970～1979 in the period of 28.02/10 ten thousand, 1960～1969 between 1954～nineteen fifty-nine, and this also is rare in the whole world.The famous oncologist R.Peto prophesy of Britain: if untimely control smoking of China and atmospheric pollution will become the first in the world lung cancer big country above 1,000,000 to the annual lung cancer of China in 2025.

The cause of disease and pathogenesis are not clear and definite as yet so far.Consistently think that the morbidity of lung cancer is relevant with following factors: 1. smoking (comprising initiatively smoking and passive smoking); 2. professional carcinogen, the occupational factor that causes Human Lung Cancer that has been identified comprise the polycyclic aromatic hydrocarbons in asbestos, inorganic arsenic chemicals, dichlormethyl ether, chromium and compound thereof, nickel, radon, yperite, vinylchlorid, coal smoke, tar and the oil, the heating product of tobacco etc.; 3. atmospheric pollution (comprising the pollution of indoor subenvironment and outdoor overall situation); 4. ionizing rays; 5. the presentation of results that the perspective crowd that diet and nutrition, USA New York and Chicago are carried out observes, in the food intake of natural vitamin A class, beta carotene and after the more than ten years cancer be negative correlation, wherein the most outstanding is lung cancer; 6. other, ACS classifies tuberculosis as one of pathogenic factors of lung cancer.Having the tuberculosis patient to suffer from risk of lung cancer is 10 times of normal population.Its main histological type is a gland cancer.In addition, factors such as virus infection, mycotoxins (flavus), body's immunity are low, endocrine disturbance and familial inheritance may also play certain comprehensive action to the generation of lung cancer.

In recent years studies show that the inactivation of the generation of lung cancer and activation of some oncogene and cancer suppressor gene is closely related.Verified several oncogene family comprises bcl-2, Kit that the ras family of mutagenesis, the myc family of amplifying gene, C-erB2, mechanism are not clear and by antioncogene p53, the p16 of wild-type variation and RB etc.Studies show that in a large number C-myc, L-myc, N-myc and RAF all have overexpression in small cell lung cancer.The then overexpression of Chang Kejian ras family gene in the nonsmall-cell lung cancer.These deep researchs will and be treated lung cancer and provide fundamental basis for the gene prevention.

The PARP1 gene is positioned at karyomit(e) 1q41-q42 position No. 1, total length 47.3kb, and mRNA total length 3861bp has 23 exons, the 1015 amino acid whose poly ADP ribose transferase proteins of encoding.This kind of enzyme is revised multiple nucleoprotein by poly-ADP ribosylation reaction.This correction participates in multiple important cellular activity based on DNA, for example: differentiation, propagation, metastases etc. also participate in the activity of molecular level simultaneously, for example DNA plerosis damaged cells or the like.The major function of PARP1 is described to 3 aspects usually: 1.DNA repairing effect (BER); 2. suppress transcribing of damaged dna; 3. exhaust the energy in the cell, cause apoptosis; 4. participate in the transcriptional control of portion gene.These functions can repair cell in impaired DNA, suppress transcribing of damaged DNA, even kill the cell that can't repair, reach the effect that cancer takes place that suppresses.Discover that the PARP1 afunction can improve mammary cancer, the sickness rate of cancers such as lung cancer.

SNP (single nucleotide polymorphism), promptly the single nucleotide polymorphism mark is the dna polymorphism that a class causes based on single nucleotide variation, is called third generation genetic marker by hereditary educational circles.Mainly be meant the dna sequence polymorphism that causes by the variation on the genome nucleotide level, comprise single base conversion, transversion, and the insertion/disappearance of single base etc.It is a class polymorphism mark that the most extensively exists in the genome, accounts for about 90%.The variation of these genome sequences can cause the difference of phenotype between individuality and Different Individual to disease, particularly the susceptibility of complex disease and to the difference of environmental factors, drug reaction.

Rs1136410 is that to be positioned on No. 1 karyomit(e) a T/C on the PARP1 gene polymorphic, causes that the 762nd amino acid of albumen of PARP1 genes encoding becomes Ala by Val.This polymorphic frequency distribution among the world crowd that NCB1 announces, T accounts for 0.808, and C accounts for about 0.192, and heterozygosity is 0.311.Molecular biology research shows that last No. 762 amino acid of PARP1 are converted into Ala by Val, have reduced the activity of enzyme, causes the occurrence risk of multiple cancer to raise.The association analysis of the Chinese population of large sample shows that the polymorphism in this site is one of lung cancer influence factor important in the Chinese population.Rs1136410 carries in the site C type and is lung cancer susceptible type.

Summary of the invention

Can be used to assess on the basis of individuality to the susceptibility of lung cancer based on the rs1136410 SNP site on the PARP1 gene, the invention provides a kind of test kit of detection of lung cancer susceptibility.

This test kit comprises: the Auele Specific Primer that detects the 161st SNP site among the PARP1 genes of SEQ ID NO:1 is to reaching specificity fluorescent probe, Taq enzyme, dNTP mixed solution, MgCl ₂Solution, quantitative fluorescent PCR reaction buffer, deionized water.

Auele Specific Primer described in this test kit designs being meant at the 161st SNP site among the SEQ ID NO:1, and it is right that the energy specific amplification goes out the primer of the dna fragmentation that comprises the 161st SNP site among the SEQ ID NO:1.Design this class primer to being that those skilled in the art can be unlabored.Preferably, it is right to comprise the primer with sequence shown in SEQ ID NO:2 and 3 in the test kit.Auele Specific Primer synthesizes the synthetic technology of available routine.Those skilled in the art will appreciate that primer of the present invention is not limited to this two pairs of primers.

Specificity fluorescent probe described in this test kit is meant at the 161st SNP site among the SEQ ID NO:1 and designs, can go out the probe of this SNP site lung cancer tumor susceptibility gene type by the fluorescent quantitative PCR technique specific detection.Designing this class probe is that those skilled in the art can be unlabored.Preferably, comprise Taqman probe in the test kit with sequence shown in the SEQ ID NO:4.The specificity fluorescent probe can synthesize with the synthetic technology of routine.Those skilled in the art will appreciate that specificity fluorescent probe of the present invention is not limited to this probe, all probes that can be used for the 161st SNP site among the fluorescence quantitative PCR detection SEQ ID NO:1 all within the scope of the present invention.

The component and the content of test kit of the present invention comprise:

1 μ l10 X quantitative fluorescent PCR reaction buffer,

0.1 μ l25mM dNTP mixed solution,

0.6 μ l25mM MgCl ₂Solution,

0.025 μ l (5units/ μ l) Taq archaeal dna polymerase,

20 μ M Auele Specific Primers are to (two) each 0.225 μ l,

10 μ M specificity fluorescent probes, 0.25 μ l,

Deionized water 5.575 μ l.

This test kit detects for a person-portion and uses, and the storage temperature of test kit is-20 ℃.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.

The use of embodiment 1. detection kit

The extraction of step 1:DNA template

Genomic dna with silica gel adsorption extracting mouth epithelial cells.

Step 2: quantitative fluorescent PCR reaction

But use the fluorescent quantificationally PCR detecting kit of detection of lung cancer susceptibility, wherein, contain following primer to and fluorescent probe:

It is 58 ℃ that adopted primer: 5 '-TTTGCCATTCACTGTGTTGG-3 ' (SEQ ID NO:2) Tm value is arranged

Antisense primer: 5 '-ATCCTTGCTGCTATCATCA-3 ' (SEQ ID NO:3) Tm value is 54 ℃

Fluorescent probe: 5 '-CAGGCCAAGGCGGAAATGCT-3 ' (SEQ ID NO:4) Tm value is 64 ℃

The quantitative fluorescent PCR reaction system is cumulative volume 10 μ l, and comprising concentration is dna profiling 2 μ l, 1 μ l10 X quantitative fluorescent PCR reaction buffer, 0.1 μ l25mM dNTP mixed solution, the 0.6 μ l25mM MgCl of 20ng/ μ l ₂The fluorescent probe 0.25 μ l that adopted primer and antisense primer each 0.225 μ l, 10 μ M are arranged of solution, 0.025 μ l (5units/ μ l) Taq archaeal dna polymerase, 20 μ M, deionized water 5.575 μ l.

React on ABI9700 type pcr amplification instrument, reaction conditions is 50 ℃, 2 minutes, 95 ℃, 10 minutes, carries out 95 ℃ of 60 round-robin, 30 seconds, 60 ℃, 1 minute.Reaction finishes the back and read the fluorescent amount on ABI7900 type quantitative real time PCR Instrument.

Step 3:SNP gene type assay

The final sample fluorescence volume and the normal control that show on the quantitative real time PCR Instrument are compared, the final fluorescent signal of fluorescent probe is apparently higher than normal control, illustrate that rs1136410 SNP loci gene type carries the C type on the PARP1 gene of detected DNA, be lung cancer susceptible type.

Embodiment 2. instructs people initiatively to prevent the service of lung cancer

Carried out this service in Shanghai Zhujian Biological Engineering Co., Ltd at present.

Step 1:DNA extracts

The detected person is served preceding consulting, sign Informed Consent Form, fill in living and diet custom questionnaire by the detected person.Using the oral cavity sampling to wipe away according to the indication in the sampling box carries out the mouth epithelial cells sampling, adopts silica gel adsorption to carry out the DNA extracting of mouth epithelial cells.

Step 2: genotype tests

Use test kit provided by the invention, fluorescence quantitative PCR detection is carried out in the rs1136410 SNP site on the PARP1 gene of detected person DNA, determine the genotype in this SNP site.

Step 3: instruct people initiatively to prevent lung cancer

By to the genotypic analysis of detected person SNP, provide genotype tests report and the report of detected person's individuation health guidance.The gene test report describes the height of detected person's lung cancer susceptible degree and ill probability size in detail.The report of individuation health guidance is based on detected person's genotype tests result, in conjunction with its living and diet custom survey result, the relative risk of assessment detected person lung cancer inheritance susceptible.Make the personalized healthy action scheme that is directed to the detected person simultaneously, scheme is included in the recommendation on improvement on food habits, the mode of life etc., and popularizes the health knowledge of prevention lung cancer for the detected person.

Sequence table

＜110〉Shanghai Zhujian Biological Engineering Co., Ltd

＜120〉a kind of test kit by PARP1 gene test lung cancer susceptibility

<160>4

<210>1

<211>300

<212>DNA

＜213〉Genus Homo, and ethnic group (Homo sapiens, human)

<400>1

tgtgcagcag?gtaacaggct?ggccctgacc?agcaggaggg?tttgccattc?actgtgttgg 60

accttctctg?catgtaggtt?ttctctgcca?cctgggtgag?tctgtctcat?tcaccatgat 120

acctaagtcg?ggggctttct?tttgctcctc?caggccaagg?tggaaatgct?tgacaacctg 180

ctggacatcg?aggtggccta?cagtctgctc?aggggagggt?ctgatgatag?cagcaaggat 240

cccatcgatg?tcaactatga?gaagctcaaa?actgacatta?aggtaacagg?gtcaggcctt 300

<210>2

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>2

tttgccattc?actgtgttgg 20

<210>3

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>3

atccttgctg?ctatcatca 19

<210>4

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉probe

<400>4

caggccaagg?cggaaatgct 20

Claims (6)

1. a test kit that is used for the detection of lung cancer susceptibility is characterized in that: comprise that the Auele Specific Primer that detects the 161st SNP site among the PARP1 genes of SEQ ID NO:1 is to reaching specificity fluorescent probe, Taq enzyme, dNTP mixed solution, MgCl ₂Solution, quantitative fluorescent PCR reaction buffer, deionized water.

2. test kit according to claim 1, it is characterized in that: described Auele Specific Primer designs being meant at the 161st SNP site among the SEQ ID NO:1, and it is right that the energy specific amplification goes out to comprise the primer of the dna fragmentation in the 161st SNP site among the SEQ ID NO:1.

3. test kit according to claim 1, it is characterized in that: described specificity fluorescent probe is meant at the 161st SNP site among the SEQ ID NO:1 and designs, can go out the probe of this SNP site lung cancer tumor susceptibility gene type by the fluorescent quantitative PCR technique specific detection.

4. test kit according to claim 1 is characterized in that: contained Auele Specific Primer is right to being selected from the primer with sequence shown in SEQ ID NO:2 and 3.

5. test kit according to claim 1 is characterized in that: contained specificity fluorescent probe is selected from the Taqman probe with sequence shown in the SEQ ID NO:4.

6. test kit according to claim 1 is characterized in that: the component of test kit and content comprise 1 μ l 10X quantitative fluorescent PCR reaction buffer, 0.1 μ l 25mM dNTP mixed solution, 0.6 μ l 25mM MgCl ₂Solution, 0.025 μ l (5units/ μ l) Taq archaeal dna polymerase, 20 μ M Auele Specific Primers are to (two) each 0.225 μ l, 10 μ M specificity fluorescent probes, 0.25 μ l, deionized water 5.575 μ l.This test kit detects for a person-portion and uses, and the storage temperature of test kit is-20 ℃.