CN102534009A

CN102534009A - SNP (Single Nucleotide Polymorphism) marker correlated to assistant diagnosis of primary lung cancer and application thereof

Info

Publication number: CN102534009A
Application number: CN2012100130909A
Authority: CN
Inventors: 沈洪兵; 胡志斌; 马红霞; 靳光付; 董静; 孙重期
Original assignee: Nanjing Medical University
Current assignee: Nanjing Jiangbei New Area Biomedical Public Service Platform Co., Ltd.
Priority date: 2012-01-16
Filing date: 2012-01-16
Publication date: 2012-07-04
Anticipated expiration: 2032-01-16
Also published as: CN102534009B

Abstract

The invention belongs to the fields of gene engineering and tumor medicines, and discloses an SNP (Single Nucleotide Polymorphism) marker correlated to assistant diagnosis of primary lung cancer and application thereof. The marker is a combination of the following 63 SNP: rs107401, rs465498, rs663414, rs622917, rs636586, rs741061, rs855641, rs948783, rs1544694, rs1663689, rs2030378, rs2225588, rs2317683, rs2395166, rs2405271, rs2788713, rs3130975, rs4344933, rs4488809 and the like. The marker can be used for preparing an assistant diagnosis kit for the primary lung cancer.

Description

A kind of SNP mark and the application thereof relevant with the primary lung cancer auxiliary diagnosis

Invention field

The invention belongs to genetically engineered and tumour medical field, relate to a kind of SNP mark and the application thereof relevant with the primary lung cancer auxiliary diagnosis.

Background technology

Lung cancer is the present whole world maximum malignant tumour of number of getting involved.Estimate lung cancer new cases 160.7 ten thousand people in 2008, dead 137.5 ten thousand people, the first place that occupies interior tumor incidence of global range and mortality ratio according to IARC (IARC).Because the change of tobacco consumption, environmental pollution and the mode of life of sustainable growth, recent two decades comes, and China's lung cancer morbidity and death are in rising trend always; Only in the period of 2000 to 2005; The number of the infected of China's lung cancer and because of the number of lung cancer death has increased by 120,000 and 100,000 respectively, according to estimates, China's male lung cancer sickness rate was 45.9/10 ten thousand in 2008; The women is 21.3/10 ten thousand, is higher than M.L. and approaching flourishing industrialized country's level in the world.The famous oncologist R.Peto prophesy of Britain: if Chinese untimely control smoking 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.This shows that lung cancer is one of important diseases that threatens the whole world and China resident life and health, is the great public health problem that needs to be resolved hurrily.

Since the sixties in 20th century, Doll and Hill found the relation of smoking and lung cancer first through case-control study and cohort study, tobacco exposed and has been considered to the main environmental risk factor that lung cancer takes place.Research shows that the smoker the comparable non-smoker of risk for lung cancer is taken place exceeded 10 times.The research of lung cancer clustering of disease in family shows that inherited genetic factors is played an important role equally in the generation of lung cancer.Twin study shows that lung cancer is the coefficient result of nature-nurture.Epidemiological study shows that the lung cancer more than 80% can be laid the blame on for tobacco exposes, but only have less than 20% smoker lung cancer takes place, and explains under equal environmental exposure, and the individuality with different genetic backgrounds is different to the susceptibility of lung cancer.Wherein (single nucleotide polymorphism's mononucleotide polymorphic SNP) plays an important role.

SNP is one type of dna polymorphism that 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 the one type of polymorphism mark that the most extensively exists in the genome, accounts for about 90%.The variation of these genome sequences can cause difference and the Different Individual of phenotype between individuality to disease, particularly the susceptibility of complex disease and to the difference of environmental factors, drug reaction.Therefore SNP causes individual important hereditary basis to common disease morbidity and prognosis susceptibility difference.Utilize the generation of the SNP spectrum prediction disease of disease-susceptible humans, not only sensitivity, accurate and quick has broad application prospects, and through the structure that the SNP prediction is composed, can also make prospective diagnosis to disease.In recent years, utilize the incidence and development of SNP prediction disease to become clinical and research focus researcher, the using value first meeting clue in common major disease prediction such as tumour and cardiovascular and cerebrovascular diseases.

Yet; Also SNP is not applied at present the report of primary pulmonary cancerous diagnose; If the SNP that can filter out the primary lung cancer susceptible is as biomarker; And develop corresponding diagnostic kit, and will be once strong promotion to China's primary pulmonary cancerous diagnose present situation, also opened up new approach for its drug screening, evaluating drug effect and targeted therapy.

Summary of the invention

The objective of the invention is to propose a kind of SNP mark and the application thereof relevant with the primary lung cancer auxiliary diagnosis to above-mentioned technical problem.

Second purpose of the present invention provides the Auele Specific Primer of above-mentioned SNP mark.

The specificity fluorescent probe that the 3rd purpose of the present invention provides above-mentioned SNP mark is right.

The 4th purpose of the present invention provides the application in preparation primary lung cancer auxiliary diagnostic box of above-mentioned SNP mark and Auele Specific Primer and specificity fluorescent probe thereof.

The 5th purpose of the present invention provides primary lung cancer auxiliary diagnostic box.

The contriver through separate and research primary lung cancer patient and with the normal healthy controls peripheral blood DNA of its age-matched in SNP; Seek one group with the high specific of primary lung cancer height correlation and the SNP of susceptibility; And develop the primary lung cancer auxiliary diagnostic box that to be convenient to clinical application; For the examination and the diagnosis of primary lung cancer provides the data support, the data support is provided for finding new small molecule medicine with potential therapeutic value.

The objective of the invention is to realize through following technical proposal:

A kind of SNP mark relevant with the primary lung cancer auxiliary diagnosis, this mark is the combination of rs107401, rs465498, rs663414, rs622917, rs636586, rs741061, rs855641, rs948783, rs1544694, rs1663689, rs2030378, rs2225588, rs2317683, rs2395166, rs2405271, rs2788713, rs3130975, rs4344933, rs4488809, rs4493136, rs4669621, rs4697406, rs4749794, rs4809957, rs6102795, rs6490820, rs9317873, rs9373105, rs9397087, rs9463490, rs10066153, rs11144475, rs11155286, rs11264579, rs11907465, rs12050469, rs12992554, rs13000907, rs16883398, rs16959825, rs17058190, rs17075390, rs17728461, rs1014588, rs1039119, rs1726744, rs2285947, rs2895680, rs3969178, rs4693311, rs4766994, rs4894545, rs4940849, rs6490294, rs6780858, rs9439527, rs9556016, rs12002185, rs12793370, rs1549058, rs2736100, rs6043705 and rs11699642.

The Auele Specific Primer of described SNP mark, these primers are:

The primer sequence of rs107401 is SEQ ID No:1 and SEQ ID No:2; The primer sequence of rs465498 is SEQID No:5 and SEQ ID No:6; The primer sequence of rs663414 is SEQ ID No:9 and SEQ ID No:10; The primer sequence of rs622917 is SEQ ID No:13 and SEQ ID No:14; The primer sequence of rs636586 is SEQID No:17 and SEQ ID No:18; The primer sequence of rs741061 is SEQ ID No:21 and SEQ ID No:22; The primer sequence of rs855641 is SEQ ID No:25 and SEQ ID No:26; The primer sequence of rs948783 is SEQID No:29 and SEQ ID No:30; The primer sequence of rs1544694 is SEQ ID No:33 and SEQ ID No:34; The primer sequence of rs1663689 is SEQ ID No:37 and SEQ ID No:38; The primer sequence of rs2030378 is SEQID No:41 and SEQ ID No:42; The primer sequence of rs2225588 is SEQ ID No:45 and SEQ ID No:46; The primer sequence of rs2317683 is SEQ ID No:49 and SEQ ID No:50; The primer sequence of rs2395166 is SEQID No:53 and SEQ ID No:54; The primer sequence of rs2405271 is SEQ ID No:57 and SEQ ID No:58; The primer sequence of rs2788713 is SEQ ID No:61 and SEQ ID No:62; The primer sequence of rs3130975 is SEQID No:65 and SEQ ID No:66; The primer sequence of rs4344933 is SEQ ID No:69 and SEQ ID No:70; The primer sequence of rs4488809 is SEQ ID No:73 and SEQ ID No:74; The primer sequence of rs4493136 is SEQ ID No:77 and SEQ ID No:78; The primer sequence of rs4669621 is SEQ ID No:81 and SEQ IDNo:82; The primer sequence of rs4697406 is SEQ ID No:85 and SEQ ID No:86; The primer sequence of rs4749794 is SEQ ID No:89 and SEQ ID No:90; The primer sequence of rs4809957 is SEQ ID No:93 and SEQ ID No:94; The primer sequence of rs6102795 is SEQ ID No:97 and SEQ ID No:98; The primer sequence of rs6490820 is SEQ ID No:101 and SEQ ID No:102; The primer sequence of rs9317873 is SEQ IDNo:105 and SEQ ID No:106; The primer sequence of rs9373105 is SEQ ID No:109 and SEQ IDNo:110; The primer sequence of rs9397087 is SEQ ID No:113 and SEQ ID No:114; The primer sequence of rs9463490 is SEQ ID No:117 and SEQ ID No:118; The primer sequence of rs10066153 is SEQ IDNo:121 and SEQ ID No:122; The primer sequence of rs11144475 is SEQ ID No:125 and SEQ IDNo:126; The primer sequence of rs11155286 is SEQ ID No:129 and SEQ ID No:130; The primer sequence of rs11264579 is SEQ ID No:133 and SEQ ID No:134; The primer sequence of rs11907465 is SEQ IDNo:137 and SEQ ID No:138; The primer sequence of rs12050469 is SEQ ID No:141 and SEQ IDNo:142; The primer sequence of rs12992554 is SEQ ID No:145 and SEQ ID No:146; The primer sequence of rs13000907 is SEQ ID No:149 and SEQ ID No:150; The primer sequence of rs16883398 is SEQ IDNo:153 and SEQ ID No:154; The primer sequence of rs16959825 is SEQ ID No:157 and SEQ IDNo:158; The primer sequence of rs17058190 is SEQ ID No:161 and SEQ ID No:162; The primer sequence of rs17075390 is SEQ ID No:165 and SEQ ID No:166; The primer sequence of rs17728461 is SEQ IDNo:169 and SEQ ID No:170; The primer sequence of rs1014588 is SEQ ID No:173 and SEQ IDNo:174; The primer sequence of rs1039119 is SEQ ID No:177 and SEQ ID No:178; The primer sequence of rs1726744 is SEQ ID No:181 and SEQ ID No:182; The primer sequence of rs2285947 is SEQ ID No:185 and SEQ ID No:186; The primer sequence of rs2895680 is SEQ ID No:189 and SEQ ID No:190; The primer sequence of rs3969178 is SEQ ID No:193 and SEQ ID No:194; The primer sequence of rs4693311 is SEQ ID No:197 and SEQ ID No:198; The primer sequence of rs4766994 is SEQ ID No:201 and SEQ IDNo:202; The primer sequence of rs4894545 is SEQ ID No:205 and SEQ ID No:206; The primer sequence of rs4940849 is SEQ ID No:209 and SEQ ID No:210; The primer sequence of rs6490294 is SEQ ID No:213 and SEQ ID No:214; The primer sequence of rs6780858 is SEQ ID No:217 and SEQ ID No:218; The primer sequence of rs9439527 is SEQ ID No:221 and SEQ ID No:222; The primer sequence of rs9556016 is SEQ ID No:225 and SEQ ID No:226; The primer sequence of rs12002185 is SEQ ID No:229 and SEQID No:230; The primer sequence of rs12793370 is SEQ ID No:233 and SEQ ID No:234; The primer sequence of rs1549058 is SEQ ID No:237 and SEQ ID No:238; The primer sequence of rs2736100 is SEQ IDNo:241 and SEQ ID No:242; The primer sequence of rs6043705 is SEQ ID No:245 and SEQ IDNo:246; The primer sequence of rs11699642 is SEQ ID No:249 and SEQ ID No:250.

The specificity fluorescent probe of described SNP mark is right, and these fluorescent probes are:

The fluorescent probe of rs107401 is SEQ ID No:3 and SEQ ID No:4 to sequence; The fluorescent probe of rs465498 is SEQ ID No:7 and SEQ ID No:8 to sequence; The fluorescent probe of rs663414 is SEQ ID No:11 and SEQ ID No:12 to sequence; The fluorescent probe of rs622917 is SEQ ID No:15 and SEQ ID No:16 to sequence; The fluorescent probe of rs636586 is SEQ ID No:19 and SEQ ID No:20 to sequence; The fluorescent probe of rs741061 is SEQ ID No:23 and SEQ ID No:24 to sequence; The fluorescent probe of rs855641 is SEQ ID No:27 and SEQ ID No:28 to sequence; The fluorescent probe of rs948783 is SEQ ID No:31 and SEQ ID No:32 to sequence; The fluorescent probe of rs1544694 is SEQ ID No:35 and SEQ ID No:36 to sequence; The fluorescent probe of rs1663689 is SEQ ID No:39 and SEQ ID No:40 to sequence; The fluorescent probe of rs2030378 is SEQ ID No:43 and SEQ IDNo:44 to sequence; The fluorescent probe of rs2225588 is SEQ ID No:47 and SEQ ID No:48 to sequence; The fluorescent probe of rs2317683 is SEQ ID No:51 and SEQ ID No:52 to sequence; The fluorescent probe of rs2395166 is SEQ IDNo:55 and SEQ ID No:56 to sequence; The fluorescent probe of rs2405271 is SEQ ID No:59 and SEQ ID No:60 to sequence; The fluorescent probe of rs2788713 is SEQ ID No:63 and SEQ ID No:64 to sequence; The fluorescent probe of rs3130975 is SEQ ID No:67 and SEQ ID No:68 to sequence; The fluorescent probe of rs4344933 is SEQ ID No:71 and SEQ ID No:72 to sequence; The fluorescent probe of rs4488809 is SEQ ID No:75 and SEQ ID No:76 to sequence; The fluorescent probe of rs4493136 is SEQ ID No:79 and SEQ ID No:80 to sequence; The fluorescent probe of rs4669621 is SEQ ID No:83 and SEQ ID No:84 to sequence; The fluorescent probe of rs4697406 is SEQ IDNo:87 and SEQ ID No:88 to sequence; The fluorescent probe of rs4749794 is SEQ ID No:91 and SEQ ID No:92 to sequence; The fluorescent probe of rs4809957 is SEQ ID No:95 and SEQ ID No:96 to sequence; The fluorescent probe of rs6102795 is SEQ ID No:99 and SEQ ID No:100 to sequence; The fluorescent probe of rs6490820 is SEQ IDNo:103 and SEQ ID No:104 to sequence; The fluorescent probe of rs9317873 is SEQ ID No:107 and SEQ IDNo:108 to sequence; The fluorescent probe of rs9373105 is SEQ ID No:111 and SEQ ID No:112 to sequence; The fluorescent probe of rs9397087 is SEQ ID No:115 and SEQ ID No:116 to sequence; The fluorescent probe of rs9463490 is SEQ ID No:119 and SEQ ID No:120 to sequence; The fluorescent probe of rs10066153 is SEQ ID No:123 and SEQ ID No:124 to sequence; The fluorescent probe of rs11144475 is SEQ ID No:127 and SEQ ID No:128 to sequence; The fluorescent probe of rs11155286 is SEQ ID No:131 and SEQ ID No:132 to sequence; The fluorescent probe of rs11264579 is SEQ ID No:135 and SEQ ID No:136 to sequence; The fluorescent probe of rs11907465 is SEQID No:139 and SEQ ID No:140 to sequence; The fluorescent probe of rs12050469 is SEQ ID No:143 and SEQ IDNo:144 to sequence; The fluorescent probe of rs12992554 is SEQ ID No:147 and SEQ ID No:148 to sequence; The fluorescent probe of rs13000907 is SEQ ID No:151 and SEQ ID No:152 to sequence; The fluorescent probe of rs16883398 is SEQ ID No:155 and SEQ ID No:156 to sequence; The fluorescent probe of rs16959825 is SEQ ID No:159 and SEQ ID No:160 to sequence; The fluorescent probe of rs17058190 is SEQ ID No:163 and SEQ ID No:164 to sequence; The fluorescent probe of rs17075390 is SEQ ID No:167 and SEQ ID No:168 to sequence; The fluorescent probe of rs17728461 is SEQ ID No:171 and SEQ ID No:172 to sequence; The fluorescent probe of rs1014588 is SEQID No:175 and SEQ ID No:176 to sequence; The fluorescent probe of rs1039119 is SEQ ID No:179 and SEQ IDNo:180 to sequence; The fluorescent probe of rs1726744 is SEQ ID No:183 and SEQ ID No:184 to sequence; The fluorescent probe of rs2285947 is SEQ ID No:187 and SEQ ID No:188 to sequence; The fluorescent probe of rs2895680 is SEQ ID No:191 and SEQ ID No:192 to sequence; The fluorescent probe of rs3969178 is SEQ ID No:195 and SEQ ID No:196 to sequence; The fluorescent probe of rs4693311 is SEQ ID No:199 and SEQ ID No:200 to sequence; The fluorescent probe of rs4766994 is SEQ ID No:203 and SEQ ID No:204 to sequence; The fluorescent probe of rs4894545 is SEQ ID No:207 and SEQ ID No:208 to sequence; The fluorescent probe of rs4940849 is SEQ IDNo:211 and SEQ ID No:212 to sequence; The fluorescent probe of rs6490294 is SEQ ID No:215 and SEQ IDNo:216 to sequence; The fluorescent probe of rs6780858 is SEQ ID No:219 and SEQ ID No:220 to sequence; The fluorescent probe of rs9439527 is SEQ ID No:223 and SEQ ID No:224 to sequence; The fluorescent probe of rs9556016 is SEQ ID No:227 and SEQ ID No:228 to sequence; The fluorescent probe of rs12002185 is SEQ ID No:231 and SEQ ID No:232 to sequence; The fluorescent probe of rs12793370 is SEQ ID No:235 and SEQ ID No:236 to sequence; The fluorescent probe of rs1549058 is SEQ ID No:239 and SEQ ID No:240 to sequence; The fluorescent probe of rs2736100 is SEQ ID No:243 and SEQ ID No:244 to sequence; The fluorescent probe of rs6043705 is SEQ IDNo:247 and SEQ ID No:248 to sequence; The fluorescent probe of rs11699642 is SEQ ID No:251 and SEQ IDNo:252 to sequence.

The application of described SNP mark in preparation primary lung cancer auxiliary diagnostic box.

The application of the Auele Specific Primer of described SNP mark in preparation primary lung cancer auxiliary diagnostic box.

The specificity fluorescent probe of described SNP mark is to the application in preparation primary lung cancer auxiliary diagnostic box.

A kind of primary lung cancer auxiliary diagnostic box, this test kit is used for detecting peripheral blood DNA rs107401, rs465498, rs663414, rs622917, rs636586, rs741061, rs855641, rs948783, rs1544694, rs1663689, rs2030378, rs2225588, rs2317683, rs2395166, rs2405271, rs2788713, rs3130975, rs4344933, rs4488809, rs4493136, rs4669621, rs4697406, rs4749794, rs4809957, rs6102795, rs6490820, rs9317873, rs9373105, rs9397087, rs9463490, rs10066153, rs11144475, rs11155286, rs11264579, rs11907465, rs12050469, rs12992554, rs13000907, rs16883398, rs16959825, rs17058190, rs17075390, rs17728461, rs1014588, rs1039119, rs1726744, rs2285947, rs2895680, rs3969178, rs4693311, rs4766994, rs4894545, rs4940849, rs6490294, rs6780858, rs9439527, rs9556016, rs12002185, rs12793370, rs1549058, rs2736100, rs6043705 and rs11699642.

Described diagnostic kit, this test kit contain the Auele Specific Primer and/or the specificity fluorescent probe of above-mentioned SNP mark.

Described diagnostic kit, the Auele Specific Primer of the SNP mark that this test kit contains is:

Described diagnostic kit, the specificity fluorescent probe of the SNP mark that this test kit contains is to being:

Said diagnostic kit, this test kit can also comprise PCR reaction enzyme and reagent commonly used, like Taq enzyme, dNTP mixed solution, Mgcl2 solution, deionized water etc.; Can also contain standard substance and/or reference substance.

Specifically, the technical scheme that the present invention deals with problems comprises: the sample storehouse and the DB of unified standard set up in (1): (SOP) gathers standard compliant blood sample with Standard operation procedure SOP, demography data and clinical data that systematic collection is complete.(2) genotype detection: select the primary lung cancer case,, utilize high-density SNP chip, in full genome range, find out the SNP relevant with primary lung cancer with the contrast of primary lung cancer case age, gender matched.(3) the positive related SNP to filtering out further detects in other sample, with the stability of judging that it is related.(4) development of primary lung cancer auxiliary diagnostic box: according to the genotype distribution frequency SNP that there were significant differences exploitation SNP auxiliary diagnostic box in primary lung cancer case and the normal healthy controls.

The inventor gathers standard compliant blood sample with Standard operation procedure SOP (SOP); The demography data that systematic collection is complete, clinical data etc.; And having adopted the Affymetrix6.0 chip to carry out full genome scanning, the TaqMan gene type carries out the detection in single site etc.

The experimental technique of research mainly comprises following components specifically:

1. research choice of sample

(1) clinical definite is a primary lung cancer;

(2) with the contrast of case age, gender matched;

This research adopts 9934 routine standard compliant samples to study altogether.

2. phenol-chloroform method extracts the peripheral blood genomic dna, by the ordinary method operation.Usually can obtain 20-50ng/ μ lDNA, purity (ultraviolet 260OD and 280OD ratio) is at 1.6-2.0.

3.Affymetrix 6.0 chip detection

(1) gets experimenter's complete genome DNA sample;

(2) on Affymetrix 6.0 chips (purchasing the high company that flies, down together), carry out full genome scanning in the U.S.;

(3) detection and relatively the difference difference of each genotype in primary lung cancer case and normal healthy controls.

4. the TaqMan gene type of single SNP

(1) gets experimenter's dna sample;

(2) Auele Specific Primer and the specificity fluorescent probe of the single SNP of design are right;

(3) carry out the PCR reaction;

(4) distributional difference of different genotype in detection and comparison primary lung cancer case and the normal healthy controls.

5. diagnostic reagent box preparation method

The Affymetrix6.0 chip carries out full genome scanning and single SNP detects the genotype distribution frequency SNP that there were significant differences in definite primary lung cancer case in back and the normal healthy controls, as the index of primary pulmonary cancerous diagnose.The SNP relevant with the primary lung cancer morbidity that filters out at last forms auxiliary diagnostic box rs107401; Rs465498; Rs663414; Rs622917; Rs636586; Rs741061; Rs855641; Rs948783; Rs1544694; Rs1663689; Rs2030378; Rs2225588; Rs2317683; Rs2395166; Rs2405271; Rs2788713; Rs3130975; Rs4344933; Rs4488809; Rs4493136; Rs4669621; Rs4697406; Rs4749794; Rs4809957; Rs6102795; Rs6490820; Rs9317873; Rs9373105; Rs9397087; Rs9463490; Rs10066153; Rs11144475; Rs11155286; Rs11264579; Rs11907465; Rs12050469; Rs12992554; Rs13000907; Rs16883398; Rs16959825; Rs17058190; Rs17075390; Rs17728461; Rs1014588; Rs1039119; Rs1726744; Rs2285947; Rs2895680; Rs3969178; Rs4693311; Rs4766994; Rs4894545; Rs4940849; Rs6490294; Rs6780858; Rs9439527; Rs9556016; Rs12002185; Rs12793370; Rs1549058; Rs2736100; Rs6043705 and rs11699642).Diagnostic reagent can comprise that the Auele Specific Primer of these SNP and specificity fluorescent probe are right, and reagent such as Taq enzyme, dNTP.

6. statistical analysis technique

The difference that utilization χ2Jian Yan (being used for classified variable) or student check (being used for the continuous variable) comparison demographic characteristics etc. distribute between the research object group.Additive model with in the logistic regression analysis carries out association analysis.

The comprehensive indication that constitutes for further these 63 SNP of research is used for the effect of early diagnosis, and we have made up a mathematical formula, take all factors into consideration positive and negative association and relation intensity that each SNP and primary lung cancer are fallen ill.Specifically; We mark to three kinds of genotype of each SNP; Wild homozygous=" 0 ", heterozygous=" 1 ", homozygous=" 2 " make a variation; Regression coefficient under the additive model during with single snp analysis is a weight, takes all factors into consideration the situation of each SNP and confirms a dangerous score value to each research research contrast.Risk score is calculated as follows: risk score = (-0.15 × rs9439527 score) + (0.33 × rs17728461 score) + (0.14 × rs2225588 score) + (0.25 × rs510321 score) + (-0.12 × rs663414 score) + (0.14 × rs4669621 score) + (0.25 × rs6490294 score) + (0.13 × rs4894545 score) + (0.14 × rs6780858 score) + (-0.16 × rs107401 score) + (-0.16 × rs1663689 score) + (0.16 × rs4697406 score) + (0.18 × rs1014588 score) + (-0.13 × rs2405271 score) + (-0.16 × rs6490820 score) + (-0.20 × rs9463490 score) + (0.27 × rs2788713 score) + (0.22 × rs855641 score) + (-0.22 × rs3130975 score) + (-0.16 × rs9397087 score) + (0.20 × rs622917 score) + (0.20 × rs12793370 score) + (0.14 × rs11155286 score) + (0.15 × rs2285947 score) + (0.21 × rs2395166 score) + (0.22 × rs741061 score) + (-0.17 × rs17058190 score) + (-0.17 × rs11144475 score) + (-0.14 × rs12002185 score) + (0.16 × rs4749794 score) + (-0.14 × rs16883398 score) + (0.13 × rs1726744 score) + (-0.17 × rs2030378 score) + (0.19 × rs2895680 score) + (0.25 × rs4940849 score) + (-0.22 × rs3969178 score) + (0.19 × rs6102795 score) + (-0.15 × rs17075390 score) + (0.14 × rs9317873 score) + (0.13 × rs9556016 score) + (0.17 × rs4809957 score) + (-0.24 × rs12050469 score) + (0.14 × rs16959825 score) + (0.26 × rs9373105 score) + (0.12 × rs948783 score) + (-0.13 × rs11907465 score) + (- 0.22 × rs6043705 score) + (0.16 × rs2317683 score) + (0.17 × rs11264579 score) + (0.17 × rs636586 score) + (-0.14 × rs13000907 score) + (-0.17 × rs1544694 score) + ( 0.25 × rs12992554 score) + (0.18 × rs11699642 score) + (-0.17 × rs4493136 score) + (0.18 × rs4766994 score) + (0.26 × rs4344933 score) + (0.18 × rs4488809 score) + (0.28 × rs1039119 score) + (-0.15 × rs4693311 score) + (0.16 × rs2736100 score) + (-0.28 × rs465498 score) + (-0.17 × rs10066153 score)), to obtain the risk factor score and boundaries values are directly applied to genome-wide association study of 5408 cases in the sample.

Statistical analysis is all accomplished (PLINK1.07) through special statistical analysis software.The horizontal P value of significance,statistical is made as 0.05, and all statistical test are two-tailed test.

Below be that the present invention further explains:

In above-mentioned 2331 routine qualified primary lung cancer cases and 3077 routine normal healthy controls, two groups of ages, sex equilibrium are comparable.We carry out full genome scanning with these two groups of crowds through the Affymetrix6.0 chip and obtain correlated results.

According to the Affymetrix6.0 chip detection, the inventor detects the SNP that the genotype distribution frequency there are differences in " primary lung cancer case " group and " normal healthy controls " are organized and comprises: rs107401, rs465498, rs663414, rs622917, rs636586, rs741061, rs855641, rs948783, rs1544694, rs1663689, rs2030378, rs2225588, rs2317683, rs2395166, rs2405271, rs2788713, rs3130975, rs4344933, rs4488809, rs4493136, rs4669621, rs4697406, rs4749794, rs4809957, rs6102795, rs6490820, rs9317873, rs9373105, rs9397087, rs9463490, rs10066153, rs11144475, rs11155286, rs11264579, rs11907465, rs12050469, rs12992554, rs13000907, rs16883398, rs16959825, rs17058190, rs17075390, rs17728461, rs1014588, rs1039119, rs1726744, rs2283947, rs2893680, rs3969178, rs4693311, rs4766994, rs4894343, rs4940849, rs6490294, rs6780858, rs9439527, rs9556016, rs12002185, rs12793370, rs1549058, rs2736100, rs6043705 and rs11699642.

According to above-mentioned detected result, we with these 63 SNPs relevant with primary lung cancer morbidity other 2283 routine primary lung cancer cases and with 2243 routine normal healthy controls of its age, gender matched in carried out the detection of single SNP, the result is consistent with chip detection.

Single factor and logistic Regression Analysis result show that all these 63 SNP exist remarkable related with the morbidity of primary lung cancer.

The combination of further analyzing these 63 SNP is used for the effect of primary pulmonary cancerous diagnose, finds that its combination can be good at distinguishing case and contrast.

According to above-mentioned experimental result, the inventor has prepared a kind of test kit that can be used for the primary lung cancer auxiliary diagnosis, comprise the Auele Specific Primer of measuring above-mentioned SNP among experimenter's blood specimen DNA, specificity fluorescent probe to other detection reagent.

Particularly; The combination of these 63 SNP; Perhaps the right dependent diagnostic test kit that constitutes of the Auele Specific Primer of these 63 SNP and specificity fluorescent probe helps the auxiliary diagnosis of primary lung cancer; For the clinician quick and precisely grasps the disease of patient state and is in a bad way degree, in time take the scheme of preventing and treating of more personalized to provide support.

Beneficial effect of the present invention:

SNP mark provided by the invention is as the meliority of the mark of primary lung cancer auxiliary judgment:

(1) SNP is a kind of novel gene biomarker; Be different from the traditional biological mark; Stable, Wicresoft, be easy to detect; With susceptibility that improves medical diagnosis on disease greatly and specificity, the successful exploitation of such biomarker will be started brand-new situation for the diagnosis and the treatment of primary lung cancer, for the development of other diseases biomarker is offered reference.

(2) the SNP test kit is a kind of system, comprehensive diagnostic kit; The auxiliary diagnosis that can be used for primary lung cancer; Help to reflect the disease of patient state, for the clinician quick and precisely grasps conditions of patients, in time takes the scheme of preventing and treating of more personalized to provide support.

(3) adopt tight checking and appraisement system, the inventor adopts full genome chip scanning to compose with the SNP that obtains disease-related at the initial stage, and uses the TaqMan methods of genotyping and in large sample, verify; The application of above method and strategy is quickened and has been guaranteed SNP biomarker and diagnostic kit application clinically, also for the development of other diseases biomarker the reference on method and the strategy is provided.

The present invention is through the influence factor to disease progression such as control age, sex, smoking, and research SNP sets forth the influence of SNP for the primary lung cancer progress in the application prospect of primary lung cancer auxiliary diagnosis, discloses its diagnostic value.Therefore, the present invention has obtained primary lung cancer morbidity related SNP spectrum and specificity marker thing; Development and application through SNP biomarker and diagnostic kit; Can make that the diagnosis of primary lung cancer is more convenient and easy; For the clinician quick and precisely grasps conditions of patients; For the clinical therapeutic efficacy evaluation lays the foundation, and for finding that the new small molecule drug targets with potential therapeutic value offers help.

Description of drawings

Fig. 1: the ROC curve that shows full genome association study case group and control group.

Show that primary lung cancer case group is the ROC curve of reference to the normal healthy controls group.

Embodiment

The collection of embodiment 1 sample and the arrangement of sample data

The contriver began to have collected in January, 2011 from tumor center of Nanjing Medical University a large amount of primary lung cancer patient blood specimens 2005 month April; Through the arrangement to the sample data, the contriver has therefrom selected 9934 examples to meet the full genome chip scanning of sample of standards and the laboratory sample of single SNP TaqMan gene type:

1, the primary lung cancer patient that makes a definite diagnosis of histopathology;

2, with the normal healthy controls of case age, gender matched;

And system acquisition the situation such as demography data and clinical data of these samples.

The full genome scanning of SNP in embodiment 2 peripheral blood DNAs

In above-mentioned qualified 2331 routine primary lung cancer patients and 3077 routine normal healthy controls, two groups of ages, gender matched.These two groups of crowds are obtained correlated results through the Affymetrix6.0 chip detection.Concrete steps are:

1, the white corpuscle in being stored in the frozen pipe of 2ml adds haemolysis reagent, changes over to fully after putting upside down mixing.

2, remove red corpuscle: with haemolysis reagent the 5ml centrifuge tube is mended to 4ml, put upside down mixing, centrifugal 10 minutes of 4000rpm abandons supernatant.In deposition, add 4ml haemolysis reagent, put upside down mixing once more and clean once, centrifugal 10 minutes of 4000rpm abandons supernatant.

3, extracting DNA: in deposition, add the 1ml extract and (contain 122.5ml 0.2M sodium-chlor among every 300ml; 14.4ml 0.5M YD 30; 15ml 10% sodium lauryl sulphate, the 148.1ml distilled water, down together) and 8 μ l Proteinase Ks; Fully shake mixing on the oscillator, 37 ℃ of water-baths are spent the night.

4, remove protein: add the abundant mixing of the saturated phenol of 1ml (have gentle hands was shaken 15 minutes), centrifugal 10 minutes of 4000rpm gets supernatant and changes in the new 5ml centrifuge tube.Adding equal-volume chloroform and primary isoamyl alcohol mixed solution in supernatant (chloroform: primary isoamyl alcohol=24: 1, v/v, down together), fully behind the mixing (hand 15 minutes), centrifugal 10 minutes of 4000rpm gets supernatant (branch is gone into the centrifuge tube of two 1.5ml).

5, DNA deposition: in supernatant, add the sodium-acetate 60 μ l of 3M, add and the isopyknic ice absolute ethyl alcohol of supernatant again, jog up and down, visible white flocculent precipitate is again with the centrifugal 10min of 12000rpm.

6, DNA washing: in deposition, add ice absolute ethyl alcohol 1ml, the centrifugal 10min of 12000rpm abandons the supernatant final vacuum and drains or place clean dry environment evaporate to dryness.

7, measure concentration: can obtain 20-50ng/ μ l DNA usually, purity (ultraviolet 260OD and 280OD ratio) is at 1.6-2.0.

8, carry out full genome scanning;

9, data analysis and processing: the genotype distribution frequency SNP that there were significant differences that in " primary lung cancer case " group and " normal healthy controls " are organized, finds is enumerated out hereinbefore, and the result sees table 1.

The full genome association analysis of table 1 case group and control group result

The TaqMan gene type of embodiment 3 single SNP

Above-mentioned full genome scanning is found to detect in other 2283 primary lung cancer cases and 2243 normal healthy controls with the relevant SNP of primary lung cancer morbidity, and concrete steps are:

3, extracting DNA: in deposition, add 1ml extract and 8 μ l Proteinase Ks, fully shake mixing on the oscillator, 37 ℃ of water-baths are spent the night.

4, remove protein: add the abundant mixing of the saturated phenol of 1ml (have gentle hands was shaken 15 minutes), centrifugal 10 minutes of 4000rpm gets supernatant and changes in the new 5ml centrifuge tube.(chloroform: primary isoamyl alcohol=24: 1), fully behind the mixing (hand 15 minutes), centrifugal 10 minutes of 4000rpm gets supernatant (branch is gone into the centrifuge tube of two 1.5ml) in supernatant, to add equal-volume chloroform and primary isoamyl alcohol mixed solution.

8, carry out the TaqMan gene type.63 positive related SNP design specific primers and specificity fluorescent probes to full genome scanning is found are right.Reaction system comprises mixture 0.25 μ l (20pmol/ μ l), 0.125 μ l TaqMan fluorescent probe (10pmol/ μ l contains corresponding two kinds of allelic fluorescent probes simultaneously) and the 1.25 μ l distilled waters of 2.5 μ l 2X TaqMan gene type Master Mix, the every pair of forward and reverse primer.Add 0.5 μ lDNA.What instrument used is ABI Prism 7900 quantitative real time PCR Instruments, and the PCR reaction conditions is: carried out 1 circulation → 95 ℃, 15 seconds in 95 ℃, 5 minutes, carried out 40 circulations in 60 ℃, 1 minute.

9, genotype interpretation: adopt ABI Prism 7900 quantitative real time PCR Instruments to carry out.

10, data processing and analysis: utilize the difference of three kinds of genotype distribution frequency in case combination control group of each SNP of addtive model comparison in the logistic regression model, the result no longer lists with genome scanning is similar entirely.

Embodiment 4 utilizes the risk level methods of marking further to analyze SNP and primary lung cancer morbidity

According to The above results; The inventor is through the comparison to 2 groups of samples (" primary lung cancer case group " and " normal healthy controls group ") genotype distribution frequency; Selecting positive related SNP, is weight with single SNP regression coefficient in the full genome scanning sample, further tries to achieve dangerous score value; Draw ROC and come the susceptibility and the specificity of evaluation prediction, and then assess the judgement of these SNP the primary lung cancer morbidity.Conjoint Analysis to 63 SNP marks finds that these 63 SNP open normal healthy controls group and primary lung cancer case component with 72.47% AUC, and the sensitivity of best stagnation point is 64.41%, specific degree: 69.71% (Fig. 1).

Therefore, the inventor has proved that the combination of adopting rs107401, rs465498, rs663414, rs622917, rs636586, rs741061, rs855641, rs948783, rs1544694, rs1663689, rs2030378, rs2225588, rs2317683, rs2395166, rs2405271, rs2788713, rs3130975, rs4344933, rs4488809, rs4493136, rs4669621, rs4697406, rs4749794, rs4809957, rs6102795, rs6490820, rs9317873, rs9373105, rs9397087, rs9463490, rs10066153, rs11144475, rs11155286, rs11264579, rs11907465, rs12050469, rs12992554, rs13000907, rs16883398, rs16959825, rs17058190, rs17075390, rs17728461, rs1014588, rs1039119, rs1726744, rs2285947, rs2895680, rs3969178, rs4693311, rs4766994, rs4894545, rs4940849, rs6490294, rs6780858, rs9439527, rs9556016, rs12002185, rs12793370, rs1549058, rs2736100, rs6043705 and rs11699642 can be well with normal healthy controls and primary lung cancer patient differentiation.

Embodiment 5 is used for the making of primary lung cancer auxiliary diagnosis SNP test kit

The making of SNP test kit and operating process are based on Affymetrix6.0 chip detection and TaqMan genotyping technique.The value of this test kit is only to need peripheral blood and does not need other tissue sample, through simplify most with special primer and fluorescent probe to detecting SNP, again through SNP spectrum auxiliary judgment primary lung cancer; Not only stable; Easy to detect, and accurately, improve the susceptibility and the specificity of medical diagnosis on disease greatly; Therefore with this test kit input practice, can help to instruct diagnosis and more effective individualized treatment.

Claims

1. a SNP mark relevant with the primary lung cancer auxiliary diagnosis is characterized in that this mark is the combination of rs107401, rs465498, rs663414, rs622917, rs636586, rs741061, rs855641, rs948783, rs1544694, rs1663689, rs2030378, rs2225588, rs2317683, rs2395166, rs2405271, rs2788713, rs3130975, rs4344933, rs4488809, rs4493136, rs4669621, rs4697406, rs4749794, rs4809957, rs6102795, rs6490820, rs9317873, rs9373105, rs9397087, rs9463490, rs10066153, rs11144475, rs11155286, rs11264579, rs11907465, rs12050469, rs12992554, rs13000907, rs16883398, rs16959825, rs17058190, rs17075390, rs17728461, rs1014588, rs1039119, rs1726744, rs2285947, rs2895680, rs3969178, rs4693311, rs4766994, rs4894545, rs4940849, rs6490294, rs6780858, rs9439527, rs9556016, rs12002185, rs12793370, rs1549058, rs2736100, rs6043705 and rs11699642.

2. the Auele Specific Primer of the described SNP mark of claim 1 is characterized in that this primer is:

The primer sequence of rs107401 is SEQ ID No:1 and SEQ ID No:2;

The primer sequence of rs465498 is SEQ ID No:5 and SEQ ID No:6;

The primer sequence of rs663414 is SEQ ID No:9 and SEQ ID No:10;

The primer sequence of rs622917 is SEQ ID No:13 and SEQ ID No:14;

The primer sequence of rs636586 is SEQ ID No:17 and SEQ ID No:18;

The primer sequence of rs741061 is SEQ ID No:21 and SEQ ID No:22;

The primer sequence of rs855641 is SEQ ID No:25 and SEQ ID No:26;

The primer sequence of rs948783 is SEQ ID No:29 and SEQ ID No:30;

The primer sequence of rs1544694 is SEQ ID No:33 and SEQ ID No:34;

The primer sequence of rs1663689 is SEQ ID No:37 and SEQ ID No:38;

The primer sequence of rs2030378 is SEQ ID No:41 and SEQ ID No:42;

The primer sequence of rs2225588 is SEQ ID No:45 and SEQ ID No:46;

The primer sequence of rs2317683 is SEQ ID No:49 and SEQ ID No:50;

The primer sequence of rs2395166 is SEQ ID No:53 and SEQ ID No:54;

The primer sequence of rs2405271 is SEQ ID No:57 and SEQ ID No:58;

The primer sequence of rs2788713 is SEQ ID No:61 and SEQ ID No:62;

The primer sequence of rs3130975 is SEQ ID No:65 and SEQ ID No:66;

The primer sequence of rs4344933 is SEQ ID No:69 and SEQ ID No:70;

The primer sequence of rs4488809 is SEQ ID No:73 and SEQ ID No:74;

The primer sequence of rs4493136 is SEQ ID No:77 and SEQ ID No:78;

The primer sequence of rs4669621 is SEQ ID No:81 and SEQ ID No:82;

The primer sequence of rs4697406 is SEQ ID No:85 and SEQ ID No:86;

The primer sequence of rs4749794 is SEQ ID No:89 and SEQ ID No:90;

The primer sequence of rs4809957 is SEQ ID No:93 and SEQ ID No:94;

The primer sequence of rs6102795 is SEQ ID No:97 and SEQ ID No:98;

The primer sequence of rs6490820 is SEQ ID No:101 and SEQ ID No:102;

The primer sequence of rs9317873 is SEQ ID No:105 and SEQ ID No:106;

The primer sequence of rs9373105 is SEQ ID No:109 and SEQ ID No:110;

The primer sequence of rs9397087 is SEQ ID No:113 and SEQ ID No:114;

The primer sequence of rs9463490 is SEQ ID No:117 and SEQ ID No:118;

The primer sequence of rs10066153 is SEQ ID No:121 and SEQ ID No:122;

The primer sequence of rs11144475 is SEQ ID No:125 and SEQ ID No:126;

The primer sequence of rs11155286 is SEQ ID No:129 and SEQ ID No:130;

The primer sequence of rs11264579 is SEQ ID No:133 and SEQ ID No:134;

The primer sequence of rs11907465 is SEQ ID No:137 and SEQ ID No:138;

The primer sequence of rs12050469 is SEQ ID No:141 and SEQ ID No:142;

The primer sequence of rs12992554 is SEQ ID No:145 and SEQ ID No:146;

The primer sequence of rs13000907 is SEQ ID No:149 and SEQ ID No:150;

The primer sequence of rs16883398 is SEQ ID No:153 and SEQ ID No:154;

The primer sequence of rs16959825 is SEQ ID No:157 and SEQ ID No:158;

The primer sequence of rs17058190 is SEQ ID No:161 and SEQ ID No:162;

The primer sequence of rs17075390 is SEQ ID No:165 and SEQ ID No:166;

The primer sequence of rs17728461 is SEQ ID No:169 and SEQ ID No:170;

The primer sequence of rs1014588 is SEQ ID No:173 and SEQ ID No:174;

The primer sequence of rs1039119 is SEQ ID No:177 and SEQ ID No:178;

The primer sequence of rs1726744 is SEQ ID No:181 and SEQ ID No:182;

The primer sequence of rs2285947 is SEQ ID No:185 and SEQ ID No:186;

The primer sequence of rs2895680 is SEQ ID No:189 and SEQ ID No:190;

The primer sequence of rs3969178 is SEQ ID No:193 and SEQ ID No:194;

The primer sequence of rs4693311 is SEQ ID No:197 and SEQ ID No:198;

The primer sequence of rs4766994 is SEQ ID No:201 and SEQ ID No:202;

The primer sequence of rs4894545 is SEQ ID No:205 and SEQ ID No:206;

The primer sequence of rs4940849 is SEQ ID No:209 and SEQ ID No:210;

The primer sequence of rs6490294 is SEQ ID No:213 and SEQ ID No:214;

The primer sequence of rs6780858 is SEQ ID No:217 and SEQ ID No:218;

The primer sequence of rs9439527 is SEQ ID No:221 and SEQ ID No:222;

The primer sequence of rs9556016 is SEQ ID No:225 and SEQ ID No:226;

The primer sequence of rs12002185 is SEQ ID No:229 and SEQ ID No:230;

The primer sequence of rs12793370 is SEQ ID No:233 and SEQ ID No:234;

The primer sequence of rs1549058 is SEQ ID No:237 and SEQ ID No:238;

The primer sequence of rs2736100 is SEQ ID No:241 and SEQ ID No:242;

The primer sequence of rs6043705 is SEQ ID No:245 and SEQ ID No:246;

The primer sequence of rs11699642 is SEQ ID No:249 and SEQ ID No:250.

3. the specificity fluorescent probe of the described SNP mark of claim 1 is right, it is characterized in that this fluorescent probe is:

The fluorescent probe of rs107401 is SEQ ID No:3 and SEQ ID No:4 to sequence;

The fluorescent probe of rs465498 is SEQ ID No:7 and SEQ ID No:8 to sequence;

The fluorescent probe of rs663414 is SEQ ID No:11 and SEQ ID No:12 to sequence;

The fluorescent probe of rs622917 is SEQ ID No:15 and SEQ ID No:16 to sequence;

The fluorescent probe of rs636586 is SEQ ID No:19 and SEQ ID No:20 to sequence;

The fluorescent probe of rs741061 is SEQ ID No:23 and SEQ ID No:24 to sequence;

The fluorescent probe of rs855641 is SEQ ID No:27 and SEQ ID No:28 to sequence;

The fluorescent probe of rs948783 is SEQ ID No:31 and SEQ ID No:32 to sequence;

The fluorescent probe of rs1544694 is SEQ ID No:35 and SEQ ID No:36 to sequence;

The fluorescent probe of rs1663689 is SEQ ID No:39 and SEQ ID No:40 to sequence;

The fluorescent probe of rs2030378 is SEQ ID No:43 and SEQ ID No:44 to sequence;

The fluorescent probe of rs2225588 is SEQ ID No:47 and SEQ ID No:48 to sequence;

The fluorescent probe of rs2317683 is SEQ ID No:51 and SEQ ID No:52 to sequence;

The fluorescent probe of rs2395166 is SEQ ID No:55 and SEQ ID No:56 to sequence;

The fluorescent probe of rs2405271 is SEQ ID No:59 and SEQ ID No:60 to sequence;

The fluorescent probe of rs2788713 is SEQ ID No:63 and SEQ ID No:64 to sequence;

The fluorescent probe of rs3130975 is SEQ ID No:67 and SEQ ID No:68 to sequence;

The fluorescent probe of rs4344933 is SEQ ID No:71 and SEQ ID No:72 to sequence;

The fluorescent probe of rs4488809 is SEQ ID No:75 and SEQ ID No:76 to sequence;

The fluorescent probe of rs4493136 is SEQ ID No:79 and SEQ ID No:80 to sequence;

The fluorescent probe of rs4669621 is SEQ ID No:83 and SEQ ID No:84 to sequence;

The fluorescent probe of rs4697406 is SEQ ID No:87 and SEQ ID No:88 to sequence;

The fluorescent probe of rs4749794 is SEQ ID No:91 and SEQ ID No:92 to sequence;

The fluorescent probe of rs4809957 is SEQ ID No:95 and SEQ ID No:96 to sequence;

The fluorescent probe of rs6102795 is SEQ ID No:99 and SEQ ID No:100 to sequence;

The fluorescent probe of rs6490820 is SEQ ID No:103 and SEQ ID No:104 to sequence;

The fluorescent probe of rs9317873 is SEQ ID No:107 and SEQ ID No:108 to sequence;

The fluorescent probe of rs9373105 is SEQ ID No:111 and SEQ ID No:112 to sequence;

The fluorescent probe of rs9397087 is SEQ ID No:115 and SEQ ID No:116 to sequence;

The fluorescent probe of rs9463490 is SEQ ID No:119 and SEQ ID No:120 to sequence;

The fluorescent probe of rs10066153 is SEQ ID No:123 and SEQ ID No:124 to sequence;

The fluorescent probe of rs11144475 is SEQ ID No:127 and SEQ ID No:128 to sequence;

The fluorescent probe of rs11155286 is SEQ ID No:131 and SEQ ID No:132 to sequence;

The fluorescent probe of rs11264579 is SEQ ID No:135 and SEQ ID No:136 to sequence;

The fluorescent probe of rs11907465 is SEQ ID No:139 and SEQ ID No:140 to sequence;

The fluorescent probe of rs12050469 is SEQ ID No:143 and SEQ ID No:144 to sequence;

The fluorescent probe of rs12992554 is SEQ ID No:147 and SEQ ID No:148 to sequence;

The fluorescent probe of rs13000907 is SEQ ID No:151 and SEQ ID No:152 to sequence;

The fluorescent probe of rs16883398 is SEQ ID No:155 and SEQ ID No:156 to sequence;

The fluorescent probe of rs16959825 is SEQ ID No:159 and SEQ ID No:160 to sequence;

The fluorescent probe of rs17058190 is SEQ ID No:163 and SEQ ID No:164 to sequence;

The fluorescent probe of rs17075390 is SEQ ID No:167 and SEQ ID No:168 to sequence;

The fluorescent probe of rs17728461 is SEQ ID No:171 and SEQ ID No:172 to sequence;

The fluorescent probe of rs1014588 is SEQ ID No:175 and SEQ ID No:176 to sequence;

The fluorescent probe of rs1039119 is SEQ ID No:179 and SEQ ID No:180 to sequence;

The fluorescent probe of rs1726744 is SEQ ID No:183 and SEQ ID No:184 to sequence;

The fluorescent probe of rs2285947 is SEQ ID No:187 and SEQ ID No:188 to sequence;

The fluorescent probe of rs2895680 is SEQ ID No:191 and SEQ ID No:192 to sequence;

The fluorescent probe of rs3969178 is SEQ ID No:195 and SEQ ID No:196 to sequence;

The fluorescent probe of rs4693311 is SEQ ID No:199 and SEQ ID No:200 to sequence;

The fluorescent probe of rs4766994 is SEQ ID No:203 and SEQ ID No:204 to sequence;

The fluorescent probe of rs4894545 is SEQ ID No:207 and SEQ ID No:208 to sequence;

The fluorescent probe of rs4940849 is SEQ ID No:211 and SEQ ID No:212 to sequence;

The fluorescent probe of rs6490294 is SEQ ID No:215 and SEQ ID No:216 to sequence;

The fluorescent probe of rs6780858 is SEQ ID No:219 and SEQ ID No:220 to sequence;

The fluorescent probe of rs9439527 is SEQ ID No:223 and SEQ ID No:224 to sequence;

The fluorescent probe of rs9556016 is SEQ ID No:227 and SEQ ID No:228 to sequence;

The fluorescent probe of rs12002185 is SEQ ID No:231 and SEQ ID No:232 to sequence;

The fluorescent probe of rs12793370 is SEQ ID No:235 and SEQ ID No:236 to sequence;

The fluorescent probe of rs1549058 is SEQ ID No:239 and SEQ ID No:240 to sequence;

The fluorescent probe of rs2736100 is SEQ ID No:243 and SEQ ID No:244 to sequence;

The fluorescent probe of rs6043705 is SEQ ID No:247 and SEQ ID No:248 to sequence;

The fluorescent probe of rs11699642 is SEQ ID No:251 and SEQ ID No:252 to sequence.

4. the application of the described SNP mark of claim 1 in preparation primary lung cancer auxiliary diagnostic box.

5. the application of the Auele Specific Primer of the described SNP mark of claim 2 in preparation primary lung cancer auxiliary diagnostic box.

6. the specificity fluorescent probe of the described SNP mark of claim 3 is to the application in preparation primary lung cancer auxiliary diagnostic box.

7. a primary lung cancer auxiliary diagnostic box is characterized in that this test kit is used for detecting peripheral blood DNA rs107401, rs465498, rs663414, rs622917, rs636586, rs741061, rs855641, rs948783, rs1544694, rs1663689, rs2030378, rs2225588, rs2317683, rs2395166, rs2405271, rs2788713, rs3130975, rs4344933, rs4488809, rs4493136, rs4669621, rs4697406, rs4749794, rs4809957, rs6102795, rs6490820, rs9317873, rs9373105, rs9397087, rs9463490, rs10066153, rs11144475, rs11155286, rs11264579, rs11907465, rs12050469, rs12992554, rs13000907, rs16883398, rs16959825, rs17058190, rs17075390, rs17728461, rs1014588, rs1039119, rs1726744, rs2285947, rs2895680, rs3969178, rs4693311, rs4766994, rs4894545, rs4940849, rs6490294, rs6780858, rs9439527, rs9556016, rs12002185, rs12793370, rs1549058, rs2736100, rs6043705 and rs11699642.

8. diagnostic kit according to claim 7, it is right to it is characterized in that this test kit contains the specificity fluorescent probe of Auele Specific Primer and/or the said SNP mark of claim 3 of the said SNP mark of claim 2.

9. according to claim 7 or 8 said diagnostic kits, it is characterized in that this test kit can also comprise the reagent that round pcr is commonly used.