CN101956014A - Kit for detecting 7 genetic markers of peripheral blood in early diagnosis of nasopharyngeal darcinoma - Google Patents
Kit for detecting 7 genetic markers of peripheral blood in early diagnosis of nasopharyngeal darcinoma Download PDFInfo
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Abstract
The invention provides a kit for detecting 7 genetic markers of peripheral blood in early diagnosis of nasopharyngeal darcinoma. The kit comprises a peripheral blood total RNA extracting reagent, RT-PCR reaction liquid, time fluorescence quantitative RT-PCR reaction liquid and an SVM nasopharyngeal darcinoma diagnosis model, wherein the time fluorescence quantitative RT-PCR reaction liquid comprises primers of which the sequences are shown as SEQ IDNO:1-16; the primer sequences are SYBR Green fluorescence quantitative RT-PCR detection primers of the 7 genetic markers and reference gene GAPD; and the 7 genetic markers are HERC5, DLG7, PPARG, PLK1, KIF15, KLHL25 and MYST4. The invention also provides a using method for the kit. The kit has the advantages of high sensitivity, specificity and convenience, and can be used as aided diagnosis and early diagnosis technology for the nasopharyngeal darcinoma.
Description
Technical field
The present invention relates to biology field, relate in particular to a kind of detection kit that is used for 7 gene markers of peripheral blood of nasopharyngeal carcinoma early diagnosis.
Background technology
Nasopharyngeal carcinoma (Nasopharyngeal carcinoma is a kind of in China south and south east asia common malignancy NPC), its progressivity development, and normal a little later generation is shifted and diffusion.At present nasopharyngeal carcinoma mainly relies on radiation treatment, and the prognosis of middle and advanced stage tumour patient is still very poor, and 5 years survival rates can reach 90% after the I phase patient radiotherapy, 5 years survival rates of IV phase patient only 30%, so early discovery is the key that improves curative ratio.It is the important channel of finding early stage nasopharyngeal carcinoma that the nasopharyngeal carcinoma high risk population is carried out the serology examination.Because the change of serological index is usually prior to the appearance of clinical symptom, sign.Serological index commonly used at present is VCA-IgA, EA-IgA, the normal titre of using indirect enzyme dyeing process mensuration serum VCA-IgA, EA-IgA antibody of clinical labororatory is carried out auxiliary diagnosis to nasopharyngeal carcinoma, but still have the every index of part patient EBV serology all negative, this type of patient can not come out by the examination of EBV serology.Simultaneously because the VCA-IgA false positive causes psychological pressure also can for the Epstein-Barr virus carrier.And imaging examination is mainly used at present and assists diagnosis, determines extent of disease, and accurately by stages, the correct treatment target area of formulating, the design radiation is wild, observe after the radiotherapy tumor regression situation and with examining inspection.Therefore, for examining rate the morning of improving nasopharyngeal carcinoma, be necessary to research and develop new diagnostic method.
The appearance of genome-based technologies makes people can more fully understand the change of gene level in the tumour generating process.The chip of expression spectrum technology is applied at first to disease this field of classifying.Golub research group at first is applied to this technology seek the difference of two kinds of disease express spectras in thin leukemia of acute marrow and the thin leukemic discriminating of acute grain.This subsequently method is widely used among the research of mammary cancer, diffuse large B cell lymphoma, colorectal carcinoma, lung cancer, ovarian cancer and malignant melanoma, and a large amount of significant tumour molecular markers is provided.Wherein van ' t Veer etc. has identified the prognosis that 70 expression of gene spectrums can be predicted patient, and this achievement in research is in conjunction with clinical stages, and histological type has been applied to clinical.Simultaneously share the possibility that can predict effectively that patient takes place to shift in 5 years with other clinical indices, this is the typical case representative of chip of expression spectrum in clinical diagnosis and predicting function.But these samples are mainly derived from tumor tissues, and the nucleic acid of tumor tissues is difficult for obtaining, and utilize the Noninvasive detection means to find that the new molecular marker of tumour has become the focus that people more and more are concerned about.Scholars such as Liew propose so-called outpost's principle (Sentinel Principle), be that peripheral blood cells exists unique genetic expression spectral pattern, can reflect the variation of (heredity) and dynamic (influence of environment and disease) that tissue is stable, they are by comparing the gene expression profile of 9 kinds of histoorgans such as peripheral blood cells and heart, liver, kidney, brain, the proof peripheral blood can be expressed the gene of human genome more than 80%, wherein comprises tissue-specific gene.This just points out us can filter out the gene with diagnostic value, and to diagnose nasopharyngeal carcinoma with real-time fluorescence quantitative RT-PCR by detecting chip of expression spectrum in the peripheral blood.
Summary of the invention
In order to overcome above-mentioned technological deficiency, the invention provides a kind of detection kit that is used for 7 gene markers of peripheral blood of nasopharyngeal carcinoma early diagnosis.
The detection kit that is used for 7 gene markers of peripheral blood of nasopharyngeal carcinoma early diagnosis provided by the present invention comprises the peripheral blood total RNA extraction reagent, the RT-PCR reaction solution, real-time fluorescence quantitative RT-PCR reaction solution and SVM nasopharyngeal carcinoma diagnosis model, described real-time fluorescence quantitative RT-PCR reaction solution comprises primer, the sequence of described primer sequence shown in SEQ IDNO:1-16, these primer sequences are that the SYBRGreen fluorescence quantitative RT-RCR of 7 gene markers and internal control gene GAPDH detects primer, and the nucleotide sequence of its upstream primer (sense) and downstream primer (antisense) is composed as follows:
HERC5
Sense：TTTCTTACAGGAACTGACAGACTACAA
Antisense：ATGTCAGTGCTCTTATAGGGTCTCTT
DLG7
Sense：TCCATTTACTCAGCTGGAGAGGA
Antisense：ATCAGGTTACCACCAAAAGAAATGT
PPARG
Sense：GCTTCATGACAAGGGAGTTTCTAA
Antisense：CTGGGCGGTCTCCACTGA
PLK1
Sense：CATCCTCTACAATGATGGTGACAG
Antisense：CGCTCATGTAATTGCGGAAA
KIF15
Sense：GAAAAGTTGCGTGCCGAAAA
Antisense：GCCAGGAGTAGGTCTTACTTGTAAAAG
KLHL25
Sense：GGGCTGTGCACATACCAGTTAC
Antisense：AAGGTCCCACCCCCAAGAG
MYST4
Sense：GAGCCTACCTGTGAGATTGAAGTG
Antisense：AGGGTCAGCATTCTTGAAGCAA
GAPDH
Sense：CTCCTCCTGTTCGACAGTCAGC
Antisense：CCCAATACGACCAAATCCGTT。
Described 7 gene markers are HERC5, DLG7, PPARG, PLK1, KIF15, KLHL25 and MYST4.
PAXgeneTM BloodRNA Kit (QIAGEN, 762174) and the miRNeasy Mini Kit (QIAGEN, 217004) of peripheral blood total RNA extraction reagent in the test kit for providing by QIAGEN company.
The M-MLV reverse transcription test kit that RT-PCR reaction solution in the test kit is provided by Invitrogen company (Invitrogen, Carlsbad, CA.C28025-032).
Real-time fluorescence quantitative RT-PCR reaction solution in the test kit comprise ddH2O,
(Invitrogen, Carlsbad CA) use downstream primer with 7 gene markers and internal control gene GAPDH detection to GreenqPCR SuperMix with ROX.
The foundation of the SVM nasopharyngeal carcinoma diagnosis model that above-mentioned 7 gene markers are formed may further comprise the steps:
1) sending the total RNA sample of some routine nasopharyngeal cancer patients and normal people's peripheral blood to carry out the full chip gene expression profile of the mankind detects;
2) filter out differential gene in nasopharyngeal carcinoma and the normal people's peripheral blood with t-testunequal unpaired algorithm;
3) filter out characterizing gene with the SVM feature selection approach with discriminating power;
4) utilize SYBR Green fluorescence quantitative RT-RCR that the characterizing gene that filters out is verified;
5) utilize SVM to select suitable kernel function and penalty factor to set up the nasopharyngeal carcinoma diagnosis model, but and form schedule of operation with canned program;
Specifically, comprise the steps:
1) serves marine life chip companies 20 routine nasopharyngeal carcinoma and the total RNA samples of 20 routine normal people's peripheral bloods (dehemoglobinize mRNA) respectively and carry out human full gene oligonucleotide chip (the Agilent Whole Human GeneExpression Microarray 4 * 44K array) detection of Agilent;
2) utilize the Quantile method that each sample raw data is carried out stdn place a kind of jade;
Whether 3) utilize Agilent biochip platform to adopt the variation coefficient (CV) and recall rate to estimate chip data reliable;
4) utilize GeneSpring 10.0 softwares to adopt t-test unequal unpaired algorithm, P＜0.05 and difference multiple are defined as differential gene at the gene more than 1.5 times;
5) method by the SVM feature selection filters out totally 7 of characterizing genes with discriminating power, and sets up the SVM discrimination model of being made up of 7 gene markers;
6) whether consistent with 7 characterizing genes of Real-time PCR checking with chip data;
7) utilize SVM to select suitable kernel function and penalty factor to set up the nasopharyngeal carcinoma diagnosis model, but and form schedule of operation with canned program.
The invention allows for the using method of mentioned reagent box, may further comprise the steps
1) collection of sample blood sample to be detected;
2) the extraction purifying of total RNA in the sample blood sample to be detected;
3) RT-PCR: with Olig (dT) is reverse transcriptase primer, and total RNA is a template, carries out the synthetic cDNA of reverse transcription reaction;
4) SYBR Green fluorescence quantitative RT-RCR detects: utilize cDNA as template, fluorescent quantitation with described 7 gene markers of claim 2 detects primer as primer, carry out the real-time fluorescence quantitative PCR reaction, obtain the relative expression quantity of 7 gene markers in the peripheral blood sample basis;
5) judgement of sample results to be detected:, utilize the described SVM nasopharyngeal carcinoma diagnosis of claim 3 model to draw the diagnostic result of peripheral blood sample according to the relative expression quantity of 7 gene markers in the sample peripheral blood sample to be detected.
Specifically, may further comprise the steps:
1) collection of sample peripheral blood sample to be detected: use the BD PAXgeneRNA heparin tube of QIAGEN company to collect patient's peripheral blood sample;
2) the extraction purifying of total RNA in the sample peripheral blood sample to be detected: use the PAXgene Blood RNA Kit of QIAGEN company and the total RNA in the miRNeasy Mini Kit extraction purifying peripheral blood simultaneously, and identify the total RNA that extracts with 1% agarose electrophoresis;
3) reverse transcription reaction: using the M-MLV reverse transcription test kit of Invitrigen company, use total RNA to be template, is reverse transcriptase primer with Olig (dT), carries out the synthetic cDNA of reverse transcription reaction;
4) SYBR Green fluorescence quantitative RT-RCR PCR detects: 7 gene markers that provide according to GenBank and the correlated series of internal control gene GAPDH, use Primer 5 software designs, selected specificity fine through the analysis of BLAST software homology, and can successfully detect the relative expression quantity of 7 gene markers in sample peripheral blood to be detected through the experimental identification primer, detect the upstream and downstream primer as primer with these 7 gene marker fluorescent quantitations, utilize cDNA as template, carry out the real-time fluorescence quantitative RT-PCR reaction, obtain the mRNA relative content of 7 gene markers in the peripheral blood sample basis;
5) sample donor result's to be detected diagnosis: according to the mRNA relative content of 7 gene markers in the peripheral blood sample basis in the SYBR Green fluorescence quantitative RT-RCR, the SVM nasopharyngeal carcinoma diagnosis prototype software that utilizes our laboratory to create draws diagnostic result.
Test kit of the present invention has very high susceptibility and specificity; This test kit utilization clinically the peripheral blood of easy collection detect, be a kind of Noninvasive means; With respect to obtain by operation or conchoscope biopsy tumor tissues be used for detecting compare more convenient; The detected result of this test kit is not influenced by the EBV yin and yang attribute, can form complementation with existing screening indexes serum VCA-IgA and EA-IgA, can be used as the auxiliary diagnosis and the early diagnosis technology of nasopharyngeal carcinoma.
Description of drawings
Fig. 1 is SYBR Green quantitative fluorescent PCR proofing chip result;
Fig. 2 is the ROC curve of SVM nasopharyngeal carcinoma discrimination model;
Fig. 3 is the total RNA agarose gel electrophoresis of sample segment figure.
Embodiment
The present invention is based upon on the human full chip gene expression profile technical foundation, pick out 7 gene markers and set up the SVM nasopharyngeal carcinoma diagnosis model of being made up of these 7 gene markers by the method for SVM feature selection, the foundation of this model comprises the following aspects:
1.Agilent human full gene oligonucleotide chip detects
Serve marine life chip companies 20 routine nasopharyngeal carcinoma and the 20 total RNA samples of routine normal people's peripheral blood (dehemoglobinize) respectively and carry out human full gene oligonucleotide chip (the Agilent Whole Human Gene ExpressionMicroarray 4 * 44K array) detection of Agilent.
2. the Quantile stdn of chip data
Utilize the Quantile method that each sample raw data is carried out standardization (Biochip company finishes by Shanghai), obtain the P value of each gene in each gene normal people/rhinitis carninomatosis people's difference multiple and these two groups of samples.
3. chip data fail-safe analysis
Utilize Agilent biochip platform to adopt the variation coefficient (CV) and recall rate to estimate the reliability of chip data, judgement criteria be CV less than 15% and recall rate greater than 65% for reliable, evaluation result is the chip detection reliable results.
4. differential gene screening
Utilize GeneSpring 10.0 softwares to adopt t-test unequal unpaired algorithm, P＜0.05 and difference multiple are defined as differential gene at the gene more than 1.5 times, filter out 1257 probes (raise 687, reduce 570) altogether.
5. characterizing gene screening and SVM discrimination model are set up
Method by the SVM feature selection filters out totally 7 of characterizing genes with discriminating power from above-mentioned differential gene, with 100 routine modelings and 60 routine samples checkings, wherein get penalty coefficient C=1000 and nuclear parameter=9, and concrete steps are as follows:
1) selects suitable kernel function (being generally the radially basic kernel function RBF of Gauss), set the parameter of penalty factor and kernel function;
2) training sample set is mapped to higher dimensional space from lower dimensional space, by the kernel function skill non-linear separable sample is transformed into and in higher dimensional space, makes its linear separability;
3) still can not linear separability learning sample to be classified if be mapped to behind the higher dimensional space lineoid, then can adopt the method at soft interval that learning sample is separated as much as possible.This method has been introduced slack variable ξ
i, can regulate slack variable ξ
iSize make the training set sample satisfy the constraint condition of classification lineoid;
4) optimization problem that will find the solution optimum lineoid adopts Lagrange (Lagrange) multiplier method can obtain Lagrangian, and then converts former optimization problem to its Wolfe dual form;
5) solve separating of above-mentioned optimization problem, draw b ° of corresponding support vector and classification lineoid decision function threshold value;
6) utilize classification lineoid decision function that the checking sample of unknown label is differentiated, draw the prediction label for the treatment of classification samples, promptly predictor is judged to be the normal people greater than 0, is judged to be rhinitis carninomatosis people less than 0.
6.SYBR 7 gene markers of Green fluorescence quantitative RT-RCR checking
The total RNA of all blood samples of used 40 exceptions in this carries out SYBR Green fluorescence quantitative PCR detection when utilizing chip detection, and Fig. 1 is SYBR Green quantitative fluorescent PCR proofing chip result.As seen from Figure 1, the result is consistent with chip results in the Real-time PCR of these 7 gene markers checking, can be used for making up the nasopharyngeal carcinoma diagnosis model.
7. identify the discriminating power of SVM nasopharyngeal carcinoma diagnosis model
In the training set that 128 routine samples are formed, utilizing SVM nasopharyngeal carcinoma diagnosis model to draw each detects the predictor of sample and draws diagnostic result, and the diagnostic result and the pathological diagnosis result of the SVM diagnostic model that we are set up compare, obtain susceptibility, specificity, positive predictive value and the negative predictive value of SVM model, the results are shown in Table 1, table 1 is the diagnostic result of 7 gene marker diagnostic kits in 60 routine samples in the peripheral blood of real-time fluorescence quantitative RT-PCR diagnosis nasopharyngeal carcinoma; Obtain the ROC curve of this model simultaneously, see Fig. 2.As seen from Figure 2, the AUC of this model ROC curve is 0.82111, and this model has higher classification accuracy rate.
Table 1
Susceptibility: 83.3% specificity: 76.6%
Positive predictive value: 78.1% negative predictive value: 82.1%
ROC?Area：82.1％
Youden index: 0.599 LR+:3.56
Below introduce the mentioned reagent box respectively and specifically use step by specific embodiment.
One. experimental technique
1. 7 gene markers that provide according to GenBank and the correlated series of internal control gene GAPDH, use Primer 5 software designs, selected the good SYBR Green of specificity fluorescent quantitation to detect primer through the analysis of BLAST software homology, its nucleotide sequence is composed as follows:
HERC5
Sense：TTTCTTACAGGAACTGACAGACTACAA
Antisense：ATGTCAGTGCTCTTATAGGGTCTCTT
DLG7
Sense：TCCATTTACTCAGCTGGAGAGGA
Antisense：ATCAGGTTACCACCAAAAGAAATGT
PPARG
Sense：GCTTCATGACAAGGGAGTTTCTAA
Antisense：CTGGGCGGTCTCCACTGA
PLK1
Sense：CATCCTCTACAATGATGGTGACAG
Antisense：CGCTCATGTAATTGCGGAAA
KIF15
Sense：GAAAAGTTGCGTGCCGAAAA
Antisense：GCCAGGAGTAGGTCTTACTTGTAAAAG
KLHL25
Sense：GGGCTGTGCACATACCAGTTAC
Antisense：AAGGTCCCACCCCCAAGAG
MYST4
Sense：GAGCCTACCTGTGAGATTGAAGTG
Antisense：AGGGTCAGCATTCTTGAAGCAA
GAPDH
Sense：CTCCTCCTGTTCGACAGTCAGC
Antisense：CCCAATACGACCAAATCCGTT
2. the collection of peripheral blood sample to be detected
Use BD PAXgene RNA heparin tube (BD, 762165) to collect peripheral blood sample 193 examples to be detected, the concrete steps that this experiment blood sample is collected comprise:
1) BD PAXgene RNA heparin tube being placed room temperature (18 ℃-25 ℃) preserves;
2) prepare blood taking needle and BD PAXgene RNA heparin tube;
3) gather 2.5ml blood with BD PAXgene RNA heparin tube;
To prevent the refluence of BD PAXgene RNA blood sampling liquid in pipe when 4) taking a blood sample;
Patient's arm is placed suitable position, during blood sampling, BD PAXgene RNA heparin tube is vertically placed, be lower than patient's arm plane, unclamp the blood sampling band, allow blood flow into automatically in the BD PAXgene RNA heparin tube, note BD PAXgene RNA blood sampling liquid in pipe not being blowed back in the patient body;
5) after blood stopped to flow out 10 seconds, BD PAXgene RNA heparin tube is taken off.BD PAXgene RNA heparin tube designs for negative pressure, can draw 2.5ml blood automatically.Blood sampling is turned upside down BD PAXgene RNA heparin tube 8-10 time after finishing immediately;
6) with the upright placement of BD PAXgene RNA heparin tube, room temperature (18 ℃-25 ℃) was placed 2 hours.
7) put into 4 ℃ of refrigerators and preserve, will be kept in the transportation in the middle of 4 ℃ of ice chests;
8) can not carry out the extraction of RNA if do not need to transport the room temperature placement after 2 hours, preserve elder generation if desired and place-20 ℃ of refrigerators preservations to change-80 ℃ of refrigerators preservations after 24 hours over to BDPAXgene RNA heparin tube.
3. the extraction purifying of the total RNA of peripheral blood sample to be detected
The PAXgene of QIAGEN company is used in this experiment simultaneously
TMBlood RNAKit (QIAGEN, 762174) and miRNeasy Mini Kit (QIAGEN, 217004) extract the total RNA of peripheral blood, and concrete steps comprise;
1) the centrifugal PAXgene RNA of room temperature 5000rpm heparin tube is 10 minutes;
2) careful supernatant discarded adds the water that 4ml does not have the RNA enzyme, covers tight Jiao Gai;
3) on the vortex vibrator, will precipitate again and hang, centrifugal 10 minutes of room temperature 5000rpm, careful supernatant discarded;
4) add the resuspended buffer of 350 μ l (BR1), hanged precipitation with pipettor, mixing is till no visible precipitate;
5) resuspended liquid is moved in the 1.5mlEP pipe, adds 300 μ l binding buffer liquid (BR2) and 40 μ l Proteinase Ks, 5 seconds of concussion on the vortex vibrator, the EP pipe placed 55 ℃ of shaking baths among 400-1400rpm hatched 10 minutes;
6) aforesaid liquid is moved to PAXgene Shredder spin column (PSC), centrifugal 3 minutes of room temperature 12000rpm;
7) carefully supernatant is moved in the new 1.5EP pipe, be not drawn onto the precipitation at the bottom of the centrifuge tube with pipettor;
8) the liquid average mark in the EP pipe is gone in 2 EP pipes, add 875 μ l dehydrated alcohols (96-100%, analytical pure) in every pipe, the fastening lid turns upside down 8-10 time, and is briefly centrifugal, makes liquid all be collected in the pipe end;
9) earlier 700 μ l liquid are moved in the RNeasy mini spin column post, centrifugal 1 minute of room temperature 12000rpm, discard the filter liquide in the collection tube, in RNeasy mini spin column post, continue to add 700 μ l liquid room temperature 12000rpm centrifugal 1 minute;
10) with behind whole centrifugal the finishing of liquid, discard collection tube and liquid wherein;
11) add in 350 μ l Buffer RWT to the RNeasy spin column posts, cover lid gently, centrifugal 15 seconds of room temperature 12000rpm discards the liquid after centrifugal;
12) 10 μ l are added DNA enzyme (DNase I stock solution) and add among the 70 μ l RDD, turn upside down 8-10 time gently, briefly centrifugal;
13) above-mentioned 80 μ l liquid are directly splashed into the central authorities of RNasy spin column post, room temperature is placed 15 minutes to remove DNA;
14) add in 350 μ l Buffer RWT to the RNeasy spin column posts, cover lid gently, centrifugal 15 seconds of room temperature 12000rpm discards the liquid after centrifugal;
15) add in 500 μ l Buffer RPE to the RNeasy spin column posts, cover lid gently, centrifugal 15 seconds of room temperature 12000rpm discards the liquid after centrifugal;
16) add in 500 μ l Buffer RPE to the RNeasy spin column posts, cover lid gently, centrifugal 2 minutes of room temperature 12000rpm discards the liquid after centrifugal;
17) RNeasy spin column post is moved in the new collection tube centrifugal 1 minute of room temperature 12000rpm;
18) RNeasy spin column post is moved in the EP pipe of new no RNA enzyme, add the water that 30 μ l remove the RNA enzyme, left standstill after 1 minute room temperature 12000rpm centrifugal 1 minute to the central authorities of film;
19) 30 μ l after centrifugal are contained the central authorities that RNA solution splashes into film again, repeating step 18 once;
20) RNA that extracting is good analyzes RNA concentration by ultraviolet spectrophotometer, 260 and 280nm measure concentration and the purity that absorbancy is determined sample, A260/A280 should be purer RNA (ratio 1.9-2.1 also can) near 2.0, pass through agarose electrophoresis, detect RNA28S, 18S, the integrity of detection RNA.A260/A280 greater than 1.90 and the electrophoretic band of 28S think that with the ratio of the electrophoretic band of 18S the RNA quality inspection is qualified about 2: 1, if the ratio of the electrophoretic band of 28S and the electrophoretic band of 18S is less than 1 and exist a large amount of 5S bands to think that degraded or part degraded appear in RNA, the RNA quality inspection is defective.
4. reverse transcription reaction
The M-MLV reverse transcription test kit of use Invitrigen company (Invitrogen, Carlsbad, CA.C28025-032), by specification is a reverse transcriptase primer with Oligo (dT), get the total RNA of 0.5 μ g and carry out reverse transcription, reverse transcription system totally 20 μ l is operated on ice, and configuration scheme is as follows:
Totally 10 μ l systems are finished denaturation process in the PCR instrument, 65 ℃ of 5min, 4 ℃ of 1min at least; Take out reaction system and place immediately on ice, dispose following reaction system immediately:
Add each 10 μ l in the good EP pipe of mark, in the PCR instrument, finish following steps:
25 ℃ of annealing 10min extend 50min for 37 ℃; 70 ℃ of termination reaction 15min; 4 ℃ of forever are standby with-20 ℃ of the cDNA packing preservations that above-mentioned reverse records.
5. real-time fluorescence quantitative PCR (Real-time PCR) reaction
The cDNA that gets 10ng with
Green qPCR SuperMix with ROX (Invitrogen, Carlsbad CA) carry out the real-time fluorescence quantitative PCR reaction, and 10 μ l reaction systems are as follows:
(7900HT Fast Real-Time PCR System, Applied Biosystems finish following PCR process in USA): the first step, 50 ℃ of 2min to the real-time fluorescence quantitative PCR instrument; Second step, 95 ℃ of pre-sex change 10min; The 3rd step, 95 ℃ of sex change 15sec, 60 ℃ of annealing with extend 1min, inferior step repeats 40 circulations.Solubility curve elementary reaction process: 95 ℃ of 15sec, 60 ℃ of 15sec, 95 ℃ of 15sec.Utilize solubility curve to observe to have or not dimer to occur and primer whether special, utilize amplification curve to observe the situation of product amplification.
6.Real-time PCR interpretation of result
The genetic expression value is calculated with the method for Delta Ct, hypothesis goal and reference gene amplification efficiency all near 100% and relative deviation be no more than 1Ct; Δ Ct=goal gene CT mean-reference gene GAPDH CT mean; The relative expression quantity of goal gene is=2
-Δ Ct
7. result's diagnosis
The relative expression quantity of 7 gene markers in the sample peripheral blood to be detected is imported in the SVM model of our foundation, draw predictor, wherein predictor is judged to be the nasopharyngeal carcinoma patient greater than 0, is judged to be the normal people less than 0
Two. the result
1. peripheral blood sample to be detected extracts concentration and the quality inspection result of total RNA
Extract the total RNA of purifying respectively with the peripheral blood method for extracting total RNA to be detected in the above-mentioned steps 3, measure concentration and adopt 1% sepharose that the total RNA that extracts is carried out electrophoresis detection by ultraviolet spectrophotometer, representational electrophoresis result as shown in Figure 3, Fig. 3 result shows in the 193 routine blood samples that we collect altogether have 160 routine quality inspections qualified.
2. real-time fluorescence quantitative RT-PCR detected result
Adopt step 1,4,5 testing conditions, utilize real-time fluorescence quantitative PCR to detect the relative expression quantity of 7 gene markers in all blood sample bases of 128 exceptions, the relative expression quantity of 7 gene markers of part peripheral blood sample to be detected sees Table 2.Table 2 is that 7 gene markers utilize the relative expression quantity of real-time fluorescence quantitative RT-PCR in 21 routine samples.
Table 2
sample?ID DLG7 HERC5 KIF15 KLHL25 MYST4 PLK1 PPARG
245N -9.63275 -6.14797 -12.9488 -8.73773 -6.2762 -7.8321 -11.424
246N -10.5411 -3.84481 -10.6825 -8.40598 -6.02662 -7.51958 -13.6739
249N -8.84851 -2.28473 -10.0054 -8.74988 -5.92592 -7.30188 -11.8067
250N -6.89907 -2.88941 -8.8747 -7.91521 -5.15156 -8.62095 -11.3441
251N -7.96687 -2.9414 -10.3947 -7.91141 -5.20621 -8.5137 -11.8386
253N -8.61895 -2.76431 -14.0459 -8.2471 -5.49629 -8.00587 -11.1182
253N -9.75357 -4.51152 -12.1569 -7.77648 -5.14216 -7.36495 -7.1607
254N -10.246 -4.33283 -9.60099 -7.19688 -5.6933 -7.26334 -11.7167
255N -6.98285 -2.4603 -13.0917 -8.21804 -3.81378 -8.33888 -12.9491
286A -10.4316 -4.01077 -9.59847 -9.16007 -6.34879 -8.27269 -13.4484
292A -10.8484 -2.71021 -10.4234 -9.11477 -6.51336 -8.29897 -12.351
X315A -10.261 -2.40335 -9.17933 -8.72617 -5.86805 -7.85801 -11.9828
290A -8.50984 -4.61881 -13.9344 -9.12934 -4.63738 -8.26637 -12.8307
295A -5.7753 -2.42522 -11.8134 -9.45148 -3.93666 -8.21373 -10.8093
Q2149A -9.05609 -3.60027 -11.9244 -8.05503 -5.18967 -7.93216 -11.4298
Q2304A -8.64905 -1.79376 -12.4209 -8.80572 -5.91699 -6.97031 -10.2746
W1317A -9.28648 -1.59159 -13.2774 -8.0039 -5.32719 -7.45794 -9.95548
244A -9.91415 -4.12282 -12.4789 -8.58479 -6.23197 -7.79324 -10.6035
C305A -9.30214 -1.70409 -12.7773 -8.9345 -5.75611 -7.51454 -11.3477
T2325A -9.42673 -3.70955 -12.5497 -8.47398 -5.96041 -7.69738 -11.4079
291A -9.98792 -1.47794 -14.1254 -8.09047 -5.47992 -7.61917 -8.02616
3. real-time fluorescence quantitative RT-PCR nasopharyngeal carcinoma diagnosis test kit diagnostic result is analyzed
The relative expression quantity of 7 gene markers of every example sample to be detected is imported in the SVM model of our foundation, drawing each detects the predictor of sample and draws diagnostic result (the part diagnostic result sees Table 3, and table 3 is SVM nasopharyngeal carcinoma diagnosis model diagnostic results in 15 routine nasopharyngeal carcinoma samples); The diagnostic result and the pathological diagnosis result of the SVM diagnostic model that this laboratory is set up compare simultaneously, and the accuracy that draws this SVM discrimination model diagnosis nasopharyngeal carcinoma reaches 80%.
Table 3
Sample predicts knot
ID HERC5 KIF15 KLHL25 MYST4 PPARG DLG7P LK1 predictor fruit
868A 0.19927 0.00024 0.0037 0.0258 0.00054 0.00196 0.00555 0.059207 NPC
323A 0.05129 0.00024 0.00174 0.0149 0.0128 0.00119 0.0019 0.114853 NPC
951A 0.08563 0.00027 0.00498 0.0403 0.00075 0.00161 0.00791 0.06154 NPC
899A 0.13348 0 0.00465 0.0238 0.00052 0.00187 0.00254 0.062789 NPC
1677A 0.05427 0.00031 0.00206 0.021 0.0006 0.00095 0.00307 0.012505 NPC
1643A 0.0237 0.00025 0.00333 0.0356 0.00082 0.00169 0.00797 0.063197 NPC
1092A 0.05877 4.7E-05 0.00257 0.0176 0.00023 0.0008 0.00201 -0.04095 Normal
Z1556A 0.28676 0.0003 0.00309 0.0254 0.00051 0.00525 0.00238 0.062997 NPC
Z1878A 0.29429 9.8E-05 0.00224 0.0188 0.00068 0.00068 0.00516 0.057952 NPC
1688A 0.05102 0.00108 0.00201 0.0232 0.00062 0.00075 0.00361 0.062997 NPC
1924A 0.03851 2.7E-05 0.00163 0.0268 0.00071 0.00055 0.00412 0.071208 NPC
1164A 0.03916 0.00022 0.00537 0.0322 0.00545 0.001 0.00358 0.055237 NPC
1776A 0.01842 8.3E-05 0.00164 0.0152 0.00584 0.00087 0.00383 0.112238 NPC
Q2304A 0.28847 0.00019 0.00226 0.0166 0.01581 0.0025 0.00799 0.063384 NPC
1458A 0.05548 0.00011 0.00144 0.0172 0.00039 0.00063 0.00058 0.258697 NPC
7 gene marker diagnostic kits have very hypersensitivity, specificity in the peripheral blood that utilizes real-time fluorescence quantitative RT-PCR diagnosis nasopharyngeal carcinoma of the present invention; With obtain tumor tissues by operation or conchoscope biopsy and be used for detecting and compare more conveniently, and be the Noninvasive detection means; Diagnostic result is not influenced by the EBV yin and yang attribute, can form complementation with existing screening indexes serum VCA-IgA and EA-IgA; Can make patient obtain early examining with obvious clinical symptom.Therefore the present invention has made significant headway in exploration nasopharyngeal carcinoma diagnosis novel method field, can be used as the auxiliary diagnosis and the early diagnosis technology of nasopharyngeal carcinoma.
Claims (4)
1. detection kit that is used for 7 gene markers of peripheral blood of nasopharyngeal carcinoma early diagnosis, it is characterized in that, described test kit comprises peripheral blood total RNA extraction reagent, RT-PCR reaction solution, real-time fluorescence quantitative RT-PCR reaction solution and SVM nasopharyngeal carcinoma diagnosis model, described real-time fluorescence quantitative RT-PCR reaction solution comprises the detection primer of 7 gene markers and internal control gene GAPDH, the sequence of described primer sequence shown in SEQ ID NO:1-16.
2. test kit according to claim 1 is characterized in that, described 7 gene markers are HERC5, DLG7, PPARG, PLK1, KIF15, KLHL25 and MYST4.
3. test kit according to claim 1 is characterized in that, the foundation of described SVM nasopharyngeal carcinoma diagnosis model may further comprise the steps:
1) sending the total RNA sample of some routine nasopharyngeal cancer patients and normal people's peripheral blood to carry out the full chip gene expression profile of the mankind detects;
2) filter out differential gene in nasopharyngeal carcinoma and the normal people's peripheral blood with t-test unequal unpaired algorithm;
3) filter out characterizing gene with the SVM feature selection approach with discriminating power;
4) utilize SYBR Green fluorescence quantitative RT-RCR that the characterizing gene that filters out is verified;
5) utilize SVM to select suitable kernel function and penalty factor to set up the nasopharyngeal carcinoma diagnosis model, but and form schedule of operation with canned program.
4. the using method of the described test kit of one of claim 1-3 is characterized in that, may further comprise the steps:
1) collection of sample blood sample to be detected;
2) the extraction purifying of total RNA in the sample blood sample to be detected;
3) RT-PCR: with Olig (dT) is reverse transcriptase primer, and total RNA is a template, carries out the synthetic cDNA of reverse transcription reaction;
4) SYBR Green fluorescence quantitative RT-RCR detects: utilize cDNA as template, fluorescent quantitation with described 7 gene markers of claim 2 detects primer as primer, carry out the real-time fluorescence quantitative PCR reaction, obtain the relative expression quantity of 7 gene markers in the peripheral blood sample basis;
5) judgement of sample results to be detected:, utilize the described SVM nasopharyngeal carcinoma diagnosis of claim 3 model to draw the diagnostic result of peripheral blood sample according to the relative expression quantity of 7 gene markers in the sample peripheral blood sample to be detected.
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