CN106399534A - Tumor blood platelet RNA quantitative detection model and method for tumor early screening - Google Patents

Tumor blood platelet RNA quantitative detection model and method for tumor early screening Download PDF

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CN106399534A
CN106399534A CN201610911677.XA CN201610911677A CN106399534A CN 106399534 A CN106399534 A CN 106399534A CN 201610911677 A CN201610911677 A CN 201610911677A CN 106399534 A CN106399534 A CN 106399534A
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tumor
primer
platelet
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卞胜超
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Shanghai Hou Cheng Medical Science And Technology Co Ltd
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract

The invention discloses a tumor blood platelet RNA quantitative detection model for tumor early screening. The model comprises PCR (polymerase chain reaction) detection specific primers. The PCR detection specific primers include an F-end primer and an RT primer which are shown as SEQ ID NO.1, an F-end primer and a RT primer shown as SEQ ID NO.2, an F-end primer and an RT primer which are shown as SEQ ID NO.3, an F-end primer and an RT primer which are shown as SEQ ID NO.4, an F-end primer and an RT primer which are shown as SEQ ID NO.5, an F-end primer and an RT primer which are shown as SEQ ID NO.6, an F-end primer and an RT primer which are shown as SEQ ID NO.7, an F-end primer and an RT primer which are shown as SEQ ID NO.8 and an F-end primer and an RT primer which are shown as SEQ ID NO.9. The invention further discloses a tumor blood platelet RNA quantitative detection method for tumor early screening. The tumor blood platelet RNA quantitative detection model and method has the advantages that tumor early screening is realized, tumor pathological identification and clinical diagnosis are assisted, and survival rate of patients is increased.

Description

Tumor platelet RNA detection by quantitative model for tumor early screening and method
Technical field
The present invention relates to molecular biology and medical domain, especially relate to a kind of tumor blood for tumor early screening Platelet RNA detection by quantitative model and method.
Background technology
With the increase of cancer morbidity and mortality rate, cancer is not only Chinese's main causes of death, is also serious Threaten human health and the great public health problem of restriction socio-economic development.The early diagnosiss of tumor mean to do sth. in advance Therapeutic intervention, great to the prognosis meaning of patient.The diagnosis being presently used for tumor depends on traditional tumor tissues clinic The aspects such as symptom, irradiation image, biochemistry detection and pathology, and wherein most method invasive, bring misery to patient, And the doctor implementing technique is had higher requirements.Blood serum tumor markers, example are mainly to the noninvasive test method of tumor As AFP, CEA, CA199 etc., but the sensitivity of diagnosis and specificity are relatively low.Therefore, find and have compared with Gao Ling Sensitivity and specific tumor markerses the morning of tumor patient is examined early control significant.
In order to reduce the tumor tissues limitation of acquisition, the liquid biopsy method based on blood becomes to tissue samples analysis Fashion trend in recent years, mainly includes plasma DNA at present（cfDNA）Detection with circulating tumor cell (CTC).So far Till, tumor is carried out to the liquid biopsy method not yet full maturity of early screening, reason is that the molecule in blood cannot be special The feature of opposite sex description primary tumor.Research in recent years shows, to tumor correlation platelet (tumor educated Platelet detection) may make to become more efficiently based on the cancer diagnosis of tumor blood.Platelet, is in peripheral blood Two abundant cell types, from the circulation no fragment of bone marrow megakaryocyte, it is noteworthy characterized by can stop blooding and promote Wound healing.In prior art, it is more accurate that also not corresponding ripe technology is carried out to tumor using tumor correlation platelet Diagnosis.
Content of the invention
In order to solve above-mentioned the deficiencies in the prior art, the invention provides a kind of swollen for tumor early screening Tumor platelet RNA detection by quantitative model and method, can be used as early diagnosis of tumor and the biological marker of risk assessment Thing combines, and detection can obtain good tumors, be capable of tumor early screening, adjuvant therapy pathological identification simultaneously And clinical diagnosises, improve the survival rate of patient.
In order to realize foregoing invention purpose, the technical scheme that the present invention provides is as follows：
A kind of tumor platelet RNA detection by quantitative model for tumor early screening, this model includes PCR detection specificity and draws Thing, described PCR detection specific primer includes the F end primer of SEQ ID NO.1 and the F end primer of RT primer, SEQ ID NO.2 With RT primer, the F end primer of SEQ ID NO.3 and RT primer, the F end primer of SEQ ID NO.4 and RT primer, SEQ ID The F end primer of NO.5 and RT primer, the F end primer of SEQ ID NO.6 and RT primer, the F end primer of SEQ ID NO.7 and RT draw The F end primer of thing, the F end primer of SEQ ID NO.8 and RT primer and SEQ ID NO.9 and RT primer.
Further, described PCR detection specific primer is in order to clinical diagnosises tumor platelet RNA biomarker group Close, described tumor platelet RNA biomarker combinations include following platelet RNA：
CD79A(ENSG00000105369),CD81(ENSG00000110651),SYTL1(ENSG00000142765),CENPC (ENSG00000145241),TTN(ENSG00000155657),RHOH(ENSG00000168421),ZNF101 (ENSG00000181896), TRABD2A (ENSG00000186854) and TRAC (ENSG00000229164).
Further, described tumor platelet RNA biomarker combinations are carried out according to below equation coefficient when using Distribute, described formula is：
Y=0.0148108×ACD79A(ENSG00000105369)+0.07848907× BCD81(ENSG00000110651)+ 0.1531495×CSYTL1(ENSG00000142765)+ 0.08874548×DCENPC(ENSG00000145241)+ 0.05914998×ETTN(ENSG00000155657) + 0.22891517×FRHOH(ENSG00000168421) + 0.5210493×GZNF101(ENSG00000181896) + 0.05066299× HTRABD2A(ENSG00000186854)+ 0.31206518×ITRAC(ENSG00000229164)；Wherein,
ACD79A (ENSG00000105369) is expression (2 in tumor platelet for the CD79A (ENSG00000105369)ΔCtValue),
BCD81 (ENSG00000110651) is expression (2 in tumor platelet for the CD81 (ENSG00000110651)ΔCt Value),
CSYTL1 (ENSG00000142765) is expression (2 in tumor platelet for the SYTL1 (ENSG00000142765)ΔCtValue),
DCENPC (ENSG00000145241) is expression (2 in tumor platelet for the CENPC (ENSG00000145241)ΔCtValue),
ETTN (ENSG00000155657) is expression (2 in tumor platelet for the TTN (ENSG00000155657)ΔCt Value),
FRHOH (ENSG00000168421) is expression (2 in tumor platelet for the RHOH (ENSG00000168421)ΔCt Value),
GZNF101 (ENSG00000181896) is expression (2 in tumor platelet for the ZNF101 (ENSG00000181896)ΔCtValue),
HTRABD2A (ENSG00000186854) is expression in tumor platelet for the TRABD2A (ENSG00000186854) (2ΔCtValue)
ITRAC (ENSG00000229164) is expression (2 in tumor platelet for the TRAC (ENSG00000229164)ΔCt Value).
Present invention additionally comprises a kind of tumor platelet RNA quantitative detecting method for tumor early screening, the method bag Include following steps：
1) prepare sample：Prepare human plasma sample to be measured, using human normal plasma sample as normal control；
2) extract RNA：Extract platelet total serum IgE；
3) reverse transcription：Platelet total serum IgE reverse transcription is become cDNA；
4) PCR detection：With cDNA as template, detect specific primer and real-time quantitative chimeric fluorescent method PCR detectable in PCR Carry out Real-Time Fluorescent Quantitative PCR Technique detection SEQ ID NO.1 SEQ ID NO.9 little in human blood to be measured under the use in conjunction of box Expression in plate and healthy human blood platelets, and adopt 2ΔCtRepresent that in sample to be tested, purpose RNA expression is with respect to house keeper The multiple that gene GAPDH (ENSG00000111640) changes；
5) calculate Y value with formula：
Y=0.0148108×ACD79A(ENSG00000105369)+0.07848907× BCD81(ENSG00000110651)+ 0.1531495×CSYTL1(ENSG00000142765)+ 0.08874548×DCENPC(ENSG00000145241)+ 0.05914998×ETTN(ENSG00000155657) + 0.22891517×FRHOH(ENSG00000168421) + 0.5210493×GZNF101(ENSG00000181896) + 0.05066299× HTRABD2A(ENSG00000186854)+ 0.31206518×ITRAC(ENSG00000229164)；Wherein,
ACD79A (ENSG00000105369) is expression (2 in tumor platelet for the CD79A (ENSG00000105369)ΔCtValue),
BCD81 (ENSG00000110651) is expression (2 in tumor platelet for the CD81 (ENSG00000110651)ΔCt Value),
CSYTL1 (ENSG00000142765) is expression (2 in tumor platelet for the SYTL1 (ENSG00000142765)ΔCtValue),
DCENPC (ENSG00000145241) is expression (2 in tumor platelet for the CENPC (ENSG00000145241)ΔCtValue),
ETTN (ENSG00000155657) is expression (2 in tumor platelet for the TTN (ENSG00000155657)ΔCt Value),
FRHOH (ENSG00000168421) is expression (2 in tumor platelet for the RHOH (ENSG00000168421)ΔCt Value),
GZNF101 (ENSG00000181896) is expression (2 in tumor platelet for the ZNF101 (ENSG00000181896)ΔCtValue),
HTRABD2A (ENSG00000186854) is expression in tumor platelet for the TRABD2A (ENSG00000186854) (2ΔCtValue)
ITRAC (ENSG00000229164) is expression (2 in tumor platelet for the TRAC (ENSG00000229164)ΔCt Value)；
ITRAC (ENSG00000229164) is TRAC (expression (2 in platelet for the ENSG00000229164ΔCtValue), Δ Ct value reference gene (GAPDH) Average Ct values of Ct=genes of interest, genes of interest Ct value is to use real-time fluorescence quantitative PCR Purpose CD79A (ENSG00000105369) that technology for detection arrives, CD81 (ENSG00000110651), SYTL1 (ENSG00000142765),CENPC(ENSG00000145241),TTN(ENSG00000155657),RHOH (ENSG00000168421), ZNF101 (ENSG00000181896), TRABD2A (ENSG00000186854) and TRAC (ENSG00000229164) the Ct value of gene；
6) result is passed judgment on：It is judged to positive (suffering from tumor) when Y value is less than 5.28, otherwise be negative (not suffering from tumor).
Based on technique scheme, compared to prior art, there is advantages below：
The present invention acts not only as early diagnosis of tumor and the biomarker combinations of risk assessment, and detection can obtain good Good tumors, can realize tumor early screening, adjuvant therapy pathological identification and clinical diagnosises simultaneously, improve patient Survival rate.
Brief description
Fig. 1 is the workflow diagram of the present invention.
Fig. 2 is the ROC curve figure of tumor platelet RNA biomarker combinations joint-detection in the present invention.
Fig. 3 is the Y value scattergram of tumor patient and Healthy People.
Specific embodiment
Tumor platelet RNA carrying out the present invention is used for tumor early screening below in conjunction with the accompanying drawings with specific embodiment is fixed Amount detection model and method are done and are further elaborated, in the hope of providing a clearer understanding of its structure type and user Formula, but the protection domain of patent of the present invention can not be limited with this.
A kind of tumor platelet RNA detection by quantitative model for tumor early screening, it is special that this model includes PCR detection Property primer, described PCR detection specific primer includes the F end primer of SEQ ID NO.1 and the F end of RT primer, SEQ ID NO.2 Primer and RT primer, the F end primer of SEQ ID NO.3 and RT primer, the F end primer of SEQ ID NO.4 and RT primer, SEQ ID The F end primer of NO.5 and RT primer, the F end primer of SEQ ID NO.6 and RT primer, the F end primer of SEQ ID NO.7 and RT draw The F end primer of thing, the F end primer of SEQ ID NO.8 and RT primer and SEQ ID NO.9 and RT primer.
Described PCR detection specific primer is in order to clinical diagnosises tumor platelet RNA biomarker combinations, described swollen Tumor platelet RNA biomarker combinations include following blood plasma：
CD79A(ENSG00000105369),CD81(ENSG00000110651),SYTL1(ENSG00000142765),CENPC (ENSG00000145241),TTN(ENSG00000155657),RHOH(ENSG00000168421),ZNF101 (ENSG00000181896), TRABD2A (ENSG00000186854) and TRAC (ENSG00000229164).
Described tumor platelet RNA biomarker combinations are allocated according to below equation coefficient when using, described Formula is：
Y=0.0148108×ACD79A(ENSG00000105369)+0.07848907× BCD81(ENSG00000110651)+ 0.1531495×CSYTL1(ENSG00000142765)+ 0.08874548×DCENPC(ENSG00000145241)+ 0.05914998×ETTN(ENSG00000155657) + 0.22891517×FRHOH(ENSG00000168421) + 0.5210493×GZNF101(ENSG00000181896) + 0.05066299× HTRABD2A(ENSG00000186854)+ 0.31206518×ITRAC(ENSG00000229164)；Wherein,
ACD79A (ENSG00000105369) is expression (2 in tumor platelet for the CD79A (ENSG00000105369)ΔCtValue),
BCD81 (ENSG00000110651) is expression (2 in tumor platelet for the CD81 (ENSG00000110651)ΔCt Value),
CSYTL1 (ENSG00000142765) is expression (2 in tumor platelet for the SYTL1 (ENSG00000142765)ΔCtValue),
DCENPC (ENSG00000145241) is expression (2 in tumor platelet for the CENPC (ENSG00000145241)ΔCtValue),
ETTN (ENSG00000155657) is expression (2 in tumor platelet for the TTN (ENSG00000155657)ΔCt Value),
FRHOH (ENSG00000168421) is expression (2 in tumor platelet for the RHOH (ENSG00000168421)ΔCt Value),
GZNF101 (ENSG00000181896) is expression (2 in tumor platelet for the ZNF101 (ENSG00000181896)ΔCtValue),
HTRABD2A (ENSG00000186854) is expression in tumor platelet for the TRABD2A (ENSG00000186854) (2ΔCtValue)
ITRAC (ENSG00000229164) is expression (2 in tumor platelet for the TRAC (ENSG00000229164)ΔCt Value).
The SEQ-ID of RNA described in table 1 and its forward and reverse primer
SEQ-ID Gene name Forward primer Reverse primer
1 CD79A(ENSG00000105369) CATTGATGGTGAGCCTGGG TCGGCTGTGATGATTCGGT
2 CD81(ENSG00000110651) GTATCTGGAGCTGGGAGACAA TTGGCGATCTGGTCCTTGTT
3 SYTL1(ENSG00000142765) TCTCTCGACCGCATGCTCA TCGTAGTGCAGCGCGAAGT
4 CENPC(ENSG00000145241) TCCGGTTTTCAACGAGACTCT TTCAACTTCGCCCAGAAAGA
5 TTN(ENSG00000155657) CCTTCGAATTCCGCCTAAAAT TTTTCAATGTGGAACCTCCC
6 RHOH(ENSG00000168421) TTTCTTCGGCATTCTGCAAC CCTCCAAAGCCTAGTCTTCAA
7 ZNF101(ENSG00000181896) TGCTGGACACAAACGATCTGA TTGGTGTTACTGTGCGCCGT
8 TRABD2A(ENSG00000186854) TGCTCCCCAGGGACATCTACT TCCGGCAATAGCATTGAAGA
9 TRAC(ENSG00000229164) TCAGCGATTCAGCCTCCTA TCAGGCCAGACAGTCAACTGA
As shown in figure 1, a kind of tumor platelet RNA quantitative detecting method for tumor early screening, the method includes following Step：
1) prepare sample：Prepare human plasma sample to be measured, using human normal plasma sample as normal control；
2) extract RNA：Extract platelet total serum IgE；
3) reverse transcription：Platelet total serum IgE reverse transcription is become cDNA；
4) PCR detection：With cDNA as template, detect specific primer and real-time quantitative chimeric fluorescent method PCR detectable in PCR Carry out Real-Time Fluorescent Quantitative PCR Technique detection SEQ ID NO.1 SEQ ID NO.9 little in human blood to be measured under the use in conjunction of box Expression in plate and healthy human blood platelets, and adopt 2ΔCtRepresent that in sample to be tested, purpose RNA expression is with respect to house keeper The multiple that gene GAPDH (ENSG00000111640) changes；
5) calculate Y value with formula：
Y=0.0148108×ACD79A(ENSG00000105369)+0.07848907× BCD81(ENSG00000110651)+ 0.1531495×CSYTL1(ENSG00000142765)+ 0.08874548×DCENPC(ENSG00000145241)+ 0.05914998×ETTN(ENSG00000155657) + 0.22891517×FRHOH(ENSG00000168421) + 0.5210493×GZNF101(ENSG00000181896) + 0.05066299× HTRABD2A(ENSG00000186854)+ 0.31206518×ITRAC(ENSG00000229164)；Wherein,
ACD79A (ENSG00000105369) is expression (2 in tumor platelet for the CD79A (ENSG00000105369)ΔCtValue),
BCD81 (ENSG00000110651) is expression (2 in tumor platelet for the CD81 (ENSG00000110651)ΔCt Value),
CSYTL1 (ENSG00000142765) is expression (2 in tumor platelet for the SYTL1 (ENSG00000142765)ΔCtValue),
DCENPC (ENSG00000145241) is expression (2 in tumor platelet for the CENPC (ENSG00000145241)ΔCtValue),
ETTN (ENSG00000155657) is expression (2 in tumor platelet for the TTN (ENSG00000155657)ΔCt Value),
FRHOH (ENSG00000168421) is expression (2 in tumor platelet for the RHOH (ENSG00000168421)ΔCt Value),
GZNF101 (ENSG00000181896) is expression (2 in tumor platelet for the ZNF101 (ENSG00000181896)ΔCtValue),
HTRABD2A (ENSG00000186854) is expression in tumor platelet for the TRABD2A (ENSG00000186854) (2ΔCtValue)
ITRAC (ENSG00000229164) is expression (2 in tumor platelet for the TRAC (ENSG00000229164)ΔCt Value)；
ITRAC (ENSG00000229164) is TRAC (expression (2 in platelet for the ENSG00000229164ΔCtValue), Δ Ct value reference gene (GAPDH) Average Ct values of Ct=genes of interest, genes of interest Ct value is to use real-time fluorescence quantitative PCR Purpose CD79A (ENSG00000105369) that technology for detection arrives, CD81 (ENSG00000110651), SYTL1 (ENSG00000142765),CENPC(ENSG00000145241),TTN(ENSG00000155657),RHOH (ENSG00000168421), ZNF101 (ENSG00000181896), TRABD2A (ENSG00000186854) and TRAC (ENSG00000229164) the Ct value of gene；
6) result is passed judgment on：It is judged to positive (suffering from tumor) when Y value is less than 5.28, otherwise be negative (not suffering from tumor).
Embodiment
Using above-mentioned tumor platelet RNA diagnosing tumor detection method to 50 tumor patients and 20 Healthy Peoples (just Often compareing) expression of purpose tumor platelet RNA is detected in blood plasma, i.e. CD79A (ENSG00000105369), CD81 (ENSG00000110651),SYTL1(ENSG00000142765),CENPC(ENSG00000145241),TTN (ENSG00000155657),RHOH(ENSG00000168421),ZNF101(ENSG00000181896),TRABD2A (ENSG00000186854), the expression (2 of TRAC (ENSG00000229164)△CtValue).
Concrete detection method, comprises the following steps:
1. the collection of tumor platelet RNA and preservation
Experimenter venous blood 4ml is in EDTA anticoagulant tube for collection, gently overturns and stands 10min for several times afterwards（In 1h under room temperature environment Interior centrifugal separation plasma）, it is centrifuged 20min in room temperature 120g, point two-layer, draw upper plasma（Clear pale yellow color liquid, blood fat is high Sample be milkiness sample）, every 400 μ l are transferred in a new no RNAse EP pipe, -80 DEG C of preservations.
The blood plasma of preservation is placed in and dissolves on ice, under room temperature, 360g centrifugation 20min, draws upper plasma（I.e. platelet） 300 μ l are simultaneously transferred to the RNAlater containing 30 μ l（Life Technologies）Guan Zhong, 4 DEG C of overnight incubation.Use mirVana RNA extracts kit (Life Technologies) by specification extracts.After lower floor blood plasma 5000rmp centrifugation 10min, -80 ° C is frozen stand-by, and method is summarized as follows：
(1) blood plasma is mixed with equal-volume 2X Denaturing Solution, ice-water bath is incubated 5min.
(2) add and the isopyknic Acid-Phenol of mixing liquid:Chloroform, the 30～60s that is vortexed mixes, 12000r/min is centrifuged 20min, divides three layers, draws upper transparent liquid 500 μ l, is transferred in new no RNAse EP pipe.
(3) add 100% ethanol of 1.25 times of volumes, transfer in centrifugal column after mixing（≤700μl）And it is loaded into collection Guan Shang, 4000r/min are centrifuged 30s, abandon collection liquid, repeating this step makes all liq pass through centrifugal column.
(4) add washing liquid 1700 μ l on centrifugal column, stand 1min, be centrifuged 15s.
(5) add washing liquid 2/3500 μ l on centrifugal column, stand 1min, be centrifuged 15s, be repeated once.
(6) add the Elution Solution 50 μ l after heat shock on centrifugal column, stand 1min, be centrifuged 1min, collect Centrifugal liquid.
(7) RNA 6000 Picochip (Agilent) is measured to the concentration of RNA and quality.
2.cDNA synthesizes
The standard operating procedure specifying according to test kit operating instruction, carries out reverse transcription to the RNA sample being extracted, and obtains cDNA；
Take in clean EP pipe plus 1 μ L random 6mers, 1 μ L dNTP, 7 μ L RNase free dH2O,1μL Temp RNA, Blow even, put in PCR instrument, setting program：65℃,5min；Again plus 4 μ L 5*buffer, 0.5 μ L RNase inhibitor, 1 μL RTase,4.5μL RNase free dH2O, blows even, puts in PCR instrument, setting program：30℃,10min 42℃, 50min 95℃,5min.
3.RT-QPCR
Using SYBR@Premix Ex TaqTM (the Tli RNaseH Plus) test kit of Takara company, according to test kit The standard operating procedure of operating instruction defined：30 μ L systems, 15 μ L SYBR MIX+0.6 μ L primer-R+11.8 μ L ddH2O+2 μ L cDNA, standard two-step method response procedures are：60 DEG C of 95 DEG C of denaturation 10min 95 DEG C of 15s of 40 cycles 1min；Afterwards according to 2△CtMethod calculate in a organized way in 9 entries RNA and internal reference mRNA GAPDH expression.
4. interpretation of result
As shown in Figures 2 and 3, wherein, enumerate above-mentioned rna gene joint-detection and apply in personal diagnosing tumor, its nine The expression of RNA is respectively：
CD79A(ENSG00000105369)=4.367212,CD81(ENSG00000110651)=3.154837,SYTL1 (ENSG00000142765)=7.553681,CENPC(ENSG00000145241)=6.658126,TTN (ENSG00000155657)=6.224972,RHOH(ENSG00000168421)=5.448671, ZNF101 (ENSG00000181896)=5.385524,TRABD2A(ENSG00000186854)=3.559716,TRAC (ENSG00000229164)= 6.235487.Can be obtained using sharp Y value computing formula：Y=8.6155 are more than 5.28, then show this Volunteer does not suffer from tumor.
Analyze the Combining diagnosis efficiency of RNA by tested worker's curve (ROC), using formula Y=0.0148108 × ACD79A(ENSG00000105369)+ 0.07848907 × BCD81(ENSG00000110651)+ 0.1531495 × CSYTL1(ENSG00000142765)+ 0.08874548 × DCENPC(ENSG00000145241)+ 0.05914998 × ETTN(ENSG00000155657)+ 0.22891517 × FRHOH(ENSG00000168421)+ 0.5210493 × GZNF101(ENSG00000181896)+ 0.05066299 × HTRABD2A(ENSG00000186854)+ 0.31206518 × ITRAC(ENSG00000229164)The ROC curve that 9 RNA detection datas of matching obtain is as shown in Fig. 2 ROC area under curve is 92.5%, diagnostic is high.
Using 2△CtTo quantitative method analytical data, Logistic regression equation (judgment formula)：Y= 0.0148108 × ACD79A(ENSG00000105369)+ 0.07848907 × BCD81(ENSG00000110651)+ 0.1531495 × CSYTL1(ENSG00000142765)+ 0.08874548 × DCENPC(ENSG00000145241)+ 0.05914998 × ETTN(ENSG00000155657)+ 0.22891517 × FRHOH(ENSG00000168421)+ 0.5210493 × GZNF101(ENSG00000181896)+ 0.05066299 × HTRABD2A(ENSG00000186854)+ 0.31206518 × ITRAC(ENSG00000229164), wherein ACD79A(ENSG00000105369)It is expression in blood plasma for the CD79A (ENSG00000105369) (2ΔCtValue), BCD81(ENSG00000110651)It is expression (2 in blood plasma for the CD81 (ENSG00000110651)ΔCtValue), CSYTL1(ENSG00000142765)It is expression (2 in blood plasma for the SYTL1 (ENSG00000142765)ΔCtValue), DCENPC(ENSG00000145241)It is expression (2 in blood plasma for the CENPC (ENSG00000145241)ΔCtValue), ETTN(ENSG00000155657)It is expression (2 in blood plasma for the TTN (ENSG00000155657)ΔCtValue), FRHOH(ENSG00000168421) It is expression (2 in blood plasma for the RHOH (ENSG00000168421)ΔCtValue), GZNF101(ENSG00000181896)It is ZNF101 (ENSG00000181896) expression (2 in blood plasmaΔCtValue), HTRABD2A(ENSG00000186854)It is TRABD2A (ENSG00000186854) expression (2 in blood plasmaΔCtValue), ITRAC(ENSG00000229164)It is TRAC (expression (2 in blood plasma for the ENSG00000229164ΔCtValue), Δ Ct=genes of interest Ct value reference gene Ct value, purpose Gene C t value refers to the Ct value of the genes of interest that real-time fluorescence quantitative PCR technology for detection arrives, and genes of interest refers to CD79A (ENSG00000105369),CD81(ENSG00000110651),SYTL1(ENSG00000142765),CENPC (ENSG00000145241),TTN(ENSG00000155657),RHOH(ENSG00000168421),ZNF101 (ENSG00000181896), TRABD2A (ENSG00000186854) and TRAC (ENSG00000229164).50 are swollen as a result The Y value of tumor patient and 20 Healthy Peoples (normal control) is as shown in table 2 it is seen that the Y value of 71% tumor patient is respectively less than 5.28. Therefore, work as Y<When 5.28, i.e. prediction probability value<Just it is judged to when 5.28 positive (suffering from tumor), otherwise be negative (being not suffering from tumor), Prove the joint-detection of said gene can be obtained good tumors, therefore CD79A (ENSG00000105369), CD81(ENSG00000110651),SYTL1(ENSG00000142765),CENPC(ENSG00000145241),TTN (ENSG00000155657),RHOH(ENSG00000168421),ZNF101(ENSG00000181896),TRABD2A (ENSG00000186854), TRAC (ENSG00000229164) combination can be assessed as early diagnosis of tumor and risk Biomarker.
2 50 tumor patients of table and 20 Healthy Peoples（Normal control）Y value
Numbering Scoring (Y value) Group
1 2.428121 Tumor patient
2 2.48863 Tumor patient
3 2.503916 Tumor patient
4 2.799115 Tumor patient
5 2.849653 Tumor patient
6 2.97098 Tumor patient
7 2.997302 Tumor patient
8 3.065475 Tumor patient
9 3.170404 Tumor patient
10 3.296256 Tumor patient
11 3.323515 Tumor patient
12 3.352802 Tumor patient
13 3.356327 Healthy People
14 3.379242 Tumor patient
15 3.406207 Tumor patient
16 3.468006 Tumor patient
17 3.489337 Tumor patient
18 3.500599 Tumor patient
19 3.502231 Tumor patient
20 3.580153 Tumor patient
21 3.598405 Tumor patient
22 3.611938 Tumor patient
23 3.716721 Tumor patient
24 3.781669 Tumor patient
25 3.852145 Tumor patient
26 3.857838 Tumor patient
27 3.919833 Tumor patient
28 3.932668 Healthy People
29 3.941747 Tumor patient
30 3.985031 Tumor patient
31 4.092127 Tumor patient
32 4.096202 Tumor patient
33 4.106992 Tumor patient
34 4.141384 Tumor patient
35 4.14155 Tumor patient
36 4.365938 Tumor patient
37 4.375088 Tumor patient
38 4.516429 Tumor patient
39 4.564401 Tumor patient
40 4.588491 Tumor patient
41 4.633284 Tumor patient
42 4.7872 Tumor patient
43 4.793377 Tumor patient
44 4.79555 Tumor patient
45 4.815989 Tumor patient
46 4.869563 Tumor patient
47 4.933796 Tumor patient
48 5.046824 Tumor patient
49 5.211091 Tumor patient
50 5.256569 Tumor patient
51 5.285875 Healthy People
52 6.790469 Healthy People
53 6.876531 Healthy People
54 7.083108 Tumor patient
55 7.151574 Healthy People
56 7.553954 Healthy People
57 7.56079 Healthy People
58 7.772101 Healthy People
59 7.80082 Healthy People
60 8.036599 Healthy People
61 8.310503 Healthy People
62 8.670087 Tumor patient
63 9.05824 Healthy People
64 9.270311 Healthy People
65 9.615263 Healthy People
66 9.858009 Healthy People
67 9.939603 Healthy People
68 10.3842 Healthy People
69 10.6205 Healthy People
70 10.88067 Healthy People
Clinical at present conventional biomarker such as CEA etc. is only 4.7～20.8% for the sensitivity of diagnosing tumor, even if connection Close using sensitivity during other mark also only have 69. 1 % (He CZ, Zhang KH, Li Q, etal.Combined use of AFP, CEA, CA125 and CA19-9 improves the sensitivity for thediagnosis of gastric cancer.BMC Gastroenterol 2013.13:87.).Data above shows, makes 92.5% can be reached with the sensitivity of RNA joint mark diagnosing tumour, sensitive higher than current clinical conventional biomarker Degree.Deviation in the data being detected by the method despite 2 tumor patients and 2 normal persons, but heterogeneous in view of tumor Property and individuality between difference (as clinic in often have following situation：Normal person's CEA value is higher than upper limits of normal, tumor patient CEA value is but within normal range), this result meets clinical medicine rule, shows mark combinatorial association screening of the present invention Method is true, effective, can be accepted and implement.
Certainly, the present invention be used for the tumor platelet RNA detection by quantitative model of tumor early screening and method except Beyond the type told about in above-described embodiment and mode, also include other similar structure composition modes and occupation mode.Total and Yan Zhi, present invention additionally comprises other conversion that will be apparent to those skilled in the art and replacement.
<110>Shanghai Hou Cheng medical science and technology company limited
<120>Tumor platelet RNA detection by quantitative model for tumor early screening and method
<130>（Numbering：）
<160>10
<210> 1
<211> 19
<212> DNA
<213>CD79A
<214>Synthetic
<220>Forward primer
<400> 1
CATTGATGGTGAGCCTGGG
<220>Reverse primer
TCGGCTGTGATGATTCGGT
<210> 2
<211> 21
<212> DNA
<213> CD81
<214>Synthetic
<220>Forward primer
<400> 2
GTATCTGGAGCTGGGAGACAA
<220>Reverse primer
TTGGCGATCTGGTCCTTGTT
<210> 3
<211> 19
<212> DNA
<213> SYTL1
<214>Synthetic
<220>Forward primer
<400> 3
TCTCTCGACCGCATGCTCA
<220>Reverse primer
TCGTAGTGCAGCGCGAAGT
<210> 4
<211> 21
<212> DNA
<213> CENPC
<214>Synthetic
<220>Forward primer
<400> 4
TCCGGTTTTCAACGAGACTCT
<220>Reverse primer
TTCAACTTCGCCCAGAAAGA
<210> 5
<211> 21
<212> DNA
<213> TTN
<214>Synthetic
<220>Forward primer
<400> 5
CCTTCGAATTCCGCCTAAAAT
<220>Reverse primer
TTTTCAATGTGGAACCTCCC
<210> 6
<211> 20
<212> DNA
<213> RHOH
<214>Synthetic
<220>Forward primer
<400> 6
TTTCTTCGGCATTCTGCAAC
<220>Reverse primer
CCTCCAAAGCCTAGTCTTCAA
<210> 7
<211> 21
<212> DNA
<213> ZNF101
<214>Synthetic
<220>Forward primer
<400> 7
TGCTGGACACAAACGATCTGA
<220>Reverse primer
TTGGTGTTACTGTGCGCCGT
<210> 8
<211> 21
<212> DNA
<213> TRABD2A
<214>Synthetic
<220>Forward primer
<400> 8
TGCTCCCCAGGGACATCTACT
<220>Reverse primer
TCCGGCAATAGCATTGAAGA
<210> 9
<211> 19
<212> DNA
<213> TRAC
<214>Synthetic
<220>Forward primer
<400> 9
TCAGCGATTCAGCCTCCTA
<220>Reverse primer
TCAGGCCAGACAGTCAACTGA

Claims (4)

1. a kind of tumor platelet RNA detection by quantitative model for tumor early screening is it is characterised in that this model includes PCR detects specific primer, and described PCR detects that specific primer includes F end primer and RT primer, the SEQ of SEQ ID NO.1 The F end primer of ID NO.2 and RT primer, the F end primer of SEQ ID NO.3 and RT primer, the F end primer of SEQ ID NO.4 and RT primer, the F end primer of SEQ ID NO.5 and RT primer, the F end primer of SEQ ID NO.6 and RT primer, SEQ ID NO.7 F end primer and the F end primer of RT primer, the F end primer of SEQ ID NO.8 and RT primer and SEQ ID NO.9 and RT draw Thing.
2. the tumor platelet RNA detection by quantitative model for tumor early screening according to claim 1, its feature exists In, in order to clinical diagnosises tumor platelet RNA biomarker combinations, described tumor blood is little for described PCR detection specific primer Plate RNA biomarker combinations include following platelet RNA：
CD79A(ENSG00000105369),CD81(ENSG00000110651),SYTL1(ENSG00000142765),CENPC (ENSG00000145241),TTN(ENSG00000155657),RHOH(ENSG00000168421),ZNF101 (ENSG00000181896), TRABD2A (ENSG00000186854) and TRAC (ENSG00000229164).
3. the tumor platelet RNA detection by quantitative model for tumor early screening according to claim 2, its feature It is, described tumor platelet RNA biomarker combinations are allocated according to below equation coefficient when using, described public affairs Formula is：
Y=0.0148108×ACD79A(ENSG00000105369)+0.07848907× BCD81(ENSG00000110651)+ 0.1531495×CSYTL1(ENSG00000142765)+ 0.08874548×DCENPC(ENSG00000145241)+ 0.05914998×ETTN(ENSG00000155657) + 0.22891517×FRHOH(ENSG00000168421) + 0.5210493×GZNF101(ENSG00000181896) + 0.05066299× HTRABD2A(ENSG00000186854)+ 0.31206518×ITRAC(ENSG00000229164)；Wherein,
ACD79A (ENSG00000105369) is expression (2 in tumor platelet for the CD79A (ENSG00000105369)ΔCtValue),
BCD81 (ENSG00000110651) is expression (2 in tumor platelet for the CD81 (ENSG00000110651)ΔCt Value),
CSYTL1 (ENSG00000142765) is expression (2 in tumor platelet for the SYTL1 (ENSG00000142765)ΔCt Value),
DCENPC (ENSG00000145241) is expression (2 in tumor platelet for the CENPC (ENSG00000145241)ΔCt Value),
ETTN (ENSG00000155657) is expression (2 in tumor platelet for the TTN (ENSG00000155657)ΔCtValue),
FRHOH (ENSG00000168421) is expression (2 in tumor platelet for the RHOH (ENSG00000168421)ΔCt Value),
GZNF101 (ENSG00000181896) is expression (2 in tumor platelet for the ZNF101 (ENSG00000181896)ΔCtValue),
HTRABD2A (ENSG00000186854) is expression in tumor platelet for the TRABD2A (ENSG00000186854) (2ΔCtValue)
ITRAC (ENSG00000229164) is expression (2 in tumor platelet for the TRAC (ENSG00000229164)ΔCt Value).
4. a kind of tumor platelet RNA quantitative detecting method for tumor early screening it is characterised in that the method include with Lower step：
1) prepare sample：Prepare human plasma sample to be measured, using human normal plasma sample as normal control；
2) extract RNA：Extract platelet total serum IgE；
3) reverse transcription：Platelet total serum IgE reverse transcription is become cDNA；
4) PCR detection：With cDNA as template, detect specific primer and real-time quantitative chimeric fluorescent method PCR detectable in PCR Carry out Real-Time Fluorescent Quantitative PCR Technique detection SEQ ID NO.1 SEQ ID NO.9 little in human blood to be measured under the use in conjunction of box Expression in plate and healthy human blood platelets, and adopt 2ΔCtRepresent that in sample to be tested, purpose RNA expression is with respect to house keeper The multiple that gene GAPDH (ENSG00000111640) changes；
5) calculate Y value with formula：
Y=0.0148108×ACD79A(ENSG00000105369)+0.07848907× BCD81(ENSG00000110651)+ 0.1531495×CSYTL1(ENSG00000142765)+ 0.08874548×DCENPC(ENSG00000145241)+ 0.05914998×ETTN(ENSG00000155657) + 0.22891517×FRHOH(ENSG00000168421) + 0.5210493×GZNF101(ENSG00000181896) + 0.05066299× HTRABD2A(ENSG00000186854)+ 0.31206518×ITRAC(ENSG00000229164)；Wherein,
ACD79A (ENSG00000105369) is expression (2 in tumor platelet for the CD79A (ENSG00000105369)ΔCtValue),
BCD81 (ENSG00000110651) is expression (2 in tumor platelet for the CD81 (ENSG00000110651)ΔCt Value),
CSYTL1 (ENSG00000142765) is expression (2 in tumor platelet for the SYTL1 (ENSG00000142765)ΔCt Value),
DCENPC (ENSG00000145241) is expression (2 in tumor platelet for the CENPC (ENSG00000145241)ΔCt Value),
ETTN (ENSG00000155657) is expression (2 in tumor platelet for the TTN (ENSG00000155657)ΔCtValue),
FRHOH (ENSG00000168421) is expression (2 in tumor platelet for the RHOH (ENSG00000168421)ΔCt Value),
GZNF101 (ENSG00000181896) is expression (2 in tumor platelet for the ZNF101 (ENSG00000181896)ΔCtValue),
HTRABD2A (ENSG00000186854) is expression in tumor platelet for the TRABD2A (ENSG00000186854) (2ΔCtValue)
ITRAC (ENSG00000229164) is expression (2 in tumor platelet for the TRAC (ENSG00000229164)ΔCt Value)；
ITRAC (ENSG00000229164) is TRAC (expression (2 in platelet for the ENSG00000229164ΔCtValue), Δ Ct value reference gene (GAPDH) Average Ct values of Ct=genes of interest, genes of interest Ct value is to use real-time fluorescence quantitative PCR Purpose CD79A (ENSG00000105369) that technology for detection arrives, CD81 (ENSG00000110651), SYTL1 (ENSG00000142765),CENPC(ENSG00000145241),TTN(ENSG00000155657),RHOH (ENSG00000168421), ZNF101 (ENSG00000181896), TRABD2A (ENSG00000186854) and TRAC (ENSG00000229164) the Ct value of gene；
6) result is passed judgment on：It is judged to positive (suffering from tumor) when Y value is less than 5.28, otherwise be negative (not suffering from tumor).
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