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

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 quantitative detection model and method for early tumor screening

Technical Field

The invention relates to the fields of molecular biology and medicine, in particular to a tumor platelet RNA quantitative detection model and a method for early tumor screening.

Background

With the increase of cancer morbidity and mortality, cancer is not only a leading cause of death of Chinese people, but also a significant public health problem that seriously threatens human health and restricts the development of economic society. Early diagnosis of tumors means that early intervention and treatment can be achieved, and is of great significance to the prognosis of patients. At present, the diagnosis of tumors mainly depends on the aspects of traditional clinical signs of tumor tissues, radiographic images, biochemical detection, pathology and the like, and most of the methods are invasive, bring pain to patients and have higher requirements on doctors implementing the technology. The non-invasive detection method for the tumor is mainly serum tumor markers, such as AFP, carcinoembryonic antigen CEA, CA199 and the like, but the sensitivity and the specificity of diagnosis are low. Therefore, the search for tumor markers with higher sensitivity and specificity is of great significance to early diagnosis and early treatment tools for tumor patients.

To reduce the limitations of tumor tissue procurement, blood-based liquid biopsy methods for tissue sample analysis have become a popular trend in recent years, and currently mainly involve the detection of plasma free dna (cfdna) and Circulating Tumor Cells (CTCs). To date, liquid biopsy methods for early screening of tumors have not been fully developed because molecules in the blood do not specifically characterize the primary tumor. Recent studies have shown that detection of tumor-associated platelets (tumor-associated platelets) may make tumor blood-based cancer diagnosis more efficient. Platelets, the second most abundant cell type in peripheral blood, are derived from the circulating anucleated fragments of bone marrow megakaryocytes, and are characterized by hemostasis and wound healing promotion. In the prior art, no corresponding mature technology utilizes tumor-related platelets to diagnose tumors more accurately.

Disclosure of Invention

In order to solve the defects in the prior art, the invention provides a tumor platelet RNA quantitative detection model and a method for early tumor screening, which can be used as a biomarker combination for early tumor diagnosis and risk assessment, can obtain good tumor diagnosis value by detection, can realize early tumor screening, auxiliary tumor pathological identification and clinical diagnosis, and improve the survival rate of patients.

In order to achieve the above object, the present invention provides the following technical solutions:

a tumor platelet RNA quantitative detection model for early tumor screening comprises PCR detection specific primers, wherein the PCR detection specific primers comprise an F-terminal primer and an RT primer of SEQ ID NO.1, an F-terminal primer and an RT primer of SEQ ID NO.2, an F-terminal primer and an RT primer of SEQ ID NO.3, an F-terminal primer and an RT primer of SEQ ID NO.4, an F-terminal primer and an RT primer of SEQ ID NO.5, an F-terminal primer and an RT primer of SEQ ID NO.6, an F-terminal primer and an RT primer of SEQ ID NO.7, an F-terminal primer and an RT primer of SEQ ID NO.8 and an F-terminal primer and an RT primer of SEQ ID NO. 9.

Further, the PCR detection specific primers are used for clinically diagnosing a tumor platelet RNA biomarker combination, and the tumor platelet RNA biomarker combination comprises the following platelet RNAs:

CD79A (ENSG00000105369), CD81(ENSG00000110651), SYTL1(ENSG00000142765), CENPC (ENSG00000145241), TTN (ENSG00000155657), RHOH (ENSG00000168421), ZNF101(ENSG00000181896), TRABD2A (ENSG00000186854) and TRAC (ENSG 00000229164).

Further, the tumor platelet RNA biomarker combinations are assigned, when used, according to the following formula coefficients:

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 (ENSG 000086854) +0.31206518 × ITRAC (ENSG 00009102264); wherein,

ACD79A (ENSG00000105369) is the expression level of CD79A (ENSG00000105369) in tumor platelets (2)^ΔCtA value),

BCD81(ENSG00000110651) is the expression level of CD81(ENSG00000110651) in tumor platelets (2)^ΔCtA value),

CSYTL1(ENSG00000142765) is the expression level of SYTL1(ENSG00000142765) in tumor platelets (2)^ΔCtA value),

DCENPC (ENSG00000145241) is the expression level of CENPC (ENSG00000145241) in tumor platelets (2)^ΔCtA value),

ETTN (ENSG00000155657) is the expression level of TTN (ENSG00000155657) in tumor platelets (2)^ΔCtA value),

FRHOH (ENSG00000168421) is the expression level of RHOH (ENSG00000168421) in tumor platelets (2)^ΔCtA value),

GZNF101(ENSG00000181896) is the expression quantity of ZNF101(ENSG00000181896) in tumor platelets (2)^ΔCtA value),

HTRABD2A (ENSG00000186854) is the expression level of TRABD2A (ENSG00000186854) in tumor platelets (2)^ΔCtValue)

ITRAC (ENSG00000229164) is the expression level of TRAC (ENSG00000229164) in tumor platelets (2)^ΔCtValue).

The invention also comprises a tumor platelet RNA quantitative detection method for early tumor screening, which comprises the following steps:

1) preparing a sample: preparing a human plasma sample to be detected, and taking a healthy human plasma sample as a normal control;

2) and (3) RNA extraction: extracting platelet total RNA;

3) reverse transcription: reverse transcribing platelet total RNA into cDNA;

4) and (3) PCR detection: using cDNA as template, detecting the expression quantity of SEQ ID NO. 1-SEQ ID NO.9 in human blood platelet to be detected and healthy human blood platelet by real-time fluorescent quantitative PCR technology under the combined application of PCR detection specific primer and real-time quantitative chimeric fluorescent PCR detection kit, and adopting 2^ΔCtExpressing the fold of the change of the expression quantity of the target RNA in the sample to be detected relative to housekeeping gene GAPDH (ENSG 00000111640);

5) calculating the Y value by using the 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 (ENSG 000086854) +0.31206518 × ITRAC (ENSG 00009102264); wherein,

ACD79A (ENSG00000105369) is the expression level of CD79A (ENSG00000105369) in tumor platelets (2)^ΔCtA value),

BCD81(ENSG00000110651) is the expression level of CD81(ENSG00000110651) in tumor platelets (2)^ΔCtA value),

CSYTL1(ENSG00000142765) is the expression level of SYTL1(ENSG00000142765) in tumor platelets (2)^ΔCtA value),

DCENPC (ENSG00000145241) is the expression level of CENPC (ENSG00000145241) in tumor platelets (2)^ΔCtA value),

ETTN (ENSG00000155657) is the expression level of TTN (ENSG00000155657) in tumor platelets (2)^ΔCtA value),

FRHOH (ENSG00000168421) is the expression level of RHOH (ENSG00000168421) in tumor platelets (2)^ΔCtA value),

GZNF101(ENSG00000181896) isExpression level of ZNF101(ENSG00000181896) in tumor platelets (2)^ΔCtA value),

HTRABD2A (ENSG00000186854) is the expression level of TRABD2A (ENSG00000186854) in tumor platelets (2)^ΔCtValue)

ITRAC (ENSG00000229164) is the expression level of TRAC (ENSG00000229164) in tumor platelets (2)^ΔCtA value);

ITRAC (ENSG00000229164) is the expression level of TRAC (ENSG00000229164) in platelets (2)^ΔCtValue), Δ Ct = Ct value of target gene-average Ct value of reference Gene (GAPDH), Ct value of target gene being Ct value of target CD79A (ENSG00000105369), CD81(ENSG00000110651), SYTL1(ENSG00000142765), CENPC (ENSG00000145241), TTN (ENSG00000155657), RHOH (ENSG00000168421), ZNF101(ENSG00000181896), TRABD2A (ENSG00000186854) and TRAC (ENSG00000229164) genes detected by real-time fluorescence quantitative PCR technique;

6) and (4) judging a result: when the Y value is less than 5.28, the sample is judged to be positive (suffering from tumor), and vice versa, the sample is negative (not suffering from tumor).

Based on the technical scheme, compared with the prior art, the invention has the following advantages:

the invention can be used as a biomarker combination for early diagnosis of tumors and evaluation of risk of the tumors, can obtain good tumor diagnosis value by detection, can realize early screening of the tumors, can assist pathological identification and clinical diagnosis of the tumors, and can improve the survival rate of patients.

Drawings

FIG. 1 is a flow chart of the operation of the present invention.

FIG. 2 is a ROC graph of the combined detection of tumor platelet RNA biomarkers in the invention.

FIG. 3 is a Y-value distribution graph of tumor patients and healthy persons.

Detailed Description

The model and method for quantitative detection of tumor platelet RNA for early stage tumor screening according to the present invention are further described in detail with reference to the accompanying drawings and specific examples, so as to clearly understand the structure type and usage manner, but the scope of the present invention is not limited thereby.

A tumor platelet RNA quantitative detection model for early tumor screening comprises PCR detection specific primers, wherein the PCR detection specific primers comprise an F-terminal primer and an RT primer of SEQ ID NO.1, an F-terminal primer and an RT primer of SEQ ID NO.2, an F-terminal primer and an RT primer of SEQ ID NO.3, an F-terminal primer and an RT primer of SEQ ID NO.4, an F-terminal primer and an RT primer of SEQ ID NO.5, an F-terminal primer and an RT primer of SEQ ID NO.6, an F-terminal primer and an RT primer of SEQ ID NO.7, an F-terminal primer and an RT primer of SEQ ID NO.8 and an F-terminal primer and an RT primer of SEQ ID NO. 9.

The PCR detection specific primer is used for clinically diagnosing a tumor platelet RNA biomarker combination, and the tumor platelet RNA biomarker combination comprises the following blood plasma:

CD79A (ENSG00000105369), CD81(ENSG00000110651), SYTL1(ENSG00000142765), CENPC (ENSG00000145241), TTN (ENSG00000155657), RHOH (ENSG00000168421), ZNF101(ENSG00000181896), TRABD2A (ENSG00000186854) and TRAC (ENSG 00000229164).

The tumor platelet RNA biomarker combinations are assigned, in use, according to the following formula coefficients:

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 (ENSG 000086854) +0.31206518 × ITRAC (ENSG 00009102264); wherein,

ACD79A (ENSG00000105369) is the expression level of CD79A (ENSG00000105369) in tumor platelets (2)^ΔCtA value),

BCD81(ENSG00000110651) is the expression level of CD81(ENSG00000110651) in tumor platelets (2)^ΔCtA value),

CSYTL1(ENSG00000142765) is the expression level of SYTL1(ENSG00000142765) in tumor platelets (2)^ΔCtA value),

DCENPC (ENSG00000145241) is the expression level of CENPC (ENSG00000145241) in tumor platelets (2)^ΔCtA value),

ETTN (ENSG00000155657) is the expression level of TTN (ENSG00000155657) in tumor platelets (2)^ΔCtA value),

FRHOH (ENSG00000168421) is the expression level of RHOH (ENSG00000168421) in tumor platelets (2)^ΔCtA value),

GZNF101(ENSG00000181896) is the expression quantity of ZNF101(ENSG00000181896) in tumor platelets (2)^ΔCtA value),

HTRABD2A (ENSG00000186854) is the expression level of TRABD2A (ENSG00000186854) in tumor platelets (2)^ΔCtValue)

ITRAC (ENSG00000229164) is the expression level of TRAC (ENSG00000229164) in tumor platelets (2)^ΔCtValue).

SEQ-ID of RNA described in Table 1 and forward and reverse primers thereof

SEQ-ID	Name of gene	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 fig. 1, a method for quantitatively detecting tumor platelet RNA for early tumor screening, the method comprises the following steps:

1) preparing a sample: preparing a human plasma sample to be detected, and taking a healthy human plasma sample as a normal control;

2) and (3) RNA extraction: extracting platelet total RNA;

3) reverse transcription: reverse transcribing platelet total RNA into cDNA;

4) and (3) PCR detection: using cDNA as template, detecting the expression quantity of SEQ ID NO. 1-SEQ ID NO.9 in human blood platelet to be detected and healthy human blood platelet by real-time fluorescent quantitative PCR technology under the combined application of PCR detection specific primer and real-time quantitative chimeric fluorescent PCR detection kit, and adopting 2^ΔCtExpressing the fold of the change of the expression quantity of the target RNA in the sample to be detected relative to housekeeping gene GAPDH (ENSG 00000111640);

5) calculating the Y value by using the 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 (ENSG 000086854) +0.31206518 × ITRAC (ENSG 00009102264); wherein,

ACD79A (ENSG00000105369) is the expression level of CD79A (ENSG00000105369) in tumor platelets (2)^ΔCtA value),

BCD81(ENSG00000110651) is the expression level of CD81(ENSG00000110651) in tumor platelets (2)^ΔCtA value),

CSYTL1(ENSG00000142765) is the expression level of SYTL1(ENSG00000142765) in tumor platelets (2)^ΔCtA value),

DCENPC (ENSG00000145241) is the expression level of CENPC (ENSG00000145241) in tumor platelets (2)^ΔCtA value),

ETTN (ENSG00000155657) is the expression level of TTN (ENSG00000155657) in tumor platelets (2)^ΔCtA value),

FRHOH (ENSG00000168421) is the expression level of RHOH (ENSG00000168421) in tumor platelets (2)^ΔCtA value),

GZNF101(ENSG00000181896) is the expression quantity of ZNF101(ENSG00000181896) in tumor platelets (2)^ΔCtA value),

HTRABD2A (ENSG00000186854) is the expression level of TRABD2A (ENSG00000186854) in tumor platelets (2)^ΔCtValue)

ITRAC (ENSG00000229164) is the expression level of TRAC (ENSG00000229164) in tumor platelets (2)^ΔCtA value);

ITRAC (ENSG00000229164) is the expression level of TRAC (ENSG00000229164) in platelets (2)^ΔCtValue), Δ Ct = Ct value of target gene-mean Ct value of reference Gene (GAPDH), target geneBecause the Ct value is the Ct value of the target CD79A (ENSG00000105369), CD81(ENSG00000110651), SYTL1(ENSG00000142765), CENPC (ENSG00000145241), TTN (ENSG00000155657), RHOH (ENSG00000168421), ZNF101(ENSG00000181896), TRABD2A (ENSG00000186854) and TRAC (ENSG 00009164) genes detected by a real-time fluorescent quantitative PCR technology;

6) and (4) judging a result: when the Y value is less than 5.28, the sample is judged to be positive (suffering from tumor), and vice versa, the sample is negative (not suffering from tumor).

Examples

The expression levels of the target tumor platelet RNA in the plasma of 50 tumor patients and 20 healthy persons (normal controls) were examined by the above-mentioned examination method for tumor platelet RNA tumor diagnosis, namely, CD79A (ENSG00000105369), CD81(ENSG00000110651), SYTL1(ENSG00000142765), CENPC (ENSG00000145241), TTN (ENSG00000155657), RHOH (ENSG00000168421), ZNF101(ENSG00000181896), TRABD2A (ENSG00000186854), and TRAC (ENSG00000229164) (2 SG 00000186864)^△CtValue).

The specific detection method comprises the following steps:

1. collection and preservation of tumor platelet RNA

Collecting venous blood of a subject 4ml in an EDTA anticoagulation tube, slightly inverting for several times, standing for 10min (centrifuging plasma in 1h at room temperature), centrifuging for 20min at room temperature for 120g, separating into two layers, sucking upper layer plasma (transparent light yellow liquid, sample with high blood lipid is opacified), transferring every 400 μ l into a new RNAse-free EP tube, and storing at-80 ℃.

The preserved plasma was dissolved in ice, centrifuged at 360g for 20min at room temperature, 300. mu.l of the upper plasma (i.e., platelets) was aspirated and transferred to a tube containing 30. mu.l of RNAlater (Life technologies), and incubated overnight at 4 ℃. Extracted using mirVanaRNA extraction kit (Life Technologies) as per instructions. Centrifuging the lower layer plasma at 5000rmp for 10min, and freezing and storing at-80 ℃ for later use, wherein the method is briefly as follows:

(1) plasma was mixed with equal volume of 2 × Denaturing Solution and incubated in an ice water bath for 5 min.

(2) Adding Acid-Phenol which is equal in volume to the mixed liquid and is Chloroform, uniformly mixing by swirling for 30-60 s, centrifuging for 20min at 12000r/min, dividing into three layers, sucking 500 mu l of upper layer transparent liquid, and transferring to a new RNAse-free EP tube.

(3) Adding 100% ethanol 1.25 times volume, mixing, transferring to centrifugal column (less than 700 μ l), loading onto collecting tube, centrifuging at 4000r/min for 30s, discarding the collected liquid, and repeating the above steps to allow all the liquid to pass through the centrifugal column.

(4) Adding 1700 μ l of washing solution to the centrifugal column, standing for 1min, and centrifuging for 15 s.

(5) Add 2/3500 μ l of washing solution to the column, let stand for 1min, centrifuge for 15s, repeat once.

(6) Adding 50 μ l of the heat-shocked Solution to a centrifugal column, standing for 1min, centrifuging for 1min, and collecting the centrifugate.

(7) RNA 6000 Picochip (Agilent) RNA concentration and quality were determined.

cDNA Synthesis

Performing reverse transcription on the extracted RNA sample according to standard operation steps specified by the kit operation instruction to obtain cDNA;

adding 1 μ L random 6mers,1 μ L dNTP, and 7 μ L RNase free dH into clean EP tube₂O, 1. mu.L of Temp RNA, blown uniformly, put into a PCR instrument, and set the program: 5min at 65 ℃; then 4. mu.L of 5-buffer, 0.5. mu.L of RNase inhibitor, 1. mu.L of RTase, 4.5. mu.L of RNase free dH₂And O, uniformly blowing, putting into a PCR instrument, and setting a program: 30 deg.C, 10min 42 deg.C, 50min 95 deg.C, 5 min.

3.RT-QPCR

Using the SYBR @ Premix Ex TaqTM (Tli RNaseH Plus) kit from Takara, the standard procedures as specified in the kit instructions were followed: 30 μ L system, 15 μ L SYBR MIX + 0.6μL primer-R+11.8μLddH₂O + 2 μ L cDNA, standard two-step reaction procedure: pre-denaturation at 95 ℃ for 10min, 40 cycles at 95 ℃ for 15s at 60 ℃ for 1 min; then according to 2^△CtThe expression amounts of RNA and internal reference mRNA GAPDH of 9 items in all tissues were calculated.

4. Analysis of results

As shown in FIGS. 2 and 3, the above-mentioned RNA gene combination assay is applied to individual tumor diagnosis, and the expression levels of nine RNAs are:

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 (ENSG 00000186886854) =3.559716, TRAC (ENSG00000229164) = 6.235487. The Y value calculation formula is utilized to obtain: y = 8.6155 greater than 5.28, indicating that the volunteer did not have a tumor.

Analysis of the combined diagnostic potency of RNA by test worker curves (ROC) using the formula Y =0.0148108 × a_{CD79A(ENSG00000105369)}+ 0.07848907 × B_{CD81(ENSG00000110651)}+ 0.1531495 ×C_{SYTL1(ENSG00000142765)}+ 0.08874548 × D_{CENPC(ENSG00000145241)}+ 0.05914998 ×E_{TTN(ENSG00000155657)}+ 0.22891517 × F_{RHOH(ENSG00000168421)}+ 0.5210493 ×G_{ZNF101(ENSG00000181896)}+ 0.05066299 × H_{TRABD2A(ENSG00000186854)}+ 0.31206518 ×I_{TRAC(ENSG00000229164)}The ROC curve obtained by fitting 9 RNA detection data is shown in FIG. 2, the area under the ROC curve is 92.5%, and the diagnosis efficiency is high.

By using 2^△CtFor quantitative analysis data, Logistic regression equation (judgment formula) Y =0.0148108 × A_{CD79A(ENSG00000105369)}+ 0.07848907 × B_{CD81(ENSG00000110651)}+ 0.1531495 ×C_{SYTL1(ENSG00000142765)}+ 0.08874548 × D_{CENPC(ENSG00000145241)}+ 0.05914998 ×E_{TTN(ENSG00000155657)}+ 0.22891517 × F_{RHOH(ENSG00000168421)}+ 0.5210493 ×G_{ZNF101(ENSG00000181896)}+ 0.05066299 × H_{TRABD2A(ENSG00000186854)}+ 0.31206518 ×I_{TRAC(ENSG00000229164)}Wherein A is_{CD79A(ENSG00000105369)}Is the expression level of CD79A (ENSG00000105369) in plasma (2)^ΔCtValue), B_{CD81(ENSG00000110651)}Is the expression level of CD81(ENSG00000110651) in plasma (2)^ΔCtValue), C_{SYTL1(ENSG00000142765)}Is the expression level of SYTL1(ENSG00000142765) in plasma (2)^ΔCtValue), D)_{CENPC(ENSG00000145241)}Is the expression level of CENPC (ENSG00000145241) in plasma (2)^ΔCtValue), E)_{TTN(ENSG00000155657)}Is the expression level of TTN (ENSG00000155657) in plasma (2)^ΔCtValue), F)_{RHOH(ENSG00000168421)}Is the expression level of RHOH (ENSG00000168421) in plasma (2)^ΔCtValue), G)_{ZNF101(ENSG00000181896)}Is the expression level of ZNF101(ENSG00000181896) in plasma (2)^ΔCtValue) H_{TRABD2A(ENSG00000186854)}Is the expression level of TRABD2A (ENSG00000186854) in plasma (2)^ΔCtValue), I)_{TRAC(ENSG00000229164)}Is TRAC (expression level of ENSG00000229164 in plasma (2)^ΔCtValue), Δ Ct = Ct value of target gene-Ct value of internal reference gene, Ct value of target gene refers to Ct value of target gene detected by real-time fluorescence quantitative PCR technique, target gene refers to CD79A (ENSG00000105369), CD81(ENSG00000110651), SYTL1(ENSG00000142765), CENPC (ENSG00000145241), TTN (ENSG00000155657), RHOH (ENSG00000168421), ZNF101(ENSG00000181896), TRABD2A (ENSG00000186854) and TRAC (ENSG 00009102264). Results Y values for 50 tumor patients and 20 healthy persons (normal controls) are shown in table 2, and it can be seen that the Y value is less than 5.28 for 71% of tumor patients. Therefore, when Y is<At 5.28, i.e. predicted probability value<The combination of CD79A (ENSG00000105369), CD81(ENSG00000110651), SYTL1(ENSG00000142765), CENPC (ENSG00000145241), TTN (ENSG00000155657), RHOH (ENSG00000168421), ZNF101(ENSG00000181896), TRABD2A (ENSG00000186854) and TRAC (ENSG 00009102264) can be used as a combination for judging the combination to be positive (suffering from tumor) and negative (not suffering from tumor) and proving that the combined detection of the genes can obtain good tumor diagnosis valueBiomarkers for early diagnosis of tumors and risk assessment of development.

TABLE 250Y values of tumor patients and 20 healthy persons (Normal control)

Numbering	Scoring (Y value)	Group of
			1	2.428121	Tumor patients
2	2.48863	Tumor patients
			3	2.503916	Tumor patients
4	2.799115	Tumor patients
			5	2.849653	Tumor patients
6	2.97098	Tumor patients
			7	2.997302	Tumor patients
8	3.065475	Tumor patients
			9	3.170404	Tumor patients
10	3.296256	Tumor patients
			11	3.323515	Tumor patients
12	3.352802	Tumor patients
			13	3.356327	Healthy person
14	3.379242	Tumor patients
			15	3.406207	Tumor patients
16	3.468006	Tumor patients
			17	3.489337	Tumor patients
18	3.500599	Tumor patients
			19	3.502231	Tumor patients
20	3.580153	Tumor patients
			21	3.598405	Tumor patients
22	3.611938	Tumor patients
			23	3.716721	Tumor patients
24	3.781669	Tumor patients
			25	3.852145	Tumor patients
26	3.857838	Tumor patients
			27	3.919833	Tumor patients
28	3.932668	Healthy person
			29	3.941747	Tumor patients
30	3.985031	Tumor patients
			31	4.092127	Tumor patients
32	4.096202	Tumor patients
			33	4.106992	Tumor patients
34	4.141384	Tumor patients
			35	4.14155	Tumor patients
36	4.365938	Tumor patients
			37	4.375088	Tumor patients
38	4.516429	Tumor patients
			39	4.564401	Tumor patients
40	4.588491	Tumor patients
			41	4.633284	Tumor patients
42	4.7872	Tumor patients
			43	4.793377	Tumor patients
44	4.79555	Tumor patients
			45	4.815989	Tumor patients
46	4.869563	Tumor patients
			47	4.933796	Tumor patients
48	5.046824	Tumor patients
			49	5.211091	Tumor patients
50	5.256569	Tumor patients
			51	5.285875	Healthy person
52	6.790469	Healthy person
			53	6.876531	Healthy person
54	7.083108	Tumor patients
			55	7.151574	Healthy person
56	7.553954	Healthy person
			57	7.56079	Healthy person
58	7.772101	Healthy person
			59	7.80082	Healthy person
60	8.036599	Healthy person
			61	8.310503	Healthy person
62	8.670087	Tumor patients
			63	9.05824	Healthy person
64	9.270311	Healthy person
			65	9.615263	Healthy person
66	9.858009	Healthy person
			67	9.939603	Healthy person
68	10.3842	Healthy person
			69	10.6205	Healthy person
70	10.88067	Healthy person

Currently, the sensitivity of clinical biomarkers such as CEA for tumor diagnosis is only 4.7-20.8%, and even if other markers are used in combination, the sensitivity is only 69.1% (He CZ, Zhang KH, Li Q, equivalent. combined use of AFP, CEA, CA125 and CA19-9 improvisations the sensitivity for the diagnosis of cancer. BMC Gastroenterol 2013.13: 87.). The data show that the sensitivity of tumor diagnosis by using the RNA combined marker can reach 92.5 percent, which is higher than the sensitivity of the current common clinical biomarker. Although there are 2 tumor patients and 2 normal people with deviation of the data detected by the method, the result accords with the clinical medical law by considering the heterogeneity of tumors and the difference between individuals (for example, the clinical condition is often that the CEA value of the normal people is higher than the upper normal limit, and the CEA value of the tumor patients is in the normal range), which shows that the marker combination combined screening method is real and effective and can be accepted and implemented.

It goes without saying that the tumor platelet RNA quantitative detection model and method for early tumor screening of the present invention include other similar structural composition modes and use modes besides the types and modes described in the above examples. In summary, the present invention also includes other variations and alternatives that will be apparent to those skilled in the art.

<110> Shanghai Thick bearing medical science and technology Limited

<120> tumor platelet RNA quantitative detection model and method for early tumor screening

<130> (numbering:)

<160>10

<210>1

<211>19

<212>DNA

<213>CD79A

<214> Artificial Synthesis

<220> Forward primer

<400>1

CATTGATGGTGAGCCTGGG

<220> reverse primer

TCGGCTGTGATGATTCGGT

<210>2

<211>21

<212>DNA

<213>CD81

<214> Artificial Synthesis

<220> Forward primer

<400>2

GTATCTGGAGCTGGGAGACAA

<220> reverse primer

TTGGCGATCTGGTCCTTGTT

<210>3

<211>19

<212>DNA

<213>SYTL1

<214> Artificial Synthesis

<220> Forward primer

<400>3

TCTCTCGACCGCATGCTCA

<220> reverse primer

TCGTAGTGCAGCGCGAAGT

<210>4

<211>21

<212>DNA

<213>CENPC

<214> Artificial Synthesis

<220> Forward primer

<400>4

TCCGGTTTTCAACGAGACTCT

<220> reverse primer

TTCAACTTCGCCCAGAAAGA

<210>5

<211>21

<212>DNA

<213>TTN

<214> Artificial Synthesis

<220> Forward primer

<400>5

CCTTCGAATTCCGCCTAAAAT

<220> reverse primer

TTTTCAATGTGGAACCTCCC

<210>6

<211>20

<212>DNA

<213>RHOH

<214> Artificial Synthesis

<220> Forward primer

<400>6

TTTCTTCGGCATTCTGCAAC

<220> reverse primer

CCTCCAAAGCCTAGTCTTCAA

<210>7

<211>21

<212>DNA

<213>ZNF101

<214> Artificial Synthesis

<220> Forward primer

<400>7

TGCTGGACACAAACGATCTGA

<220> reverse primer

TTGGTGTTACTGTGCGCCGT

<210>8

<211>21

<212>DNA

<213>TRABD2A

<214> Artificial Synthesis

<220> Forward primer

<400>8

TGCTCCCCAGGGACATCTACT

<220> reverse primer

TCCGGCAATAGCATTGAAGA

<210>9

<211>19

<212>DNA

<213>TRAC

<214> Artificial Synthesis

<220> Forward primer

<400>9

TCAGCGATTCAGCCTCCTA

<220> reverse primer

TCAGGCCAGACAGTCAACTGA

Claims (4)

1. A tumor platelet RNA quantitative detection model for early tumor screening is characterized by comprising PCR detection specific primers, wherein the PCR detection specific primers comprise an F-end primer and an RT primer of SEQ ID NO.1, an F-end primer and an RT primer of SEQ ID NO.2, an F-end primer and an RT primer of SEQ ID NO.3, an F-end primer and an RT primer of SEQ ID NO.4, an F-end primer and an RT primer of SEQ ID NO.5, an F-end primer and an RT primer of SEQ ID NO.6, an F-end primer and an RT primer of SEQ ID NO.7, an F-end primer and an RT primer of SEQ ID NO.8 and an F-end primer and an RT primer of SEQ ID NO. 9.

2. The quantitative detection model of tumor platelet RNA for screening early in tumor as claimed in claim 1, wherein the PCR detection specific primers are used to clinically diagnose the biomarker combination of tumor platelet RNA, which comprises the following platelet RNA:

CD79A (ENSG00000105369), CD81(ENSG00000110651), SYTL1(ENSG00000142765), CENPC (ENSG00000145241), TTN (ENSG00000155657), RHOH (ENSG00000168421), ZNF101(ENSG00000181896), TRABD2A (ENSG00000186854) and TRAC (ENSG 00000229164).

3. The quantitative detection model of tumor platelet RNA for screening of early tumor according to claim 2, wherein the combination of tumor platelet RNA biomarkers is assigned according to the following formula coefficients when in use:

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 (ENSG 000086854) +0.31206518 × ITRAC (ENSG 00009102264); wherein,

ACD79A (ENSG00000105369) is the expression level of CD79A (ENSG00000105369) in tumor platelets (2)^ΔCtA value),

BCD81(ENSG00000110651) is the expression level of CD81(ENSG00000110651) in tumor platelets (2)^ΔCtA value),

CSYTL1(ENSG00000142765) is the expression level of SYTL1(ENSG00000142765) in tumor platelets (2)^ΔCtA value),

DCENPC (ENSG00000145241) is the expression level of CENPC (ENSG00000145241) in tumor platelets (2)^ΔCtA value),

ETTN (ENSG00000155657) is the expression level of TTN (ENSG00000155657) in tumor platelets (2)^ΔCtA value),

FRHOH (ENSG00000168421) is RHOH (RHOHExpression level of ENSG00000168421) in tumor platelets (2)^ΔCtA value),

GZNF101(ENSG00000181896) is the expression quantity of ZNF101(ENSG00000181896) in tumor platelets (2)^ΔCtA value),

HTRABD2A (ENSG00000186854) is the expression level of TRABD2A (ENSG00000186854) in tumor platelets (2)^ΔCtValue)

ITRAC (ENSG00000229164) is the expression level of TRAC (ENSG00000229164) in tumor platelets (2)^ΔCtValue).

4. A quantitative detection method of tumor platelet RNA for early tumor screening is characterized by comprising the following steps:

1) preparing a sample: preparing a human plasma sample to be detected, and taking a healthy human plasma sample as a normal control;

2) and (3) RNA extraction: extracting platelet total RNA;

3) reverse transcription: reverse transcribing platelet total RNA into cDNA;

4) and (3) PCR detection: using cDNA as template, detecting the expression quantity of SEQ ID NO. 1-SEQ ID NO.9 in human blood platelet to be detected and healthy human blood platelet by real-time fluorescent quantitative PCR technology under the combined application of PCR detection specific primer and real-time quantitative chimeric fluorescent PCR detection kit, and adopting 2^ΔCtExpressing the fold of the change of the expression quantity of the target RNA in the sample to be detected relative to housekeeping gene GAPDH (ENSG 00000111640);

5) calculating the Y value by using the 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 (ENSG 000086854) +0.31206518 × ITRAC (ENSG 00009102264); wherein,

ACD79A (ENSG00000105369) is the expression level of CD79A (ENSG00000105369) in tumor platelets (2)^ΔCtA value),

BCD81(ENSG00000110651) is CD81(ENSG00000110651) in tumor plateletsExpression amount of (2)^ΔCtA value),

CSYTL1(ENSG00000142765) is the expression level of SYTL1(ENSG00000142765) in tumor platelets (2)^ΔCtA value),

DCENPC (ENSG00000145241) is the expression level of CENPC (ENSG00000145241) in tumor platelets (2)^ΔCtA value),

ETTN (ENSG00000155657) is the expression level of TTN (ENSG00000155657) in tumor platelets (2)^ΔCtA value),

FRHOH (ENSG00000168421) is the expression level of RHOH (ENSG00000168421) in tumor platelets (2)^ΔCtA value),

GZNF101(ENSG00000181896) is the expression quantity of ZNF101(ENSG00000181896) in tumor platelets (2)^ΔCtA value),

HTRABD2A (ENSG00000186854) is the expression level of TRABD2A (ENSG00000186854) in tumor platelets (2)^ΔCtValue)

ITRAC (ENSG00000229164) is the expression level of TRAC (ENSG00000229164) in tumor platelets (2)^ΔCtA value);

ITRAC (ENSG00000229164) is the expression level of TRAC (ENSG00000229164) in platelets (2)^ΔCtValue), Δ Ct = Ct value of target gene-average Ct value of reference Gene (GAPDH), Ct value of target gene being Ct value of target CD79A (ENSG00000105369), CD81(ENSG00000110651), SYTL1(ENSG00000142765), CENPC (ENSG00000145241), TTN (ENSG00000155657), RHOH (ENSG00000168421), ZNF101(ENSG00000181896), TRABD2A (ENSG00000186854) and TRAC (ENSG00000229164) genes detected by real-time fluorescence quantitative PCR technique;

6) and (4) judging a result: when the Y value is less than 5.28, the sample is judged to be positive (suffering from tumor), and vice versa, the sample is negative (not suffering from tumor).