CN109988845B - A kind of application of new stomach cancer marker circ-EIF4G3 - Google Patents

A kind of application of new stomach cancer marker circ-EIF4G3 Download PDF

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CN109988845B
CN109988845B CN201910403455.0A CN201910403455A CN109988845B CN 109988845 B CN109988845 B CN 109988845B CN 201910403455 A CN201910403455 A CN 201910403455A CN 109988845 B CN109988845 B CN 109988845B
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circ
eif4g3
reagent
gastric cancer
marker
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CN109988845A (en
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康宁
朱桂芳
曹秀峰
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Jiangsu Wancheng Biomedical Research Institute Co Ltd
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Abstract

The invention discloses the applications of new stomach cancer marker circ-EIF4G3 a kind of, are related to detection, the application of circ-EIF4G3 molecular marker, design and synthesize out the detection primer for being specifically used for real-time quantitative PCR.It is poor to gastric cancer Endoscopic Screening compliance for target group, and the patients with gastric cancer overwhelming majority clinically made a definite diagnosis is the status proposition of middle and advanced stage, ideal molecule pre-warning signal is found in gastric cancer canceration process, targetedly carry out endoscopy, definitive pathological diagnosis, to mitigate patient's pain, over-treatment is avoided, medical resource is saved.Present invention discover that circ-EIF4G3 molecular marker can be used as the marker of patients with gastric cancer detection and monitoring prognosis.

Description

A kind of application of new stomach cancer marker circ-EIF4G3
Technical field
The present invention relates to oncomolecularbiology fields, and a kind of specially new stomach cancer marker circ-EIF4G3's answers With.
Background technique
Currently, gastric cancer is still number three position in global cancer related mortality, it is one of major health burden of society.? China, gastric cancer are second to be very popular cancer, and lead to the second largest reason of cancer mortality, and it is new to be estimated to be within 2015 679,100 Case and 498,000 people are dead.Although surgical technic and Combination chemotherapy are improved, in most countries, the 5 of gastric cancer Year, total existence was still below 30%, in default of early gastric caacer diagnosis marker.Therefore, it is necessary to carefully explore gastric cancer relevant molecule Feature, and develop reasonable early gastric caacer detection method.
Circular rna (circRNA) is considered as being generated by the non-classical montage of linear precursor mRNA, is infected in virus-like Property particle such as viroid, round satellite virus and Hepatitis D virus found extensively.Circular rna s seemingly wrong RNA is cut It is connecing as a result, do not find its key effect and function always, find recently and for good and all change us To the viewpoint of cancer, especially canceration and cancer progression.So far, it has discovered that in mammalian cells some endogenous Property circRNA, and they in tumour progression can with high abundance exist and evolutionary conservatism.In addition, having had a large amount of Research confirms that circular rna can be adjustable transcription and manipulate the access of microRNA.Having several circRNA is considered as causing cancer thin The reason of born of the same parents' malignant behaviors.However, many can be used as since we are limited the understanding of circRNA The circRNA that microRNA works may be found not yet.More and more evidences show that circRNA may include It plays an important role in the occurrence and development of cancer including gastric cancer, and is likely to become the newborn marker of cancer.Zhang J's Team discovery circLARP4 is lowered in stomach organization, and is inhibited by amplification miR-424 targeting in LATS1 gene The biological behaviour of stomach cancer cell, this is the independent prognostic factor that patients with gastric cancer is always survived.
Currently, in the prior art, crossing the Tumor invasions such as CEA/CA199/CA153 in detection blood samples of patients all and suffering to assess The state of an illness of person, these index specificity and sensitivity are poor.
Summary of the invention
To solve the above problems, the invention discloses the applications of new stomach cancer marker circ-EIF4G3 a kind of, for mesh Mark crowd is poor to gastric cancer Endoscopic Screening compliance, and the patients with gastric cancer overwhelming majority clinically made a definite diagnosis is the status of middle and advanced stage It proposes, ideal molecule pre-warning signal is found in gastric cancer canceration process, targetedly carry out endoscopy, definitive pathological diagnosis, To mitigate patient's pain, over-treatment is avoided, medical resource is saved.Present invention discover that blood circ-EIF4G3 can be used as gastric cancer trouble The marker of person's lymphatic metastasis and monitoring prognosis.
In order to reach the goals above, the present invention supplies following technical solution:
The invention discloses a kind of circ-EIF4G3 markers to prepare the application in stomach cancer diagnosis reagent, the circ- The nucleotides sequence of EIF4G3 marker is classified as SEQ ID NO.1.
It in the present invention, is real-time quantitative PCR reagent for stomach cancer diagnosis reagent.
A kind of stomach cancer diagnosis reagent, the reagent including detecting circ-EIF4G3 marker, the reagent include detection The primer of circ-EIF4G3 marker and/probe.
In the present invention, 1 pair of primer is devised according to the circ-EIF4G3 that nucleotides sequence is classified as SEQ ID NO.1 to be used for The sequence is detected, primer nucleotide sequences are shown in SEQ ID NO.4, SEQ ID NO.5.
Use GAPDH as internal contrast, GAPDH (F), 5'-GCATCCTGGGCTACACTG-3', nucleotides sequence is classified as SEQ ID NO.2；GAPDH (R), 5'-ACTTCAGGAGCATCTGAAATAGGT-3', nucleotides sequence are classified as SEQ ID NO.3.
Using primer pair, upstream primer: 5 '-CCTACCCCATCCCCTTATTC-3 ', nucleotides sequence are classified as SEQ ID NO.4.Downstream primer, 5 '-ACCGTGCTGTAGACTGCTGAG-3 ', nucleotides sequence are classified as SEQ ID NO.5.
A kind of detection method of circ-EIF4G3 marker, including Total RNAs extraction；By RNA reverse transcription at cDNA；It carries out QRT-PCR reaction.Specifically: total serum IgE in tissue is extracted by using TRIzol reagent, and passes through TIANamp Virus RNA kit extracts the total serum IgE in blood plasma.RNA reverse transcription is tried at cDNA TM One step RT-PCR using Prime-Script Agent box, glyceraldehyde 3 phosphate dehydrogenase (GAPDH) are used as internal contrast.Use primer pair, upstream primer: 5 '- CCTACCCCATCCCCTTATTC-3 ', nucleotides sequence are classified as SEQ ID NO.4.Downstream primer, 5 '- ACCGTGCTGTAGACTGCTGAG-3 ', nucleotides sequence are classified as SEQ ID NO.5.Circ-CC2D1A table is determined by qRT-PCR Up to level.All qRT-PCR reactions are carried out using ABI7500 system and SYBR Green PCR Master Mix.In order to accurate The multiple variation for verifying circ-CC2D1A expression in GC tissue, by the Ct value of calculating relative to the GAPDH expanded from same sample (Δ Ct=Cttested-CtGAPDH) standardization, and use-Δ Ct method come estimate variation value.Each sample repeats Three times, all reactions are independent in triplicate to ensure the repeatabilities of all data.The Cutoff value of circ-EIF4G3 be- 7.374, when detect-Δ Ct be greater than this value when for gastric cancer, specificity is 81.25%, sensibility 60.94%.
The invention also discloses a kind of real time quantitative PCR detecting reagent kits for diagnosing gastric cancer, including according to nucleotide Sequence is that the circ-EIF4G3 molecular marker of SEQ ID NO.1 designs and synthesizes out the inspection for being specifically used for real-time quantitative PCR Survey primer.
In the present invention, further, the specific primer includes upstream primer and SEQ shown in SEQ ID NO.4 Downstream primer shown in ID NO.5.
The invention also discloses a kind of molecular marker for diagnosing gastric cancer, the molecular marker is nucleotide sequence For the circ-EIF4G3 molecular marker of SEQ ID NO.1.
For detecting the specific primer of circ-EIF4G3 molecular marker, the specific primer includes SEQ ID Downstream primer shown in upstream primer shown in NO.4 and SEQ ID NO.5.
The present invention has the advantage that
(1) purposes of circ-EIF4G3 is proposed.(2) circular rna has mechanism stable, abundance degree and tissue specificity The features such as expression.And circ-EIF4G3 has differences in gastric cancer and expression quantity in cancer beside organism, and to it is by stages related, therefore Circ-EIF4G3 has the application prospect as gastric cancer biomarker.
The present invention be directed to target group is poor to gastric cancer Endoscopic Screening compliance, and the patients with gastric cancer clinically made a definite diagnosis is exhausted Most of is all that the status of middle and advanced stage proposes, it would be desirable to find ideal molecule pre-warning signal in gastric cancer canceration process, there is needle To the carry out endoscopy of property, definitive pathological diagnosis avoids over-treatment, saves medical resource to mitigate patient's pain.Present invention hair Existing blood circ-EIF4G3 can be used as the marker of patients with gastric cancer diagnosis and monitoring prognosis.
Detailed description of the invention
Fig. 1, with the abundance of circ-EIF4G3 and EIF4G3mRNA in the GC cell of RNase R processing；
Fig. 2, with the abundance of circ-EIF4G3 and EIF4G3mRNA in the GC cell of D actinomycin D processing；
The significant expression higher than the adjacent non-cancer tissue of GC patient of expression of Fig. 3, circ-EIF4G3 expression；
Fig. 4, ROC curve have been used for the assessment potential diagnostic value of circ-EIF4G3, and area (AUC) is under ROC curve 0.7158；
Fig. 5, according to the total survivorship curve of Kaplan-Meier of circ-EIF4G3 level.
In figure: the decreased survival time of height expression patient, P < 0.0001 * * *.
Specific embodiment
With reference to the accompanying drawings and detailed description, the present invention is furture elucidated, it should be understood that following specific embodiments are only For illustrating the present invention rather than limiting the scope of the invention.
The invention discloses the application of new stomach cancer marker circ-EIF4G3 a kind of, circ-EIF4G3 molecular markers Nucleotides sequence be classified as SEQ ID NO.1, CCCTACCCCATCCCCTTATTCAGCACATGAAATAAACAAGGGGCATCCAA ATCTTGCGGCAACGCCCCCGGGACATGCATCGTCCCCTGGACTCTCTCAA AATTCCTAGAGGACCTGTGCAACAA CCTCTTGAGGATCGAATCTTCACTCCCGCTGTCTCAGCAGTCTACAGCACGGTAACACAAGTGGCAAGACAGCCG。
Marker of the circ-EIF4G3 as diagnosing gastric cancer and prognosis.Following experimental method is specific embodiment, real It is as shown in Figures 1 to 5 to test result.
Embodiment 1
1.RNase R digestion and actinomycin D digestion
2mg total serum IgE is incubated 20 minutes at 37 DEG C, is added or is added without 3U/mg RNase R, then uses RNeasy MinElute kit.For actinomycin D treatment, by 2mg total serum IgE at 37 DEG C with or be added without 1mg D actinomycin D (unwrapping wire Rhzomorph D, Sigma, the best Reagent Company in Chengdu) it incubates together, and the detection gained RNA respectively at 0,6,12,18 hour.
2. patient and case screening
It is collected into 64 GC tissues altogether from GC patient and corresponding adjacent non-tumor tissue sample, all tissue samples is equal From department of general surgery, attached Nanjing hospital, Nanjing Medical University (in January, 2011 is to during in December, 2017).All patients in group exist Preoperative row radiation and chemotherapy, and their tissue samples are stored in immediately in RNA fixating reagent, and be immediately placed in -80 DEG C It is saved in refrigerator.The adjacent nonneoplastic tissue of pairing need to not have tumour cell by pathological analysis, and be located in apart from knurl At edge 5cm.According to tumour-lymphatic metastasis (TNM) Staging System of International Union Against Cancer, all tumours it is accurate by stages. Every patient signs Written informed consent, and the local medical ethics committee has approved the research approach.
3. clinical tissue sample samples
(1) preparatory items:
Liquid nitrogen container (checking liquid nitrogen volume, fill liquid nitrogen in time in advance), ice chest, ice physiological saline, sterile no enzyme cryopreservation tube (dispensed RNA protection liquid and numbered spare), sterile glove (without talcum powder), mask, cap, tweezers, scissors, low temperature mark Pen etc..
(2) operating process:
A. determine and left and taken its blood sample patient is preoperative, and extract flow processing according to blood sample, after will extract Serum move into Greiner cryopreservation tube in and with red low temperature marking pen marker number, be stored in low temperature refrigerator, and be recorded in disease In example data sheet and its electronic version.
B. it is pre-filled with tissue specimen case-data table (recording effective contact method in order to follow-up), pays attention to filling in interior The integrality of appearance.The number for leaving and taking the cryopreservation tube of cancer and cancer beside organism is predefined, and is numbered and is recorded in case-data table In.
C. be immediately placed in kidney basin after tissue is in vitro, rapidly with brine ice shower (low temperature can temporarily inhibit RNA enzyme activity with Strive for sample time；The blood mixed in washing tissue samples, reduces pollution；Maintain the permeability of tissue samples in favor of RNA Protection liquid permeates rapidly and plays Inhibitory activity).
D. it chooses mucosa tissue and is broken down into the fritter packing of about soya bean size, pay attention to sampling sequence: taking 2~4 Block Carcinoma side normal tissue and 2~4 pieces of tumor tissues, thickness are no more than 5mm, first take normal tissue, then take tumor tissues, a jelly Depositing pipe only is suitable for one piece of sample of storage, and the cryopreservation tube for not obscuring tumour and peri- tumorous normal tissues is numbered.
E. cryopreservation tube is tightened after the completion of sampling (to penetrate into cryopreservation tube as do not screwed bottle cap liquid nitrogen, take out and freeze under normal temperature state Deposit Guan Shiyi initiation freeze pipe explosion), be softly inverted and rock cryopreservation tube, make tissue and protection liquid mix, after be immediately placed in liquid In nitrogen tank.
F. whether inspection tissue specimen case-data table is filled in perfect, predefines and records in " refrigerator sample positions table " Enter storage location of the sample in profound hypothermia refrigerator, the sample in liquid nitrogen container is moved into profound hypothermia refrigerator in time.It avoids because of carelessness Forget, causes sample waste after liquid nitrogen volatilization totally unavailable.
G. postoperative one week tracking pathological replacement determines whether sample can enter as a result, simultaneously in timely typing case-data table Group research.
Extraction, reverse transcription and the real-time quantitative PCR of 4.RNA
Total serum IgE in tissue is extracted by using TRIzol reagent, and passes through TIANamp according to the manufacturer's instructions Virus RNA kit extracts the total serum IgE in blood plasma.Using Prime-Script by RNA reverse transcription at cDNA TM one-step method RT-PCR kit, glyceraldehyde 3 phosphate dehydrogenase (GAPDH) are used as internal contrast.Use primer pair, upstream primer: 5 '- CCTACCCCATCCCCTTATTC-3 ', nucleotides sequence are classified as SEQ ID NO.4.Downstream primer, 5 '- ACCGTGCTGTAGACTGCTGAG-3 ', nucleotides sequence are classified as SEQ ID NO.5.Use ABI7500 system and SYBR Green PCR Master Mix carries out all qRT-PCR reactions.In order to which the multiple of circ-CC2D1A expression in accurate validation GC tissue becomes Change, the Ct value of calculating is standardized relative to the GAPDH (Δ Ct=Cttested-CtGAPDH) expanded from same sample, and The value of variation is estimated using-Δ Ct method.In triplicate, all reactions are independent in triplicate to ensure to own for each sample The repeatability of data.The Cutoff value of circ-EIF4G3 is -7.374, when detect-Δ Ct be greater than this value when for stomach Cancer, specificity are 81.25%, sensibility 60.94%.
Laboratory operating procedures:
(1) Trizol extracts tissue RNA:
A. it wears masks and gloves, opens 4 DEG C of refrigerated centrifuges, it is spare to set temperature.The sample number extracted needed for calculating, The sterile no enzyme EP pipe of 1.5ml for preparing double quantity, is placed in spare on EP pipe support.
B. it grinds in vessel and pours into appropriate Liquid nitrogen precooler, take in 50-100mg tissue merging liquid nitrogen, tissue abrasion to powder Shape.
C. 1ml Trizol is added into grinding vessel, continues the mixture of the tissue and Trizol of grinding solidification shape.
D. it stands to its liquefaction, is transferred in Rnase free 1.5ml EP pipe.
E. 5min is stood at room temperature, and 200ul chloroform is added in every 1ml Trizol.
F. 15s is acutely shaken, stands 2-3min at room temperature.
G.4 DEG C, 12000g-14000g is centrifuged 15min.
H. for transfer water phase into new Rnase free 1.5ml EP, 500ul isopropanol, room is added in every 1ml Trizol Temperature places 10min.
I.4 DEG C, 12000g is centrifuged 10min.75% ethyl alcohol (need to be prepared with DEPC water) of prewired cleaning RNA.
J. it carefully discards supernatant, at least 75% ethanol washing RNA precipitate of 1ml, vortex mixed is added in every pipe.
K.4 DEG C, 7500-10000g is centrifuged 5-10min.Carefully discard supernatant.
L. RNA precipitate (RNA is colourless after air-drying) is air-dried, (is added according to precipitating size using DEPC- water dissolution precipitating 10-80ul or so), with pipette tips suction beat blow it is even.
(2) Trizol extracts cell RNA:
A. cell to be extracted is cleaned twice with PBS.
B. appropriate Trizol (bottle/six orifice plates every hole 1ml, big ware 3ml, big bottle 5ml) into culture vessel is added.
C. it blows and beats several times, is transferred in Rnase free 1.5ml EP pipe repeatedly.
D. 5min is stood at room temperature, and 200ul chloroform is added in every 1ml Trizol.
E. 15s is acutely shaken, stands 2-3min at room temperature.
F.4 DEG C, 12000-14000g is centrifuged 15min.
G. for transfer water phase into new Rnase free 1.5ml EP, 500ul isopropanol, room is added in every 1ml Trizol Temperature places 10min.
H.4 DEG C, 12000g is centrifuged 10min.
I. it discards supernatant, at least 75% ethanol washing RNA precipitate of 1ml, vortex mixed is added.
J.4 DEG C, 7500-10000g is centrifuged 5-10min.
K. RNA precipitate is air-dried, is used DEPC- water dissolution precipitating (10-80ul or so is added according to precipitating size).
(3) extracted sample rna concentration is detected:
A. it is (each to be detected each sample to be tested to be configured in new Rnase free200ul EP pipe according to 1% concentration Sample 1ul+99ulDEPC water).
B. ultraviolet specrophotometer is opened, cuvette is cleaned, takes 100ulDEPC water that blank control is set.
C. it is implanted sequentially sample to be tested and detects and record concentration of specimens, OD260/280 ratio.
(4) RNA reverse transcription:
A. Reverse Transcriptase kit is taken out from -20 DEG C of refrigerators, by 2 kinds of enzyme insertion ice chest ice, other reagents melt on ice, RNase Free water room temperature melts spare.
B. according to surveyed RNA concentration, RNA volume needed for calculating the every pipe of reverse transcription, and DEPC water volume is calculated simultaneously.
C. it takes PCR pipe to be placed on ice, be put into sterile no enzyme 200ul PCR pipe as needed and mark by number.Pipettor After drawing appropriate RNA, pipette tips are inserted perpendicularly into PCR pipe bottom, at the uniform velocity softly get liquid.
D. pipettor draws appropriate DEPC water, and pipette tips are squeezed into PCR pipe close to rear wall.
E. reverse transcription Step1: gDNA Eraser 1ul and 5x gDNA Eraser Buffer 2ul is added in every pipe.
F. it is placed on eight union centrifuges, gently bullet falls tube bottom or tube wall bubble, is centrifuged in short-term.
42 DEG C 2 minutes on G.RT-PCR instrument, product is placed in spare on ice after reaction.
H. it reverse transcription Step2: takes 1.5ml EP to manage, according to formula as below, configures every tube reaction Mix
Setting reaction condition: 37 DEG C, 15min；85 DEG C, 5sec；4 DEG C of maintenances.
I. pipe lid is fastened, it is taken off from pipe support, is placed on centrifuge tube shelf, having bubble, person can flick tube bottom, in short-term From
The heart resets after confirming that the liquid of every pipe equals the interior bubble-free of consistent and pipe on ice.
J., reaction condition is set: 37 DEG C, 15min；85 DEG C, 5sec；4 DEG C of maintenances.Operate the computer, by cDNA after 15-20min It is placed in -20 DEG C of preservations.
(5) qRT-PCR step:
A. ice chest is got out, cDNA, the primer, PCR reagent of sample to be tested are taken out from -20 DEG C of refrigerators, is thawed on ice.
B. the liquid relief of eight unions and Guan Gai, eight union framves, tweezers, sterile 1.5ml EP pipe, EP pipe support, suitable range is got ready Device, aseptic thin-film gloves.
C. by the cDNA to have thawed, primer, PCR reagent, first successively oscillation is centrifuged in short-term, on ice stand for standby use.
D. according to detection sample and index quantity, different target gene sample application regions are divided.Eight unions are placed on ice, Eight unions are clamped with tweezers and are laid down on eight union framves.
E. it takes 1.5ml EP to manage, prepares eight union PCR Mix by every hole 19ul below, and shake centrifugation.
F. by each sample and multiple holes are preset in the position of eight unions, the 1ul cDNA and 19ul of sample are sequentially added PCR Mix。
G. bubble is removed in centrifugation, operates the computer, and reaction condition is arranged.
(6) qRT-PCR reaction system:
QRT-PCR reaction condition:
6, data are analyzed:
Experimental result is as Figure 1-Figure 5, as shown in table 1, table 2, using t inspection and Chi-square Test and the side of survival analysis The correlation of method circ-EIF4G3 and patients with gastric cancer clinical stages data and prognosis.Carry out Receiver Operating Characteristics (ROC) curve To assess its diagnostic value.V.17.0, all statistical analysis are carried out using SPSS for Windows.For all as a result, P < 0.05 is considered statistically significant.
The expression of table 1.circ-EIF4G3 to by stages and lymphatic metastasis is related
Table 1.Clinicopathological characteristics and expression of circ- EIF4G3
*P<0.05
The single argument and multi-variables analysis of the comprehensive existence of table 2.
Table 2.Univariate and multivariate analysis for overall survival
* P < 0.05
The technical means disclosed in the embodiments of the present invention is not limited only to technological means disclosed in above embodiment, further includes Technical solution consisting of any combination of the above technical features.It should be pointed out that for those skilled in the art For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as Protection scope of the present invention.
Sequence table
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Claims (4)

1. the reagent of circ-EIF4G3 marker expression level is preparing answering in stomach cancer diagnosis reagent in a kind of detection blood With the nucleotides sequence of the circ-EIF4G3 marker is classified as SEQ ID NO.1.
2. the reagent of circ-EIF4G3 marker expression level is examined in preparation gastric cancer in detection blood as described in claim 1 Application in disconnected reagent, it is characterised in that: the reagent of circ-EIF4G3 marker expression level is real-time in the detection blood Quantitative PCR reagent.
3. the reagent of circ-EIF4G3 marker expression level is preparing gastric cancer in detection blood as claimed in claim 1 or 2 Application in diagnostic reagent, it is characterised in that: the reagent of circ-EIF4G3 marker expression level includes in the detection blood 1 pair for detecting the primer of SEQ ID NO.1, the nucleotides sequence of the primer is classified as SEQ ID NO.4, SEQ ID NO.5 institute Show.
4. the reagent of circ-EIF4G3 marker expression level is examined in preparation gastric cancer in detection blood as described in claim 1 Application in disconnected reagent, it is characterised in that: the reagent of circ-EIF4G3 marker expression level includes inspection in the detection blood Survey the primer and/or probe of circ-EIF4G3 marker.
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