CN110079603A - A kind of detection primer, probe and its application of nasopharynx carcinoma marker - Google Patents
A kind of detection primer, probe and its application of nasopharynx carcinoma marker Download PDFInfo
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Abstract
The present invention relates to gene therapies and medical diagnostic techniqu field, the present invention provides a kind of application of biological markers EBV-miR-BART13 in the diagnostic kit of the early diagnosis of preparation nasopharyngeal carcinoma and prognosis prediction, invention further provides a kind of to carry out the kit of nasopharyngeal carcinoma diagnosis for EBV-miR-BART13 detection, comprising: (1) in blood plasma total serum IgE extraction；(2) cDNA of EBV-miR-BART13 is synthesized in blood plasma；(3) sonde method detection EBV-miR-BART13 expression.Biological markers EBV-miR-BART13 of the present invention can be used as early diagnosis and predict the biological markers of prognosis, and the susceptibility of the kit diagnosis of nasopharyngeal carcinoma is high, high specificity.
Description
Technical field
The present invention relates to gene therapies and medical diagnostic techniqu field, and in particular to a kind of biological markers EBV-miR-
Application of the BART13 in the diagnostic kit of the early diagnosis of preparation nasopharyngeal carcinoma and prognosis prediction.
Background technique
Nasopharyngeal carcinoma is one of malignant tumour of south China, is apt to occur between twenty and fifty (35-55 year old), year Standardized incidence rate can height
Up to 15-30/100000.5 years survival rates of early stage nasopharyngeal carcinoma are up to 90%, but patients with terminal only 50% or so.Due to region of anatomy spy
Very, early symptom is hidden, and when 70% or more patient assessment has been middle and advanced stage.Therefore, the pathogenesis of nasopharyngeal carcinoma is studied, is found
Simply, effective tumor markers are to prevent and treat in nasopharyngeal carcinoma future to improve nasopharyngeal carcinoma early diagnostic rate and prediction prognostic capabilities
Major tasks.
Epstein-Barr virus (Epstein-Barr virus, EBV) infection is one of main pathogenic factor of nasopharyngeal carcinoma.Research
It was found that: almost 100% non-keratinization type nasopharyngeal carcinoma is infected with EBV, therefore, can assist nasopharyngeal carcinoma by detecting EBV gene product
Diagnosis.Discussion about nasopharyngeal carcinoma peripheral blood marker is shown, persistently increases crowd in EBV EBNA-IgA and VCA-IgA
In, the onset risk of nasopharyngeal carcinoma is in increase year by year, but as NPC early screening and diagnosis index, sensibility and specificity are very
It is low, such as the recall rate of NPC is only 3.19% in EBV VCA-IgA Positive Populations.Blood plasma EBV DNA is current most study
Nasopharyngeal carcinoma tumor marker has clinical value in the screening of NPC, early diagnosis, treatment and prognosis context of detection.Utilize blood plasma
In EBV DNA screening early stage asymptomatic NPC patient, sensibility and specificity are respectively 97.1%, 98.6%, and data are shown
NPC patient makes a definite diagnosis the time and obviously shifts to an earlier date, and prognosis is more preferably.But due to each laboratory EBV DNA detection technique, detection platform,
Threshold value etc. is inconsistent, currently without standardized process, practicability, repeatability, the generalization of its diagnosis of nasopharyngeal carcinoma is caused to be deposited
In very big difficulty.Therefore, whether blood plasma EBV DNA can need further to be studied as ideal nasopharyngeal carcinoma tumor marker.
Microrna (microRNA, miRNA) is the small molecule non-coding RNA that a kind of length is about 20~23 nucleotide,
It by with target gene is complete or incomplete complementary pairing, degrade and target gene or check its transcription or translation.MiRNA can pass through
Play the part of oncogene or tumor suppressor gene role, to participate in the proliferation of tumour cell, differentiation, apoptosis, invasion transfer, immunologic escape etc.
Process is the research hotspot of tumor markers in recent years.It has now been found that before EBV encodes 22 EBV-miR-BARTs altogether
Body forms 44 mature miRNA, and EBV-miR-BARTs great expression is in tissues of nasopharyngeal carcinoma, and in healthy person or non-
It low expression or is not expressed in nasopharyngeal carcinoma cancer patient.Therefore, specifically expressed EBV-miR-BARTs in Screening of Nasopharyngeal Carcinoma, as
The tumour of potential nasopharyngeal carcinoma early sieve check, early diagnosis, therapeutic regimen decision, prediction prognosis, detection recurrence etc.
Biomarkers have extremely important clinical meaning.
Summary of the invention
The purpose of the present invention is to provide a kind of biological markers EBV-miR-BART13 to examine in preparation nasopharyngeal carcinoma early stage
Application in disconnected and prognosis prediction diagnostic kit.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of diagnostic kit of biological markers EBV-miR-BART13 in preparation nasopharyngeal carcinoma early diagnosis and prognosis prediction
In application, the sequence of the EBV-miR-BART13 is as shown in SEQ ID No.1: UGUAACUUGCCAGGGACGGCUGA.
The kit is the expression quantity that EBV-miR-BART13 in biological sample is detected by round pcr.
The kit includes the primer sequence and probe for having detection specificity to EBV-miR-BART13, and sequence is such as
Under:
Upstream primer is miR-BART13-F:5 '-GTCGTTGTAACTTGCCAGG-3 ';
Downstream primer is miR-BART13-R:5 '-GTGCAGGGTCCGAGGTAT-3 ';
Probe are as follows: 5 '-FAM-CTGGATACGACTCAGCCG-BHQX-3 ', the fluorescent reporter group of 5 ' end labels are FAM, 3 ' ends
The fluorescent quenching group of label is BHQX.
The kit includes:
(1) in blood plasma total serum IgE extraction；
(2) cDNA of EBV-miR-BART13 is synthesized in blood plasma；
(3) sonde method detection EBV-miR-BART13 expression.
The present invention has the advantages that
The present invention by specifically expressed EBV-miR-BART13 in Screening of Nasopharyngeal Carcinoma, as potential nasopharyngeal carcinoma early sieve check,
The tumor marker of early diagnosis, therapeutic regimen decision, prediction prognosis, detection recurrence etc. has extremely heavy
The clinical meaning wanted.
Detailed description of the invention
Fig. 1 is Nasopharyngeal Carcinoma Patients and healthy population EBV-miR-BART13 expression quantity measurement result.
Fig. 2 is the measurement result of EBV-miR-BART13 expression quantity before and after Radiotherapy for Nasopharyngeal Carcinoma.
Fig. 3 is the relationship of EBV-miR-BART13 expression quantity and tumor load in blood plasma；Wherein 3a is the T1-T4 stage；3b
For the N0-N3 stage；3c is I-IV phase of clinical pathology.
Fig. 4 is prognostic value analysis chart.
Specific embodiment
Embodiment 1
1, in blood plasma total serum IgE extraction
(1) prepare blood plasma, in the RNA enzyme free/Clear EP pipe for inhaling the AXYGEN that 480ul blood plasma is fitted into 2ml；
(2) every pipe, which is protected from light, is added 1ml TRAZOL plus, is vortexed 15 seconds, cracks static 30 minutes；
(3) every pipe is protected from light the chloroform that 200ul is added, and is vortexed 30 seconds, stands 30 minutes, and layering half is comparatively ideal state；
(4) centrifugation 15000g, 25 minutes, 4 DEG C, it is seen that sample is layered 3 layers, collects top layer's clarified solution (500-800ul) and puts
In the 1.5ml-2.0ml EP pipe of RNA enzyme free.
(5) isopropanol, vortex 15s is added according to 1:1 volume, -20 DEG C of refrigerators are stood overnight；
(6) it is taken out from -20 DEG C of refrigerators, 4 DEG C of centrifugation 20000g, 30 minutes, it is seen that precipitating；
(7) supernatant is abandoned, is upside down on filter paper, dries, 1.4ml-1.8ml is added and (sees the size of pipe, attaching is closely full, 1:1's
Amount) fresh configuration 75% alcohol, vortex 15s is centrifuged 20000g, 30 minutes, 4 DEG C.
(8) supernatant is abandoned, is upside down on filter paper, is dried 5-10 minutes, do not dry, it is reserved tube wall can be drawn with rifle point
Alcohol, or blotted the alcohol of the EP pipe upper half with filter paper, prevent alcohol residue from influencing subsequent experiment.30 minutes dried,
Guarantee that alcohol has volatilized completely.
(9) rifle point gos deep into tube wall bottom, be added 15uL without enzyme water, vortex 15s gets rid of the liquid on lower tube wall with hand, low
Fast (< 2000g) is centrifuged several seconds, can continue to place on ice 10 minutes or so, RNA is allowed sufficiently to dissolve, -80 DEG C of refrigerators of postposition save to
With.
2, the cDNA of EBV-miR-BART13 is synthesized in blood plasma
Using TaqMan Small RNA Assays by RNA reverse transcription be cDNA.
(1) Mix reaction solution configuration (being loaded in order)
1 Mix reaction solution table of table
Mir-BART13-RT primer sequence are as follows:
5’- GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAGCC-3’。
(2) reaction condition is set
Reverse transcription reaction is carried out by the reaction condition of table 2 in PCR instrument.
2 reaction condition of table
3, sonde method detection EBV-miR-BART13 expression
Using TaqMan Universal Master Mix II detect EBV-miR-BART13 expression quantity, by 3 component of table into
Row reaction solution is prepared.
3 sonde method detection reagent table of table
2 Analysis of test results of embodiment
207 healthy body volunteers, 12 benign disease patients, 24 non-nasopharyngeal cancer patients are detected using the method for embodiment 1
With 479 NPC patients.Patient's basic document is as shown in table 4.
4 NPC patient's basic document table of table
As shown in Figure 1, EBV-miR-BART13-3p high expression can be detected in the blood plasma of NPC, and it is healthy volunteer, benign
It can not be detected in disease patient and other tumour patients.Wherein make by EBV-miR-BART13-3p in blood plasma of 980copie/ml
For the critical value of nasopharyngeal carcinoma diagnosis, sensitivity 95.37%, specificity 98.82%.After carrying out radiotherapy in the treatment, patients blood plasma
In EBV-miR-BART13-3p expression quantity be reduced to normal level (Fig. 2) substantially.Illustrate that EBV-miR-BART13-3p is expressed
Amount can be used as NPC early screening and diagnosis index, can assist the diagnosis of nasopharyngeal carcinoma.
The blood in NPC patient tumors T1-T4 stage, I-IV stage of N0-N3 stage and clinicopathologic stage TNM is detected respectively
EBV-miR-BART13-3p expression quantity in slurry, as a result as shown in figure 3, in blood plasma EBV-miR-BART13-3p expression quantity and nose
The tumor load of pharynx cancer is related.
Table 5: the diagnostic test of plasma circulation miR-BART13-3p
The prognostic value of 3 EBV-miR-BART13-3p of embodiment
The EBV-miR-BART13-3p expression quantity for detecting 497 NPC patients, using median 10122copies/ml as critical value,
Survival analysis finds total existence (OS) of high expression quantity patient in blood plasma and is substantially less than blood plasma without transfer survival rate (DMFS)
The patient of middle EBV-miR-BART13-3p low expression, as a result sees Fig. 4.
As shown in Figure 4, EBV-miR-BART13-3p high expresses the patient of (> 10122copies/ml) in blood plasma, total raw
It deposits (OS) and no distant transfer survival rate (DMFS) is substantially less than low expression group (≤10122copies/ml), illustrate EBV-miR-
BART13-3p high expression prompts worse prognosis, and difference has statistics statistical significance (p is respectively 0.005 and 0.022), shows
The expression quantity of EBV-miR-BART13-3p can be used as the marker of nasopharyngeal carcinoma diagnosis prognosis.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
SEQUENCE LISTING
<110>Fujian Cancer Hospital
<120>a kind of detection primer, probe and its application of nasopharynx carcinoma marker
<130> 5
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213>artificial sequence
<400> 1
uguaacuugc cagggacggc uga 23
<210> 2
<211> 19
<212> DNA
<213>artificial sequence
<400> 2
gtcgttgtaa cttgccagg 19
<210> 3
<211> 18
<212> DNA
<213>artificial sequence
<400> 3
gtgcagggtc cgaggtat 18
<210> 4
<211> 18
<212> DNA
<213>artificial sequence
<400> 4
ctggatacga ctcagccg 18
<210> 5
<211> 50
<212> DNA
<213>artificial sequence
<400> 5
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgactcagcc 50
Claims (4)
1. a kind of biological markers EBV-miR-BART13 is in the diagnostic reagent of preparation nasopharyngeal carcinoma early diagnosis and prognosis prediction
Application in box, which is characterized in that the sequence of the EBV-miR-BART13 is as shown in SEQ ID No.1.
2. a kind of biological markers EBV-miR-BART13 according to claim 1 preparation nasopharyngeal carcinoma early diagnosis and
Application in the diagnostic kit of prognosis prediction, which is characterized in that the kit is detected in biological sample by round pcr
The expression quantity of EBV-miR-BART13.
3. a kind of biological markers EBV-miR-BART13 according to claim 2 preparation nasopharyngeal carcinoma early diagnosis and
Application in the diagnostic kit of prognosis prediction, which is characterized in that the kit includes having inspection to EBV-miR-BART13
The primer sequence and probe of specificity are surveyed, sequence is as follows:
Upstream primer is miR-BART13-F:5 '-GTCGTTGTAACTTGCCAGG-3 ';
Downstream primer is miR-BART13-R:5 '-GTGCAGGGTCCGAGGTAT-3 ';
Probe are as follows: 5 '-FAM-CTGGATACGACTCAGCCG-BHQX-3 ', the fluorescent reporter group of 5 ' end labels are FAM, 3 ' ends
The fluorescent quenching group of label is BHQX.
4. a kind of biological markers EBV-miR-BART13 according to claim 1 preparation nasopharyngeal carcinoma early diagnosis and
Application in the diagnostic kit of prognosis prediction, which is characterized in that the kit includes:
(1) in blood plasma total serum IgE extraction；
(2) cDNA of EBV-miR-BART13 is synthesized in blood plasma；
(3) sonde method detection EBV-miR-BART13 expression.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114058705A (en) * | 2021-11-24 | 2022-02-18 | 中南大学湘雅医院 | METTL3 as nasopharyngeal carcinoma prognosis prediction marker, detection primer and kit |
| WO2023072076A1 (en) * | 2021-10-25 | 2023-05-04 | 杭州诺辉健康科技有限公司 | Reagent and method for diagnosis of nasopharyngeal carcinoma |
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| US20140296319A1 (en) * | 2011-05-25 | 2014-10-02 | Catholic University Industry Academic Cooperation Foundation | Composition For Promoting Apoptosis Or Inhibiting Cell Growth, Comprising Epstein-Barr Virus Microrna |
| CN109321655A (en) * | 2018-05-17 | 2019-02-12 | 福建省肿瘤医院（福建省肿瘤研究所、福建省癌症防治中心） | NKIRAS2 gene regulation region sequence, regulating and controlling sequence and its application in nasopharyngeal carcinoma |
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2019
- 2019-06-14 CN CN201910517452.XA patent/CN110079603A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140296319A1 (en) * | 2011-05-25 | 2014-10-02 | Catholic University Industry Academic Cooperation Foundation | Composition For Promoting Apoptosis Or Inhibiting Cell Growth, Comprising Epstein-Barr Virus Microrna |
| CN103930563A (en) * | 2011-06-01 | 2014-07-16 | 医学预后研究所 | Methods and devices for prognosis of cancer relapse |
| CN102268477A (en) * | 2011-06-15 | 2011-12-07 | 中山大学肿瘤防治中心 | Application of nasopharyngeal carcinoma related EB (Epstein-Barr) virus miRNAs (microribonucleic acids) |
| CN109321655A (en) * | 2018-05-17 | 2019-02-12 | 福建省肿瘤医院（福建省肿瘤研究所、福建省癌症防治中心） | NKIRAS2 gene regulation region sequence, regulating and controlling sequence and its application in nasopharyngeal carcinoma |
Non-Patent Citations (1)
| Title |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023072076A1 (en) * | 2021-10-25 | 2023-05-04 | 杭州诺辉健康科技有限公司 | Reagent and method for diagnosis of nasopharyngeal carcinoma |
| CN114058705A (en) * | 2021-11-24 | 2022-02-18 | 中南大学湘雅医院 | METTL3 as nasopharyngeal carcinoma prognosis prediction marker, detection primer and kit |
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