CN110229904A - Marker and its kit for chromoma diagnosis - Google Patents
Marker and its kit for chromoma diagnosis Download PDFInfo
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- CN110229904A CN110229904A CN201910569686.9A CN201910569686A CN110229904A CN 110229904 A CN110229904 A CN 110229904A CN 201910569686 A CN201910569686 A CN 201910569686A CN 110229904 A CN110229904 A CN 110229904A
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Abstract
The present invention specifically provides the marker and its kit for chromoma diagnosis, by to hTERT gene-correlation SNP site, mRNA expression and the determination of associated regulatory miRNA detection label, accurate detection can be carried out to chromoma, it can be used as perspective biomarker, to reach early diagnosis, treatment, risk stratification and prognosis evaluation.
Description
Technical field
The present invention relates to biomedicine fields, and in particular to for the marker and kit of chromoma diagnosis, more
It is specifically related to hTERT gene and its application in preparation chromoma diagnosis and treatment drug.
Background technique
Malignant mela noma (malignantmelanoma, MM), abbreviation melanoma are initiated by epidermal melanophore
A kind of high malignancy tumour, mostly occurs in skin, mucous membrane, is also found in skin-mucous membrane intersection, ocular choroid and pia mater etc.
Place.The disease incidence of melanoma is still in sustainable growth trend in the world in recent years.National Cancer Institute
The data of (National Cancer Institute, NCI) show that the U.S. in 2017 newly sends out melanoma case load and is up to eight
More than ten thousand examples, death number is close to 10,000.China's tumour registration annual report and the data of China national Cancer center show, China
The disease incidence of melanoma still shows an increasing trend year by year, and annual neopathy number of cases reaches 20,000 people or so, is that China falls ill in recent years
Rate increases one of faster malignant tumour.
Currently, surgical resection treatment is still the preferred treatment means of melanoma.Early stage melanoma prognosis is preferable, and 5 years
Survival rate can be of about 90% or more.But once tumour shifts, the treatment of melanoma just becomes very intractable, and suffers from
The prognosis of person is often very poor.The median survival time of metastatic melanoma patient only has 8-9 months, 3 years overall survivals less than
15%.Although currently used for the chemotherapeutics of clinical treatment, immunotherapy medicaments, molecular biosciences target therapeutic agent and joint
The overall survival (overall survival rate, OS) of improvement metastatic melanoma that can be different degrees of such as treat, but
The problems such as following tumor drug resistance, drug resistance increase brings bigger challenge to the treatment of melanoma.Melanoma is tight
The health that threaten the mankind again causes great stress and economic impact to sufferers themselves and its family, to social band
Lot of unstable factor is carried out.Therefore, melanoma accurately diagnosis marker and its its relevant targeted therapy problem are studied, is mentioned
Become the common target of current domestic and foreign scholars for more effective, safer diagnosing and treating strategy.
Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of Telomerase, can extend telomere, promotes the unlimited of cell
Proliferation.There is scholar to think, the expression of hTERT mRNA up-regulation will lead to telomerase activation raising, the proliferation of cell, differentiation and
It plays an important role in aging and the occurrence and development of tumour.2013, Susanne Hom and Huang etc. were in succession at " Science "
On publish an article and point out, the occurrence and development of hTERT gene promoter region mutation and melanoma are closely related, hTERT gene promoter
Sub-district mutation rate is up to 70% or more in melanoma, most common with C228T and C250T mutation.HTERT base is pointed out in multinomial research
Because promoter region mutation is related with Several Kinds of Malignancy, such as central nerve neuroma, bladder cancer, thyroid cancer.HTERT base
Because promoter region mutation can be used as diagnosis, antidiastole and prison of the important biomolecule marker for Patients with Urinary System Tumors
It surveys, diagnosis, treatment and prognosis for Patients with Urinary System Tumors especially for bladder cancer and carcinoma of renal pelvis provide new approaches.Melanoma is
A kind of multifactor related disease, the phases such as mechanism and environmental factor, genetic predisposition, gene mutation and abnormal cell proliferation
It closes, hTERT gene plays an important role in the occurrence and development of melanoma.HTERT gene and Several Kinds of Malignancy have certain relationship,
The occurrence and development of the especially mutation of the gene promoter area and methylation state and tumour are more close.
Currently, for the clinical diagnosis of melanoma be based primarily upon histopathological criteria (including tumor depth, invasion water
Put down, whether have ulcer and lymphatic metastasis), but histopathological criteria cannot be distinguished from benign melanotic nevus and be changed into melanin
The hypotype of tumor can not also prejudge which patient is easy to happen transfer.The present invention provides chromoma diagnosis marker and its examination
Agent box provides theoretical foundation for chromoma early diagnosis, invasion, transfer, Index for diagnosis and targeted therapy.
Summary of the invention
For the deficiencies in the prior art problem, the purpose of the present invention is to provide what is diagnosed for chromoma
Marker and kit, by hTERT gene-correlation SNP site, mRNA expression and associated regulatory miRNA detection label
It determines, accurate detection can be carried out to chromoma, can be used as perspective biomarker, to reach early diagnosis, control
Treatment, risk stratification and prognosis evaluation.
What the invention is realized by the following technical scheme:
The present invention is provided to the markers of chromoma diagnosis, and the marker includes the upstream hTERT Gene A TG-
The site 245bpSNP, hTERT gene mRNA, hTERT gene-correlation regulate and control miRNAs.
The present invention provides a kind of kits for chromoma diagnosis, are able to detect hTERT gene in peripheral blood
The upstream the ATG site -245bpSNP.
Preferably, mentioned reagent box contains the primer in the upstream the hTERT Gene A TG site -245bpSNP.
Meanwhile the present invention also provides the upstream the hTERT Gene A TG sites -245bpSNP to prepare one kind for pernicious black
Application in the kit of plain tumor diagnosis.
Preferably, the reverse transcription primer in the above-mentioned upstream the hTERT Gene A TG site -245bpSNP such as SEQ ID NO.1 institute
Show, the qPCR primer in the upstream the hTERT Gene A TG site -245bpSNP is as shown in SEQ ID NO.2.
The present invention provides a kind of kits for chromoma diagnosis, are able to detect hTERT base in skin histology
Because of the expression quantity of associated regulatory miRNAs, the hTERT gene-correlation regulation miRNAs is hsa_miR-497-5p, hsa_
MiR-195-5p, hsa_miR-455-3p, the hsa_miR-497-5p, hsa_miR-195-5p, hsa_miR-455-3p,
For the miRNA code name in Relational database, can retrieve to obtain in the database.
Preferably, mentioned reagent box contain hsa_miR-497-5p, hsa_miR-195-5p, hs a_miR-455-3p,
Primer.
Meanwhile the present invention also provides hsa_miR-497-5p, hsa_miR-195-5p, hsa_miR-455-3p, making
Application in a kind of standby kit for chromoma diagnosis.
Preferably, the reverse transcription primer of above-mentioned hsa_miR-497-5p is as shown in SEQ ID NO.3, hsa_miR-497-5p
QPCR primer as shown in SEQ ID NO.4；The reverse transcription primer of above-mentioned hsa_miR-195-5p as shown in SEQ ID NO.5,
The qPCR primer of hsa_miR-195-5p is as shown in SEQ ID NO.6；The reverse transcription primer of above-mentioned hsa_miR-455-3p such as SEQ
Shown in ID NO.7, the qPCR primer of hsa_miR-455-3p is as shown in SEQ ID NO.8.
The present invention provides a kind of kits for chromoma diagnosis, are able to detect hTERT base in skin histology
Because of mrna expression amount.
Preferably, mentioned reagent box contains the primer of hTERT gene mRNA.
Meanwhile the present invention also provides hTERT gene mRNAs to prepare a kind of kit for chromoma diagnosis
In application.
Preferably, the reverse transcription primer of above-mentioned hTERT gene mRNA is as shown in SEQ ID NO.9, hTERT gene mRNA
QPCR primer is as shown in SEQ ID NO.10.
It further include that PCR reacts common agents and some other auxiliary reagent in the chromoma diagnostic kit, this
The characteristic and their preparation method of a little reagents are well-known to those skilled in the art；In specific embodiment party of the invention
In case, the method that has used quantitative fluorescent PCR.
The present invention provides the marker and kit for chromoma diagnosis obtained by above-mentioned preparation process.It is logical
Cross the specific summary of the invention of the implementation present invention, can achieve it is following the utility model has the advantages that
(1) marker provided by the present invention for chromoma diagnosis and kit have high stability, special
Property, extremely significant correlation is all had by the correlation analysis of experimental result and clinical data and related coefficient is above
0.98, there is higher clinical reference value.
(2) sieve of chromoma can be made provided by the present invention for marker, the kit of chromoma diagnosis
It looks into, early diagnose not against subjective experience judgement, but SNP site, mRNA and associated regulatory miRNAs can be passed through and express water
This objective indicator inspection is put down, accuracy rate of diagnosis, Cultivation process are improved.
Detailed description of the invention
Fig. 1 is shown as DNA agarose gel electrophoresis detection figure.
Fig. 2 is shown as RNA agarose gel electrophoresis detection figure.
Fig. 3 is shown as the gene promoter area hTERT mutational site testing result figure, wherein figure A is pcr amplification product agar
Sugared detected through gel electrophoresis is as a result, figure B is pcr amplification product sequencing result figure.
Specific embodiment
In the following, illustrating the present invention for embodiment, still, the present invention is not limited to following embodiments.
There is material in the present invention: QIAGEN miRNeasy FFPE Kit extracts kit, QIAamp DNA FFPE
Tissue Kit (50) kit (Germany), miScript II RT Ki t (QIAGEN) kit, miScript SYBR
Green PCR Kit (QIAGEN) kit, TaKaRa LA Taq with GC Buffer, 6 DNA × loading
The bis- dyestuffs of Buffer, 1 × TAE electrophoretic buffer, agar Icing Sugar, bromophenol blue, ethidium bromide, dehydrated alcohol, dimethylbenzene,
QIAamp MinElut Columns、Collection Tubes(2ml)、Buffer AL、Buffer ATL、Buffer AWl、
Buffer AW2, Buffer ATE, Proteinase K.
Instrument used in the present invention has: the manual rotary microtome of Leica RM2235 (Leica), CHK Olympus
Microscope (Olympus), NaNO Drop ND-1000 UV detector (NanoDrop), Biosens SC720 gel figure
As imager (upper seamount richness scientific instrument Co., Ltd), real-time fluorescence fluorescent quantitative PCR instrument (BIO-RAD), YXQ-LS-
50G vertical pressure steam sterilizer (Shanghai Boxun Industrial Co., Ltd.), miniature whirlpool mixed instrument (Shanghai Luxi analysis instrument
Factory), AF-10 type automatic ice maker (Scotsman), micropipettor (Eppdorff), ultra-clean laboratory bench (win fast in Shanghai
Industrial Co., Ltd.), -20 DEG C of refrigerators (Haier Group, Co), 4 DEG C of supercentrifuges (Eppdorff), U.S. water chestnut BCD-238 type ice
Case (Mei Ling Group company), 192 type ultra low temperature freezer (Sanyo) electromagnetic ovens (beautiful), pressure cooker (beautiful), constant temperature roaster
(Beijing light instrument plant), electronic thermostatic three water tank (Beijing forever bright Medical Instruments factory), the water tank (gold of digital display constant temperature three
Altar city Guo Wang laboratory apparatus factory).The reagent, material can be bought by public channel, equipment employed in technique and instrument
Device is the common equipment in this field.
What all material, reagent and the instrument selected in the present invention were all well known in the art, but reality of the invention is not limited
It applies, other some reagents well known in the art and equipment are applied both to the implementation of following implementation of the present invention.
Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case where spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to
Range.
Embodiment one: the marker for chromoma diagnosis
The present invention is provided to the markers of chromoma diagnosis, and the marker includes the upstream hTERT Gene A TG-
The site 245bpSNP, hTERT gene mRNA, hTERT gene-correlation regulate and control miRNAs.
Embodiment two: the kit for chromoma diagnosis
The present invention provides a kind of kits for chromoma diagnosis, are able to detect hTERT gene in peripheral blood
The upstream the ATG site -245bpSNP.
Preferably, mentioned reagent box contains the primer in the upstream the hTERT Gene A TG site -245bpSNP.
Meanwhile the present invention also provides the upstream the hTERT Gene A TG sites -245bpSNP to prepare one kind for pernicious black
Application in the kit of plain tumor diagnosis.
Preferably, the reverse transcription primer in the above-mentioned upstream the hTERT Gene A TG site -245bpSNP such as SEQ ID NO.1 institute
Show, the q PCR primer in the upstream the hTERT Gene A TG site -245bpSNP is as shown in SEQ ID NO.2.
The present invention provides a kind of kits for chromoma diagnosis, are able to detect hTERT base in skin histology
Because of the expression quantity of associated regulatory miRNAs, the hTERT gene-correlation regulation miRNAs is hsa_miR-497-5p, hsa_
MiR-195-5p, hsa_miR-455-3p, the hsa_miR-497-5p, hsa_miR-195-5p, hsa_miR-455-3p,
For the miRNA code name in Relational database, can retrieve to obtain in the database.
Preferably, mentioned reagent box contain hsa_miR-497-5p, hsa_miR-195-5p, hsa_miR-455-3p,
Primer.
Meanwhile the present invention also provides hsa_miR-497-5p, hsa_miR-195-5p, hsa_miR-455-3p, making
Application in a kind of standby kit for chromoma diagnosis.
Preferably, the reverse transcription primer of above-mentioned hsa_miR-497-5p is as shown in SEQ ID NO.3, hsa_miR-497-5p
QPCR primer as shown in SEQ ID NO.4；The reverse transcription primer of above-mentioned hsa_miR-195-5p as shown in SEQ ID NO.5,
The qPCR primer of hsa_miR-195-5p is as shown in SEQ ID NO.6；The reverse transcription primer of above-mentioned hsa_miR-455-3p such as SEQ
Shown in ID NO.7, the qPCR primer of hsa_miR-455-3p is as shown in SEQ ID NO.8.
The present invention provides a kind of kits for chromoma diagnosis, are able to detect hTERT base in skin histology
Because of mrna expression amount.
Preferably, mentioned reagent box contains the primer of hTERT gene mRNA.
Meanwhile the present invention also provides hTERT gene mRNAs to prepare a kind of kit for chromoma diagnosis
In application.
Preferably, the reverse transcription primer of above-mentioned hTERT gene mRNA is as shown in SEQ ID NO.9, hTERT gene mRNA
QPCR primer is as shown in SEQ ID NO.10.
It further include that PCR reaction common agents and quantitative fluorescent PCR reaction are common in the chromoma diagnostic kit
Reagent.
Embodiment three: clinical verification test
Research object
Case group:
By January nineteen ninety in October, 2014 to pathology department, Xinjiang Autonomous Region the People's Hospital, dermatology, department of plastic surgery and other
The chromoma patient cases data and its Follow-up Data of hospital collect one by one, typing, establishes Xinjiang region chromoma
Clinical case database.
Currently, Xinjiang chromoma specimen resource library is collected altogether: 120 parts of paraffin-embedded tissue；128 parts of blood sample；It is fresh
25 parts of tissue by chromoma tissue and tumor.
Inclusion criteria:
(1) diagnosis is clear: the pathological replacement of patient is classified by two or more pathology professors according to the World Health Organization
Standard carries out morphological observation and makes diagnosis；
(2) Paraffin-embedded tissue sample of patient is intact；
(3) has complete clinical and pathological data.
Exclusion criteria:
(1) patient is associated with disease of immune system, connective tissue disease and other important organ diseases；
(2) patient carried out radiotherapy, chemotherapy, freezing, immune, laser and other treatment preoperative March；
(3) patient or its legal guardian disagree signature informed consent form person.
Control group: 36 specific Uygur nationality's mole patient's paraffin organization sample standard deviations of diagnosis are from Xinjiang Uygur
People's Hospital of the Autonomous Region's Dermatology & STD Dept. in May, 2016 is to underwent operative in November, 2016 is cut off, 10% formalin is fixed, paraffin
The archive sample of embedding.
The acquisition for all paraffin-embedded tissue samples used in this research acquirement patient's or its legal guardian knows
Feelings are agreed to, and sign informed consent form.The acquisition of case group and control group paraffin organization sample passes through Xinjiang Uygur Autonomous Regions
The approval of Ethics Committee, the People's Hospital.In addition, all research objects fill in the melanoma sample collection application form of standard, in detail
Thin record every patient and essential information, historical clinical information, the pathological data of collator etc..
Research method
1. data collection
It is included in standard and exclusion criteria in strict accordance with formulation, collects People's Hospital of Xinjiang Uyghur Autonomous Region's Dermatology
The Uygur nationality's melanoma sample and Xinjiang Uygur that section and pathology department clarify a diagnosis in November, -2016 in January, 2010 are autonomous
The People's Hospital, area Dermatology & STD Dept. in May, 2016 in November, 2016 diagnoses specific Uygur nationality's mole sample.To own
Meet the essential information (such as gender, age) and clinical pathology information of the standard of being included in and exclusion criteria patient (such as: melanoma point
Whether type occurs ulcer, lymphatic metastasis whether occurs, DISTANT METASTASES IN whether occurs etc.) it is entered into specified Excel table,
In case subsequent analysis.
Illustrate: referring to the definition of " Chinese melanoma diagnosis and treatment guide 2015 editions ", melanoma parting refers to current state herein
The partings of general four classification on border, comprising: acra type, mucous membrane type, chronic solar damage type (Chronic sundamaged,
) and non-chronic solar damage type (Non chronic sun damaged, Non-CSD, including the unknown type of primary lesion CSD.Separately
Outside, the definition of " DISTANT METASTASES IN " is revised in " malignant mela noma AJCC the 8th edition is by stages " of in November, 2017 publication,
But the sample collection arrangement stage (in November, -2016 in January, 2010) of this research, " malignant mela noma AJCC the 8th edition is by stages "
It not yet issues, therefore " DISTANT METASTASES IN " in this research still uses the definition of AJCC the 7th edition by stages: i.e. skin, subcutaneous tissue or remote
Locate lymphatic metastasis, Lung metastases, other visceral metastases or any DISTANT METASTASES IN to increase with lactic dehydrogenase (LDH).
2.DNA is extracted and RNA is extracted
One, DNA extraction and quality inspection
It is extracted in paraffin tissue sections using QIAamp DNA FFPE Tissue Kit (50) kit (Germany)
The DNA obtained in DNA, EP pipe is put in -20 DEG C of refrigerators after packing and saves, is spare.Take the DNA purity extracted of experiment and dense
Degree measurement.Before measurement, Yao Xianyong ddH2O returns to zero NanoDrop spectrophotometer, and surveyed DNA concentration is all larger than 50ng/ μ l,
Purity (A260/A280 value) as meets the requirement of experiment between 1.8-2.0；The Ago-Gel of configuration 1.5%, by 2 μ l
DNA and 3 μ l bromophenol blue mix after be added in well, the steady electrophoresis of 100V 15 minutes；With Biosens SC720 gel
Image imager acquisition image is simultaneously marked, is saved, in case subsequent analysis.DNA agarose gel electrophoresis testing result such as Fig. 1 institute
Show.
Two, RNA extraction and quality inspection
RNA in paraffin tissue sections is extracted using QIAGEN miRNeasy FFPE Kit extracts kit, is obtained
RNA detects its concentration and purity (A260/A280) with 8000 spectrophotometer of Nanodrop.The RNA for meeting quality inspection result is put
In -80 DEG C of refrigerators, save backup.The RNA2 μ l for taking experiment to extract is for purity and concentration mensuration.Before measurement, Yao Xianyong
ddH2O returns to zero NanoDrop spectrophotometer, and adjusted numerical value is between -1 to+1 when detecting RNA.Surveyed RNA concentration is big
In 100ng/ μ l, purity (A260/A280 value) as meets the requirement of experiment between 1.7-2.0.The agar of configuration 0.4%
Sugared gel is added in well, the steady electrophoresis of 100V 15 minutes after mixing the bromophenol blue of the RNA of 2 μ l and 3 μ l；With
Biosens SC720 gel images imager acquisition image is simultaneously marked, is saved, in case subsequent analysis.RNA agarose gel electrophoresis
Testing result is as shown in Figure 2.
The gene promoter area 4.hTERT PCR amplification
Melanoma Tissue DNA is taken, selects primer shown in SEQ ID NO.1 and SEQ ID NO.2 according to the reaction system of table 1
And the response procedures of table 2 carry out PCR amplification, while replacing template DNA with sterilizing distilled water, as negative control.It detects part
As a result as shown in Figure 3.
1 gene promoter area hTERT pcr amplification reaction system of table
| Reagent | Volume is added |
| Upstream primer | 1μl |
| Downstream primer | 1μl |
| Template DNA | 1μl |
| dNTPMix | 3μl |
| LATaq | 0.25μl |
| 2×GC Buffer II | 12.5μl |
| ddH2O | 6.25μl |
| Total volume | 25.0μl |
2 gene promoter area hTERT pcr amplification reaction program of table
By the gene promoter area hTERT PCR amplification and sequencing experimental result, correlation analysis is carried out in conjunction with case information
It was found that the mutation of A → G has occurred in the gene promoter area the hTERT upstream-ATG -245bp, and it is mutated and occurs to send out with chromoma
Disease is in extremely significant positive correlation, related coefficient 0.989, it is seen that the gene promoter area hTERT-ATG upstream -245bp is mutated position
Point can be used as the biomarker of effective chromoma clinical diagnosis or the target spot of disease treatment has a high potential.
5.hTERT gene-correlation regulates and controls miRNA verifying
Melanoma skin tissue RNA is taken, carries out cDNA synthesis according to miScript II RT Kit (QIAGEN) kit,
Primer shown in SEQ ID NO.3 to SEQ ID NO.8 is selected to try according to miScript SYBR Green PCR Kit (QIAGEN)
Agent box illustrates to carry out fluorescent quantitative PCR, using U6 as reference gene.Normal skin tissue RNA is as control sample, simultaneously
Replace template DNA with sterilizing distilled water, as negative control.Quantitative fluorescent PCR verifying analysis find, chromoma with compare
Group miRNA expression compare, chromoma patient skin tissue hsa_miR-497-5p, hsa_miR-195-5p and
The expression of hsa_miR-455-3p is significantly lowered, and the results are shown in Table 3.
Table 3: case group (△ Ct ± SD) compared with control group miRNA expression
| miRNAs | 2Δ Δ Ct(△Ct±SD) | t | p |
| hsa_miR-497-5p | 0.492±0.371 | -6.152 | <0.0001* |
| hsa_miR-195-5p | 0.629±0.317 | -5.427 | <0.0001* |
| hsa_miR-455-3p | 0.271±0.266 | -8.781 | <0.0001* |
By fluorescent quantitative PCR experiment as a result, carrying out correlation analysis discovery, hsa_miR-497- in conjunction with case information
The expression of 5p, hsa_miR-195-5p and hsa_miR-455-3p significantly lower and expression significantly raise with it is pernicious
Melanoma morbidity is in extremely significant positive correlation, related coefficient 0.988, it is seen that hsa_miR-497-5p, hsa_miR-195-5p,
Hsa_miR-455-3p can be used as the biomarker of effective chromoma clinical diagnosis or the target spot of disease treatment is dived
Power is huge.
The verifying of 6.hTERT mrna expression
Melanoma skin tissue RNA is taken, carries out cDNA synthesis according to miScript II RT Kit (QIAGEN) kit,
Select primer shown in SEQ ID NO.9 to SEQ ID NO.10 according to miScript SYBR Green PCR Kit (QIAGEN)
Kit illustrates to carry out fluorescent quantitative PCR, using GAPDH as reference gene normal skin tissue RNA as control sample,
Replace template DNA with sterilizing distilled water simultaneously, as negative control.Quantitative fluorescent PCR verifying analysis find, chromoma with
The hTERT mrna expression of control group compares, chromoma patient skin tissue hTERT mrna expression
It is extremely significant to be higher than normal tissue, it the results are shown in Table 4.
Table 4: case group (△ Ct ± SD) compared with control group hTERT mrna expression
| mRNA | 2Δ Δ Ct(△Ct±SD) | t | p |
| Chromoma group | 1.962±0.509 | 7.134 | <0.001 |
By fluorescent quantitative PCR experiment as a result, carrying out correlation analysis discovery, chromoma group in conjunction with case information
Expression and the chromoma morbidity of hTERT gene mRNA are in extremely significant positive correlation, related coefficient 0.99, it is seen that
The expression of hTERT gene mRNA can be used as biomarker or the disease treatment of effective chromoma clinical diagnosis
Target spot have a high potential.
In conclusion provided by the present invention for chromoma diagnosis marker, kit have high degree of accuracy,
Specificity has higher clinical reference value；In addition, marker, reagent provided by the present invention for chromoma diagnosis
Box can be such that the screening of chromoma, early diagnosis judges not against subjective experience, but can provide through the invention
This objective indicator inspection of marker of chromoma diagnosis, with raising accuracy rate of diagnosis, Cultivation process.
As described above, the present invention can be realized preferably, the above embodiments are only to preferred implementation side of the invention
Formula is described, and is not intended to limit the scope of the present invention, and without departing from the spirit of the design of the present invention, this field is general
The various changes and improvement that logical technical staff makes technical solution of the present invention, should all fall into present invention determine that protection scope
It is interior.
Sequence table
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Claims (6)
1. a kind of kit for chromoma diagnosis, which is characterized in that be able to detect hTERT Gene A TG in peripheral blood
The upstream site -245bpSNP.
2. a kind of kit for chromoma diagnosis as described in claim 1, which is characterized in that hTERT Gene A TG
The reverse transcription primer in the upstream site -245bpSNP is as shown in SEQ ID NO.1, the upstream the hTERT Gene A TG site -245bpSNP
QPCR primer as shown in SEQ ID NO.2.
3. a kind of kit for chromoma diagnosis, which is characterized in that be able to detect hTERT gene phase in skin histology
The expression quantity of regulation miRNAs is closed, the hTERT gene-correlation regulation miRNAs is hsa_miR-497-5p, hsa_miR-
195-5p, hsa_miR-455-3p, described hsa_miR-497-5p, hsa_miR-195-5p, hsa_miR-455-3p are correlation
MiRNA code name in database.
4. a kind of kit in chromoma diagnosis as claimed in claim 2, which is characterized in that hsa_miR-497-5p
Reverse transcription primer as shown in SEQ ID NO.3, the qPCR primer of hsa_miR-497-5p is as shown in SEQ ID NO.4；hsa_
The reverse transcription primer of miR-195-5p is as shown in SEQ ID NO.5, the qPCR primer of hsa_miR-195-5p such as SEQ ID NO.6
It is shown；The reverse transcription primer of hsa_miR-455-3p is as shown in SEQ ID NO.7, and the qPCR primer of hsa_miR-455-3p is such as
Shown in SEQ ID NO.8.
5. a kind of kit for chromoma diagnosis, which is characterized in that be able to detect hTERT gene in skin histology
Mrna expression amount.
6. a kind of kit for chromoma diagnosis as claimed in claim 5, it is characterised in that hTERT gene mRNA
Reverse transcription primer as shown in SEQ ID NO.9, the qPCR primer of hTERT gene mRNA is as shown in SEQ ID NO.10.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111088361A (en) * | 2020-01-20 | 2020-05-01 | 西安交通大学医学院第一附属医院 | Long-chain non-coding RNA marker for early diagnosis of malignant melanoma and application thereof |
| CN112941185A (en) * | 2021-03-26 | 2021-06-11 | 杭州医学院 | Application of miR-29a as marker in preparation of malignant mesothelioma detection kit |
| CN113637763A (en) * | 2021-10-15 | 2021-11-12 | 北京百奥思科生物医学技术有限公司 | Application of miRNA biomarker in early diagnosis and treatment of melanoma |
| CN114703283A (en) * | 2022-04-07 | 2022-07-05 | 海南源轩生物科技有限公司 | Application of MNS16A genotype as biomarker for predicting cancer chemotherapy sensitivity |
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2019
- 2019-06-27 CN CN201910569686.9A patent/CN110229904A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111088361A (en) * | 2020-01-20 | 2020-05-01 | 西安交通大学医学院第一附属医院 | Long-chain non-coding RNA marker for early diagnosis of malignant melanoma and application thereof |
| CN112941185A (en) * | 2021-03-26 | 2021-06-11 | 杭州医学院 | Application of miR-29a as marker in preparation of malignant mesothelioma detection kit |
| CN112941185B (en) * | 2021-03-26 | 2023-06-23 | 杭州医学院 | Application of miR-29a as marker in preparation of malignant mesothelioma detection kit |
| CN113637763A (en) * | 2021-10-15 | 2021-11-12 | 北京百奥思科生物医学技术有限公司 | Application of miRNA biomarker in early diagnosis and treatment of melanoma |
| CN113637763B (en) * | 2021-10-15 | 2024-04-26 | 北京百奥思科生物医学技术有限公司 | Use of miRNA biomarker in early diagnosis and treatment of melanoma |
| CN114703283A (en) * | 2022-04-07 | 2022-07-05 | 海南源轩生物科技有限公司 | Application of MNS16A genotype as biomarker for predicting cancer chemotherapy sensitivity |
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Application publication date: 20190913 |