CN109652535A - Identify human thyroid tubercle good pernicious kit and its application method and application - Google Patents
Identify human thyroid tubercle good pernicious kit and its application method and application Download PDFInfo
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Abstract
The invention belongs to kit technical field, in particular to identification human thyroid tubercle good pernicious kit and its application method and application, the kit includes sequencing primer system, PCR buffer solution system, Taq enzyme, UNG enzyme and negative quality-control product；The sequencing primer system include measure BRAF gene, KRAS gene, NRAS gene, on HRAS gene and TERT gene 19 mutational sites primer pair BRAF-2, primer pair KRAS-2, primer pair NRAS-2, primer pair HRAS-3, primer pair HRAS-4 and primer pair TERT-2.Kit of the invention can detect several genes variation simultaneously, its testing result can be used as intermediate result, and for clinical diagnosis Benign Thyroid Nodules, pernicious, judging prognosis provides auxiliary, whether thyroid cancer can be suffered from further combined with other result verifications, effectively reduce the probability of misjudgement, erroneous judgement.
Description
Technical field
The invention belongs to kit technical fields, in particular to identify the good pernicious kit of human thyroid tubercle and its make
Use methods and applications.
Background technique
Thyroid nodule (Thyroid nodule) is a kind of thyroid disease generally existing in crowd, and most tubercles are
It is benign, the thyroid nodule that conservative therapy can be taken, but have 5%-10% be it is pernicious, need Operation in early stage,
Good prognosis can be obtained.If giving the human thyroid benign nodules patients surgery treatment for not needing operation largely, it will generate
Huge medical insurance burden；, whereas if the real malignant tumour of identification cannot be identified from a large amount of human thyroid benign nodules, then prolong
The slow treatment to these patients, jeopardizes the life and health of patient.Therefore, the good of detection thyroid nodule promptly and accurately pernicious be
It is vital.
With the progress of Protocols in Molecular Biology, molecular diagnosis is in the pernicious antidiastole of Benign Thyroid Nodules, gradually
It is concerned by people.The theoretical basis of the good pernicious molecular diagnosis of tubercle is the Disease-causing gene based on some thyroid cancers,
The somatic mutation of specificity or the appearance of fusion, this mutation or new fusion only occur in the cancerous tissue of cancer patient
Gene is not present in the normal thyroid cell of patient and the parathyroid tissue of Non-thyrogenous cancer patient.Therefore, with this
A little Disease-causing genes are as molecular marker, in the malignant and benign lesion antidiastole of thyroid cancer, have specificity.
Wherein, the patient of the thyroid nodule 100% of the Val600Glu mutation of the 600th amino acids is in BRAF gene
Malignant Nodules, it is each although these gene mutations have extraordinary specificity in Benign Thyroid Nodules differential diagnosis of malignant
The frequency of mutation of the Disease-causing gene in thyroid cancer patient be it is indefinite, therefore, depend merely on the molecular mutation in this way to individual gene
It is detected, although has important Identification Significance to positive patient, but there is the possibility failed to pinpoint a disease in diagnosis to negative patient, delay patient's
Treatment.Therefore this predicament of thyroid cancer molecular diagnosis is solved, needs to the multiple genes for leading to thyroid cancer while carries out
Abrupt climatic change can improve the specificity of Benign Thyroid Nodules differential diagnosis of malignant, reduce false negative rate.
But, it is desirable in thyroid nodule sample, while the body for carrying out multiple thyroid cancer specific pathogenetic genes is thin
Born of the same parents' abrupt climatic change needs to break through the Science and Technology difficult point of the following aspects: although firstly, existing literature reports a large amount of first
The Disease-causing gene of shape gland cancer is mutated, but in the mutated gene of these reports, which is that real Disease-causing gene is unclear.
Secondly, the combination of which gene is positive in thyroid nodule antidiastole in the thyroid cancer Disease-causing gene reported at present
It is also ignorant that it is small, which to fail to pinpoint a disease in diagnosis possibility, for rate height and feminine gender.Therefore, the group of such a group thyroid cancer Disease-causing gene is found
It closes, being applied in the pernicious identification of Benign Thyroid Nodules reduces the probability of mistake, erroneous judgement, is very important.
Summary of the invention
In view of the deficienciess of the prior art, the first purpose of this invention is to provide a kind of identification human thyroid tubercle
Good pernicious kit, the Disease-causing gene based on thyroid cancer exist only in cancer patient and specialized cells mutation occur
This feature in tissue measures the mutational site on BRAF gene, KRAS gene, NRAS gene, HRAS gene and TERT gene,
Pernicious good booster action is provided to identify the good of thyroid nodule.
Second object of the present invention is to provide a kind of user for identifying the good pernicious kit of human thyroid tubercle
Method still can normally amplify target gene even if the sample size of thyroid nodule is less by two-wheeled PCR amplification.
Third object of the present invention is to provide a kind of application for identifying the good pernicious kit of human thyroid tubercle, be somebody's turn to do
Kit can provide booster action for the good pernicious and judging prognosis of clinical diagnosis thyroid nodule.
To achieve the above object, the present invention provides the following technical scheme that
The good pernicious kit of a kind of identification human thyroid tubercle, which is characterized in that the kit includes PCR buffering liquid
System, sequencing primer system, Taq enzyme, UNG enzyme and negative quality-control product；
The PCR buffer solution system is by PCR buffer 1, PCR buffer 2, PCR buffer 3, PCR buffer 4, PCR buffer
5, PCR buffer 6 and PCR buffer 7 form, and PCR buffer 1 includes primer pair BRAF-1, primer pair KRAS-1, primer pair
NRAS-1, primer pair HRAS-1, primer pair HRAS-2 and primer pair TERT-1, PCR buffer 2 include primer pair BRAF-2, PCR
Buffer 3 includes primer pair KRAS-2, and PCR buffer 4 includes primer pair NRAS-2, and PCR buffer 5 includes primer pair HRAS-
3, PCR buffers 6 include primer pair HRAS-4, and PCR buffer 7 includes primer pair TERT-2；
The sequencing primer system includes 19 on measurement BRAF gene, KRAS gene, NRAS gene, HRAS gene and TERT gene
Primer pair BRAF-2, primer pair KRAS-2, primer pair NRAS-2, primer pair HRAS-3, the primer pair HRAS-4 in a mutational site
With primer pair TERT-2；
19 kinds of mutational sites are successively are as follows: the T1799A and A1801G of BRAF gene, G34A, G34T of KRAS gene, G35A,
G35T, G37C, G37T and G38A, C181A, A182G, A182T, A183T and A183C of NRAS gene, the G35T of HRAS gene,
The C250T and C228T of C181A and A182G, TERT gene.
Further, BRAF-1 is as shown in SEQ ID NO.26 and SEQ ID NO.27, BRAF-2 such as SEQ ID NO.28
With shown in SEQ ID NO.29, KRAS-1 is as shown in SEQ ID NO.30 and SEQ ID NO.31, KRAS-2 such as SEQ ID
Shown in NO.32 and SEQ ID NO.33, NRAS-1 is as shown in SEQ ID NO.34 and SEQ ID NO.35, NRAS-2 such as SEQ ID
Shown in NO.36 and SEQ ID NO.37, HRAS-1 is as shown in SEQ ID NO.38 and SEQ ID NO.38, HRAS-2 such as SEQ ID
Shown in NO.40 and SEQ ID NO.41, HRAS-3 is as shown in SEQ ID NO.42 and SEQ ID NO.43, HRAS-4 such as SEQ ID
Shown in NO.44 and SEQ ID NO.45, TERT-1 is as shown in SEQ ID NO.46 and SEQ ID NO.47, TERT-2 such as SEQ ID
Shown in NO.48 and SEQ ID NO.49.
Further, the PCR buffer 1, PCR buffer 2, PCR buffer 3, PCR buffer 4, PCR buffer 5,
PCR buffer 6 and PCR buffer 7 further include having PCR buffer, dNTP or dUTP, nuclease-free water.
Further, the concentration of the PCR buffer be 10 ×.
Further, the concentration of the dNTP is 10mM-20mM.
Further, the PCR buffer 1, PCR buffer 2, PCR buffer 3, PCR buffer 4, PCR buffer 5,
PCR buffer 6 and PCR buffer 7 further include having Q-Solution.
Further, the storage temperature of the kit is -20 DEG C.
To realize above-mentioned second purpose, the present invention provides the following technical scheme that
A kind of application method identifying the good pernicious kit of human thyroid tubercle, comprising the following steps:
1., PCR amplification
1. -1, first round PCR reaction system: sample to be tested DNA1/ feminine gender quality-control product, 1-5 μ L；PCR buffer 1,14-18 μ L；
UNG enzyme, 0.5 μ L；0.5 μ L of Taq enzyme；
1. the -2, second wheel PCR reaction system: sample to be tested DNA2/ feminine gender quality-control product, 1-5 μ L, PCR buffer 2/3/4/5/6/
7,14-18 μ L, Taq enzyme, 0.5 μ L；
Wherein, sample to be tested DNA1 is the sample DNA of thyroid nodule, and the content of DNA is 20-100ng；Sample to be tested DNA2
10 times of gained are diluted for the negative quality-control product of first round PCR product；
2., PCR product electroresis appraisal
It takes step 1. to handle obtained PCR product using PCR buffer 2-PCR buffer 7 in -2, loading race successively is carried out to it
Glue obtains electrophoretogram, indicates that PCR amplification is normal if the race adhesive tape in electrophoretogram clearly becomes clear；
3., PCR product sequencing
2. step of learning from else's experience identifies the normal PCR product of PCR amplification, corresponding to use primer pair BRAF-2, primer pair KRAS-2, primer
Generation sequencing is carried out to NRAS-2, primer pair HRAS-3, primer pair HRAS-4 and primer pair TERT-2.
Further, first round PCR reaction condition are as follows:
Second wheel PCR reaction condition are as follows:
To realize above-mentioned third purpose, the present invention provides the following technical scheme that
A kind of application identifying the good pernicious kit of human thyroid tubercle, the kit are clinical diagnosis thyroid nodule
Good pernicious and judging prognosis provides booster action.
The invention has the following advantages:
1, phase is arranged in the mutational site of present invention selection BRAF gene, KRAS gene, NRAS gene, HRAS gene and TERT gene
The primer answered realizes and detects several genes variation simultaneously, pernicious provides good auxiliary work to identify the good of thyroid nodule
With the means being sequenced using a generation, so that detection becomes simple, easy to operate；
2, the present invention is reacted by two-wheeled PCR, and the sample size of even thyroid nodule is less, still can normally amplify mesh
Gene；
3, the present invention can provide booster action for the good pernicious and judging prognosis of clinical diagnosis thyroid nodule, be effectively reduced
The probability of misjudgement, erroneous judgement.
Detailed description of the invention
Fig. 1 is the analysis chart of the common mutated gene of Cosmic lane database thyroid cancer；
Fig. 2 is the electrophoretogram of embodiment 1；
Fig. 3 is the electrophoretogram of embodiment 2；
Fig. 4 is the electrophoretogram of embodiment 3；
Fig. 5 is the electrophoretogram of embodiment 4.
In figure, Fig. 2 race adhesive tape of number 1-8 into Fig. 5 respectively refer on behalf of: BRAF gene, KRAS gene, NRAS gene,
TERT gene, HRAS gene 1, the PCR product of HRAS gene 2, negative control PCR product and Marker.
Specific embodiment
Below in conjunction with specific attached drawing, invention is further described in detail.
1, the analysis and selection of mutated gene
In the art, the common mutated gene of Cosmic lane database thyroid cancer is referring to Fig. 1, wherein BRAF gene, RET
Gene, RAS gene family and TERT gene are located at forefront.
BRAF gene encoding serine-threonine kinase can change after being integrated to plasma membrane by RAS protein activation and transhipment
Attenuate the position of born of the same parents.If BRAF gene mutates, it will be caused the disorder of MEK/REK signal transduction by sustained activation, be led
Cell transition increment is caused, vicious transformation occurs.The RRAF gene and lymphatic metastasis, thyroid gland external diffusion, cancer of late stage (III phase
With IV phase), cancer return etc. it is closely related.
RET gene is the closest in medullary carcinoma of thyroid gland kind and germline or somatic mutation, and thyroid papillary carcinoma
It may also be on No. 10 chromosome in the presence of the gene rearrangement of concealment RET/PTC1 and PTC3, RET/PTC genetic mutation and because of year
The cancer that age or radioactive exposure are suffered from is related, related to lymphatic metastasis in terms of prognosis, but low point for making a variation with tumour
Change or undifferentiated uncorrelated.
KRAS gene, NRAS gene, HRAS gene belong to RAS gene, and in close relations, mediation junket ammonia is passed to signal
Acid kinase adjusts the differentiation of cell, is proliferated and withers to G-protein Ou Lian receptor to the signal path of MAPK and PI3K-AKT effector
It dies.The mutation of RAS gene can make it closer with the relationship of GTP (codon 12,13), or inhibit autocatalytic
The function of GTPase (codon 61).Both mechanism can all continue, activate MAPK the and PI3/AKT signal in downstream logical singularly
Oncogenic generation is then led on road.RAS gene all exists in thyroid tumors, and with tumour inertia and invasion
Clinical disease course is related, can be used as a label of cancer cell hypotype, display morphological differentiation is good but has potential transfer and goes
The cancer stove of differentiation.
TERT is reverse transcriptase of telomere, refers to the catalytic subunit in Telomerase with reverse transcriptase activity, belongs to telomere
A part of enzyme, and Telomerase can say that the telomere of DNA replication dna loss carries out reparation extension, so that fissional number increases
Add.The mutation of TERT gene is a kind of tumor cells marker of discovered in recent years, and mutation is concentrated mainly on promoter region
Two sites C250T and C228T, mutation probability reach 70%-80%.Since the mutation of TERT gene is in promoter region, make
The activity for obtaining TERT promoter increases, and causes cell transition to rise in value, promotes the amplification of mutated gene, aggravates the disease of thyroid cancer
Feelings.
In addition, it has been investigated that, carry TERT mutated gene (C250T, C228T) and BRAFV600EThe patient of mutated gene
Prognosis mala, and BRAFV600EGene mutation and TERT gene mutation are again (easy with the high mortality of thyroid papillary carcinoma (PTC)
Iodine affinity missing is radiated after metastatic, cancer return) it is closely related, specific PTC specificity mortality risk sequence is merging
The mono- BRAF of dual-gene mutation ﹥ ﹥V600EGene mutation=two gene of mono- TERT gene mutation ﹥ is without mutation, as shown in following table one,
After adjusting each factor, the mortality risk for merging dual-gene mutation is still 9 times or so of two genes without mutation.
The level of significance of table one every 1000 person/year of PTC specific mortality and different genes group
It can to sum up obtain, the mutation of BRAF gene, TERT gene and RAS gene family is the most key cause of thyroid cancer
Ospc gene, if thering is any one to be mutated in the combination of the gene, then determine thyroid nodule sample to be tested be it is pernicious,
If the combination of the gene without mutation, illustrates that a possibility that thyroid nodule is pernicious is smaller, as identification first
The good of shape gland tubercle pernicious provides good booster action.
Kit of the invention selects BRAF gene, RAS gene, NRAS gene, HRAS gene and TERT gene as a result,
Corresponding sequencing primer is arranged as molecular marker, with this in mutational site, realizes while detecting several genes variation, so that this examination
The combination of agent box has excellent specificity, reduces the probability of misjudgement, erroneous judgement.
2, embodiment
Below in conjunction with specific embodiment, invention is further described in detail.
It should be noted that it is disclosed by the invention for identifying the good pernicious kit of human thyroid tubercle, for 24
The measurement of person-portion thyroid nodule, including sequencing primer system, PCR buffer solution system, Taq enzyme, UNG enzyme and negative Quality Control
Product；PCR buffer solution system is by PCR buffer 1, PCR buffer 2, PCR buffer 3, PCR buffer 4, PCR buffer 5, PCR
Buffer 6 and PCR buffer 7 form；
PCR buffer 1 includes primer pair BRAF-1, primer pair KRAS-1, primer pair NRAS-1, primer pair HRAS-1, primer pair
HRAS-2 and primer pair TERT-1；
PCR buffer 2 includes primer pair BRAF-2, and PCR buffer 3 includes primer pair KRAS-2, and PCR buffer 4 includes primer
To NRAS-2, PCR buffer 5 includes primer pair HRAS-3, and PCR buffer 6 includes primer pair HRAS-4, and PCR buffer 7 includes
Primer pair TERT-2；
Wherein, sequencing primer series includes on measurement BRAF gene, KRAS gene, NRAS gene, HRAS gene and TERT gene
Primer pair BRAF-2, primer pair KRAS-2, primer pair NRAS-2, primer pair HRAS-3, the primer pair HRAS- in 19 mutational sites
4 and primer pair TERT-2, and each primer pair and PCR buffer 2, PCR buffer 3, PCR buffer in sequencing primer series
4, PCR buffer 5, PCR buffer 6 are consistent with the primer pair in PCR buffer 7.Kit of the invention is stored at -20 DEG C
Environment in, to guarantee the excellent performance of each reagent.
19 kinds of mutational sites are successively are as follows: the T1799A and A1801G of BRAF gene, G34A, G34T of KRAS gene, G35A,
G35T, G37C, G37T and G38A, C181A, A182G, A182T, A183T and A183C of NRAS gene, the G35T of HRAS gene,
The gene sequencing of the C250T and C228T of C181A and A182G, TERT gene, the wild types of 5 kinds of genes and saltant type is such as
Following table two, the sequence of each sequencing primer pair such as following table three, and following primer are outer to committee to be synthesized.
The wild type of two or five kinds of genes of table and the gene sequencing table of saltant type
The sequence table of the sequencing primer pair of three or five kinds of genes of table
In addition, PCR buffer 1, PCR buffer 2, PCR buffer 3, PCR buffer 4, PCR buffer 5, PCR buffer
It further include having PCR buffer, dNTP or dUTP, nuclease-free water and Q-Solution with PCR buffer 7, and PCR
Buffer, dNTP, dUTP, Q-Solution are obtained from purchase on the market.The concentration of PCR buffer is preferably 10 ×, remove this
Except can also be other concentration；The concentration of dNTP is preferably 10mM-20mM, but in addition to this also other concentration；Q-
Solution concentration is preferably 5 ×, but in addition to this also other concentration；Nuclease-free water, which is that the homemade sterilizing in laboratory is double, steams
Water.
The sample of thyroid nodule in the present invention is mainly thyroid needle sample, fresh pathological tissue, postoperative frost
In addition to this tissue and paraffin-embedded tissue can also be the sample of other forms, obtaining channel is hospital through pathological analysis
Remaining tissue samples afterwards.
1.1, embodiment 1
It is a kind of identify the good pernicious kit of human thyroid tubercle application method, including PCR amplification, PCR product electroresis appraisal,
The operating procedure of PCR product sequencing.
1., PCR amplification:
The component and content of the kit are configured by the PCR reaction system of table four on ice.
Four PCR reaction system of table
Wherein, sample to be tested DNA1 is the sample DNA of thyroid nodule, and benign first is specially determined as after pathological analysis
The puncture sample of shape gland tubercle, the sample DNA are extracted using the cell DNA extracts kit that Sigma company sells and are obtained, DNA
Content be 20ng；Negative quality-control product is through the resulting nuclease-free water of high pressure sterilization, and every kind of sample DNA is correspondingly arranged on one
Negative control group；Sample to be tested DNA2 is that the negative quality-control product of first round PCR product dilutes 10 times of gained；PCR buffer 1-PCR
The component and content of buffer 7 are referring to table five；The reaction condition of the kit is as shown in following table six.
The component and content of five PCR buffer 1-PCR buffer 7 of table
Six PCR reaction condition of table
2., PCR product electroresis appraisal
2. -1, configuring TAE: by concentration be 50 × TAE storing liquid saved in 4 DEG C, take 100ml storing liquid to be configured to 5L concentration to be
1 × TAE working solution, it is spare；
2. -2, glue: the Agarose powder of 1.4g being dissolved in 1 × TAE working solution of 120mL, micro-wave oven height fire 2-3min is molten
Solution, is decreased to about 60 DEG C to fluid temperature, and the Gel-Red of 12 μ L is added for 10000:1 by volume, mixes well, encapsulating；
2. -3, preparing loading:
A, 6 × Loading of loading Buffer is diluted with 1 × TAE working solution, and Loading Buffer and TAE working solution
Volume ratio is 1:2, obtains Loading Buffer dilution；
B, take 2 μ L steps are 1. middle to handle obtained PCR product and 6 part of 1 μ L using PCR buffer 2-PCR buffer 7 respectively
Step 2. Loading Buffer dilution obtained in -3, the corresponding portion Loading Buffer dilution of every part of PCR product,
6 parts of samples are formed after mixing two-by-two, and glue successively is run to its loading；
C, loading sequence are as follows: the PCR product of BRAF gene, the PCR product of KRAS gene, the PCR product of NRAS gene, TERT base
The PCR product of cause, the PCR product of HRAS gene 1, the PCR product of HRAS gene 2, negative control PCR product, number consecutively are
1-7；The Marker (2000bp) that 3 μ L are saved at 4 DEG C on 8th swimming lane；
2. -4, electrophoresis: black interface terminates cathode, red interface termination anode, and 120V electrophoresis 15-20min is obtained such as Fig. 2 institute
The electrophoretogram shown, the race adhesive tape of each genetic fragment is limpid in sight in electrophoretogram；
It can be obtained by Fig. 2, it can be preferably by BRAF gene, KRAS gene, NRAS gene, HRAS gene 1, HRAS base in this implementation
Because the mutational site of 2 and TERT gene is effectively expanded, amplification length 100-200bp, and negative control object is without amplification,
Subsequent sequencing procedures can be carried out to the genetic fragment.
3., PCR product sequencing
Take step 1. in using PCR buffer 2 to the obtained PCR product of the processing of PCR buffer 7, it is corresponding using BRAF-2,
KRAS-2, NRAS-2, HRAS-3, HRAS-4 and TERT-2 carry out generation sequencing, the gene in the gene order measured and table three
Sequence is compared.
Obtain through sequencing: NRAS gene is mutated on A182G, and TERT gene is mutated on C250T；Separately
Outside, BRAF gene, KRAS gene, HRAS gene 1 and HRAS gene 2 are wild type, therefore the thyroid nodule should be evil
Property and it is non-benign, corresponding therapeutic scheme is made convenient for patient and doctor with this in time.
1.2, embodiment 2
Difference from example 1 is that PCR reaction system is as shown in following table seven, the group of PCR buffer 1-PCR buffer 7
Divide and content is as shown in following table eight, the sample DNA of thyroid nodule is specially to be determined as benign thyroid knot after pathological analysis
The fresh pathological tissue of section, the content of DNA are 40ng, and measurand is the same patient, and electrophoretogram is as shown in figure 3, electricity
The race adhesive tape of each genetic fragment is limpid in sight in swimming figure, but obscures a bit than embodiment 1, and electrophoretogram is basic with embodiment 1
Unanimously, the gene order measured is also same as Example 1.
Seven PCR reaction system of table
The component and content of eight PCR buffer 1-PCR buffer 7 of table
1.3, embodiment 3
Difference from example 1 is that PCR reaction system is as shown in following table nine, the group of PCR buffer 1-PCR buffer 7
Divide and content is as shown in following table ten, the sample DNA of thyroid nodule is specially to be determined as benign thyroid knot after pathological analysis
The postoperative frozen tissue of section, the content of DNA are 100ng, and measurand is the same patient, and electrophoretogram is as shown in figure 4, electricity
The race adhesive tape of each genetic fragment is limpid in sight in swimming figure, and almost the same, the gene order measured of its electrophoretogram and embodiment 1
Also identical with embodiment 1.
Nine PCR reaction system of table
The component and content of ten PCR buffer 1-PCR buffer 7 of table
1.4, embodiment 4
Difference from example 1 is that PCR reaction system is as shown in following table 11, PCR buffer 1-PCR buffer 7
As shown in following table 12, the sample DNA of thyroid nodule is specially to be determined as benign first shape after pathological analysis for component and content
The puncture sample of gland tubercle, the content of DNA are 60ng, and measurand is the same patient, and electrophoretogram is as shown in figure 5, electricity
The clarity of the race adhesive tape of each genetic fragment is slightly poor to embodiment 3 relative to embodiment 1 in swimming figure, but its electrophoretogram and embodiment
1 it is almost the same, the gene order measured is also same as Example 1.
11 PCR reaction system of table
The component and content of 12 PCR buffer 1-PCR buffer 7 of table
To sum up, kit of the invention can detect several genes variation, the even sample size of thyroid nodule simultaneously
It is less, target gene still can be normally amplified, while detection being made to become simple, easy to operate, can be clinical diagnosis
The good pernicious and judging prognosis of thyroid nodule provides booster action, effectively reduces the probability of misjudgement, erroneous judgement.
This specific embodiment is only explanation of the invention, is not limitation of the present invention, those skilled in the art
Member can according to need the modification that not creative contribution is made to the present embodiment after reading this specification, but as long as at this
All by the protection of Patent Law in the scope of the claims of invention.
Sequence table
<110>Town in Shanghai first Biotechnology Co., Ltd
<120>identify human thyroid tubercle good pernicious kit and its application method and application
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gtggtagttg gagctggtgg cgtaggcaag agtgc 35
<210> 5
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gtggtagttg gagctagtgg cgtaggcaag agtgc 35
<210> 6
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gtggtagttg gagcttgtgg cgtaggcaag agtgc 35
<210> 7
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gtggtagttg gagctgatgg cgtaggcaag agtgc 35
<210> 8
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
gtggtagttg gagctgttgg cgtaggcaag agtgc 35
<210> 9
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
gtggtagttg gagctggtcg cgtaggcaag agtgc 35
<210> 10
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gtggtagttg gagctggttg cgtaggcaag agtgc 35
<210> 11
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
gtggtagttg gagctggtga cgtaggcaag agtgc 35
<210> 12
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
ctggatacag ctggacaaga agagtacagt gcc 33
<210> 13
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
ctggatacag ctggaaaaga agagtacagt gcc 33
<210> 14
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
ctggatacag ctggacgaga agagtacagt gcc 33
<210> 15
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
ctggatacag ctggactaga agagtacagt gcc 33
<210> 16
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
ctggatacag ctggacatga agagtacagt gcc 33
<210> 17
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
ctggatacag ctggacacga agagtacagt gcc 33
<210> 18
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
gtggtggtgg gcgccggcgg tgtgggcaag agt 33
<210> 19
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
gtggtggtgg gcgccgtcgg tgtgggcaag agt 33
<210> 20
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
ctggataccg ccggccagga ggagtacagc gcc 33
<210> 21
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
ctggataccg ccggcaagga ggagtacagc gcc 33
<210> 22
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
ctggataccg ccggccggga ggagtacagc gcc 33
<210> 23
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
cctcccgggt ccccggccca gccccctcc 29
<210> 24
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
cctcccgggt ccccggccca gccccttcc 29
<210> 25
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
ccttccgggt ccccggccca gccccctcc 29
<210> 26
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
ttgctctgat aggaaaatga gatctact 28
<210> 27
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
tcagtggaaa aatagcctca attct 25
<210> 28
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
tactgttttc ctttacttac tacacctcag 30
<210> 29
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
ggaaaaatag cctcaattct taccat 26
<210> 30
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
tggagtattt gatagtgtat taaccttatg tgt 33
<210> 31
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
tttacctcta ttgttggatc atattcg 27
<210> 32
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
accttatgtg tgacatgttc taatatagtc a 31
<210> 33
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
ggatcatatt cgtccacaaa atg 23
<210> 34
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
tccctgcccc cttaccct 18
<210> 35
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
ggttaatatc cgcaaatgac ttgc 24
<210> 36
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
gcccccttac cctccaca 18
<210> 37
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
ccgcaaatga cttgctatta ttga 24
<210> 38
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
cacggaaggt cctgaggg 18
<210> 39
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
aagacttggt gttgttgatg gc 22
<210> 40
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
cttggcaggt ggggcaggag ac 22
<210> 41
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
ctagaggaag caggagacag 20
<210> 42
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
gaggagcgat gacggaatat 20
<210> 43
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 43
tgctggcacc tggacg 16
<210> 44
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 44
tccctgagcc ctgtcctc 18
<210> 45
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 45
aaacacacac aggaagccct c 21
<210> 46
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 46
cgattcgacc tctctccgct 20
<210> 47
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 47
gcgctgcctg aaactcgc 18
<210> 48
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 48
agtggattcg cgggcacaga 20
<210> 49
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 49
cagcgctgcc tgaaactc 18
Claims (10)
1. a kind of good pernicious kit of identification human thyroid tubercle, which is characterized in that the kit includes PCR buffering liquid
System, sequencing primer system, Taq enzyme, UNG enzyme and negative quality-control product；
The PCR buffer solution system is by PCR buffer 1, PCR buffer 2, PCR buffer 3, PCR buffer 4, PCR buffer
5, PCR buffer 6 and PCR buffer 7 form, and PCR buffer 1 includes primer pair BRAF-1, primer pair KRAS-1, primer pair
NRAS-1, primer pair HRAS-1, primer pair HRAS-2 and primer pair TERT-1, PCR buffer 2 include primer pair BRAF-2, PCR
Buffer 3 includes primer pair KRAS-2, and PCR buffer 4 includes primer pair NRAS-2, and PCR buffer 5 includes primer pair HRAS-
3, PCR buffers 6 include primer pair HRAS-4, and PCR buffer 7 includes primer pair TERT-2；
The sequencing primer system includes 19 on measurement BRAF gene, KRAS gene, NRAS gene, HRAS gene and TERT gene
Primer pair BRAF-2, primer pair KRAS-2, primer pair NRAS-2, primer pair HRAS-3, the primer pair HRAS-4 in a mutational site
With primer pair TERT-2；
19 kinds of mutational sites are successively are as follows: the T1799A and A1801G of BRAF gene, G34A, G34T of KRAS gene, G35A,
G35T, G37C, G37T and G38A, C181A, A182G, A182T, A183T and A183C of NRAS gene, the G35T of HRAS gene,
The C250T and C228T of C181A and A182G, TERT gene.
2. a kind of good pernicious kit of identification human thyroid tubercle according to claim 1, which is characterized in that BRAF-1
As shown in SEQ ID NO.26 and SEQ ID NO.27, BRAF-2 is as shown in SEQ ID NO.28 and SEQ ID NO.29, KRAS-
1 as shown in SEQ ID NO.30 and SEQ ID NO.31, KRAS-2 as shown in SEQ ID NO.32 and SEQ ID NO.33,
NRAS-1 is as shown in SEQ ID NO.34 and SEQ ID NO.35, NRAS-2 such as SEQ ID NO.36 and SEQ ID NO.37 institute
Show, HRAS-1 is as shown in SEQ ID NO.38 and SEQ ID NO.38, HRAS-2 such as SEQ ID NO.40 and SEQ ID NO.41
Shown, HRAS-3 is as shown in SEQ ID NO.42 and SEQ ID NO.43, HRAS-4 such as SEQ ID NO.44 and SEQ ID
Shown in NO.45, TERT-1 is as shown in SEQ ID NO.46 and SEQ ID NO.47, TERT-2 such as SEQ ID NO.48 and SEQ ID
Shown in NO.49.
3. a kind of good pernicious kit of identification human thyroid tubercle according to claim 1, which is characterized in that described
PCR buffer 1, PCR buffer 2, PCR buffer 3, PCR buffer 4, PCR buffer 5, PCR buffer 6 and PCR buffer 7
It further include having PCR buffer, dNTP or dUTP, nuclease-free water.
4. a kind of good pernicious kit of identification human thyroid tubercle according to claim 3, which is characterized in that described
The concentration of PCR buffer be 10 ×.
5. a kind of good pernicious kit of identification human thyroid tubercle according to claim 3, which is characterized in that described
The concentration of dNTP is 10mM-20mM.
6. a kind of good pernicious kit of identification human thyroid tubercle according to claim 3, which is characterized in that described
PCR buffer 1, PCR buffer 2, PCR buffer 3, PCR buffer 4, PCR buffer 5, PCR buffer 6 and PCR buffer 7
It further include having Q-Solution.
7. a kind of good pernicious kit of identification human thyroid tubercle according to claim 1, which is characterized in that the examination
The storage temperature of agent box is -20 DEG C.
8. a kind of good pernicious kit of identification human thyroid tubercle described in any one of -7 makes according to claim 1
With method, which comprises the following steps:
1., PCR amplification
1. -1, first round PCR reaction system: sample to be tested DNA1/ feminine gender quality-control product, 1-5 μ L；PCR buffer 1,14-18 μ L；
UNG enzyme, 0.5 μ L；0.5 μ L of Taq enzyme；
1. the -2, second wheel PCR reaction system: sample to be tested DNA2/ feminine gender quality-control product, 1-5 μ L, PCR buffer 2/3/4/5/6/
7,14-18 μ L, Taq enzyme, 0.5 μ L；
Wherein, sample to be tested DNA1 is the sample DNA of thyroid nodule, and the content of DNA is 20-100ng；Sample to be tested DNA2
10 times of gained are diluted for the negative quality-control product of first round PCR product；
2., PCR product electroresis appraisal
It takes step 1. to handle obtained PCR product using PCR buffer 2-PCR buffer 7 in -2, loading race successively is carried out to it
Glue obtains electrophoretogram, indicates that PCR amplification is normal if the race adhesive tape in electrophoretogram clearly becomes clear；
3., PCR product sequencing
2. step of learning from else's experience identifies the normal PCR product of PCR amplification, corresponding to use primer pair BRAF-2, primer pair KRAS-2, primer
Generation sequencing is carried out to NRAS-2, primer pair HRAS-3, primer pair HRAS-4 and primer pair TERT-2.
9. a kind of application method for identifying the good pernicious kit of human thyroid tubercle according to claim 8, feature
It is,
First round PCR reaction condition are as follows:
Second wheel PCR reaction condition are as follows:
10. a kind of identification good pernicious kit of human thyroid tubercle according to any one of claims 1-7 is answered
With, which is characterized in that the kit provides booster action for the good pernicious and judging prognosis of clinical diagnosis thyroid nodule.
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| CN201910090514.3A CN109652535A (en) | 2019-01-30 | 2019-01-30 | Identify human thyroid tubercle good pernicious kit and its application method and application |
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| CN111534599A (en) * | 2020-06-18 | 2020-08-14 | 重庆浦洛通基因医学研究院有限公司 | Thyroid cancer auxiliary molecular diagnosis kit and using method thereof |
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