CN109402123A - A kind of circular rna hsa-circ-0073004 and its specificity amplification primer and application - Google Patents
A kind of circular rna hsa-circ-0073004 and its specificity amplification primer and application Download PDFInfo
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- CN109402123A CN109402123A CN201811508153.1A CN201811508153A CN109402123A CN 109402123 A CN109402123 A CN 109402123A CN 201811508153 A CN201811508153 A CN 201811508153A CN 109402123 A CN109402123 A CN 109402123A
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- 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
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention discloses a kind of circular rna hsa-circ-0073004 and its specificity amplification primer and applications, wherein, the nucleotide sequence of the circular rna is as shown in SEQ ID NO:1, its low expression in breast cancer tissue's cell and blood with patient with breast cancer, can be used as the molecular marker of screening breast cancer；The present invention also provides a kind of for expanding the specificity amplification primer of the circular rna, the specificity amplification primer includes upstream primer and downstream primer, wherein, the nucleotide sequence of the upstream primer is as shown in SEQ ID NO:2, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO:3；The invention also discloses application of the specificity amplification primer in the kit that preparation is used for breast cancer screening and the kits including the specificity amplification primer.
Description
Technical field
The invention belongs to oncomolecularbiology field more particularly to a kind of circular rnas for breast cancer diagnosis, are used for
The specificity amplification primer of the circular rna and application.
Background technique
Breast cancer is a kind of common high-risk malignant tumour of women, seriously affects people's lives quality.In recent years, full generation
About 22.9% is patient with breast cancer in the female cancer patients on boundary, and in newfound patient with breast cancer, Chinese patients are accounted for
12.2%.Although having huge breakthrough on drug susceptibility and surgical technic, the recurrence and transfer of breast cancer are still continually
Occur, becomes the main reason for advanced breast cancer patient is dead.Early diagnosis, early treatment can greatly reduce breast cancer deterioration
Further occurrence, further ground with the development of molecular level and to mammary gland carcinogenesis, development, treatment related mechanism
Study carefully, screening to targeting diagnosis and targeted therapy molecular marker and research become breast cancer research field of greatest concern it
One.
Circular rna (Circular RNA, circRNA) is a kind of without the end 5' cap and the end 3' poly (A) tail
Bar and with covalent bond formed ring structure non-coding RNA molecule, variable sheer is mainly passed through by precursor RNA (pre-mRNA)
Processing generates.Most of circRNA is positioned in cytoplasm, and minority is positioned in nucleus, most of to be formed by exon.
CircRNA is not easy to be degraded by exonuclease, can more stably be present in organism compared with linear rna, different
There is conservative in species, while there is expression specificity in different tissues and stage of development, most of circRNA are different
Between species rich content and all have tissue specificity, have stable expression in tissue, saliva, blood and excretion body,
This makes circRNA become the ideal biological marker that tumor patient diagnosis, treatment and prognosis are tracked.
In recent years, about the existing many reports of the relationship research between circRNA and tumour, clear circRNA has
Have and adjusts the functions such as tumor cell proliferation, apoptosis, vascularization, metabolism and drug resistance.Zhang etc. passed through to different lactation periods
Two groups of mouse breast tissue carry out the detection of circRNA chip, find circRNA in different lactation mouse breast tissue
Expression have very big difference, two periods detect the most of differences of circRNA type.Therefore, circRNA may be breast cancer
The novel targets of accurate diagnosis and treatment, discovery circRNA marker relevant with screening breast cancer, have early prevention and treatment breast cancer important
Meaning and value.
Summary of the invention
The application's is designed to provide the following aspects:
In a first aspect, the application provides a kind of circular rna, the nucleotide sequence of the circular rna such as SEQ ID NO:1 institute
Show, wherein two nucleotide of starting are cyclic binding site with two nucleotide of most end.
Full name of the circular rna in circ-RNA database is hsa-circ-0073004, abbreviation circ-
0073004, No. 5 regions chromosome positive-sense strand 73153480-73181929 of the mankind are positioned at, by ARHGEF28 (alias RGNEF)
The shearing of the gene extron area (exon) 15-25 is cyclized.
Fig. 1 is the gene structure figure of the circular rna, as shown in Figure 1, the sequence of the circular rna circ-0073004
Overall length 1520bp, wherein CDS is protein coding region.
According to a second aspect of the present application, circular rna circ-0073004 described in first aspect present invention is provided as sieve
Look into the purposes of the molecular marker of breast cancer.
By Bioinformatics Prediction, inventor has found the response original part miR-21 of circular rna circ-0073004, should
Gene can influence apoptosis of tumor cells by PDCD4/MAPK/BCL-2 signal path.
Also, the research of the invention finds that relative to normal tissue by breast cancer patients, circular rna circ-0073004
It is significantly lowered in breast cancer tissue；In blood sample, relative healths crowd, circular rna circ-0073004 is in breast cancer
It is also significant in blood samples of patients to lower.
Therefore, the circular rna circ-0073004 can be used for breast cancer screening.
Second aspect, the application also provide the purposes of molecular marker of the circular rna as screening breast cancer.
The third aspect, the application also provide it is a kind of for expanding the specificity amplification primer of circular rna described in first aspect,
The specificity amplification primer includes upstream primer and downstream primer, wherein
The nucleotide sequence of the upstream primer is as shown in SEQ ID NO:2；
The nucleotide sequence of the downstream primer is as shown in SEQ ID NO:3.
Specifically, the nucleotide sequence of the upstream primer are as follows: 5 '-TGTGTTTAGGAAGCAGGCACT-3 '；
The nucleotide sequence of the downstream primer are as follows: 5 '-TCCTGAATTCTTTGAAACGACCTG-3 '.
In a kind of achievable mode, the G/C content of the upstream primer is 47.6%, and the GC of the downstream primer contains
Amount is 41.7%, wherein the G/C content refers in 4 kinds of bases of DNA, ratio shared by guanine and cytimidine.
Further, the TM value of the upstream primer is 56.6, the TM value 54.9 of the downstream primer, upstream and downstream after verifying
Most suitable TM value when primer qPCR is used is 60, wherein the TM value refers to the melting temperature of upstream primer or downstream primer
Degree.
The inventors discovered that expanded using specificity amplification primer RNA described in first aspect provided by the present application,
As described in Example 1, amplification is analyzed, obtains ROC curve, as a result as shown in figure 4, area is under ROC curve
0.861, this shows that the amplified production obtained using specificity amplification primer provided by the present application is single band, no non-specificity
Amplification, the Specific marker that the specificity amplification primer can be detected as this kind.
Fourth aspect, the application also provide a kind of method for expanding circular rna described in first aspect, which comprises
Step 1, synthesize the first chain cDNA: mixed raw material, the raw material include circular rna described in first aspect, with power traction
Object, ddH2O, dNTP mixed liquor, reverse transcription buffer, RNase inhibitor, reverse transcriptase are reacted according to the first temperature programmed control；
Step 2, Fluorescent quantitative PCR (qPCR) is carried out to the first chain cDNA made from step 1 to expand
Increase: preparing amplification system, the amplification system includes cDNA, upstream primer, third party shown in the third aspect prepared by step 1
Downstream primer, ddH shown in face2O, qPCR expands Mix (dyestuff containing SYBGREEN), reacts according to the second temperature programmed control.
In a kind of achievable mode, in step 1,
The mixing according to the following ratio of the raw material: circular rna described in 1 μ l first aspect, 1 μ l random primer, 10 μ l
ddH2O, 2 μ l dNTP mixed liquors, 4 μ l reverse transcription buffers, 1 μ l RNase inhibitor, 1 μ l reverse transcriptase, wherein described
DNTP mixed liquor includes dATP, dGTP, dCTP and dTTP；
First program are as follows: react 5min under the conditions of 25 DEG C, 60min is reacted under the conditions of 42 DEG C, is reacted under the conditions of 70 DEG C
5min.Reverse transcription is carried out using ABI PCR amplification instrument, the cDNA is placed in -80 DEG C and stores for future use.
Further, in step 2,
The amplification system is prepared according to the following ratio: the cDNA of 2 μ l steps 1 preparation, 0.5 μ l is as described in the third aspect
Upstream primer, downstream primer described in the 0.5 μ l third aspect, 7 μ l ddH2O, 10 μ l qPCR expand Mix (SYBGREEN dye
Material)；
Second program are as follows: 95 DEG C of initial denaturation 10s, then with 95 DEG C of reaction 15s, 60 DEG C of reaction 30s, 72 DEG C of reactions
40s carries out 40 circulations, with 95 DEG C of reaction 5s, 65 DEG C of reaction 1min, 37 DEG C of reaction 1min, finally terminates reaction.
5th aspect, the application also provide aforementioned specificity amplification primer in the kit that preparation is used for breast cancer screening
Application.
6th aspect, the application also provide a kind of kit for breast cancer screening, and the kit includes third party
Specificity amplification primer described in face.
In a kind of achievable mode, the kit further includes reverse transcriptase, buffer, ddH2O, archaeal dna polymerase,
At least one of fluorescent dye and dNTP mixed liquor.
The circ-0073004 in the method detection breast cancer tissue and peripheral blood of qPCR can be used in the kit, is used for
Screening breast cancer.
Applicants have discovered that low expression is presented in breast cancer tissue's cell in circular rna described herein, that is, mammary gland
The content of circular rna described in cancerous tissue is far below the rna content in cancer beside organism, therefore, the circular rna circ-
0073004 may be used as the molecular marker of breast cancer screening, moreover, provided by the present application for expanding the circular rna
Specificity amplification primer has high stability, specificity and sensibility to the circular rna, and medium sensitivity is reachable
66.7%, specificity is up to 100%.
Detailed description of the invention
Fig. 1 shows circ-0073004 gene structure figure；
Fig. 2 shows qPCR to detect circ-0073004 differential expression figure in breast cancer tissue and cancer beside organism；
Fig. 3 shows qPCR detection circ-0073004 differential expression in patient with breast cancer's blood and healthy population blood
Figure；
Fig. 4 shows the ROC curve of circ-0073004 testing result in breast cancer tissue.
Specific embodiment
The following describes the present invention in detail with reference to examples, the features and advantages of the invention will with these explanation and
It becomes more apparent from, is clear.But these examples are only exemplary, do not constitute any limit to protection scope of the present invention
System.
Embodiment
In embodiment,
The no RNA enzyme is without DNA enzymatic aqua sterilisa (ddH2O Tiangeng biotech firm, the PCR amplification mixed system) are purchased from
(Mix) and qPCR (containing fluorescent dye) amplification mixed system (Mix) is purchased from TAKARA company, and the reverse transcriptase is purchased from Thermo
Company, the dNTP mixed liquor are purchased from Invitrogen company.
Expression of the 1 qPCR reaction detection circ-0073004 of embodiment in breast cancer tissue
1, specificity amplification primer is designed:
Specificity amplification primer sequence and relevant information designed by the applicant are as shown in table 1:
1 specificity amplification primer sequence of table and relevant information
2, the extraction of total serum IgE:
(1) Trizol method extracts the total serum IgE in breast cancer tissue/blood: 0.2g breast cancer tissue/blood is taken, in liquid nitrogen
Under the conditions of ground, until the tissue/blood be in pulverulence, in liquid nitrogen be added 1mL Trizol continue to grind, to
It after Trizol is ground to dry powder, is stored at room temperature, collects all liq after dry powder turns to liquid；
(2) fluid sample made from step 1 cracks after five minutes at room temperature, adds according to the Trizol grinding homogenate of every 1mL
Chloroform is added in the ratio for entering 0.2mL chloroform, covers tightly pipe lid, is vortexed after concussion 15s, after standing layering to appear, under the conditions of 4 DEG C
12000rpm is centrifuged 15 minutes, and mixing liquid is layered as lower layer's chloroform phase, middle layer albumin layer, upper layer colourless aqueous phase, RNA after centrifugation
All it is distributed in water phase；
(3) water phase is transferred in new centrifuge tube, 0.5mL isopropanol is added according to the Trizol grinding homogenate of every 1mL
Ratio isopropanol is added into system, mix, after -20 DEG C of standings 30min, under the conditions of 4 DEG C 12000rpm centrifugation 15 minutes,
It must precipitate, RNA is all present in precipitating；
(4) supernatant is removed, 75% ethyl alcohol of 1mL is added into system and cleans RNA precipitate, the 7500rpm under the conditions of 4 DEG C
Centrifugation 5 minutes；
(5) ethanol solution is removed, is dried 5-10 minutes at room temperature, until ethyl alcohol volatilizees, but is completely dried RNA not, by nothing
The ddH of RNA enzyme2Centrifuge tube is added in O water, and 60 DEG C are incubated for 5 minutes, obtains total serum IgE；
(6) total serum IgE extracted determines the concentration of total serum IgE using the concentration and purity of NanoDrop ND-2000 measurement RNA
It can satisfy the requirement of subsequent experimental with purity, total rna solution packing is placed in -80 DEG C of preservations.
3, the synthesis of the first chain cDNA sequence:
PCR pipe is taken to configure reverse transcription system, reverse transcription system are as follows: 1 μ L total serum IgE (about 500ng), 1 μ L random primer
(Thermo company), 10 μ L ddH2O (Tiangeng biotech firm), 2 μ L dNTP mixed liquors (dATP, dGTP, dCTP and dTTP,
Invitrogen company), 4 μ L reverse transcription buffers (Thermo company), 1 μ L RNase inhibitor (Thermo company), 1 μ L is anti-
Transcriptase (Thermo company), 20 μ L of total volume；
Reaction condition are as follows: react 5min at 25 DEG C；60min is reacted at 42 DEG C, reacts 5min at 70 DEG C.Use ABI PCR
Amplification instrument carries out reverse transcription.The cDNA that reverse transcription obtains is placed in -80 DEG C and stores for future use.
4, Circ-0073004 gene cDNA amplification verifying:
The cDNA for taking 2 μ L step 3 reverse transcriptions to obtain prepares system and carries out PCR amplification；
PCR amplification system are as follows: 2 μ L cDNA, upstream primer shown in 0.5 μ L table 1, downstream primer shown in 0.5 μ table 1,7
μL ddH2O, 10 μ LPCR expand Mix, 20 μ L of total volume；
Reaction condition is 94 DEG C of initial denaturation 5min；94 DEG C of reaction 30s, 20s, 72 DEG C of reaction 20s progress 35 are reacted at 60 DEG C
A circulation；Finally with 72 DEG C of reaction 5min.Take 2 μ L reaction products in 1.5g agarose/100ml 1 × TAE buffer, 120V electricity
It presses, whether verifying PCR product is single specificity amplified band under the conditions of 25min.
5, pcr amplification reaction:
The cDNA sample obtained using reverse transcription is detected.
Reaction system are as follows: 2 μ L cDNA, 0.8 μ L upstream primer, 0.8 μ L downstream primer, 6.4 μ L ddH2O (Tiangeng biology
Company), 10 μ L qPCR expand Mix (SYBGREEN dyestuff), 20 μ L of total volume；
QPCR reaction condition: 95 DEG C of initial denaturation 10s, then with 95 DEG C of reaction 15s, 60 DEG C of reaction 30s, 72 DEG C of reaction 40s
40 circulations are carried out, with 95 DEG C of reaction 5s, 65 DEG C of reaction 1min, 37 DEG C of reaction 1min, finally terminate reaction.
Amplified reaction carries out on real-time fluorescence quantitative PCR instrument LightCyler480, expands while amplifying target genes
Increase β-actin to compare as internal reference, and passes through 2(-△△CT)The relative expression quantity of method calculating gene.
For embodiment 1, different samples are taken respectively, are repeated 6 times.
Wherein, as can be seen that amplification in internal reference β-actin gene and circ-0073004 gene qPCR solubility curve figure
Peak is single, no miscellaneous peak, it was demonstrated that primer specificity is outstanding, and amplification experiment is normal.
Expression of the 2 qPCR reaction detection circ-0073004 of embodiment in breast cancer cancer beside organism
The process of embodiment 1 is repeated, difference is: in step 2, extracting the total serum IgE in breast cancer cancer beside organism.
For embodiment 2, different samples are taken respectively, are repeated 6 times.
Expression of the 3 qPCR reaction detection circ-0073004 of embodiment in healthy population blood
The process of embodiment 1 is repeated, difference is: in step 2, extracting the total serum IgE in healthy population blood.
For embodiment 3, different samples are taken respectively, are repeated 6 times.
Expression of the 4 qPCR reaction detection circ-0073004 of embodiment in patient with breast cancer's blood
The process of embodiment 1 is repeated, difference is: in step 2, extracting the total serum IgE in patient with breast cancer's blood.
For embodiment 4, different samples are taken respectively, are repeated 6 times.
The qPCR interpretation of result of Examples 1 to 4
1, gene expression analysis
Wherein, the expression of results of circ-0073004 is as shown in figures 2-3:
(a) in 6 pairs of tissues of Fig. 2, relative to breast cancer cancer beside organism, circular rna circ-0073004 is in breast cancer
Significant downward is expressed in tissue, lowers about 2.5 times；
(b) in the 6 Peripheral Blood samples of Fig. 3, relative to 6 healthy population peripheral blood samples, circ-0073004
Significant downward is expressed in Peripheral Blood In Patients With Breast Cancer sample, lowers about 4 times.
The above result shows that circular rna circ-0073004 has difference table in breast cancer tissue and cancer beside organism
It reaches, equally, there is differential expression, specifically, the circular rna circ- in patient with breast cancer's blood and healthy population blood
0073004 in breast cancer tissue and blood is low expression, and therefore, the circular rna circ-0073004 can be used for cream
The screening of gland cancer.
2, ROC curve is analyzed
The test data obtained to embodiment 1 is analyzed, and obtains ROC curve, as shown in Figure 4, wherein area under the curve
AUC is 0.861, indicates that detection target spot can be as the Specific marker for detecting the circular rna, also, detection sensitivity can
Up to 66.7%, specificity is up to 100% or more.
One skilled in the art will appreciate that area is between 1.0 and 0.5 under ROC curve, and in the case where AUC > 0.5, AUC
Closer to 1, illustrate that diagnosis effect is better.AUC has lower accuracy in 0.5-0.7, and AUC is fixed in 0.7-0.9 Shi Youyi
True property, AUC have high accuracy at 0.9 or more, which, which is greater than 0.7, indicates the spy that detection target spot can be detected as this kind
Anisotropic marker.
Combine detailed description and exemplary example that the application is described in detail above, but these explanations are simultaneously
It should not be understood as the limitation to the application.It will be appreciated by those skilled in the art that without departing from the application spirit and scope,
A variety of equivalent substitution, modification or improvements can be carried out to technical scheme and embodiments thereof, these each fall within the application
In the range of.The protection scope of the application is determined by the appended claims.
Sequence table
<110>hospital general, Ningxia Medical University
<120>a kind of circular rna hsa-circ-0073004 and its specificity amplification primer and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1520
<212> DNA
<213>mankind (Homo sapiens)
<400> 1
aattcaggaa gaagaatggg ataaatacat catacctgcc aaatcagagt ctgaaaaata 60
taaagtgagt cgaactttca gtttcctcat gaataggatg actagccctc ggaataaatc 120
aaagacaaaa agcaaggatg ccaaagataa agagaagctg aatcgacatc agtttgcccc 180
aggaacattc tctggggttc tgcagtgttt ggtttgtgat aaaacactcc tggggaaaga 240
gtcactgcag tgttctaact gtaatgcaaa tgtgcacaaa ggttgtaaag atgctgcgcc 300
tgcatgcacc aagaaattcc aagagaaata taacaagaac aaaccacaga ccatccttgg 360
aaattcttca tttagagaca tcccacagcc tggtctctcc ttgcaccctt cttcctccgt 420
gcctgttgga ttgccgactg gaaggaggga gactgtggga caggtccatc cattgtccag 480
aagtgttcca ggcaccacct tggaaagctt caggaggtca gccacatcct tggagtctga 540
gagtgaccat aacagctgca gaagcaggtc tcattctgat gagctgctac agtccatggg 600
ctcttctccc tctacagagt ctttcataat ggaagatgtt gtggattctt ctctgtggag 660
tgacctcagc agtgatgccc aggagtttga agcagaatct tggagtcttg tggtggatcc 720
ctcattttgt aataggcagg agaaggatgt catcaaaaga caggatgtca tttttgagct 780
aatgcaaaca gagatgcatc acatccagac cctgttcatc atgtctgaga tcttcaggaa 840
aggcatgaaa gaggagctgc agctggacca cagcaccgtg gataaaattt tcccctgttt 900
agatgagttg cttgaaatcc acaggcattt cttctacagt atgaaggaac gaaggcagga 960
atcctgtgct ggcagcgaca ggaattttgt gatcgaccga attggagata ttttggtaca 1020
acagttttca gaagaaaatg caagtaaaat gaagaaaata tatggagaat tctgttgcca 1080
tcataaagaa gctgttaacc tctttaaaga actccagcag aataaaaagt ttcagaattt 1140
tattaagctc cgaaatagta atcttttggc tcgacgccga ggaattccag aatgcattct 1200
gttggtcact cagcgtatta caaaataccc tgtcttggtg gaaaggatat tgcagtacac 1260
aaaggaaaga actgaggaac ataaagactt acgcaaagcg ctttgcttaa ttaaagacat 1320
gattgcaaca gtggatttaa aagtcaatga atatgagaaa aaccaaaaat ggcttgagat 1380
cctaaataag attgaaaaca aaacatacac gaagctcaaa aatggacatg tgtttaggaa 1440
gcaggcactg atgagtgaag aaaggactct gttatatgat ggccttgttt actggaaaac 1500
tgctacaggt cgtttcaaag 1520
<210> 2
<211> 21
<212> DNA
<213>artificial sequence
<400> 2
tgtgtttagg aagcaggcac t 21
<210> 3
<211> 24
<212> DNA
<213>artificial sequence
<400> 3
tcctgaattc tttgaaacga cctg 24
Claims (9)
1. a kind of circular rna, which is characterized in that full name of the circular rna in circ-RNA database is hsa-circ-
0073004, nucleotide sequence is as shown in SEQ ID NO:1.
2. the purposes that circular rna described in claim 1 is used as the molecular marker of screening breast cancer.
3. the specificity amplification primer for expanding circular rna described in claim 1, which is characterized in that the specific amplification
Primer includes upstream primer and downstream primer, wherein
The nucleotide sequence of the upstream primer is as shown in SEQ ID NO:2；
The nucleotide sequence of the downstream primer is as shown in SEQ ID NO:3.
4. a kind of method of circular rna described in amplification claim 1, which is characterized in that the described method includes:
Step 1, synthesize the first chain cDNA: mixed raw material, the raw material include circular rna, random primer described in claim 1,
ddH2O, dNTP mixed liquor, reverse transcription buffer, RNase inhibitor, reverse transcriptase are reacted according to the first temperature programmed control；
Step 2, Fluorescent quantitative PCR amplification is carried out to the first chain cDNA made from step 1: prepares and expands
System, the amplification system include step 1 prepare cDNA, upstream primer as stated in claim 3, such as claim 3 institute
The downstream primer shown, ddH2O, qPCR amplification fluorescent dyestuff Mix is reacted according to the second temperature programmed control.
5. according to the method described in claim 4, it is characterized in that, in step 1,
The mixing according to the following ratio of the raw material: circular rna described in 1 μ l claim 1,1 μ l random primer, 10 μ l ddH2O,
2 μ l dNTP mixed liquors, 4 μ l reverse transcription buffers, 1 μ l RNase inhibitor, 1 μ l reverse transcriptase, wherein the dNTP mixing
Liquid includes dATP, dGTP, dCTP and dTTP；
First program are as follows: 5min is reacted under the conditions of 25 DEG C, and 60min is reacted under the conditions of 42 DEG C, it is finally anti-under the conditions of 70 DEG C
Answer 5min.
6. according to the method described in claim 4, it is characterized in that, in step 2,
The amplification system is prepared according to the following ratio: the cDNA of 2 μ l steps 1 preparation, on 0.5 μ l is as claimed in claim 3
Swim primer, 0.5 μ l downstream primer as claimed in claim 3,7 μ l ddH2O, 10 μ l qPCR amplification fluorescent dyestuff Mix；
Second program are as follows: 95 DEG C of initial denaturation 10s, then with 95 DEG C of reaction 15s, 60 DEG C of reaction 30s, 72 DEG C of reaction 40s into
Row 40 circulations finally terminate reaction with 95 DEG C of reaction 5s, 65 DEG C of reaction 1min, 37 DEG C of reaction 1min.
7. application of the specificity amplification primer as claimed in claim 3 in the kit that preparation is used for breast cancer screening.
8. a kind of kit for breast cancer screening, which is characterized in that the kit includes as claimed in claim 3 special
Property amplimer.
9. kit according to claim 8, which is characterized in that the kit further include reverse transcriptase, buffer,
ddH2O, at least one of archaeal dna polymerase, fluorescent dye and dNTP mixed liquor.
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