CN107400731A - Oncogene BRCA1 and BRCA2 quick diagnosis reagent kit - Google Patents
Oncogene BRCA1 and BRCA2 quick diagnosis reagent kit Download PDFInfo
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- CN107400731A CN107400731A CN201710884253.3A CN201710884253A CN107400731A CN 107400731 A CN107400731 A CN 107400731A CN 201710884253 A CN201710884253 A CN 201710884253A CN 107400731 A CN107400731 A CN 107400731A
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- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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Abstract
The invention belongs to biotechnology and medical domain, and in particular to a kind of oncogene BRCA1 and BRCA2 quick diagnosis reagent kit.The kit includes sample pretreatment buffer solution, archaeal dna polymerase, MgCl2, dNTP, sense primer, anti-sense primer, positive control, negative control, Enhence Buffer, fluorescent dye and redistilled water；The Enhence Buffer are by the μ g/mL of glycerine 20 30, formamide 0.1 0.5uL/mL, the 1.5uL/mL of 3 dioxide thiophene alkene 0.5 and the μ g/mL of seralbumin 100 150 compositions.Kit cost of the present invention is low, simple to operate；Kit of the present invention and its detection method using instrument and equipment, DNA profiling to requiring low, and the amplified production specificity obtained is high.
Description
Technical field
The invention belongs to biotechnology and medical domain, and in particular to a kind of oncogene BRCA1 and BRCA2 quick diagnosis
Kit.
Background technology
Breast cancer is one of most common malignant tumour of women, and its incidence of disease is in obvious ascendant trend in the whole world, so as to
Seriously endanger the physical and mental health of numerous women.Although the current breast cancer incidence in China is less than western countries, and is not belonging to breast
Gland cancer hotspot, but with industrialized development, the influence of environmental factor and the change of people life style, breast cancer
Total incidence increase rapidly.Statistical result showed in 2008, breast cancer is in urban area female tumor incidence of disease middle position
First is occupied, and year new breast cancer case increases to 200,000, and have age of onset rejuvenation trend.
The pathogenesis of breast cancer is unclear, and presently relevant research trend is that galactophore epithelial cell exists in its occurrence and development
A variety of lower results that gene mutation and paraplasm occurs of carcinogenic factor effect.Research finds, the about 5%- in patient with breast cancer
10% case has familial aggregation and genetic predisposition, therefore breast cancer can be divided into heredity and sporadic two major class.
First gene related to familial breast cancer and oophoroma, i.e. mammary gland/ovum were isolated by successful clones such as Miki in 1994
Nest tumor susceptibility gene 1 (Breast Cancer Susceptibility Gene 1, BRCA1), the discovery such as subsequent Wooste
Gene on human chromosomal 13q12-1, i.e. mammary gland/(Breast Cancer of ovarian neoplasm tumor susceptibility gene 2
Susceptibility Gene 2, BRCA2).BRCA1 genes are considered as the caregiver of genome, main biological characteristics
It is to participate in injury repair DNA, regulation cell cycle and transcriptional modulatory gene.When the tumor susceptibility gene is undergone mutation, show as compiling
Code protein function is reduced or lost, and influences cell differentiation, so that cell loses normal differentiation and proliferation function, is ultimately resulted in
Malignant change of cell, develop into malignant tumour.The encoding proteins of BRCA2 genes are very huge, have its expression in multiple tissues, at present
Its biological characteristics is unclear.It is reported that the regulation of BRCA2 cell cycles plays an important role.
Chinese patent application (CN101921831A) discloses a kind of PCR reaction kits of detection BRCA gene mutations,
The kit includes several primer pairs for specific amplification BRCA gene target sequences, and the primer pair contains
At least 15 in the exon 2 of BRCA1 genes or the 11st extron of the 20th extron of BRCA1 genes or BRCA2 genes
The continuous nucleotide sequence that continuous nucleotides is formed, the exon 2 of the BRCA1 genes have SEQ ID No：1 company
Continuous nucleotide sequence；20th extron of the BRCA1 genes has SEQ ID No：2 continuous nucleotide sequence；BRCA2 bases
21st extron of cause has SEQ ID No：3 continuous nucleotide sequence.It mainly carries out BRCA1/ using HRM technologies
The detection of 2 mutators, HRM technologies are a kind of brand-new the mutation scanning and the analysis of Genotyping that recent domestic rises
Method.Round pcr based on Efficient robust, HRM are not limited to by mutating alkali yl site and type, without sequence-specific probes,
Dissolving analysis directly is carried out to PCR primer after PCR terminates, you can complete paired samples mutation, SNP-SNP, first
The analysis of base, HLA distribution type etc..For in blood plasma and serum DNA content it is very low in the case of, can still be used using this method
This method carries out the detection of BRCA1/2 gene mutations, breaches micro can not detect or obstacle that detection efficiency is low.
Therefore, required for obtaining higher-quality PCR primer and being accurate detection BRCA1/2 mutators.
The content of the invention
To overcome above mentioned problem, the invention provides a kind of oncogene BRCA1 and BRCA2 quick diagnosis reagent kit.This
Invention kit cost is low, simple to operate；Kit of the present invention and its detection method are to using instrument and equipment, DNA profiling requirement
It is low, and the amplified production specificity obtained is high.Present invention also offers a kind of detection method of kit.
The present invention is achieved through the following technical solutions：
A kind of oncogene BRCA1 and BRCA2 quick diagnosis reagent kit, including archaeal dna polymerase, MgCl2, dNTP, upstream
Primer, anti-sense primer, positive control, negative control, Enhence Buffer, fluorescent dye and redistilled water.
The Enhence Buffer are by glycerine 20-30 μ g/mL, formamide 0.1-0.5uL/mL, 3- dioxide thiophene alkene
0.5-1.5uL/mL and seralbumin 100-150 μ g/mL compositions.
The sense primer such as SEQ ID NO：Shown in 1.
The anti-sense primer such as SEQ ID NO：Shown in 2.
Preferably, the Enhence Buffer are by the μ g/mL of glycerine 27.5, formamide 0.45uL/mL, 3- dioxide thiophene
The alkene 0.65uL/mL and μ g/mL of seralbumin 125 compositions.
Preferably, the negative control is redistilled water, and positive control is BRCA1 containing oncogene and BRCA2DNA samples
Product.
Preferably, the fluorescent dye is ResoLight dyestuffs.
Preferably, in the sample pretreatment buffer solution by 8mM Tris-HCl, 1.5mM EDTA-2Na, 20mM ammonium chlorides
Formed with the glycerine of volume content 10%.
Preferably, the archaeal dna polymerase, MgCl2, dNTP, sense primer, anti-sense primer, Enhence Buffer and glimmering
The PCR system and system concentration of photoinitiator dye composition, which form, is：Archaeal dna polymerase 0.5 μ L, 20mM MgCl23 μ L, l0mmol/L
The μ L of dNTP 0.5, the μ L of sense primer 0.5, the μ L of 0.5 μ L, Enhence Buffer of anti-sense primer, 2 μ L, 10x fluorescent dyes 5.
The application method of the quick diagnosis reagent kit of the oncogene BRCA1 and BRCA2, comprises the following steps：
S1 sample pretreatments, after the sample being collected into is carried out into ungrease treatment, the buffer solution of 5 times of volumes is added, up and down top
After pouring row mixing into, after standing 15min, 4 DEG C of centrifugation 15-20min clocks of 5000g/min, sample I is obtained；
Above-mentioned sample I is carried out DNA extractions by S2；
S3 PCR amplification systems expand；
S4 pcr amplification products detect；
S5 carries out mutational site detection according to high-resolution solubility curve method to BRCA1 the and BRCA2 genes after amplification.
Preferably, PCR amplification system is in the S2：Archaeal dna polymerase 0.5 μ L, 20mM MgCl23 μ L, l0mmol/L
The μ L of dNTP 0.5, the μ L of sense primer 0.5, the μ L of anti-sense primer 0.5 μ L, DNA profiling sample 100ng, Enhence Buffer 2,
The μ L of 10x fluorescent dyes 5,50 μ L are complemented to redistilled water.
Preferably, PCR amplification programs include in the S2：
(1) thermal starting：95 DEG C of denaturation 2min, add archaeal dna polymerase；
(2)Slowdown PCR：95 DEG C of denaturation 30-50s, anneal 45s~55s when temperature is 70-65 DEG C, 72 DEG C of extensions
50s~100s, common 15-20 circulation, makes annealing temperature be down to 59-55 DEG C, and each circulation reduces by 1 DEG C, suspends after every 5 circulations
Rejoin archaeal dna polymerase；Subsequent 95 DEG C of denaturation 45s~50s, anneal 30s~60s at 58-55 DEG C, and 72 DEG C of extension 50s~
120s, common 25-30 circulation；
(3) extend：72 DEG C of extension 5min；
(4) 4 DEG C of preservations.
Technical solution of the present invention compared with prior art, has following technical advantage：
(1) PCR system used in kit of the present invention, thermal starting and improved slowdown PCR methods are combined,
BRCA1 the and BRCA2 genes rapid amplifying suitable for different samples.
(2) additive therefor can aid in PCR reaction progress in kit of the present invention, expand BRCA1 and BRCA2 genes
It is high to increase specificity.
(3) kit of the present invention and its detection method using instrument and equipment, DNA profiling to requiring low, suitable for extensive
Experiment and diagnosis.
Embodiment：
The present invention will further be described in detail below.It is pointed out that following explanation is only to application claims
Protection technical scheme is for example, not to any restrictions of these technical schemes.Protection scope of the present invention is with appended
The content that claims are recorded is defined.
A kind of oncogene BRCA1 and BRCA2 quick diagnosis reagent kit of embodiment 1
The quick diagnosis reagent kit of the oncogene BRCA1 and BRCA2, including archaeal dna polymerase, MgCl2, dNTP, upstream
Primer, anti-sense primer, positive control, negative control, Enhence Buffer, fluorescent dye and redistilled water.
The Enhence Buffer are by the μ g/mL of glycerine 27.5, the μ of 0.45 μ L/mL, 3- dioxide thiophenes alkene of formamide 0.65
The L/mL and μ g/mL of seralbumin 125 compositions.
The sense primer such as SEQ ID NO：Shown in 1.
The anti-sense primer such as SEQ ID NO：Shown in 2.
The negative control is redistilled water, and positive control is BRCA1 containing oncogene and BRCA2DNA samples.
The fluorescent dye is ResoLight dyestuffs.
Buffer solution is by 8mM Tris-HCl, 1.5mM EDTA-2Na, 20mM ammonium chlorides and volume in the sample pretreatment
The glycerine composition of content 10%.
The archaeal dna polymerase, MgCl2, dNTP, sense primer, anti-sense primer, Enhence Buffer and fluorescent dye group
Into PCR system and system concentration composition be：Archaeal dna polymerase 0.5 μ L, 20mM MgCl2The μ L of 3 μ L, l0mmol/L dNTP 0.5,
The μ L of 2 μ L, 10x fluorescent dye of the μ L of sense primer 0.5,0.5 μ L, Enhence Buffer of anti-sense primer 5.
A kind of oncogene BRCA1 and BRCA2 quick diagnosis reagent kit of embodiment 2
The quick diagnosis reagent kit of the oncogene BRCA1 and BRCA2, including archaeal dna polymerase, MgCl2, dNTP, upstream
Primer, anti-sense primer, positive control, negative control, Enhence Buffer, fluorescent dye and redistilled water.
The Enhence Buffer are by the μ g/mL of glycerine 20, the μ L/mL of 0.1 μ L/mL, 3- dioxide thiophenes alkene of formamide 0.5
Formed with the μ g/mL of seralbumin 100.
Buffer solution, negative control, positive control, PCR bodies in the upstream, anti-sense primer, fluorescent dye, sample pretreatment
System's composition is similar with embodiment 1.
A kind of oncogene BRCA1 and BRCA2 quick diagnosis reagent kit of embodiment 3
The quick diagnosis reagent kit of the oncogene BRCA1 and BRCA2, including archaeal dna polymerase, MgCl2, dNTP, upstream
Primer, anti-sense primer, positive control, negative control, Enhence Buffer, fluorescent dye and redistilled water.
The Enhence Buffer are by the μ g/mL of glycerine 20, the μ L/mL of 0.5 μ L/mL, 3- dioxide thiophenes alkene of formamide 1.5
Formed with the μ g/mL of seralbumin 150.
Buffer solution, negative control, positive control, PCR bodies in the upstream, anti-sense primer, fluorescent dye, sample pretreatment
System's composition is similar with embodiment 1.
A kind of application method of oncogene BRCA1 and BRCA2 quick diagnosis reagent kit of experimental example 4
The application method of the quick diagnosis reagent kit of the oncogene BRCA1 and BRCA2, comprises the following steps：
S1 sample pretreatments, after the sample being collected into is carried out into ungrease treatment, the buffer solution of 5 times of volumes is added, up and down top
After pouring row mixing into, after standing 15min, 4 DEG C of centrifugation 15-20min clocks of 5000g/min, sample I is obtained；
Above-mentioned sample I is carried out DNA extractions by S2；
S3PCR amplification systems expand；
S4PCR amplified productions detect；
S5 carries out mutational site detection according to high-resolution solubility curve method to BRCA1 the and BRCA2 genes after amplification.
PCR amplification system is in the S2：Archaeal dna polymerase 0.5 μ L, 20mM MgCl2The μ of 3 μ L, l0mmol/L dNTP 0.5
L, the μ L of sense primer 0.5, μ L, the 10x fluorescent dyes of anti-sense primer 0.5 μ L, DNA profiling sample 100ng, Enhence Buffer 2
5 μ L, 50 μ L are complemented to redistilled water.
PCR amplification programs include in the S2：
(1) thermal starting：95 DEG C of denaturation 2min, add archaeal dna polymerase；
(2)Slowdown PCR：95 DEG C of denaturation 30-50s, anneal 45s~55s when temperature is 70-65 DEG C, 72 DEG C of extensions
50s~100s, common 15-20 circulation, makes annealing temperature be down to 59-55 DEG C, and each circulation reduces by 1 DEG C, suspends after every 5 circulations
Rejoin archaeal dna polymerase；Subsequent 95 DEG C of denaturation 45s~50s, anneal 30s~60s at 58-55 DEG C, and 72 DEG C of extension 50s~
120s, common 25-30 circulation；
(3) extend：72 DEG C of extension 5min；
(4) 4 DEG C of preservations.
A kind of oncogene BRCA1 and BRCA2 quick diagnosis reagent kit of comparative example 1
Difference with embodiment 1 is, does not contain 3- dioxide thiophene alkene in the Enhence Buffer, it is other with
Embodiment 1 is similar.
The measure of the kit minimum detection limit of test example 1
(1) test material：Embodiment 1-3 and the kit of comparative example 1
(2) test method：By positive mark product by 10 times of dilutions, concentration is diluted to respectively as 1.0 × 107、1.0×106、1.0
×105、1.0×104、1.0×103、1.0×102, 10 copies/μ L dilution.Reality is detected respectively by the application method of embodiment 4
Apply a 1-3 and the kit minimum detection limit of comparative example 1.
(3) result of the test：Result of the test is as shown in table 1.
The minimum detection limit of 1 different kits of table
As shown in Table 1, the kit lowest detection of the embodiment of the present invention 1 is limited to 1.0 × 102Copy/μ L, higher than embodiment 2,
3 kits, it is most preferred embodiment.The kit minimum detection limit of comparative example 1 illustrates its detection sensitivity apparently higher than embodiment 1
Significantly lower than the kit of embodiment 1, it follows that 3- dioxide thiophenes alkene and other compositions in Enhence Buffer of the present invention
Interaction, the sensitivity of kit of the present invention can be improved.
Test example 2, the quick diagnosis reagent kit stability for detecting oncogene BRCA1 and BRCA2
1st, accelerate the failure stability
Respectively to placing kit user as described in embodiment 4 described in the embodiments 1 of 4 DEG C, 37 DEG C preservations 10 days
Method carries out performance test, the performance indications of test kit.The stability result that accelerates the failure is as shown in table 2.
Table 2：Kit accelerates the failure stability result
The empirical method of the kit term of validity is calculated according to the experiment that accelerates the failure, is accelerated the failure 10 days at 37 DEG C, equivalent to
The term of validity 1 year half, the requirement of the kit term of validity of 1 year can be met.As can be seen from Table 2, oncogene BRCA1 of the present invention and
For BRCA2 quick diagnosis reagent kit after 4 DEG C, 37 DEG C preserve 10 days, sensitivity, accuracy and accuracy of kit etc. are full
Foot requires.
2nd, freeze-thaw stability
It is to simulate kit in transportation, kit each component multigelation is to stabilization of kit under low temperature environment
Influence, by the kit of embodiment 1 freeze overnight in a low temperature of -20 DEG C, then redissolve, then put again at room temperature
The freeze overnight in a low temperature of -20 DEG C, after such multigelation 5 times, application method described in embodiment 4 is utilized to carry out stability inspection
Survey, as a result as shown in table 3.
Table 3：Kit freeze-thaw stability result
As can be seen from Table 3, oncogene BRCA1 and BRCA2 of the present invention quick diagnosis reagent kit is through multigelation 5 times
Afterwards, the sensitivity of kit, accuracy and accuracy etc. are satisfied by requiring.This explanation, kit of the present invention, which has, preferably to be frozen
Melt stability.
3rd, transportation stability is simulated
Be simulation kit in transportation, vehicle jolt and influence of the hot environment to stabilization of kit,
The kit of embodiment 1 is fixed on micro oscillator, 37 DEG C of insulating boxs is positioned over and vibrates 7 days, using described in embodiment 4
Application method carries out Detection of Stability, as a result as shown in table 4.
Table 4：Kit simulates transportation stability result
As can be seen from Table 4, oncogene BRCA1 and BRCA2 of the present invention quick diagnosis reagent kit is positioned over 37 DEG C of constant temperature
After case vibrates 7 days, sensitivity, accuracy and accuracy of kit etc. are satisfied by requiring.
Sequence table
<110>Guangzhou Hua Hong bio tech ltd
<120>Oncogene BRCA1 and BRCA2 quick diagnosis reagent kit
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 51
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gctagaggaa acctttgagg aactattaat gaaataggtt ccagtgatga t 51
Claims (6)
- A kind of 1. oncogene BRCA1 and BRCA2 quick diagnosis reagent kit, it is characterised in that including sample pretreatment buffer solution, Archaeal dna polymerase, MgCl2, dNTP, sense primer, anti-sense primer, positive control, negative control, Enhence Buffer, fluorescence Dyestuff and redistilled water；The Enhence Buffer are by glycerine 20-30 μ g/mL, formamide 0.1-0.5uL/mL, 3- dioxide thiophene alkene 0.5- 1.5uL/mL and seralbumin 100-150 μ g/mL compositions；The sense primer such as SEQ ID NO：Shown in 1；The HER-2/neu downstream of gene primer such as SEQ ID NO：Shown in 2.
- 2. oncogene BRCA1 and BRCA2 quick diagnosis reagent kit according to claim 1, it is characterised in that described Enhence Buffer are white by the μ g/mL of glycerine 27.5, formamide 0.45uL/mL, 3- dioxide thiophene alkene 0.65uL/mL and serum The μ g/mL of protein 12 5 are formed.
- 3. oncogene BRCA1 and BRCA2 quick diagnosis reagent kit according to claim 1, it is characterised in that the feminine gender It is BRCA1 containing oncogene and BRCA2DNA samples to compare as redistilled water, positive control.
- 4. oncogene BRCA1 and BRCA2 quick diagnosis reagent kit according to claim 1, it is characterised in that the fluorescence Dyestuff is ResoLight dyestuffs.
- 5. oncogene BRCA1 and BRCA2 quick diagnosis reagent kit according to claim 1, it is characterised in that the sample Pre-treatment buffer is by 8mM Tris-HCl, the glycerine composition of 1.5mM EDTA-2Na, 20mM ammonium chlorides and volume content 10%.
- 6. oncogene BRCA1 and BRCA2 quick diagnosis reagent kit according to claim 1, it is characterised in that the DNA Polymerase, MgCl2, dNTP, sense primer, anti-sense primer, Enhence Buffer and fluorescent dye composition PCR system and body It is that concentration composition is：Archaeal dna polymerase 0.5 μ L, 20mM MgCl23 μ L, l0mmol/L dNTP 0.5 μ L, the μ L of sense primer 0.5, The μ L of 0.5 μ L, Enhence Buffer of anti-sense primer, 2 μ L, 10x fluorescent dyes 5.
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| CN201710884253.3A CN107400731A (en) | 2017-09-26 | 2017-09-26 | Oncogene BRCA1 and BRCA2 quick diagnosis reagent kit |
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| CN201710884253.3A CN107400731A (en) | 2017-09-26 | 2017-09-26 | Oncogene BRCA1 and BRCA2 quick diagnosis reagent kit |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101643762A (en) * | 2009-09-02 | 2010-02-10 | 陈依军 | PCR amplification system and PCR amplification method for high GC content gene |
| CN102998289A (en) * | 2012-11-14 | 2013-03-27 | 广西安仁欣生物科技有限公司 | Glycosylated hemoglobin kit based on nucleic acid aptamer fluorescence probe and detection method thereof |
| CN107119137A (en) * | 2017-05-26 | 2017-09-01 | 广州华弘生物科技有限公司 | A kind of detection kit and detection method of quick detection HER 2/neu gene expressions |
-
2017
- 2017-09-26 CN CN201710884253.3A patent/CN107400731A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101643762A (en) * | 2009-09-02 | 2010-02-10 | 陈依军 | PCR amplification system and PCR amplification method for high GC content gene |
| CN102998289A (en) * | 2012-11-14 | 2013-03-27 | 广西安仁欣生物科技有限公司 | Glycosylated hemoglobin kit based on nucleic acid aptamer fluorescence probe and detection method thereof |
| CN107119137A (en) * | 2017-05-26 | 2017-09-01 | 广州华弘生物科技有限公司 | A kind of detection kit and detection method of quick detection HER 2/neu gene expressions |
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Application publication date: 20171128 |