CN109295192A - A kind of composition, kit, sample treatment and application detecting people MDR1 gene pleiomorphism - Google Patents
A kind of composition, kit, sample treatment and application detecting people MDR1 gene pleiomorphism Download PDFInfo
- Publication number
- CN109295192A CN109295192A CN201811271585.5A CN201811271585A CN109295192A CN 109295192 A CN109295192 A CN 109295192A CN 201811271585 A CN201811271585 A CN 201811271585A CN 109295192 A CN109295192 A CN 109295192A
- Authority
- CN
- China
- Prior art keywords
- seq
- mdr1 gene
- primer
- hex
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The present invention relates to a kind of composition, kit, sample treatment and application for detecting people MDR1 gene pleiomorphism, the composition includes that the primer of detection 1236 loci polymorphism of people MDR1 gene includes sequence primer as shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4；The primer of 2677 loci polymorphism of people MDR1 gene includes sequence primer as shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8；The primer of 3435 loci polymorphism of people MDR1 gene includes sequence primer as shown in SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, and each primer of the invention does not interfere between each other, and specificity is high, sensitivity, reproducible.
Description
Technical field
The invention belongs to the nucleic acid detection technique fields in field of biomedicine clinical detection technique, are specifically related to one
Composition, kit, sample treatment and the application of kind detection people MDR1 gene pleiomorphism.
Background technique
Polymerase chain reaction (PCR) technology is one of most common technique in current detection of nucleic acids, wherein using fluorescence mark
The real-time fluorescent PCR technology of the taqman sonde method of note detection of nucleic acids, clinical diagnosis and in terms of
Using highly developed, which is had been applied in numerous industries such as laboratory research, food safety, medical and health, but
It is that these technologies mostly use greatly single reaction tube to detect single target nucleotide, when in the multiple target nucleotides of single reaction tube detection
When, the primer and probe sequence of different target nucleotides is detected due to introduce simultaneously, these introduce primer and probes can because
There is hybridization from each other and generate interference, simultaneously as being related to three positions being located on the same gene of mankind MDR1 gene
Point (1236 sites, 2677 sites, 3435 sites), extremely difficult find can distinguish wild type and saltant type, and again can be in list
The primed probe design that will not be interfered with each other in pipe.Meanwhile it as soon as being needed to evade in false negative needs to be added as a kit
Control target nucleic acid sequence and the primed probe that can detect internal reference target nucleic acid sequence exacerbate the tired of Single-tube multiplex-PCR technology
It is difficult.
Currently, the method for Genotyping mainly has the restricted Length Polymorphism technique of PCR sequencing PCR, polymerase chain reaction-, core
Piece method and fluorescence quantitative PCR method etc..It is higher to obtain that these technologies all have to pass through cumbersome nucleic acid extraction, purification process
The template DNA of purity.The step of increasing operation, it is time-consuming and laborious, and increase false positive rate caused by manual operation error and vacation yin
Property rate.
Summary of the invention
The technical problem to be solved in the present invention is to provide it is a kind of detect the composition of people MDR1 gene pleiomorphism, kit,
Sample treatment and application, each primer do not interfere between each other, and specificity is high, sensitivity, reproducible.
The contents of the present invention include a kind of composition for detecting people MDR1 gene pleiomorphism, and the composition includes for examining
Survey 1236 site of people MDR1 gene, 2677 sites, 3435 loci polymorphisms primer,
The primer of detection 1236 loci polymorphism of people MDR1 gene includes sequence such as SEQ ID NO.2, SEQ ID
Primer shown in NO.3 and SEQ ID NO.4；The primer of 2677 loci polymorphism of people MDR1 gene includes sequence such as SEQ
Primer shown in ID NO.6, SEQ ID NO.7 and SEQ ID NO.8；The primer of 3435 loci polymorphism of people MDR1 gene
Including sequence primer as shown in SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12.
It further include the probe for detecting 1236 loci polymorphism of people MDR1 gene, sequence is SEQ ID NO.1, detects people MDR1
The probe of 2677 loci polymorphism of gene, sequence are SEQ ID NO.5, detect the spy of 3435 loci polymorphism of people MDR1 gene
Needle, sequence are SEQ ID NO.9.
The composition further includes for detecting 1236 site of MDR1 gene, 2677 sites, in 3435 loci polymorphisms
Object and internal standard probe are indexed, the interior label primer sequence as shown in SEQ ID NO.13 and SEQ ID NO.14, visit by the internal standard
Needle sequence is as shown in SEQ ID NO.15.
The present invention also provides a kind of kit, the kit contains PCR detection reagent A and PCR detection reagent B,
The PCR detection reagent A includes sequence such as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID
NO.8、SEQ ID NO.10、SEQ ID NO.12、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.1、SEQ ID
A variety of primer and probes of NO.5, SEQ ID NO.9, SEQ ID NO.13；
The PCR detection reagent B includes sequence respectively such as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.7, SEQ
ID NO.8、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.1、SEQ
A variety of primer and probes of ID NO.5, SEQ ID NO.9, SEQ ID NO.13.
The kit further includes sample lysate, and the sample lysate includes Tris-HCl buffer, dodecyl
Sodium sulphate, EDTA, glycerol and Proteinase K.
Further include: wild type control product, saltant comparison product and blank control product；The wild type control product be include
1236 site of MDR1 gene polymorphism sites, 2677 sites, 3435 sites wild type recombinant plasmid and include internal standard base
The mixture of the recombinant plasmid of cause；The saltant comparison product be include 1236 site of MDR1 gene polymorphism sites, 2677
Site, 3435 sites saltant type recombinant plasmid and include internal standard gene recombinant plasmid mixture；The blank pair
According to for the physiological saline not comprising MDR1 gene polymorphism sites and internal standard gene.
The present invention provides a kind of sample treatment, and this method uses kit of the invention, includes the following steps:
1) 2 parts of human genome sample of nucleic acid pre-processed are taken, by 2 parts of samples respectively with the PCR detection reagent A and
PCR detection reagent B mixing, wherein SEQ ID NO.1, SEQ ID NO.5, probe shown in SEQ ID NO.9 respectively by ROX,
CY5, FAM fluorescent marker, probe shown in SEQ ID NO.15 is by VIC or HEX fluorescent marker；
Pcr amplification reaction is carried out, CY5, FAM, ROX, VIC or HEX fluorescence signal are acquired；
2) result judges:
Save after reaction as a result, according to the Start value, End value of image adjustment Baseline after analysis and
(user can voluntarily adjust Threshold value according to the actual situation, and Start value can may be provided in 5~20 in 3~15, End value, adjust
The amplification curve of whole negative control is straight or is lower than threshold line, and the threshold value of instrument is it is not recommended that suggest manually adjusting, if it is desired, uses
Family can be set as at the 1/8 of external control signal height according to amplification).
When wild type control product and saltant comparison product have CY5, FAM, ROX, VIC or HEX fluorescence signal initial line, blank pair
According to without CY5, FAM, ROX, VIC or HEX fluorescence signal initial line, when the CT≤35 of sample detection pipe VIC or HEX, judgment experiment
Success；According to point in 1236 site of Δ Ct value situation interpretation MDR1 gene of ROX fluorescence signal in two sample detection pipes A, B
Type, according to the parting in 2677 site of Δ Ct value situation interpretation MDR1 gene of CY5 fluorescence signal in two sample detection pipes A, B,
According to the parting in 3435 site of Δ Ct value situation interpretation MDR1 gene of FAM fluorescence signal in two sample detection pipes A, B.
The program of pcr amplification reaction includes:
Genotyping interpretation follows following rule:
Calculation: Δ CtFAM=CtFAM(B)-CtFAM(A)-(CtHEX(B)-CtHEX(A))
ΔCtCY5=CtCY5(B)-CtCY5(A)-(CtHEX(B)-CtHEX(A))
ΔCtROX=CtROX(B)-CtROX(A)-(CtHEX(B)-CtHEX(A)),
When the probe shown in the SEQ ID NO.15 is by VIC fluorescent marker, CtHEXFor CtVIC；
If ROX/CY5/FAM determines that without Ct value, being defaulted as Ct value is 40, is included in calculating,
The invention also includes a kind of application of kit in terms of detecting people MDR1 gene pleiomorphism.
First aspect main contents of the present invention are a kind of sample lysate of optimization design, and of short duration standing is mixed with sample
Afterwards, sampling cooperates MDR1 gene PCR detection reagent A, MDR1 gene PCR detection reagent B of the invention directly to carry out real-time fluorescence
PCR amplification, needing not move through complicated cumbersome nucleic acid extraction purification step can be realized detection to target gene.
Second aspect, main contents of the present invention are that optimization design goes out the Multiple detection sample in two kinds of single PCR reaction tubes
The reaction system in 1236 sites of middle mankind MDR1 gene, 2677 sites, 3435 sites comprising:
(1) MDR1 gene PCR detection reagent A, MDR1 gene PCR detection reagent B.MDR1 gene PCR detection reagent A detection
1236 site of MDR1 gene, 2677 sites, 3435 site wild types, MDR1 gene PCR detection reagent B detect MDR1 gene 1236
Site, 2677 sites, 3435 site mutation types.
(2) taq DNA polymerase that contains in single PCR reaction tube, magnesium chloride, dNTPs (dATP, dCTP,
DGTP and dTTP), internal reference nucleic acid, target nucleic acid primer pair, internal reference nucleic acid primer is to, two kinds of target nucleic acid probes and internal reference core
Acid probe.Wherein various probes are marked with fluorophor and quenching group, and the fluorophor of various probes label detects wave
Length is different；
(3) PCR reaction, and the fluorescence of real-time detection different wave length are carried out；
(4) it is poor to be made according to the calculated Ct value of each channel result of fluorescent quantitative PCR, is judged according to the Δ Ct value obtained
Sample MDR1 genotype (1236C > T, 2677G > T, 3435C > T).
In the second aspect of the present invention, the real time fluorescent PCR method sample detected is containing the in vitro of target nucleic acid
Sample, such as blood, blood product, cast-off cells.The real time fluorescent PCR method is only limitted to the detection to vitro samples, inspection
The direct result of survey is the Genotyping of target nucleic acid.In the method for the second aspect of the present invention, sample is by the present invention the
One side sample lysate and sample mix the mixed liquor of of short duration standing, and the nucleic acid solution of unavailable other methods enrichment is examined
It surveys, there is the risk of failure or false negative.
Herein, " single PCR reaction tube " refer to the real time fluorescent PCR method technically and be in a same vessel into
Capable, it is not necessary that replacement container.Wherein the container is PCR reaction tube for optimal selection, other can carry out PCR reaction and can
The container for carrying out real-time fluorescence detection is also within the scope of the present invention.In the real-time PCR method of first aspect present invention,
The step of can completing PCR amplification and real-time detection in a same vessel, whole process need not replace container, thus conveniently
Operator.Operator only needs that sample and various reagents are added into container, so that it may by commercially available common real-time
Fluorescent PCR instrument is automatically performed the detection of genetic loci of interest, very convenient in user, is also convenient for mechanical realizing automation.
Herein, " Multiple detection " refers to while detecting a variety of target nucleic acids, i.e., at least one.In first party of the invention
In the method in face, due at least having detected internal reference and a kind of target nucleic acid, the nucleic acid of detection at least there are two types of.When detecting
Target nucleic acid type more than one when, while detect nucleic acid increase accordingly.Wherein, every kind of nucleic acid can only be DNA.In the present invention
Specific embodiment in, preferential selection carries out two re-detections, three re-detections detection.
In the method for the second aspect of the present invention, the target nucleic acid of detection can be one such, two kinds, three kinds, point
Not Wei MDR1 gene PCR detection reagent A detect 1236 site of MDR1 gene, 2677 sites, 3435 site wild types, MDR1 gene
PCR detection reagent B detects 1236 site of MDR1 gene, 2677 sites, 3435 site mutation types.The present inventor passes through a large amount of sample
Product test, data analysis and summary preferably go out four pairs of primed probes from several pairs of primed probes of design and demonstrate optimal work
Make concentration, can satisfy the requirement of high specific, high sensitivity and repeatability that this kit detects target nucleic acid, and mixed
The case where interfering with each other is not present in three pairs of primed probes in zoarium system.Specific primed probe design are as follows:
1. being directed to 1236 site of MDR1 gene, according to the literature, preferably its highly conserved region is as amplification region, needle
Primer pair sequence to region design is wild primers SEQ ID NO.2 and mutant primers SEQ ID NO.3, and downstream is drawn
Object is that sequence is SEQ ID NO.4, and probe sequence is SEQ ID NO.1.
2. being directed to 2677 site of MDR1 gene, according to the literature, preferably its highly conserved region is as amplification region, needle
Primer pair sequence to region design is wild primers SEQ ID NO.6 and mutant primers SEQ ID NO.7, and downstream is drawn
Object is that sequence is SEQ ID NO.8, and probe sequence is SEQ ID NO.5.
3. being directed to 3435 site of MDR1 gene, according to the literature, preferably its highly conserved region is as design section, needle
Primer pair sequence to region design is wild primers SEQ ID NO.10 and mutant primers SEQ ID NO.11, downstream
Primer is that sequence is SEQ ID NO.12, and probe sequence is SEQ ID NO.9.
After 1236 site of MDR1 gene, 2677 sites, 3435 site primer probe sequences and reaction density has been determined,
Design joined the internal reference primed probe as kit control false negative.The internal reference target that this kit uses is human body
A kind of house-keeping gene of genome, corresponding primer are SEQ ID NO.13 and SEQ ID NO.14, and fluorescence probe is SEQ ID
NO.15。
In the second aspect of the present invention, in 1236 site of screening and optimizing MDR1 gene, 2677 sites, 3435 sites and interior right
After primer and fluorescence probe sequence according to nucleic acid, further upgrading has been done to single PCR reaction tube described in step (1), has been optimized
The components such as dNTPs, enzyme, magnesium chloride use concentration；Reaction reagent needed for being mixed with other PCR, such as pH buffer and salt.It is additionally added
The glycerol of certain volume, extending activity of enzyme under the conditions of PCR.In specific implementation of the invention, pH buffer is excellent
That first select is Bicine-Koh, K-acetate；That salt preferentially selects is KCl；It is also added into BSA (bovine serum albumin(BSA)) work
For albumen enzymatic protective reagent, the environmental field of each set probe primer serial response is determined respectively, is finally handed in two sets of systems
Collection, present invention determine that the system of step (1) the single multi-PRC reaction pipe is as follows:
1 MDR1 gene multiple real time fluorescence PCR reaction system A of table
| Serial number | Component | Final concentration |
| 1 | Bicine-Koh ph8.2 | 100mM |
| 2 | K-acetate | 125mM |
| 3 | glycerol | 8% (V.V) |
| 4 | BSA | 20μg/ml |
| 5 | KCl | 5mM |
| 6 | Edta | 1μM |
| 7 | Tth DNA polymerase | 1U/test |
| 8 | Taq DNA polymerase | 2U/test |
| 9 | dntps | 2mM |
| 10 | MgCl2 | 2.5mM |
| 11 | SEQ ID NO.2 | 15pmol/test |
| 12 | SEQ ID NO.4 | 15pmol/test |
| 13 | SEQ ID NO.6 | 15pmol/test |
| 14 | SEQ ID NO.8 | 15pmol/test |
| 15 | SEQ ID NO.10 | 15pmol/test |
| 16 | SEQ ID NO.12 | 15pmol/test |
| 17 | SEQ ID NO.13 | 7.5pmol/test |
| 18 | SEQ ID NO.14 | 7.5pmol/test |
| 19 | SEQ ID NO.1 | 5pmol/test |
| 20 | SEQ ID NO.5 | 5pmol/test |
| 21 | SEQ ID NO.9 | 5pmol/test |
| 22 | SEQ ID NO.15 | 3.25pmol/test |
2 MDR1 gene multiple real time fluorescence PCR reaction system B of table
| Serial number | Component | Final concentration |
| 1 | Bicine-Koh ph8.2 | 100mM |
| 2 | K-acetate | 125mM |
| 3 | glycerol | 8% (V.V) |
| 4 | BSA | 20μg/ml |
| 5 | KCl | 5mM |
| 6 | Edta | 1μM |
| 7 | Tth DNA polymerase | 1U/test |
| 8 | Taq DNA polymerase | 2U/test |
| 9 | dntps | 2mM |
| 10 | MgCl2 | 2.5mM |
| 11 | SEQ ID NO.3 | 15pmol/test |
| 12 | SEQ ID NO.4 | 15pmol/test |
| 13 | SEQ ID NO.7 | 15pmol/test |
| 14 | SEQ ID NO.8 | 15pmol/test |
| 15 | SEQ ID NO.11 | 15pmol/test |
| 16 | SEQ ID NO.12 | 15pmol/test |
| 17 | SEQ ID NO.13 | 7.5pmol/test |
| 18 | SEQ ID NO.14 | 7.5pmol/test |
| 19 | SEQ ID NO.1 | 5pmol/test |
| 20 | SEQ ID NO.5 | 5pmol/test |
| 21 | SEQ ID NO.9 | 5pmol/test |
| 22 | SEQ ID NO.15 | 3.25pmol/test |
In the method for the second aspect of the present invention, the fluorescence detection wave of the fluorophor marked between probe not of the same race
Length is different, this makes it possible to simultaneously and the rapidly different Detection wavelength of sequential scan, records various fluorescent bases respectively
The change in fluorescence of group, thus allows for detecting simultaneously.Herein, probe has well-known to those skilled in the art contain
Justice, being can be with the single stranded DNA of the single-stranded combination of target nucleic acid amplified.In general, 5 ' ends of the nucleotide sequence in every kind of probe
Mark fluorescent group, 3 ' end label quenching groups.The fluorophor and quenching group of use are commercially available, and can such as use FAM/
The conventional products such as SYBR-GREEN, VIC/JOE/HEX, NED/TAMRA/CY3, ROX/Texas Red and CY5, their detection
Wavelength is different, can also directly synthesize the probe for having fluorescent marker with authorized company.In a specific embodiment of the invention,
The fluorescent marker that each target nucleic acid uses is as follows:
Each target nucleic acid of table 3 corresponds to fluorescent species
In second aspect, the present invention provides the detection kits for second aspect of the present invention the method comprising
Archaeal dna polymerase, dNTPs, target nucleic acid primer pair, internal reference nucleic acid primer are to, one or more of target nucleic acid probes and internal reference core
Acid probe, wherein aforementioned various probes are marked with fluorophor and quenching group, and the fluorophor of various probes label
Fluorescence detection wavelength is different.Wherein, different reagents can be divided in different containers, and container can be bottle, box, note
The container of the receiving mentioned reagent such as emitter.Can also there are label or specification in detection kit, to indicate according to the present invention
Second aspect the method is operated.As needed, such as convenient transportation, storage, kit can also be packed further into more
In big packaging, such product is also within the scope of the invention.
In the third aspect, the present invention provides the detection kit described in second aspect of the present invention in preparation for the present invention
Application in the detection reagent product of first aspect the method.Detection reagent product can be detection kit itself, can also
To be to merge the more bulk goods that multiple detection kits are housed.According to described previously, those skilled in the art agree to manage very much
The solution wherein ingredient of detection kit and the wherein process of method.
4 sequence table of table
| SEQIDNO.1 | AGGTGCTGAGTGGGCAGACGGT |
| SEQIDNO.2 | CTGGTAGTTCTTGTAGCGC |
| SEQIDNO.3 | CTGGTAGTTCTTGTAGCGT |
| SEQIDNO.4 | GACTGTTGTGCTCTTCCCTC |
| SEQIDNO.5 | TGGGAAGGTGAGTCAAACTAAATGT |
| SEQIDNO.6 | GTAAGAAAGAACTAGAAGATG |
| SEQIDNO.7 | GTAAGAAAGAACTAGAAGATT |
| SEQIDNO.8 | GCATAGTAAGCAGTAGGGAGTAAC |
| SEQIDNO.9 | AGCAAAGGAGGCCAACATACATG |
| SEQIDNO.10 | GGTGGTGTCACAGGAAGAAATC |
| SEQIDNO.11 | GGTGGTGTCACAGGAAGATATT |
| SEQIDNO.12 | TTACATTAGGCAGTGACTTGAT |
| SEQIDNO.13 | TGCCACCCAGAAGACTGTGGATGG |
| SEQIDNO.14 | CTTTGGTATCGTGGAAGGACTCA |
| SEQIDNO.15 | GCCATCACGCCACAGTTTCC |
Beneficial effects of the present invention:
It 1, can be with quick release human blood sample or buccal swab suspension using the sample lysate of kit of the present invention
Genomic nucleic acids in sample, entire process of testing, only need to be directly molten with sample by sample without the DNA individually extracted in sample
Solution liquid is sufficiently mixed the template that can be used as PCR amplification, reduces operating procedure, avoids the ring in conventional nucleic acid extraction process
Border pollution.
2, parting quickly can be carried out to people's MDR1 gene using kit of the present invention, there is simple and quick, sensitivity
The advantages such as degree is high, high specificity, and as a result interpretation is objective.
3, probe used in this kit is Taqman probe, low in cost；Meanwhile all reaction whole process stopped pipes of the invention
It carries out, does not have to recycle product, purify after PCR reaction, digestion, electrophoresis, both saved testing cost, shorten
Detection cycle also reduces the pollution of false positive caused by PCR product.
4, this kit includes the primer and probe of internal standard gene, by the detection to internal standard gene, is reduced because of operation
The false negative that process generates.
5, kit of the present invention is using common fluorescent quantitative PCR platform, and platform use scope is wide, relative to sequencing
Method, chip method are easier to realize high-throughput detection, are more able to satisfy clinical needs.
Detailed description of the invention
Fig. 1 is the testing result figure of the MDR1 gene PCR detection reagent A of embodiment 1.
Fig. 2 is the testing result figure of the MDR1 gene PCR detection reagent B of embodiment 1.
Fig. 3 is the testing result figure of the MDR1 gene PCR detection reagent A of embodiment 2.
Fig. 4 is the testing result figure of the MDR1 gene PCR detection reagent B of embodiment 2.
Specific embodiment
The present invention will be described by specific embodiment herein below, wherein detection reagent used is good for base biology from Hunan
Technology Co., Ltd., probe used, the synthesis of primer commission hundred Li Ge biotech company of Shanghai, chemical reagent buying used is westerly
Lattice Ma aldrich (Shanghai) trade Co., Ltd, DNA polymerase used come from precious bioengineering (Dalian) limited public affairs
Department.Kit forms of the present invention are as follows:
The composition of 1 kit of table
The operating procedure of kit is used in implementation
(1) sample preprocessing
A. 50 μ L human mouth swab suspensions are taken with 1.5mL EP pipe, the mixing of 50 μ L sample lysates is added, pipettor is beaten
It mixes for several times, is stored at room temperature 10min；
B. 50 μ L people's anticoagulated whole bloods are taken with 1.5mL EP pipe, 100 μ L erythrocyte cracked liquids is added, pipettor piping and druming mixes for several times
Even, 10000rpm/min is centrifuged 1min and abandons supernatant, and 50 μ LddH are added2O piping and druming mixes, and adds 50uL sample lysate, shakes
It mixes, is stored at room temperature 10min；
(2) by two portions of 10ul sample mixed liquors respectively with 40ul reaction system A (MDR1 gene PCR detection reagent A 39ul+
Enzyme mixation 1ul), reaction system B (MDR1 gene PCR detection reagent B 39ul+ enzyme mixation 1ul) mixing, then by PCR eight
Pipe is placed in progress fluorescent PCR amplified reaction in fluorescent PCR instrument, acquires CY5, FAM, ROX, HEX or VIC fluorescence signal；
(3) result judges: when wild type control product and saltant comparison product are respectively in detection reagent A and detection reagent B
There are CY5, FAM, ROX, HEX or VIC fluorescence signal initial line, blank control rises without CY5, FAM, ROX, HEX or VIC fluorescence signal
Line, when the CT≤35 of sample detection pipe HEX or VIC, judgment experiment success；Believed according to ROX fluorescence in two sample detection pipes A, B
Number 1236 site of Δ Ct value situation interpretation MDR1 gene parting, according to CY5 fluorescence signal in two sample detection pipes A, B
The parting in 2677 site of Δ Ct value situation interpretation MDR1 gene, according to the Δ Ct of FAM fluorescence signal in two sample detection pipes A, B
It is worth the parting in 3435 site of situation interpretation MDR1 gene；
(4) program of pcr amplification reaction includes:
Genotyping interpretation follows following rule:
Calculation: Δ CtFAM=CtFAM(B)-CtFAM(A)-(CtHEX(B)-CtHEX(A))
ΔCtCY5=CtCY5(B)-CtCY5(A)-(CtHEX(B)-CtHEX(A))
ΔCtROX=CtROX(B)-CtROX(A)-(CtHEX(B)-CtHEX(A)),
If ROX/CY5/FAM determines that without Ct value, being defaulted as Ct value is 40, is included in calculating.
1 three re-detection of embodiment
The sample is detected using the above method, as a result as shown in Figure 1, genotype be 3435CT, 2677GG,
1236CC。
In Fig. 1, it is subject to the rightmost side lines schemed, lines from top to bottom are followed successively by fluorescent marker: ROX, FAM,
CY5、HEX。
In Fig. 2, it is subject to the rightmost side lines schemed, lines from top to bottom are followed successively by fluorescent marker: FAM, ROX,
HEX、CY5。
2 three re-detection of embodiment
The sample is detected using the above method, as a result as shown in Fig. 2, the sample genotype is 3435CC,
2677GG,1236CC。
In Fig. 3, it is subject to the rightmost side lines schemed, lines from top to bottom are followed successively by fluorescent marker: ROX, FAM,
CY5、HEX。
In Fig. 4, it is subject to the rightmost side lines schemed, lines from top to bottom are followed successively by fluorescent marker: ROX, CY5,
HEX、FAM。
<110>Hunan Jian Ji Bioisystech Co., Ltd
<120>a kind of composition, kit, sample treatment and application for detecting people MDR1 gene pleiomorphism
<160>15
<210>1
<211>22
<212>DNA
<213>artificial sequence
<400>1
aggtgctgagtgggcagacggt 22
<210>2
<211>19
<212>DNA
<213>artificial sequence
<400>2
ctggtagttcttgtagcgc 19
<210>3
<211>19
<212>DNA
<213>artificial sequence
<400>3
ctggtagttcttgtagcgt 19
<210>4
<211>20
<212>DNA
<213>artificial sequence
<400>4
gactgttgtgctcttccctc 20
<210>5
<211>25
<212>DNA
<213>artificial sequence
<400>5
tgggaaggtgagtcaaactaaatgt 25
<210>6
<211>21
<212>DNA
<213>artificial sequence
<400>6
gtaagaaagaactagaagatg 21
<210>7
<211>21
<212>DNA
<213>artificial sequence
<400>7
gtaagaaagaactagaagatt 21
<210>8
<211>24
<212>DNA
<213>artificial sequence
<400>8
gcatagtaagcagtagggagtaac 24
<210>9
<211>23
<212>DNA
<213>artificial sequence
<400>9
agcaaaggaggccaacatacatg 23
<210>10
<211>22
<212>DNA
<213>artificial sequence
<400>10
ggtggtgtcacaggaagaaatc 22
<210>11
<211>22
<212>DNA
<213>artificial sequence
<400>11
ggtggtgtcacaggaagatatt 22
<210>12
<211>22
<212>DNA
<213>artificial sequence
<400>12
ttacattaggcagtgacttgat 22
<210>13
<211>24
<212>DNA
<213>artificial sequence
<400>13
tgccacccagaagactgtggatgg 24
<210>14
<211>23
<212>DNA
<213>artificial sequence
<400>14
ctttggtatcgtggaaggactca 23
<210>15
<211>20
<212>DNA
<213>artificial sequence
<400>15
gccatcacgccacagtttcc 20
Claims (10)
1. a kind of composition for detecting people MDR1 gene pleiomorphism, characterized in that the composition includes for detecting people MDR1
1236 site of gene, 2677 sites, 3435 loci polymorphisms primer,
It is described detection 1236 loci polymorphism of people MDR1 gene primer include sequence such as SEQ ID NO.2, SEQ ID NO.3 and
Primer shown in SEQ ID NO.4；The primer of 2677 loci polymorphism of people MDR1 gene includes sequence such as SEQ ID
Primer shown in NO.6, SEQ ID NO.7 and SEQ ID NO.8；The primer packet of 3435 loci polymorphism of people MDR1 gene
Include sequence primer as shown in SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12.
2. the composition of detection people MDR1 gene pleiomorphism as described in claim 1, characterized in that further include detection people MDR1
The probe of 1236 loci polymorphism of gene, sequence are SEQ ID NO.1, detect the spy of 2677 loci polymorphism of people MDR1 gene
Needle, sequence are SEQ ID NO.5, detect the probe of 3435 loci polymorphism of people MDR1 gene, and sequence is SEQ ID NO.9.
3. composition as claimed in claim 1 or 2, characterized in that the composition further includes for detecting MDR1 gene
1236 sites, 2677 sites, the interior label primer of 3435 loci polymorphisms and internal standard probe, the interior label primer sequence such as SEQ ID
Shown in NO.13 and SEQ ID NO.14, the internal standard probe sequence is as shown in SEQ ID NO.15.
4. a kind of includes the kit of the composition as described in claim any one of 1-3, characterized in that the kit contains
PCR detection reagent A and PCR detection reagent B,
The PCR detection reagent A includes sequence such as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID
NO.8、SEQ ID NO.10、SEQ ID NO.12、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.1、SEQ ID
A variety of primer and probes of NO.5, SEQ ID NO.9, SEQ ID NO.13；
The PCR detection reagent B includes sequence respectively such as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.7, SEQ ID
NO.8、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.1、SEQ ID
A variety of primer and probes of NO.5, SEQ ID NO.9, SEQ ID NO.13.
5. kit as claimed in claim 4, characterized in that the kit further includes sample lysate, and the sample is molten
Solving liquid includes Tris-HCl buffer, lauryl sodium sulfate, EDTA, glycerol and Proteinase K.
6. kit as described in claim 4 or 5, characterized in that further include: wild type control product, saltant comparison product and
Blank control product；The wild type control product be include 1236 site of MDR1 gene polymorphism sites, 2677 sites, 3435
Point wild type recombinant plasmid and include internal standard gene recombinant plasmid mixture；The saltant comparison product be comprising
Have 1236 site of MDR1 gene polymorphism sites, 2677 sites, 3435 sites saltant type recombinant plasmid and include internal standard
The mixture of the recombinant plasmid of gene；The blank control is the physiology not comprising MDR1 gene polymorphism sites and internal standard gene
Salt water.
7. a kind of sample treatment, characterized in that this method uses the described in any item kits of claim 4-6, including
Following steps:
1) 2 parts of human genome sample of nucleic acid pre-processed are taken, 2 parts of samples are examined with PCR the detection reagent A and PCR respectively
Test agent B mixing, wherein SEQ ID NO.1, SEQ ID NO.5, probe shown in SEQ ID NO.9 are respectively by ROX, CY5, FAM
Fluorescent marker, probe shown in SEQ ID NO.15 is by VIC or HEX fluorescent marker；
Pcr amplification reaction is carried out, CY5, FAM, ROX, VIC or HEX fluorescence signal are acquired；
2) result judges:
When wild type control product and saltant comparison product have CY5, FAM, ROX, VIC or HEX fluorescence signal initial line, blank control is equal
Without CY5, FAM, ROX, VIC or HEX fluorescence signal initial line, when the CT≤35 of sample detection pipe VIC or HEX, judgment experiment success；
According to the parting in 1236 site of Δ Ct value situation interpretation MDR1 gene of ROX fluorescence signal in two sample detection pipes A, B, according to
The parting in 2677 site of Δ Ct value situation interpretation MDR1 gene of CY5 fluorescence signal in two sample detection pipes A, B, according to two
The parting in 3435 site of Δ Ct value situation interpretation MDR1 gene of FAM fluorescence signal in sample detection pipe A, B.
8. the method for claim 7, characterized in that the program of pcr amplification reaction includes:
9. method as claimed in claim 7 or 8, characterized in that Genotyping interpretation follows following rule:
Calculation: Δ CtFAM=CtFAM(B)-CtFAM(A)-(CtHEX(B)-CtHEX(A))
ΔCtCY5=CtCY5(B)-CtCY5(A)-(CtHEX(B)-CtHEX(A))
ΔCtROX=CtROX(B)-CtROX(A)-(CtHEX(B)-CtHEX(A)),
When the probe shown in the SEQ ID NO.15 is by VIC fluorescent marker, CtHEXFor CtVIC；
If ROX/CY5/FAM determines that without Ct value, being defaulted as Ct value is 40, is included in calculating,
10. a kind of application such as the kit of claim 4,5 or 6 in terms of detecting people MDR1 gene pleiomorphism.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811271585.5A CN109295192A (en) | 2018-10-29 | 2018-10-29 | A kind of composition, kit, sample treatment and application detecting people MDR1 gene pleiomorphism |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811271585.5A CN109295192A (en) | 2018-10-29 | 2018-10-29 | A kind of composition, kit, sample treatment and application detecting people MDR1 gene pleiomorphism |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109295192A true CN109295192A (en) | 2019-02-01 |
Family
ID=65158993
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811271585.5A Pending CN109295192A (en) | 2018-10-29 | 2018-10-29 | A kind of composition, kit, sample treatment and application detecting people MDR1 gene pleiomorphism |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109295192A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110643699A (en) * | 2019-10-16 | 2020-01-03 | 成都仕康美生物科技有限公司 | Cyclosporin A metabolic gene detection kit and application method thereof |
| CN113584160A (en) * | 2021-06-15 | 2021-11-02 | 湖南菲思特精准医疗科技有限公司 | Detection kit for ondansetron metabolic marker, detection method and application thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277443A (en) * | 2011-09-16 | 2011-12-14 | 协和干细胞基因工程有限公司 | Kit for detecting tacrolimus and cyclosporine A individual medicine administration associated single nucleotide polymorphism (SNP) loci and amplification method and detection method |
| CN103088156A (en) * | 2012-12-26 | 2013-05-08 | 杭州三合创新科技有限公司 | White spot syndrome virus detection kit and method |
| CN103122374A (en) * | 2011-10-03 | 2013-05-29 | 爱科来株式会社 | Probe, polymorphism detection method, method of evaluating drug efficacy or tolerance, and reagent kit |
| CN104805181A (en) * | 2015-03-26 | 2015-07-29 | 协和干细胞基因工程有限公司 | Primers for detecting polymorphism of CYP3A4, CYP3A5 and MDR1 genes with ARMs-PCR (amplification refractory mutation system-polymerase chain reaction) method and prepared kit |
| CN105274190A (en) * | 2014-07-22 | 2016-01-27 | 复旦大学附属华山医院 | HRM method for detecting genetic polymorphism of CYP3A4*1G and MDR1C1236T |
| CN105525005A (en) * | 2016-01-22 | 2016-04-27 | 广州金域检测科技股份有限公司 | Primer and method for detecting MDR1 gene polymorphism |
| CN106554995A (en) * | 2016-07-26 | 2017-04-05 | 北京京诺玛特科技有限公司 | The method and test kit of detection chemotherapy of tumors personalized medicine related gene |
| CN106755360A (en) * | 2016-12-06 | 2017-05-31 | 武汉海吉力生物科技有限公司 | Nucleic acid, kit and method for detecting mankind's CYP2D6 gene pleiomorphisms |
-
2018
- 2018-10-29 CN CN201811271585.5A patent/CN109295192A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277443A (en) * | 2011-09-16 | 2011-12-14 | 协和干细胞基因工程有限公司 | Kit for detecting tacrolimus and cyclosporine A individual medicine administration associated single nucleotide polymorphism (SNP) loci and amplification method and detection method |
| CN103122374A (en) * | 2011-10-03 | 2013-05-29 | 爱科来株式会社 | Probe, polymorphism detection method, method of evaluating drug efficacy or tolerance, and reagent kit |
| CN103088156A (en) * | 2012-12-26 | 2013-05-08 | 杭州三合创新科技有限公司 | White spot syndrome virus detection kit and method |
| CN105274190A (en) * | 2014-07-22 | 2016-01-27 | 复旦大学附属华山医院 | HRM method for detecting genetic polymorphism of CYP3A4*1G and MDR1C1236T |
| CN104805181A (en) * | 2015-03-26 | 2015-07-29 | 协和干细胞基因工程有限公司 | Primers for detecting polymorphism of CYP3A4, CYP3A5 and MDR1 genes with ARMs-PCR (amplification refractory mutation system-polymerase chain reaction) method and prepared kit |
| CN105525005A (en) * | 2016-01-22 | 2016-04-27 | 广州金域检测科技股份有限公司 | Primer and method for detecting MDR1 gene polymorphism |
| CN106554995A (en) * | 2016-07-26 | 2017-04-05 | 北京京诺玛特科技有限公司 | The method and test kit of detection chemotherapy of tumors personalized medicine related gene |
| CN106755360A (en) * | 2016-12-06 | 2017-05-31 | 武汉海吉力生物科技有限公司 | Nucleic acid, kit and method for detecting mankind's CYP2D6 gene pleiomorphisms |
Non-Patent Citations (1)
| Title |
|---|
| 付孟龙 等: "MDR1 基因多态性及其临床相关性", 《临床检验杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110643699A (en) * | 2019-10-16 | 2020-01-03 | 成都仕康美生物科技有限公司 | Cyclosporin A metabolic gene detection kit and application method thereof |
| CN110643699B (en) * | 2019-10-16 | 2020-12-01 | 成都仕康美生物科技有限公司 | Cyclosporin A metabolic gene detection kit and application method thereof |
| CN113584160A (en) * | 2021-06-15 | 2021-11-02 | 湖南菲思特精准医疗科技有限公司 | Detection kit for ondansetron metabolic marker, detection method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104630368B (en) | Relative quantitative detection method of human motor neuron gene copy numbers and kit thereof | |
| JP2010521156A (en) | System and method for detection of HIV drug resistant variants | |
| JP2008538496A (en) | Method for determining sequence variants using ultra-deep sequencing | |
| CN107385024B (en) | Rice fertility restorer gene assisted breeding molecular marker and application thereof | |
| CN111334573A (en) | Gene detection kit for hypertension medication and use method | |
| CN112941210A (en) | Kit and method for detecting drug-resistant mutation of mycobacterium tuberculosis rifampicin and isoniazid | |
| CN109321651A (en) | A kind of composition, kit, sample treatment and application detecting people CYP2D6 gene pleiomorphism | |
| CN106939334B (en) | Method for detecting fetal DNA content in plasma of pregnant woman | |
| CN109182493B (en) | Primer and kit for detecting human 16p11.2 microdeletion syndrome and detection method thereof | |
| CN109295192A (en) | A kind of composition, kit, sample treatment and application detecting people MDR1 gene pleiomorphism | |
| CN113502335B (en) | Molecular marker related to sheep growth traits and application thereof | |
| CN115029444A (en) | Molecular marker related to sheep growth traits and application thereof | |
| CN110607381B (en) | Mycobacterium tuberculosis detection kit and method | |
| CN109371113A (en) | A kind of composition, kit, sample treatment and application detecting mankind's APOE and SLCO1B1 gene pleiomorphism | |
| EP2013366B1 (en) | Sequencing of the L10 codon of the HIV gag gene | |
| CN108060213A (en) | Isothermal duplication method detection SNP site probe and kit based on the recombinase-mediated that probe is oriented to | |
| CN107287283B (en) | High-throughput detection kit for multiple SNP sites related to children susceptibility diseases and use method thereof | |
| CN114085926B (en) | Primer, probe, kit and detection method for SNP locus polymorphism of ABCB1 gene C3435T | |
| CN113913530B (en) | Molecular marker related to sheep body height and application thereof | |
| CN110904099A (en) | Digital PCR technology and kit for detecting African swine fever viruses in feed | |
| CN116083592A (en) | Molecular marker related to sheep growth traits and application thereof | |
| CN111593115B (en) | Primer and probe combination and kit for multiplex real-time fluorescence PCR detection of beta-thalassemia gene mutation | |
| CN113957176A (en) | Detection reagent, reaction system and kit for identifying new coronavirus variant strain and wild strain and application | |
| CN110241231A (en) | Detect composition, kit, method and the application of CYP2C19 gene pleiomorphism | |
| CN106566880A (en) | Hereditary hearing loss gene mutation detection kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190201 |