CN108949962A - A kind of fluorescent quantitative PCR detection method and its detection kit of KIR2DS5 mRNA - Google Patents

A kind of fluorescent quantitative PCR detection method and its detection kit of KIR2DS5 mRNA Download PDF

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CN108949962A
CN108949962A CN201810938966.8A CN201810938966A CN108949962A CN 108949962 A CN108949962 A CN 108949962A CN 201810938966 A CN201810938966 A CN 201810938966A CN 108949962 A CN108949962 A CN 108949962A
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kir2ds5
primer
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pcr
mrna
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何军
李颖
邱桥成
王甜
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First Affiliated Hospital of Suzhou University
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Abstract

The invention belongs to field of biological detection, more particularly to the fluorescent quantitative PCR detection method and its detection kit of a kind of KIR2DS5 mRNA, the design method of primer and probe filters out KIR2DS5 positive DNA the following steps are included: (1) carries out KIR Genotyping to DNA sample with PCR-SSOP method；(2) positive DNA sample is subjected to PCR amplification, electrophoresis；(3) extract KIR2DS5 specific DNA band, through rubber tapping, after purification connect with carrier T, it is inverted, select flora after extract plasmid order-checking；(4) sequencing result is compared with KIR/IPD database, it was demonstrated that its being characterized property gene order；(5) primer and probe is designed with primer-design software according to characteristic KIR2DS5 gene order.Genetic test is carried out using the primer and probe designed.The method increase the specificity and accuracy rate of PCR detection.

Description

A kind of fluorescent quantitative PCR detection method and its detection kit of KIR2DS5 mRNA
Technical field
The invention belongs to technical field of biological, and in particular to a kind of fluorescence quantitative PCR detection of KIR2DS5 mRNA Method and its detection kit.
Background technique
Killer cell immunoglobulin-like receptors (KIR) are a kind of transmembrane glycopeptides for being mainly expressed in NK cell membrane surface It is white, with ligand HLA interaction of molecules, allogenic NK cell is generated, GVL effect is played, in hematopoietic stem cell transplantation Important function is generated in terms of prognosis.KIR is located at human chromosome 19q13.4, contains 2 (KIR2D) or 3 (KIR3D) extracellular Ig Spline structure domain is divided into inhibition KIR(inhibitory KIR, iKIR) and activity KIR(activating KIR, aKIR) Two classes, iKIR gene include KIR2DL1, KIR 2DL2, the KIR3DL1 etc. of heterologous reactivity function, aKIR gene packet Include KIR2DS1, KIR2DS2, KIR2DS5, KIR2DS4, KIR2DS5, KIR3DS1 etc..DNA sequence dna 80- between each gene of KIR 90% is identical, at present for design of primers, objective gene sequence is mostly found on NCBI, or carry out full genome survey to the gene Sequence, and then according to gene order by primer-design software, after designing primer, carry out blast；These methods are in design KIR It is had some limitations when each gene primer, mainly shows low efficiency, poor specificity, accuracy rate is low.
Summary of the invention
Above of the existing technology in order to solve the problems, such as, the purpose of the present invention is to provide a kind of KIR2DS5 mRNA's Fluorescent quantitative PCR detection method and its detection kit, to improve the detection efficiency of KIR2DS5 and the specificity of detection.
To achieve the goals above, the present invention the following technical schemes are provided:
The primer and probe of fluorescence quantitative PCR detection KIR2DS5 mRNA, sequence are as follows:
2DS5F:5 '-AGAGAGGGGACGTTTAACC-3 '；
2DS5R:5 '-CACTGGGCGCTGACAACT-3 '；
Fluorescence probe: 5 '-CATCGGTCGCATGACA-3 '.
The method for obtaining primer and probe described in claim 1, comprising the following steps:
(1) normal person's DNA sample is extracted, carries out KIR Genotyping with PCR-SSOP method, filters out the KIR2DS5 positive Normal person's DNA sample；
(2) by the DNA sample filtered out using commercially available KIR2DS5 sequence specific primers amplification plate (being purchased from One Lambda) into Row PCR amplification, agarose gel electrophoresis；
(3) extract KIR2DS5 specific band in PCR product, through rubber tapping, after purification, be attached with carrier T, conversion by Body is the Escherichia coli of TOP10 competence resistance to streptomycin, after selecting flora, extracts plasmid, uses ABI 3730XL type sequenator It is sequenced；
(4) sequencing result is compared with KIR/IPD database, it was demonstrated that its being characterized property KIR2DS5 gene order, sequencing obtain Particular sequence are as follows: 5 '-AGAGAGGGGACGTTTAACCACACTTTGCGCCTCATTGGAGAGCAC
ATTGATGGGGTCTCCAAGGGCAACTTCTCCATCGGTCGCATGACACAAGACCTGGCAGG
GACCTACAGATGCTACGGTTCTGTTACTCACTCCCCCTATCAGTTGTCAGCGCCCAGTG-3'；
(5) primer as described in claim 1 is designed with primer-design software according to characteristic KIR2DS5 gene order And probe.
Further, in the method for obtaining primer and probe of the present invention, the instrument that PCR amplification uses is Perkin 9700 PCR amplification instrument of Elmer GeneAmp.
Further, in the method for obtaining primer and probe of the present invention, carrier T used in the step (3) For pMD18-T Vector.
Further, in the method for obtaining primer and probe of the present invention, the primer and probe passes through Primerexpress software is designed.
The primer and probe of fluorescence quantitative PCR detection KIR2DS5 mRNA of the present invention is in preparation KIR2DS5 mRNA Purposes in fluorescent quantificationally PCR detecting kit.
Detect KIR2DS5 mRNA method the following steps are included:
(1) total serum IgE is extracted；
It (2) is cDNA by RNA reverse transcription；
(3) mRNA of KIR2DS5 is detected with quantitative fluorescent PCR general system using the primer and probe in claim 1 Transcriptional level.
The utility model has the advantages that the present invention provides the fluorescent quantitative PCR detection method of KIR2DS5 mRNA a kind of and its detection examinations Agent box, the present invention carry out routine KIR Genotyping to normal person's DNA sample by using PCR-SSOP method, filter out After KIR2DS5 positive sample, its DNA sample is subjected to PCR amplification, electrophoresis using specific primer, by PCR product and carrier T Connection, and Clonal expansion, screening, sequencing are carried out, since PCR product is not single-minded, direct Sequencing effect is bad, and PCR product is straight Sequencing is connect, primer end does not measure to come there are about tens bases, if needing this section of sequence just, direct Sequencing will miss this Section gene order.It is connect using PCR product with T bacteriophage, can avoid the above problem.And the invention is finally directly found out Characteristic gene sequence in KIR2DS5 gene order directly avoids and amplifies other high unknown bands of other homologys. The fluorescence quantitative PCR detection that KIR2DS5 mRNA is carried out using the primer and probe that the present invention obtains, with high specificity, accurately The high feature of rate, while design primer of the present invention and the method for probe can also be used and give the high KIR gene of other homologys.
Detailed description of the invention
Fig. 1 is the quantitative fluorescent PCR canonical plotting of ABL standard items in inventive embodiments, using the data of table 1 as cross Ordinate show that standard curve, abscissa are indicated with the logarithm of ABL standard items copy number, specially log10(200, 2000,20000,200000,200000)=(2.3010,3.3010,4.3010,5.3010,6.3010), ordinate are ABL The Ct value of standard items.
Fig. 2 is the mentality of designing figure that the present invention carries out KIR2DS5 mRNA fluorescence quantitative PCR detection.
Fig. 3 is the KIR2DS5 sun that the KIR2DS5 primed probe designed with the present invention carries out that fluorescence quantitative PCR detection obtains 1.5% agarose electrophoresis figure of property product.
Specific embodiment
The invention will now be further described with reference to specific embodiments, but examples are merely exemplary, not to this hair Bright range constitutes any restrictions.It will be understood by those skilled in the art that without departing from the spirit and scope of the invention Can with the details and forms of the technical scheme of the invention are modified or replaced, but these modification and replacement each fall within it is of the invention In protection scope.
Experimental method:
1. DNA is extracted: extracting DNA according to DNA extraction agent box (being purchased from U.S. Promega company) specification whole blood.
2. PCR-SSOP carries out Genotyping:
Step experiment is operated using KIR SSO genotyping kit (being purchased from One Lambda company).
2.1 PCR reaction systems: KIR SSO amplification divides Group 1, Group 2,3 three groups of Group, and each reacting hole adds D-Mix 7.5 μ L, Primer 2 μ L, 0.1 μ L of Taq enzyme, 2.5 μ L of DNA sample, (it is public that D-Mix, Primer are purchased from One Lambda Department).
2.2 carry out PCR amplification, PCR amplification condition: 96 DEG C through 9700 PCR amplification instrument of Perkin Elmer GeneAmp 3min 1 circulation；96 DEG C of 20s, 60 DEG C of 20s, 72 DEG C of 20s are recycled for 5 totally；96 DEG C of 10s, 60 DEG C, 15s, 72 DEG C 20s, Totally 32 circulations；72 DEG C of 10min 1 circulations；4 DEG C of terminations.
After 2.3 are denaturalized amplified production using alkalies, Acidic Liquid neutralization is reused, adds microballon and fluorescence two again later It is anti-to be incubated for, after washing, using machine testing on 200 instrument of Luminex, filter out normal person's DNA sample of the KIR2DS5 positive This.
3. PCR amplification
3.1 PCR reaction systems: PCR buffer 115 μ L, ddH2152 μ L of O, the DNA for the KIR2DS5 positive that step 2 filters out 31 μ L of sample, 1.8 μ L, KIR2DS5 sequence specific primers of Taq enzyme expand plate, and (PCR buffer, KIR2DS5 sequence-specific draw Object amplification plate is purchased from One Lambda company)；
3.2 3000rpm are centrifuged 15-20s；
3.3 carry out PCR amplification through 9700 PCR amplification instrument of Perkin Elmer GeneAmp, PCR amplification condition: initial denaturation, 95℃ 5min；PCR cycle: denaturation, 95 DEG C of 30s；Annealing, 68 DEG C, 30s；Extend 72 DEG C of 90s；Totally 30 circulations；
The Ago-Gel of 3.4 configurations 1.5% carries out electrophoresis, and ultraviolet gel imager imaging takes the photograph figure and prints amplified production electricity Swimming figure sticks in progress result interpretation on KIR worksheet；
3.5, by result interpretation, the KIR2DS5 band on Ago-Gel are tapped rubber, purification process, and KIR2DS5 is obtained Gene.
4. TA is cloned
4.1 connection: in microcentrifugal tube be added 1 μ L of pMD18-T Vector, 0.2 pmol of KIR2DS5 gene, Solution I (the connection liquid in pMD 18-T Vector Cloning Kit kit Takara 6011) 5 μ L, add ddH2O to 10 μ L；16 DEG C of reaction 30min
4.2 conversion cultures: taking 50 μ L competent cells, be placed on ice, and cell even suspension is gently added 5 after thawing completely μ L connection liquid, places 30min on ice.42 DEG C of water-bath heat shock 45s, place 3min on ice.Add 500 μ L 2 × YT nonreactive culture mediums, 37 DEG C of 220 rpm shaken cultivation 1h.5000rpm is centrifuged 5min, is inhaled with pipette tips and abandons 400 μ L culture bacterium solution, and residue piping and druming, which mixes, to be applied Cloth at ampicillin plate (plus in right amount IPTG and X-gal), plate at 37 DEG C it is positive place to be inverted after 10min cultivated Night 12h connects bacterium 2mL, overnight incubation using sterile 10 μ L pipette tips picking hickie 6.
4.3 plasmid extractions: according to the small extraction reagent kit of paramagnetic particle method plasmid (Shanghai Shuo Mei Biotechnology Co., Ltd) specification Carry out plasmid extraction；
4.4 are sequenced using ABI 3730XL type sequenator, sequencing gained sequence and KIR/IPD database KIR2DS5 sequence It is compared, it was demonstrated that its being characterized property KIR2DS5 gene order,
Particular sequence after sequencing are as follows: 5 '-AGAGAGGGGACGTTTAACCACACTTTGCGCCTCATTGGAGA
GCACATTGATGGGGTCTCCAAGGGCAACTTCTCCATCGGTCGCATGACACAAGACCTG
GCAGGGACCTACAGATGCTACGGTTCTGTTACTCACTCCCCCTATCAGTTGTCAGCGCC
CAGTG-3'；
5. carrying out primer and probe design to characteristic KIR2DS5 gene order using primerexpress software, gained draws The particular sequence of object and probe is as follows,
2DS5F:5 '-AGAGAGGGGACGTTTAACC-3 '；
2DS5R:5 '-CACTGGGCGCTGACAACT-3 '；
Fluorescence probe: 5 '-CATCGGTCGCATGACA-3 '.
6. quantitative fluorescent PCR
6.1 extract RNA；
6.1.1 10mL peripheral blood is taken, 1500rpm is centrifuged 5min, abandons supernatant；
6.1.2 plus PBS buffer solution 5mL, 1500rpm are centrifuged 5min, abandon supernatant；
6.1.3 plus PBS buffer solution 1mL, 1500rpm are centrifuged 5min, abandon supernatant；
6.1.4 plus 1mL Trizol is blown and beaten, and abundant lytic cell, -20 DEG C save backup；
6.1.5 the spare Trizol sample that total serum IgE will be extracted is placed on to be melted on ice；
6.1.6 the Trizol sample after melting adds 200 μ L chloroforms, and after mixing, 4 DEG C of 12000rpm are centrifuged 15min；
6.1.7 transfer supernatant after adding 500 μ L of isopropanol to mix, precipitates 5min into 1.5mL EP pipe on ice, and 4 DEG C 12000rpm is centrifuged 15min；
6.1.8 supernatant is abandoned, 75% 600 μ L of ethyl alcohol is added, after mixing, 4 DEG C of 12000rpm are centrifuged 5min；
6.1.9 supernatant is abandoned, 800 μ L of dehydrated alcohol is added, after mixing, 4 DEG C of 12000rpm are centrifuged 5min；
6.1.10 supernatant is abandoned, and after drying, adds DEPC water to dissolve, and adjust RNA concentration value 0.5ug/ μ L.
6.2 mRNA reverse transcriptions are cDNA
6.2.1 reaction system: after 2 μ L of Random Primer, 9 μ L of DEPC water, 4 RNA μ L are mixed, 3000rpm it is of short duration from The heart (Random Primer is purchased from Shanghai bioengineering Co., Ltd)；
6.2.2 machine on PCR, reaction condition: 70 DEG C of 5min, are terminated by 4 DEG C；
6.2.3 by 5 × Buffer, 8 μ L, 1 μ L of MMLV reverse transcriptase, Rnasin0.5 μ L, 1.5 dNTP μ L, 15 μ L of DEPC water It is added into 6.2.2 (MMLV reverse transcriptase is purchased from IBM Corporation) after mixing；
6.2.4 6.2.3 is mixed, the of short duration centrifugation of 3000rpm；
6.2.5 machine on PCR, reaction condition: 37 DEG C of 60min, 95 DEG C of 5min, 4 DEG C of terminations.
6.3 quantitative fluorescent PCR
6.3.1 PCR reaction system: 96 orifice plates, every hole add MIX(to include Mg2+, dNTP, Taq enzyme, UNG enzyme and ROX reference dye Deng) 12.5 μ L, 0.5 μ L of upstream primer, 0.5 μ L of downstream primer, 7.2 μ L of 4 μ L of 0.3 μ L of probe, cDNA, ddH2O；
6.3.2 the of short duration centrifugation of 3000rpm；
6.3.3 PCR reaction condition: 50 DEG C of 2min, 1 circulation；95 DEG C of 10min 1 circulations；95 DEG C of 15s, 60 DEG C 1min, totally 40 recycle；
6.3.4 result is analyzed using 480 software of Roche Light Cycler.
The mrna expression amount analysis of fluorescence quantitative PCR detection KIR2DS5 is carried out using kit of the invention, result is such as Under: the transcriptional level of KIR target gene mRNA is calculated with following formula: (KIR target gene mRNA copy number/ABL reference gene MRNA copy number) × 10000, it is defined as in whole blood karyocyte, KIR target gene in every 10000 abl genes Copy number.
Standard curve: the carrier T of the known copy number containing one section of house-keeping gene abl segment dilutes various concentration gradient Standard curve is done, amplification efficiency is judged according to slope of a curve.ABL standard items and the mapping table of Ct value are as shown in table 1, institute The standard curve of acquisition is as shown in Figure 1, the copy number of KIR2DS5 is as shown in table 2.Fluorescence threshold: facing when fluorescence is more than background Boundary's numerical value, fluorescence threshold are 10 times that basal fluorescence signal mean value adds standard deviation, and PCR amplification reaches corresponding when fluorescence threshold Recurring number be known as Ct value.
Fig. 3 is the KIR2DS5 sun that the KIR2DS5 primed probe designed with the present invention carries out that fluorescence quantitative PCR detection obtains Property product 1.5% agarose electrophoresis figure, as seen from Figure 3, KIR2DS5 positive products are 163bp's after electrophoretogram and blast Conclusion is consistent, to illustrate that the primer and probe that the present invention designs has good identification specificity to KIR2DS5 gene.
Table 1
Table 2
Sequence table
<110>First Affiliated Hospital of Soochow University,Suzhou
<120>a kind of fluorescent quantitative PCR detection method and its detection kit of KIR2DS5 mRNA
<141> 2018-08-17
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>artificial sequence (chemical synthesis)
<400> 1
agagagggga cgtttaacc 19
<210> 2
<211> 18
<212> DNA
<213>artificial sequence (chemical synthesis)
<400> 2
cactgggcgc tgacaact 18
<210> 3
<211> 16
<212> DNA
<213>artificial sequence (chemical synthesis)
<400> 3
catcggtcgc atgaca 16

Claims (7)

1. the primer and probe of fluorescence quantitative PCR detection KIR2DS5 mRNA, which is characterized in that its sequence are as follows:
2DS5F:5 '-AGAGAGGGGACGTTTAACC-3 '；
2DS5R:5 '-CACTGGGCGCTGACAACT-3 '；
Fluorescence probe: 5 '-CATCGGTCGCATGACA-3 '.
2. the primer and probe of fluorescence quantitative PCR detection KIR2DS5 mRNA according to claim 1, which is characterized in that The method for obtaining primer and probe, comprising the following steps:
(1) normal person's DNA sample is extracted, carries out KIR Genotyping with PCR-SSOP method, filters out the KIR2DS5 positive Normal person's DNA sample；
(2) DNA sample filtered out is subjected to PCR amplification, agarose using commercially available KIR2DS5 sequence specific primers amplification plate Gel electrophoresis；
(3) extract KIR2DS5 specific band in PCR product, through rubber tapping, after purification, be attached with carrier T, conversion by Body is the Escherichia coli of TOP10 competence resistance to streptomycin, after selecting flora, extracts plasmid, uses ABI 3730XL type sequenator It is sequenced；
(4) sequencing result is compared with KIR/IPD database, it was demonstrated that its being characterized property KIR2DS5 gene order, sequencing obtain Particular sequence are as follows: 5 '-AGAGAGGGGACGTTTAACCACACTTTGCGCCTCATTGGAGAGCAC
ATTGATGGGGTCTCCAAGGGCAACTTCTCCATCGGTCGCATGACACAAGACCTGGCAGG
GACCTACAGATGCTACGGTTCTGTTACTCACTCCCCCTATCAGTTGTCAGCGCCCAGTG-3'；
(5) primer as described in claim 1 is designed with primer-design software according to characteristic KIR2DS5 gene order And probe.
3. the primer and probe of fluorescence quantitative PCR detection KIR2DS5 mRNA according to claim 2, which is characterized in that The instrument that PCR amplification uses is 9700 PCR amplification instrument of Perkin Elmer GeneAmp.
4. the primer and probe of fluorescence quantitative PCR detection KIR2DS5 mRNA according to claim 2, which is characterized in that Carrier T used in the step (3) is pMD18-T Vector.
5. the primer and probe of fluorescence quantitative PCR detection KIR2DS5 mRNA according to claim 2, which is characterized in that The primer and probe is designed by primerexpress software.
6. the primer and probe of fluorescence quantitative PCR detection KIR2DS5 mRNA described in claim 1 is in preparation KIR2DS5 Purposes in mRNA fluorescent quantificationally PCR detecting kit.
7. purposes as claimed in claim 6, which is characterized in that detection KIR2DS5 mRNA method the following steps are included:
(1) total serum IgE is extracted；
It (2) is cDNA by RNA reverse transcription；
(3) mRNA of KIR2DS5 is detected with quantitative fluorescent PCR general system using the primer and probe in claim 1 Transcriptional level.
CN201810938966.8A 2018-08-17 2018-08-17 A kind of fluorescent quantitative PCR detection method and its detection kit of KIR2DS5 mRNA Pending CN108949962A (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112708672A (en) * 2021-02-22 2021-04-27 深圳荻硕贝肯精准医学有限公司 KIR2DS5 genotyping kit and genotyping method

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