CN107058562A - The application of SNP site and its detection kit - Google Patents

The application of SNP site and its detection kit Download PDF

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Publication number
CN107058562A
CN107058562A CN201710348135.0A CN201710348135A CN107058562A CN 107058562 A CN107058562 A CN 107058562A CN 201710348135 A CN201710348135 A CN 201710348135A CN 107058562 A CN107058562 A CN 107058562A
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dna
artificial sequence
snp
site
dissociative dna
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CN107058562B (en
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魏亮
卢俊
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S&km Bio-Tech Co Ltd
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S&km Bio-Tech Co Ltd
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    • 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
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • 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

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Abstract

The present invention relates to application and its detection kit of biological technical field, more particularly to SNP site.The invention provides application of 60 SNP sites in assessing the damage of after liver transplantation graft and repelling risk, detected by 60 specific SNP sites to the total dissociative DNA of acceptor after transplanting, with reference to specific computational methods, the final content for obtaining donor dissociative DNA in acceptor dissociative DNA, that is GcfDNA supply by than, supplied according to GcfDNA by the degree than quickly and accurately judging graft rejection, it is that the postoperative graft damage of clinical detection organ transplant and repulsion risk provide a kind of simple and effective supplementary means without the detection of any intrusive mood.

Description

The application of SNP site and its detection kit
Technical field
The present invention relates to application and its detection kit of biological technical field, more particularly to SNP site.
Background technology
The health monitoring of the postoperative graft of current entity organ transplant carries out liver function test, or puncture needle frequently with blood drawing Gather tissue and carry out pathological examination.
For conventional blood drawing functional check, its indices such as creatinine, ALT, AST, bilirubin etc. sensitivity and specifically Property it is not high, it is impossible to accurately reaction graft health status.
For the tissue biopsy of current goldstandard, though it can directly react the health status of graft.But exist following Significant drawback：
1) intrusive mood is detected, larger pain is caused to patient, while causing damage to graft；
2) when detecting abnormal, graft substantive damage has occurred and that, and damages often more serious, at this moment right Graft is saved for clinician often too late；
If 3) puncture needle does not collect the lesion tissue part of graft, result accuracy can be affected greatly, because This accuracy and sensitivity is not high.
Therefore, seek it is a kind of can it is sensitive, special, it is accurate assess liver transfer operation thing damage status and repel risk it is noninvasive Hinder detection method significant.
The content of the invention
Preparing the damage of assessment after liver transplantation graft it is an object of the invention to provide SNP site and repelling risk Application and its detection kit in product so that the kit being capable of sensitive, special, the accurate damage for assessing liver transfer operation thing Situation and repulsion risk, without carrying out any wound detection.
The invention provides SNP site in the product for assessing the damage of after liver transplantation graft and repulsion risk is prepared Using the SNP site is selected from：
rs2012852、rs2071985、rs867983、rs218503、rs407930、rs2070849、rs2067003、 rs2269951、rs2269952、rs399714、rs469783、rs2074012、rs179785、rs84933、rs2076537、 rs2071277、rs2075817、rs1007311、rs400218、rs1064416、rs2076416、rs2076435、 rs2074428、rs2070776、rs2071427、rs2071502、rs2073900、rs2071484、rs518471、 rs2279096、rs2278945、rs474949、rs1381532、rs2279984、rs2279985、rs1278278、 rs2279547、rs2270181、rs2070669、rs2070759、rs1635537、rs2070876、rs624707、 rs1289315、rs2070340、rs926027、rs2070777、rs2238354、rs2076237、rs2075852、 rs2076393、rs2251252、rs362549、rs2268082、rs2070351、rs2070356、rs2251252、 Rs2070438, rs521861 and rs2282614.
Present invention research shows, using above-mentioned 60 SNP sites as detection target spot, calculates in obtained acceptor dissociative DNA The content (GcfDNA supply by than) of donor dissociative DNA can accurately reflect out the degree of graft rejection, be that clinical detection organ is moved Plant postoperative graft damage and repel risk and provide a kind of simple and effective supplementary means.
Present invention also offers a kind of kit for being used to assess the damage of after liver transplantation graft and repel risk, including Primer and sequence probe as shown in SEQ ID No.121~240 of the sequence as shown in SEQ ID No.1~120.
The mentioned reagent box that the present invention is provided, primer and SEQ ID No.121~240 shown in SEQ ID No.1~120 Shown probe is used to expand, detect 60 SNP sites, and sequence is as follows：
It is preferred that, in the probe shown in SEQ ID No.121~240,5 ' ends point of the probe pair for detecting SNP site Different fluorophors, 3 ' end mark NFQ and MGB are not marked.The present invention is not particularly limited for the species of fluorophor, this The conventional species in field.In the specific embodiment that the present invention is provided, 5 ' end difference of two probes of SNP site are detected Flag F AM fluorophors and VIC fluorophors.
The kit that the present invention is provided also includes ddPCRTMProbeSupermix and ultra-pure water.
The present invention also provides a kind of method for detecting donor dissociative DNA content in acceptor dissociative DNA, comprises the following steps：
Step 1：Total dissociative DNA using testing sample as template, add primer shown in SEQ ID No.1~120 and Probe of the sequence as shown in SEQ ID No.121~240, prepares digital pcr reaction droplet, the reaction droplet prepared is shifted Into 96 orifice plates, digital pcr reaction is carried out；
Step 2：After reaction terminates, 96 orifice plates are transferred to QX200TMDigital pcr is read in instrument, is read result and is independently divided Analyse each SNP site, calculate the ratio of donor dissociative DNA/receptor's dissociative DNA, i.e. GcfDNA supply by than, wherein, SNP site Analysis method is as follows：
1) two kinds of site base ratio (low content/high content)=0.9~1, regards the site data as invalid data；
2) two kinds of site base ratio (low content/high content)=0, i.e., only a kind of base is positive, depending on the site data For invalid data；
3) two kinds of site base ratio (low content/high content)=0~0.9, regards the site data as valid data；Having Imitate in data, by taking SNP No#1 and SNP No#2 as an example, SNP No#1 base ratio be approximately equal to SNP No#2 twice (2 ± 0.4), then judge in SNP No#1, transplant recipient and transplantation donor are homozygote in；In SNP No#2, transplant recipient For homozygote, transplantation donor is heterozygote；
4) selection judges that transplant recipient and transplantation donor are homozygous SNP site data, calculates average value, is GcfDNA supply by than.
In the specific embodiment that the present invention is provided, the reaction system of digital pcr is：
The primer and probe of the SNP site is the primer and probe in kit described in claim 3.
In the specific embodiment that the present invention is provided, the PCR response procedures are as follows：
95 DEG C, 10min pre-degenerations；95 DEG C, 15s, 57 DEG C, 1min, 40 circulations；95 DEG C of holding 10min；10 DEG C of preservations.
The invention provides application of the SNP site in assessing the damage of after liver transplantation graft and repelling risk, pass through 60 specific SNP sites to the total dissociative DNA of acceptor after transplanting are detected, with reference to digital round pcr and specific calculating Method, the final content for obtaining donor dissociative DNA in acceptor dissociative DNA, i.e. GcfDNA is supplied by than being supplied according to GcfDNA by than sentencing The degree of disconnected graft rejection, is that the postoperative graft damage of clinical detection organ transplant and repulsion risk provide one kind and simply had The supplementary means of effect, with following advantage：
(1) noninvasive, 5~10ml venous blood collections can be detected；
(2) it is sensitive, select precision highest digital pcr equipment in existing round pcr to be used as detection platform；
(3) early stage, from nucleic acid molecules as detection target spot, the change of these target spots occurs in organ lesion or damage Earliest period；
(4) specifically, the specific dissociative DNA detected is directed to liver allograft, specific can reflect liver The health status of graft；
(5) it is convenient, compared to the other technologies in the field or detection, transplant recipient and transplanting are needed such as sequencing, DSA detections The sample of donor both sides is compared, and this method is without transplantation donor sample, it is only necessary to which receptor's blood sample can be completed.
Embodiment
The invention discloses the application of SNP site and its detection kit, those skilled in the art can be used for reference in herein Hold, be suitably modified technological parameter realization.In particular, all similar replacements and change are to those skilled in the art For be it will be apparent that they are considered as being included in the present invention.The method of the present invention and application are by preferably implementing Example is described, related personnel substantially can not departing from present invention, in spirit and scope to method described herein and Using being modified or suitably changing with combining, to realize and apply the technology of the present invention.
To the explanation of the disclosed embodiments, professional and technical personnel in the field are enable to realize or using the present invention.To this A variety of modifications of a little embodiments will be apparent for those skilled in the art, as defined herein general Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, the present invention will not It can be intended to be limited to the embodiments shown herein, and be to fit to consistent with principles disclosed herein and features of novelty Most wide scope.
Agents useful for same can be bought by market in the application for the SNP site that the present invention is provided and its detection kit.Primer Sequence and probe sequence are synthesized by (please supplement composition sequence) company.
With reference to embodiment, the present invention is expanded on further：
The kit that embodiment 1 is provided using the present invention assesses the damage of after liver transplantation graft and repels risk
Total dissociative DNA is extracted in blood plasma
1st, using the Streck receptor's blood of after liver transplantation 2 weeks of noninvasive heparin tube venous collection one 10ml；
2nd, the blood by the noninvasive heparin tube collections of Streck is transferred in 15ml centrifuge tubes, 4 DEG C of 2000g centrifugations of low temperature 10min；Upper plasma about 4~6ml is taken in new 15ml centrifuge tubes, continues 4 DEG C of 4000g centrifugations 10min of low temperature；Take upper strata blood 4~6ml is starched in new 15ml centrifuge tubes；
3rd, 4ml blood plasma is taken, is carried out using the QIAamp Circulating Nucleic Acid Kit of QIAGEN companies total The extraction of dissociative DNA, adjustment DNA concentration is 10~40ng/ul.
Prepare digital pcr reaction droplet
1st, digital pcr reaction system is configured, digital pcr reaction droplet is prepared, enters performing PCR reaction；The inspection of 60 SNP sites Survey system, 60 is managed, reaction is formulated as follows altogether：
Wherein, for detecting that the primer of SNP site includes upstream and downstream primer, each 0.125 μ L, totally 0.25 μ L；Probe is two Bar, each 0.125 μ L, common 0.25ul.
2nd, the reaction system configured is added to droplet and occurs card DG8TMIn Cartridges sample cell, while 60ul droplets are added in generation oil groove and occur oil Droplet Generation Oil for Probes, sealer DG8 is finally usedTM Gaskets is sealed, and is placed on droplet generator, carries out the preparation of reaction droplet.
3rd, the reaction droplet prepared is slowly transferred in 96 orifice plates, then coordinates sealer machine to seal with aluminum sealer Good laggard performing PCR reaction, response procedures set as follows：
Interpretation of result
After PCR reactions terminate, 96 orifice plates are transferred to QX200TMDigital pcr is read in instrument, is read result, is shown in Table 1.
The initial data of 1 60 SNP sites of table
The data to table 1 are analyzed in accordance with the following methods：
1) two kinds of site base ratio (low content/high content)=0.9~1, regards the site data as invalid data；
2) two kinds of site base ratio (low content/high content)=0, i.e., only a kind of base is positive, depending on the site data For invalid data；
3) two kinds of site base ratio (low content/high content)=0~0.9, regards the site data as valid data；Having Imitate in data, by taking SNP No#1 and SNP No#2 as an example, SNP No#1 base ratio be approximately equal to SNP No#2 twice (2 ± 0.4), then judge in SNP No#1, transplant recipient and transplantation donor are homozygote in；In SNP No#2, transplant recipient For homozygote, transplantation donor is heterozygote；
4) selection judges that transplant recipient and transplantation donor are homozygous SNP site data, calculates average value, is GcfDNA supply by than.
Evaluation criterion：GcfDNA supplies, by than≤10%, to represent receptor's liver allograft relative healths, repel risk low； GcfDNA supplies, by than ＞ 10%, representing that receptor's liver allograft has heavier damage, to repel risk high.
According to the data of table 1 calculate obtain GcfDNA supply by than (donor dissociative DNA/receptor's dissociative DNA) be 35.4 ± 1.2%, represent that receptor's liver allograft has heavier damage, repel risk high.
Embodiment 2
The blood 2ml of 2 weeks receptors of after liver transplantation in embodiment 1 is gathered using EDTA heparin tubes, liver function is carried out and routinely examines Survey, as a result as shown in table 2.
The receptor's liver function conventional detection result of table 2
As a result show, the liver allograft of 2 weeks receptors of the after liver transplantation has substantive damage, and present invention detection knot Fruit coincide, in addition, detected using the kit that provides of the present invention to other 50 liver-transplantation patients, and testing result is and liver Work(conventional detection result matches, and shows that kit of the present invention is capable of the strong of in time, accurately and specifically reflection liver allograft Health situation.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should It is considered as protection scope of the present invention.
SEQUENCE LISTING
<110>Chengdu Shi Kangmei Bioisystech Co., Ltd
<120>The application of SNP site and its detection kit
<130> MP1705612
<160> 240
<170> PatentIn version 3.3
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<211> 18
<212> DNA
<213>Artificial sequence
<400> 79
ctctgaagga gacctgtg 18
<210> 80
<211> 18
<212> DNA
<213>Artificial sequence
<400> 80
cacgtagaga aaattatg 18
<210> 81
<211> 18
<212> DNA
<213>Artificial sequence
<400> 81
caccacagat gtagtcag 18
<210> 82
<211> 18
<212> DNA
<213>Artificial sequence
<400> 82
aggccttgga tgaaaatt 18
<210> 83
<211> 18
<212> DNA
<213>Artificial sequence
<400> 83
ccttgctaag acgtttag 18
<210> 84
<211> 18
<212> DNA
<213>Artificial sequence
<400> 84
caggtgagcc agggtgta 18
<210> 85
<211> 18
<212> DNA
<213>Artificial sequence
<400> 85
aatgttaact tgctattt 18
<210> 86
<211> 18
<212> DNA
<213>Artificial sequence
<400> 86
agtcaaggat ggtggata 18
<210> 87
<211> 18
<212> DNA
<213>Artificial sequence
<400> 87
taagaaagct cgcgcata 18
<210> 88
<211> 18
<212> DNA
<213>Artificial sequence
<400> 88
agcagatggt catggtga 18
<210> 89
<211> 18
<212> DNA
<213>Artificial sequence
<400> 89
ggtcaccagt caggagcc 18
<210> 90
<211> 18
<212> DNA
<213>Artificial sequence
<400> 90
atacagaaat cacaaaaa 18
<210> 91
<211> 18
<212> DNA
<213>Artificial sequence
<400> 91
ggggccatat ctccagca 18
<210> 92
<211> 18
<212> DNA
<213>Artificial sequence
<400> 92
cgagatgggt gcagaggc 18
<210> 93
<211> 18
<212> DNA
<213>Artificial sequence
<400> 93
aacctggcct ttcaacat 18
<210> 94
<211> 18
<212> DNA
<213>Artificial sequence
<400> 94
ccctctctag ctggctct 18
<210> 95
<211> 18
<212> DNA
<213>Artificial sequence
<400> 95
gcacggtgcc tgcacagc 18
<210> 96
<211> 18
<212> DNA
<213>Artificial sequence
<400> 96
tggcagctta accctgct 18
<210> 97
<211> 18
<212> DNA
<213>Artificial sequence
<400> 97
cctacaacag ggttgttg 18
<210> 98
<211> 18
<212> DNA
<213>Artificial sequence
<400> 98
tcctgcctcc aacctcag 18
<210> 99
<211> 18
<212> DNA
<213>Artificial sequence
<400> 99
agaggcccct ttcatctt 18
<210> 100
<211> 18
<212> DNA
<213>Artificial sequence
<400> 100
cctcttgcta tcccttcc 18
<210> 101
<211> 18
<212> DNA
<213>Artificial sequence
<400> 101
ccaggagaac cacttgag 18
<210> 102
<211> 18
<212> DNA
<213>Artificial sequence
<400> 102
tcaaagcaac gtgtcaat 18
<210> 103
<211> 18
<212> DNA
<213>Artificial sequence
<400> 103
gcaggaggca agaacaag 18
<210> 104
<211> 18
<212> DNA
<213>Artificial sequence
<400> 104
ggtcactctg atctcagc 18
<210> 105
<211> 18
<212> DNA
<213>Artificial sequence
<400> 105
cactgagcat gacaacca 18
<210> 106
<211> 18
<212> DNA
<213>Artificial sequence
<400> 106
catttgggga cttccctt 18
<210> 107
<211> 18
<212> DNA
<213>Artificial sequence
<400> 107
atctgcgcac aagtcctc 18
<210> 108
<211> 18
<212> DNA
<213>Artificial sequence
<400> 108
aaagaaatgt ggaggatg 18
<210> 109
<211> 18
<212> DNA
<213>Artificial sequence
<400> 109
ccttgttctc accttttc 18
<210> 110
<211> 18
<212> DNA
<213>Artificial sequence
<400> 110
agagccaggt ggccgggg 18
<210> 111
<211> 18
<212> DNA
<213>Artificial sequence
<400> 111
gaggatccac gtcgccca 18
<210> 112
<211> 18
<212> DNA
<213>Artificial sequence
<400> 112
tggctggatg tggaggag 18
<210> 113
<211> 18
<212> DNA
<213>Artificial sequence
<400> 113
ggaagcagga ggcaagaa 18
<210> 114
<211> 18
<212> DNA
<213>Artificial sequence
<400> 114
ggtcactctg atctcagc 18
<210> 115
<211> 18
<212> DNA
<213>Artificial sequence
<400> 115
tcaaagactg ccagacac 18
<210> 116
<211> 18
<212> DNA
<213>Artificial sequence
<400> 116
gtgaaacagg caaccatt 18
<210> 117
<211> 18
<212> DNA
<213>Artificial sequence
<400> 117
aaatgataac tgtaggcc 18
<210> 118
<211> 18
<212> DNA
<213>Artificial sequence
<400> 118
tagagccaat ggccaatc 18
<210> 119
<211> 18
<212> DNA
<213>Artificial sequence
<400> 119
tgttctatta ataggcag 18
<210> 120
<211> 18
<212> DNA
<213>Artificial sequence
<400> 120
tccctctcca tcactccc 18
<210> 121
<211> 15
<212> DNA
<213>Artificial sequence
<400> 121
tgcaccagaa ccatc 15
<210> 122
<211> 15
<212> DNA
<213>Artificial sequence
<400> 122
tgccccagaa ccatc 15
<210> 123
<211> 15
<212> DNA
<213>Artificial sequence
<400> 123
cacatcctcg ctcac 15
<210> 124
<211> 15
<212> DNA
<213>Artificial sequence
<400> 124
cacgtcctcg ctcac 15
<210> 125
<211> 15
<212> DNA
<213>Artificial sequence
<400> 125
ccaaattgct cctgg 15
<210> 126
<211> 15
<212> DNA
<213>Artificial sequence
<400> 126
ccagattgct cctgg 15
<210> 127
<211> 15
<212> DNA
<213>Artificial sequence
<400> 127
tcacgctatg gtgga 15
<210> 128
<211> 15
<212> DNA
<213>Artificial sequence
<400> 128
tcatgctatg gtgga 15
<210> 129
<211> 15
<212> DNA
<213>Artificial sequence
<400> 129
agagctttgg tggga 15
<210> 130
<211> 15
<212> DNA
<213>Artificial sequence
<400> 130
agatctttgg tggga 15
<210> 131
<211> 15
<212> DNA
<213>Artificial sequence
<400> 131
agtaactaac tttta 15
<210> 132
<211> 15
<212> DNA
<213>Artificial sequence
<400> 132
agtagctaac tttta 15
<210> 133
<211> 14
<212> DNA
<213>Artificial sequence
<400> 133
agggacagag gcca 14
<210> 134
<211> 14
<212> DNA
<213>Artificial sequence
<400> 134
agggactgag gcca 14
<210> 135
<211> 15
<212> DNA
<213>Artificial sequence
<400> 135
tgtcctgacc tgggt 15
<210> 136
<211> 15
<212> DNA
<213>Artificial sequence
<400> 136
tgttctgacc tgggt 15
<210> 137
<211> 15
<212> DNA
<213>Artificial sequence
<400> 137
tgtaccccaa aatga 15
<210> 138
<211> 15
<212> DNA
<213>Artificial sequence
<400> 138
tgtgccccaa aatga 15
<210> 139
<211> 15
<212> DNA
<213>Artificial sequence
<400> 139
aggcatatga actca 15
<210> 140
<211> 15
<212> DNA
<213>Artificial sequence
<400> 140
aggtatatga actca 15
<210> 141
<211> 15
<212> DNA
<213>Artificial sequence
<400> 141
actcgcccga tcatt 15
<210> 142
<211> 15
<212> DNA
<213>Artificial sequence
<400> 142
acttgcccga tcatt 15
<210> 143
<211> 15
<212> DNA
<213>Artificial sequence
<400> 143
attaagttta tttgg 15
<210> 144
<211> 15
<212> DNA
<213>Artificial sequence
<400> 144
attgagttta tttgg 15
<210> 145
<211> 15
<212> DNA
<213>Artificial sequence
<400> 145
aggcaggcgt ggagg 15
<210> 146
<211> 15
<212> DNA
<213>Artificial sequence
<400> 146
aggcgggcgt ggagg 15
<210> 147
<211> 15
<212> DNA
<213>Artificial sequence
<400> 147
cagactagtg agagc 15
<210> 148
<211> 15
<212> DNA
<213>Artificial sequence
<400> 148
caggctagtg agagc 15
<210> 149
<211> 15
<212> DNA
<213>Artificial sequence
<400> 149
atcctctttt ccaca 15
<210> 150
<211> 15
<212> DNA
<213>Artificial sequence
<400> 150
atcttctttt ccaca 15
<210> 151
<211> 15
<212> DNA
<213>Artificial sequence
<400> 151
tgacatctaa tctcc 15
<210> 152
<211> 15
<212> DNA
<213>Artificial sequence
<400> 152
tgacgtctaa tctcc 15
<210> 153
<211> 15
<212> DNA
<213>Artificial sequence
<400> 153
cgggatctgc gtgcg 15
<210> 154
<211> 15
<212> DNA
<213>Artificial sequence
<400> 154
cgggatttgc gtgcg 15
<210> 155
<211> 15
<212> DNA
<213>Artificial sequence
<400> 155
cagaaccccc tctgg 15
<210> 156
<211> 15
<212> DNA
<213>Artificial sequence
<400> 156
caggaccccc tctgg 15
<210> 157
<211> 15
<212> DNA
<213>Artificial sequence
<400> 157
cataccaaca tgaaa 15
<210> 158
<211> 15
<212> DNA
<213>Artificial sequence
<400> 158
catgccaaca tgaaa 15
<210> 159
<211> 15
<212> DNA
<213>Artificial sequence
<400> 159
accagtttga gagca 15
<210> 160
<211> 15
<212> DNA
<213>Artificial sequence
<400> 160
accggtttga gagca 15
<210> 161
<211> 15
<212> DNA
<213>Artificial sequence
<400> 161
agcccgtgca gggct 15
<210> 162
<211> 15
<212> DNA
<213>Artificial sequence
<400> 162
agccggtgca gggct 15
<210> 163
<211> 15
<212> DNA
<213>Artificial sequence
<400> 163
tttcagagca ttttt 15
<210> 164
<211> 15
<212> DNA
<213>Artificial sequence
<400> 164
ttttagagca ttttt 15
<210> 165
<211> 15
<212> DNA
<213>Artificial sequence
<400> 165
tggaagttgt tttta 15
<210> 166
<211> 15
<212> DNA
<213>Artificial sequence
<400> 166
tggacgttgt tttta 15
<210> 167
<211> 15
<212> DNA
<213>Artificial sequence
<400> 167
ctgccagcag aagtg 15
<210> 168
<211> 15
<212> DNA
<213>Artificial sequence
<400> 168
ctgtcagcag aagtg 15
<210> 169
<211> 15
<212> DNA
<213>Artificial sequence
<400> 169
tgccagaggt gtggc 15
<210> 170
<211> 15
<212> DNA
<213>Artificial sequence
<400> 170
tgccggaggt gtggc 15
<210> 171
<211> 15
<212> DNA
<213>Artificial sequence
<400> 171
tcacgcccct cattc 15
<210> 172
<211> 15
<212> DNA
<213>Artificial sequence
<400> 172
tcaggcccct cattc 15
<210> 173
<211> 15
<212> DNA
<213>Artificial sequence
<400> 173
cgtcgagtgc cagga 15
<210> 174
<211> 15
<212> DNA
<213>Artificial sequence
<400> 174
cgttgagtgc cagga 15
<210> 175
<211> 15
<212> DNA
<213>Artificial sequence
<400> 175
tctagagccc accag 15
<210> 176
<211> 15
<212> DNA
<213>Artificial sequence
<400> 176
tctggagccc accag 15
<210> 177
<211> 15
<212> DNA
<213>Artificial sequence
<400> 177
ttacctgtgc tgtgt 15
<210> 178
<211> 15
<212> DNA
<213>Artificial sequence
<400> 178
ttatctgtgc tgtgt 15
<210> 179
<211> 16
<212> DNA
<213>Artificial sequence
<400> 179
atacacctct tgtttt 16
<210> 180
<211> 16
<212> DNA
<213>Artificial sequence
<400> 180
atacgcctct tgtttt 16
<210> 181
<211> 15
<212> DNA
<213>Artificial sequence
<400> 181
tgcaacgtgt cctta 15
<210> 182
<211> 15
<212> DNA
<213>Artificial sequence
<400> 182
tgcgacgtgt cctta 15
<210> 183
<211> 15
<212> DNA
<213>Artificial sequence
<400> 183
aggctgttac agctg 15
<210> 184
<211> 15
<212> DNA
<213>Artificial sequence
<400> 184
aggttgttac agctg 15
<210> 185
<211> 15
<212> DNA
<213>Artificial sequence
<400> 185
tcgcagcatc tttga 15
<210> 186
<211> 15
<212> DNA
<213>Artificial sequence
<400> 186
tcgtagcatc tttga 15
<210> 187
<211> 15
<212> DNA
<213>Artificial sequence
<400> 187
ttcagccttt gcaaa 15
<210> 188
<211> 15
<212> DNA
<213>Artificial sequence
<400> 188
ttctgccttt gcaaa 15
<210> 189
<211> 15
<212> DNA
<213>Artificial sequence
<400> 189
tgggctttta tttta 15
<210> 190
<211> 15
<212> DNA
<213>Artificial sequence
<400> 190
tgggttttta tttta 15
<210> 191
<211> 15
<212> DNA
<213>Artificial sequence
<400> 191
cccaccagga gtggg 15
<210> 192
<211> 15
<212> DNA
<213>Artificial sequence
<400> 192
cccgccagga gtggg 15
<210> 193
<211> 15
<212> DNA
<213>Artificial sequence
<400> 193
cacagtaacc aaaaa 15
<210> 194
<211> 15
<212> DNA
<213>Artificial sequence
<400> 194
cacggtaacc aaaaa 15
<210> 195
<211> 15
<212> DNA
<213>Artificial sequence
<400> 195
agacagtggg aaggg 15
<210> 196
<211> 15
<212> DNA
<213>Artificial sequence
<400> 196
agacggtggg aaggg 15
<210> 197
<211> 15
<212> DNA
<213>Artificial sequence
<400> 197
tgagcggcga gggat 15
<210> 198
<211> 15
<212> DNA
<213>Artificial sequence
<400> 198
tgaggggcga gggat 15
<210> 199
<211> 15
<212> DNA
<213>Artificial sequence
<400> 199
ataataagca cgact 15
<210> 200
<211> 15
<212> DNA
<213>Artificial sequence
<400> 200
atactaagca cgact 15
<210> 201
<211> 15
<212> DNA
<213>Artificial sequence
<400> 201
aagcttcctg gggac 15
<210> 202
<211> 15
<212> DNA
<213>Artificial sequence
<400> 202
aagtttcctg gggac 15
<210> 203
<211> 14
<212> DNA
<213>Artificial sequence
<400> 203
agaccgttct gatg 14
<210> 204
<211> 14
<212> DNA
<213>Artificial sequence
<400> 204
agactgttct gatg 14
<210> 205
<211> 15
<212> DNA
<213>Artificial sequence
<400> 205
ccactgatcc taact 15
<210> 206
<211> 15
<212> DNA
<213>Artificial sequence
<400> 206
ccattgatcc taact 15
<210> 207
<211> 15
<212> DNA
<213>Artificial sequence
<400> 207
agaacaaaga ttttc 15
<210> 208
<211> 15
<212> DNA
<213>Artificial sequence
<400> 208
agagcaaaga ttttc 15
<210> 209
<211> 15
<212> DNA
<213>Artificial sequence
<400> 209
tgtcatcttt ttaac 15
<210> 210
<211> 15
<212> DNA
<213>Artificial sequence
<400> 210
tgtcgtcttt ttaac 15
<210> 211
<211> 15
<212> DNA
<213>Artificial sequence
<400> 211
ccaatcccgc ccgcc 15
<210> 212
<211> 15
<212> DNA
<213>Artificial sequence
<400> 212
ccagtcccgc ccgcc 15
<210> 213
<211> 15
<212> DNA
<213>Artificial sequence
<400> 213
agcagtttgt gggga 15
<210> 214
<211> 15
<212> DNA
<213>Artificial sequence
<400> 214
agctgtttgt gggga 15
<210> 215
<211> 15
<212> DNA
<213>Artificial sequence
<400> 215
tccatgtcta tgttc 15
<210> 216
<211> 15
<212> DNA
<213>Artificial sequence
<400> 216
tccttgtcta tgttc 15
<210> 217
<211> 15
<212> DNA
<213>Artificial sequence
<400> 217
gtgcgatgaa atgga 15
<210> 218
<211> 15
<212> DNA
<213>Artificial sequence
<400> 218
gtgtgatgaa atgga 15
<210> 219
<211> 15
<212> DNA
<213>Artificial sequence
<400> 219
taccggtaag aaaag 15
<210> 220
<211> 15
<212> DNA
<213>Artificial sequence
<400> 220
tactggtaag aaaag 15
<210> 221
<211> 15
<212> DNA
<213>Artificial sequence
<400> 221
agaccatacc acaaa 15
<210> 222
<211> 15
<212> DNA
<213>Artificial sequence
<400> 222
agactatacc acaaa 15
<210> 223
<211> 15
<212> DNA
<213>Artificial sequence
<400> 223
aggatagctg tagaa 15
<210> 224
<211> 15
<212> DNA
<213>Artificial sequence
<400> 224
aggatggctg tagaa 15
<210> 225
<211> 15
<212> DNA
<213>Artificial sequence
<400> 225
tggaactttc attcc 15
<210> 226
<211> 15
<212> DNA
<213>Artificial sequence
<400> 226
tgggactttc attcc 15
<210> 227
<211> 15
<212> DNA
<213>Artificial sequence
<400> 227
ccgagccaga tgcca 15
<210> 228
<211> 15
<212> DNA
<213>Artificial sequence
<400> 228
ccgtgccaga tgcca 15
<210> 229
<211> 15
<212> DNA
<213>Artificial sequence
<400> 229
tgcccgatca tgttc 15
<210> 230
<211> 15
<212> DNA
<213>Artificial sequence
<400> 230
tgcctgatca tgttc 15
<210> 231
<211> 15
<212> DNA
<213>Artificial sequence
<400> 231
cccattcttt cccag 15
<210> 232
<211> 15
<212> DNA
<213>Artificial sequence
<400> 232
cccgttcttt cccag 15
<210> 233
<211> 15
<212> DNA
<213>Artificial sequence
<400> 233
aggatagctg tagaa 15
<210> 234
<211> 15
<212> DNA
<213>Artificial sequence
<400> 234
aggatggctg tagaa 15
<210> 235
<211> 15
<212> DNA
<213>Artificial sequence
<400> 235
aaaaagagaa gggag 15
<210> 236
<211> 15
<212> DNA
<213>Artificial sequence
<400> 236
aaagagagaa gggag 15
<210> 237
<211> 15
<212> DNA
<213>Artificial sequence
<400> 237
tggccatttt atagg 15
<210> 238
<211> 15
<212> DNA
<213>Artificial sequence
<400> 238
tgggcatttt atagg 15
<210> 239
<211> 15
<212> DNA
<213>Artificial sequence
<400> 239
ctgcctctgc aaccc 15
<210> 240
<211> 15
<212> DNA
<213>Artificial sequence
<400> 240
ctgtctctgc aaccc 15

Claims (7)

1.SNP sites are preparing the application in assessing the damage of after liver transplantation graft and repelling the product of risk, described SNP Point is selected from：
rs2012852、rs2071985、rs867983、rs218503、rs407930、rs2070849、rs2067003、 rs2269951、rs2269952、rs399714、rs469783、rs2074012、rs179785、rs84933、rs2076537、 rs2071277、rs2075817、rs1007311、rs400218、rs1064416、rs2076416、rs2076435、 rs2074428、rs2070776、rs2071427、rs2071502、rs2073900、rs2071484、rs518471、 rs2279096、rs2278945、rs474949、rs1381532、rs2279984、rs2279985、rs1278278、 rs2279547、rs2270181、rs2070669、rs2070759、rs1635537、rs2070876、rs624707、 rs1289315、rs2070340、rs926027、rs2070777、rs2238354、rs2076237、rs2075852、 rs2076393、rs2251252、rs362549、rs2268082、rs2070351、rs2070356、rs2251252、 Rs2070438, rs521861 and rs2282614.
2. a kind of kit for being used to assess the damage of after liver transplantation graft and repel risk, it is characterised in that including sequence The probe of primer and sequence as shown in SEQ ID No.121~239 as shown in SEQ ID No.1~120.
3. kit according to claim 2, it is characterised in that 5 ' the end connection FAM fluorophors or VIC of the probe Fluorophor, 3 ' end mark NFQ and MGB.
4. kit according to claim 2, it is characterised in that the kit also includes ddPCRTM ProbeSupermix and ultra-pure water.
5. a kind of method for detecting donor dissociative DNA content in acceptor dissociative DNA, it is characterised in that comprise the following steps：
Step 1：Total dissociative DNA using testing sample adds the primer and sequence shown in SEQ ID No.1~120 as template Probe as shown in SEQ ID No.121~239, prepares digital pcr reaction droplet, the reaction droplet prepared is transferred into 96 In orifice plate, digital pcr reaction is carried out；
Step 2：After reaction terminates, 96 orifice plates are transferred to QX200TMDigital pcr is read in instrument, is read result and is independently analyzed every Individual SNP site, calculates the ratio of donor dissociative DNA/receptor's dissociative DNA, i.e. GcfDNA supply by than, wherein, the analysis of SNP site Method is as follows：
1) two kinds of site base ratio (low content/high content)=0.9~1, regards the site data as invalid data；
2) two kinds of site base ratio (low content/high content)=0, i.e., only a kind of base is positive, regards the site data as nothing Imitate data；
3) two kinds of site base ratio (low content/high content)=0~0.9, regards the site data as valid data；In significant figure In, by taking SNP No#1 and SNP No#2 as an example, SNP No#1 base ratio is approximately equal to SNP No#2 twice (2 ± 0.4), then Judge in SNP No#1, transplant recipient and transplantation donor are homozygote in；In SNP No#2, transplant recipient is homozygosis Son, transplantation donor is heterozygote；
4) selection judges that transplant recipient and transplantation donor are homozygous SNP site data, calculates average value, as GcfDNA For by than.
6. method according to claim 5, it is characterised in that the reaction system of the digital pcr is：
The primer and probe of the SNP site is the primer and probe in kit described in claim 2.
7. method according to claim 5, it is characterised in that the PCR response procedures are as follows：
95 DEG C, 10min pre-degenerations；95 DEG C, 15s, 57 DEG C, 1min, 40 circulations；95 DEG C of holding 10min；10 DEG C of preservations.
CN201710348135.0A 2017-05-17 2017-05-17 The application of SNP site and its detection kit Active CN107058562B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710348135.0A CN107058562B (en) 2017-05-17 2017-05-17 The application of SNP site and its detection kit

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710348135.0A CN107058562B (en) 2017-05-17 2017-05-17 The application of SNP site and its detection kit

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CN114381511A (en) * 2021-12-31 2022-04-22 上海药明奥测医疗科技有限公司 Detection method for evaluating injury and rejection risk of transplanted plant after liver transplantation
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