CN105695567A - Kit, primers, probe sequence and method for detecting fetus chromosome aneuploid - Google Patents
Kit, primers, probe sequence and method for detecting fetus chromosome aneuploid Download PDFInfo
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Abstract
The invention provides a kit, primers, probe sequences and a method for detecting fetus chromosome aneuploid. The kit comprises the multiple pairs of specific primers and a plurality of specific Taqman probes all of which are designed according to 13#, 18# and 21# chromogene sequences of the human, and further comprises a reaction system used for digital PCR, wherein the nucleotide sequence of the specific primers and the specific Taqman probes is SEQ ID NO: 1-90. The detection method comprises the steps that firstly, the specific primers and the specific Taqman probes are designed according to the 13#, 18# and 21# chromogene sequences of the human; secondly, peripheral blood of a (12-23)-week pregnant woman is collected for plasma DNA extracting; thirdly, the reaction system of the digital PCR amplification gene sequence is prepared; fourthly, the numbers of 13#, 18# and 21# chromosomes of a fetus are calculated through digital PCR detection for the number of the chromosomes, digital PCR is used for detecting FAM and VIC fluorescence of all reaction units of a chip, and the copy number of the 13# chromosome, the copy number of the 18# chromosome and the copy number of the 21# chromosome are calculated respectively. By means of the kit and the method, the number of the 13# chromosome, the number of the 18# chromosome and the number of the 21# chromosome of the fetus can be efficiently, quickly and accurately detected without invasion, and the kit and the method are beneficial to clinic application and popularization.
Description
Technical field
The present invention relates to chromosome detection, particularly to the detection of fetus dissociative nucleic acid in female blood, especially with the method for digital pcr technology for detection fetal aneuploidy。
Background technology
Birth defect is current prenatal and postnatal care problem in urgent need to solve, and chromosomal abnormality is the major reason causing birth defect。According to statistics, the incidence rate of neonatal chromosome disorder is about 0.5。In numerous chromosome diseases, deformity and life obstacle that chromosome aneuploid causes are particularly evident。Wherein most commonly seen with 21-tri-body (mongolism), 18-tri-body (Edward's syndrome) and three kinds of autosome aneuploids of 13-tri-body (handkerchief pottery Cotard) again, sickness rate in neonate respectively 1/800,1/6000 and 1/10000。Although chromosome aneuploidy disease can occur at all pregnant woman ages, but being as increasing of pregnant woman age, the incidence rate of chromosome aneuploidy disease also can substantially rise。At present, there is no effective Therapeutic Method to cure fetal chromosomal disease, and the birth of each patau syndrome infant causes huge spirit and financial burden can to family and society, carrying out Prenatal Screening and diagnosing the birth avoiding infant is the most effective measure controlling prenatal diagnosis at present。
The method detecting prenatal diagnosis at present has multiple, it is broadly divided into two big classes, one class is invasive diagnosis, main villous membrane puncture, amniocentesis and through umbilical vein puncture technology, such method determines karyotype by fetal cell, is the goldstandard of diagnosing fetal chromosomal aneuploid disease, but these technology add unnecessary intrusion, fetus and anemia of pregnant woman there is a degree of injury, the miscarriage with about 1%, intrauterine infection, premature labor equivalent risk。Due to above-mentioned risk, so the method for invasive cannot be used for extensive examination, it is only suitable for doing the confirmation that excessive risk is individual；Another kind of is non-invasive screening, the difference according to the mark of examination, and non-invasive screening divides again traditional screening serum and a new generation high-throughout accounting order-checking examination。The protein marker that screening serum is in detection maternal serum is detection object, and due to the complexity that protein expression is modulated, the accuracy of detection is very poor, so screening serum has significantly high false positive rate and loss, the practical significance of detection is little。
(the LoYM such as Lo in 1997, CorbettaN, ChamberlainPF, etal.PresenceoffetalDNAinmaternalplasmaandserum.Lancet, 1997,350 (9076): 485-487) find in pregnant woman blood plasma, there is free foetal DNA, and fetus dissociative DNA content accounts for the 3～6% of Maternal plasma STb gene, and content increases along with the growth in pregnant week, also having a process sharply increased to pregnant late period, the birth that the studies above is noinvasive prenatal diagnosis technology provides premise。In this theoretical basis, the high-flux sequence platform of illumina company and lifetechnologies company has been applied to noinvasive DNA prenatal diagnosis。Although, based on the noinvasive DNA prenatal diagnosis technology of high-flux sequence, there is accuracy height, flux advantages of higher；But high-throughout operating platform requires higher, only third-party large-scale order-checking company can complete this detection at present。High-flux sequence carries out the step of the antenatal detection of noinvasive and includes: plasma dna extracts, library construction, the quantitatively mixed storehouse of quality inspection, checks order on secondary sequenator, and information analysis processes。Think that complete independently high-flux sequence carries out the antenatal detection of noinvasive and needs the equipment investment of great number and the professional of each operating procedure。
Digital pcr technology is by micro-example is made big multiple dilutions and separatory (partitioning), until testing molecule number contained in each sample is not over 1 copy, all samples is carried out under the same conditions pcr amplification a kind of technology that the sample that there occurs amplified reaction is counted one by one again。Digital pcr is a kind of nucleic acid molecules absolute quantitation technology, more more accurate than real-time PCR, it is possible to fetus dissociative DNA to carry out accurate quantification analysis, and is independent of sex of foetus and genetic polymorphism, even can allow the pollution of mother body D NA and chimeric existence。Digital pcr has the sensitivity of brilliance, accuracy and repeatability, and the detection in rare mutation detection, copy number variation, gene relative expression etc. has obvious superiority。(the LoYM such as Lo, LunFM, ChanKC, etal.DigitalPCRforthemoleculardetectionoffetalchromosoma laneuploidy.ProcNatlAcadSciUSA, 2007,104 (32): 13116-13121) utilize digital pcr, by detecting PLAC4mRNASNP, and compare the mode diagnosis of fetal aneuploidy of the relative chromosome dosage in corresponding site on No. 21 chromosomes and No. 1 chromosome。Although said method can diagnose chimera, but it needs fetal aneuploidy DNA content need to reach more than the 25% of foetal DNA total content, greatly limits the application of the method。
On the basis of the above, digital pcr technology and the application in fetal chromosomal detects thereof are progressively deeply。Now commonly used digital pcr instrument is mainly the QuantStudio of ThermoFisherScientific companyTMThe QX200 of RaindropdigitalPCRSystem and the Bio-RAD company of 3D, RainDance company。The common feature of the digital pcr instrument of three producers is to be all a considerable number of dispersed droplets, and what be distinctive in that ThermoFisherScientific company utilizes is chip micropore, and two other company is water in oil droplet。
At present, the Fetal genome carried out based on digital pcr technology or nucleotide detection are existing multiple, such as: disclosed in patent WO2011057094A, carried out Fetal genome analysis by maternal biological sample, it passes through the analysis fetal DNA fragments from maternal sample to identify the allele at some gene locus place, the comprehensive amount analyzing these gene locus places each allelic DNA fragmentation, to determine the relative quantity of the haplotype of these locus, and determined from the heredity of parental gene group which kind of haplotype, thus at the gene mapping of the antenatal full-length genome building fetus or selected gene region, determine that fetus suffers from the risk of heredopathia or other diseases or hereditary character based on above-mentioned collection of illustrative plates。But the enforcement of said method relies primarily on and data analysis and process system, it is necessary to powerful background analysis instrument is supported, it is also desirable to the data analyst of specialty, it is unfavorable for the popularization of clinical practice。US Patent No. 2011151442A1 then discloses the molecular detecting method of fetal aneuploidy, and the method uses digital pcr technology that the single target sequence from fetal tissue's sample is expanded and detected, and is used for detecting chromosome aneuploid or variation。But, said method based on sample be fetal tissue, although overcoming the problem needing cell to cultivate in amniocentesis detection, but the acquisition of fetal tissue will necessarily bring potential infection or other adverse effects, and this patent uses single primer sequence for each target sequence in pcr amplification, will certainly affect accuracy and the sensitivity of result。Domestic patent is mainly multiple numeral round pcr application in chromosome aneuploid examination disclosed in ZL201510274271.0, it is with the pregnant object peripheral blood of fetus dissociative DNA for detection sample, leave away except cell from peripheral blood sample one's duty, and from remove cell described sample enrichment DNA, obtain DNA sample, above-mentioned sample is divided into matched group and experimental group carry out multiple numeral pcr amplification, determines chromosome number according to amplification。This patented technology avoids the acquisition of sample in above-mentioned patent and the drawback of data analysis aspect, but owing in its amplification method, primer logarithm uses less, thus in amplification, the requirement of sample DNA content is higher, it is only capable of the accurately detection trisomy 21 foetal DNA less than 5% and 10%。
Therefore, it is desired to be able to provide a kind of can Noninvasive, high sensitivity, the test kit identifying foetal chromosome aneuploidy and method high accuracy, in order to clinical popularization is quoted。
Summary of the invention:
The purpose of the present invention is contemplated to overcome screening serum technology to exist and detects inaccurate problem and high flux examination technical operation requirement height, the shortcoming that input cost is huge, there is provided a kind of simple to operate, low cost, quickly, the test kit of high accuracy, primer and using method thereof, for digital pcr detection of platform fetal chromosomal Number Variation。
In order to realize the detection Chromosome number purpose purpose of the present invention, one aspect of the present invention devises provides a kind of test kit for detecting fetal chromosomal number ploidy and primer, designs amplimer and taqman probe according to the 13rd, 18 and No. 21 chromosomal gene orders。In blood plasma, the DNA of parent and fetus dissociative is not complete chromosome and large fragment existence, it is possible to designs multiple detection site on item chromosome, so can detect 13,18 and No. 21 chromosomal numbers more accurately。On 13,18 and No. 21 chromosomes, separately design and filter out 10 qualified amplimers in site and probe in the present invention。Not exclusively being uniformly distributed 10 detection site of design on every chromosome, so design has two benefits: first, improves the accuracy of detection, will not due to the random errors affect testing result of a detection site；Second, if chromosome is partial trisomy, it is also possible to make accurate judgement。
Wherein, owing to the fragment length 2/3 of blood plasma is above at about 166bp, in order to more utilize the short-movie section existed in blood plasma, the present invention is when designing primer and probe, limit the amplification scope of detection between 60-100bp, so can guarantee that the template number of detection, improve the accuracy of monitoring。
Wherein, in order to more improve the utilization of template, make the temperature annealing temperature higher than amplimer of withdrawing from a secret society or underworld gang of taqman probe again, add LNA or MGB when designing probe and modify。
Wherein, in order to improve the accuracy of detection, reducing the interference of detection signal, the 3 of amplimer ' with thio-modification between two bases at end end, are so possible to prevent the exo-acting of polymerase, improve the specificity of amplification, reduce unnecessary amplification and produce。
Wherein, in order to improve the accuracy of detection, the selection of detection object of reference is with other chromosomes of same detection sample for the detected ploidy of reference comparing calculation。While the accuracy of detection, for the simplicity detected and economy, 13 and No. 18 chromosomal detection site probe marks are VIC, and No. 21 chromosomal detection site probe marks are FAM。Three chromosomes of one sample of detection have reacted with two digital pcrs, and a pipe is 13 and No. 21 chromosomal detection combinations, and another is 18 and No. 21 chromosomal detection combinations。The probability that 21-tri-body occurs is far above 13-tri-body and 18-tri-body, and little little again of two kind of three simultaneous probability of body, and this combination is equivalent to 21 chromosomes detected twice, and this more can promote 21 chromosome accuracys。
Particularly as follows: the present invention is claimed a kind of test kit for detecting foetal chromosome aneuploidy, including: according to the mankind No. 13, No. 18 and No. 21 chromosomal gene sequence, the multipair specific primer separately designed and a plurality of specificity T aqman probe；And for carrying out the reaction system of digital pcr。
Wherein said multipair specific primer and a plurality of specificity T aqman probe sequence are SEQIDNO:1-90。
Wherein said multipair specific primer and a plurality of specificity T aqman probe sequence are divided into two groups: first group of nucleotide sequence including SEQIDNO:1-60, second group of nucleotide sequence including SEQIDNO:1-30,61-90。
Additionally, the present invention also protects the specific primer for detecting foetal chromosome aneuploidy and Taqman probe, its nucleotides sequence is classified as SEQIDNO:1-90。
Wherein said specific primer and Taqman probe are divided into two groups, first group of nucleotide sequence including SEQIDNO:1-60, second group of nucleotide sequence including SEQIDNO:1-30,61-90。
For realizing the purpose of the present invention, another aspect of the present invention provides one to be used for detecting fetal chromosomal number method, primer and probe that the present invention detects at digital pcr carry out strict experiment screening, filter out and there is roughly the same amplification efficiency, the combination primer of primer non-interference again mixed and probe。Meanwhile, the primer of amplification and the concentration of probe are also optimized by the present invention。
Wherein, in order to filter out roughly the same amplification efficiency detection react, the present invention on item chromosome design 20 detection primers and probe, each detection is individually tested, filtering out amplification efficiency height, amplification efficiency is close simultaneously primer and probe in detecting are reacted。Single detection site performance after filtering out primer and the probe that individually amplification meets requirement, in test two groups detection mix primer and probe。First group of primer and probe combinations are used for detecting No. 21 chromosomal and No. 18 each 10 detections of Chromosome number purpose；Second group of primer and probe combinations are used for detecting No. 21 chromosomes and No. 13 each 10 detections of Chromosome number purpose。
Wherein, in order to promote accuracy and the sensitivity of detection, the primer of detection and the concentration of probe are optimized by the present invention。The concentration that primer when present invention discover that one site of detection and probe need is high, it is necessary to final concentration be 200nM。It is 20-100nM for the present invention detects No. 21 chromosomal and 10 primers detected of No. 18 Chromosome number purposes and probe concentration limits scopes；It is equally used for No. 21 chromosomes of detection and No. 13 each primers of 10 detections of Chromosome number purpose and the concentration limits scope of probe is 20-100nM。
Wherein, further, present invention discover that the performance when concentration of the combination of two groups of primers and probe is 50nM is optimum。
Specifically, the method that the present invention asks a kind of external Noninvasive detection foetal chromosome aneuploidy, comprise the steps:
(1) according to the mankind No. 13, No. 18 and No. 21 chromosomal gene sequence, 10 pairs of specific primers and 10 specificity T aqman probes are separately designed；
(2) gather pregnant 12-23 week maternal blood to extract for plasma dna；
(3) reaction system of digital pcr amplification gene sequence is prepared；
(4) detect chromosome number by digital pcr and calculate 13,18 and No. 21 chromosome numbers of fetus, utilize FAM and the VIC fluorescence of each reaction member of digital pcr detection chip, calculate 13,18 and 21 chromosomal copy numbers respectively。
The sequence of wherein said 30 pairs of specific primers and 30 specificity T aqman probes is: SEQIDNO:1-90。
The extraction step of wherein said maternal plasma DNA is: a, extraction maternal blood 10ml, in EDTA anticoagulant tube, turn upside down and mix 8 times, room temperature preservation；B, separation peripheral blood blood plasma, with 10min centrifugal under 1600g room temperature, be dispensed in 1.5ml centrifuge tube by supernatant (blood plasma) after centrifugal, again removes residual cells with 10min centrifugal under 16000g room temperature, and centrifugal the obtained supernatant of collection is also integrated with standby；C, the DNA extracted in the blood plasma that collection obtains also carry out quantitatively。
The preparation of the reaction system of wherein said digital pcr amplification gene sequence includes the step that primer and probe sequence SEQIDNO:1-90 are divided into first group and second group。
Wherein said first group of nucleotide sequence including SEQIDNO:1-60, described second group of nucleotide sequence including SEQIDNO:1-30,61-90, and first group be used for detecting 21 and No. 18 chromosomes, and second group is used for detecting 21 and No. 13 chromosomes。
For realizing the purpose of the present invention, the detection of the present invention may operate on any digital pcr instrument produced, if the fluorescence signal passage collected is abundant, two groups of detection reactions of the detection of the present invention can synthesize detection in a pipe, the design of the detection of the present invention is 13,18 and No. 21 chromosomes of detection, but and only limit detects these three chromosomes, can detect other chromosomes according to the design philosophy of the present invention。
Beneficial effects of the present invention is embodied in:
1. test kit provided by the invention can realize detecting chromosome number rapidly and accurately。Pregnant woman blood plasma DNA only need to be detected by the test kit of the present invention on digital pcr, does not need the data analysis of complexity, and whole detection process is consuming time short；The test kit of the present invention can detect every chromosomal 10 specific fragments simultaneously, it is to avoid because of the detection error that base mutation in sample quality problem and amplification procedure produces。
2. preparation method provided by the invention is simple, cost is low。Only needing the preparation of single step reaction system can go up machine testing, greatly reduce the human input in detection process, applicable situation of all-level hospitals is promoted the use of, not by the restriction of fund input and peopleware。
3. the present invention directly detects the copy number of DNA in pregnant woman blood plasma, the most accurate and directly detection fetal chromosomal ploidy method when being noinvasive。
Accompanying drawing explanation
Fig. 1. two groups of detection reaction chromosome position schematic diagrams
Fig. 2. the primer of detection position and probe design schematic diagram
Fig. 3. different primers concentration results figure
Fig. 4. without concentration and probe concentration result figure
Fig. 5 .chr18 and chr21 primer mixing fluorescent quantitation result figure
Fig. 6 .chr13 and chr21 primer mixing fluorescent quantitation result figure
Detailed description of the invention
Below in conjunction with specific embodiment, such scheme is described further。Should be understood that these embodiments to be an illustration for the present invention and be not limited to restriction the scope of the present invention。The implementation condition adopted in embodiment can do further adjustment according to the condition of concrete producer, and not marked implementation condition is generally the condition in normal experiment。
The design of embodiment 1 detection primer and probe
One aspect of the present invention devises a kind of test kit for detecting fetal chromosomal number ploidy of offer, designs amplimer and taqman probe on 13,18 and No. 21 chromosomes。In blood plasma, the DNA of parent and fetus dissociative is not complete chromosome and large fragment existence, it is possible to designs multiple detection site on item chromosome, so can detect 13,18 and No. 21 chromosomal numbers more accurately。On 13,18 and No. 21 chromosomes, separately design and filter out 10 qualified amplimers in site and probe in the present invention。
Detection in order to filter out roughly the same amplification efficiency is reacted, the primer of present invention design 20 detection on item chromosome and probe, each detection is individually tested, and filters out amplification efficiency height, and amplification efficiency is close simultaneously primer and probe in detecting are reacted。Single detection site performance after filtering out primer and the probe that individually amplification meets requirement, in test two groups detection mix primer and probe。First group of primer and probe combinations are used for detecting No. 21 chromosomal and No. 18 each 10 detections of Chromosome number purpose；Second group of primer and probe combinations are used for detecting No. 21 chromosomes and No. 13 each 10 detections of Chromosome number purpose。As it is shown in figure 1, the combination of two pipe detections and detection site distribution substantially on chromosome。
Owing to the fragment length major part of blood plasma is at about 166bp, in order to more utilize the short-movie section existed in blood plasma, the present invention, when designing primer and probe, limits the amplification scope of detection between 60-100bp, so can guarantee that the template number of detection, improve the accuracy of monitoring。As in figure 2 it is shown, the more little detected fragment of fragment of detection is more many, the accuracy of detection can be higher, and the detection segment of present invention design is all shorter than 100bp。
According to described above and experiment screening, the present invention filters out the combination of primers being suitable for detection Chr13, Chr18 and Chr21 ploidy, altogether 30 groups of primers and probe, and sequence is respectively as follows:
Chr21-3-F(SEQIDNO:1):TCATCAGGAGAACGCTGTTG
Chr21-3-R(SEQIDNO:2):GGGCAACGTTAGGTCAAGAT
Chr21-3-probe(SEQIDNO:3):FAM-CAAAAGGTGCATTACAGTTGCATGG-BHQ1
Chr21-5-F(SEQIDNO:4):CTTACAATGCTTTGATGAGGCA
Chr21-5-R(SEQIDNO:5):TGTGTGTTGATGGCAGAGTTT
Chr21-5-probe(SEQIDNO:6):FAM-CCCCACGGACTTGGGAATAAGG-BHQ1
Chr21-6-F(SEQIDNO:7):GGAAACTGAGTAAGGCCCAC
Chr21-6-R(SEQIDNO:8):GGGGGTGAGGACATAGCTT
Chr21-6-probe(SEQIDNO:9):FAM-TACCTCCAGTGAACAGCTTCAGGC-BHQ1
Chr21-8-F(SEQIDNO:10):GGCAGGACACGATTACCAAT
Chr21-8-R(SEQIDNO:11):AATGATGGATGGTTGGGCTC
Chr21-8-probe(SEQIDNO:12):FAM-CACCCCGTGTCATTTGGATTAGAC-BHQ1
Chr21-11-F(SEQIDNO:13):CAAGCGCTTCTGCTGAAAGT
Chr21-11-R(SEQIDNO:14):TCAGGCACGAAGAACTGTCC
Chr21-11-probe(SEQIDNO:15):FAM-AGGTCCCATGCTCCACCCGA-BHQ1
Chr21-14-F(SEQIDNO:16):CGGCTACATTTCCCGTGAGT
Chr21-14-R(SEQIDNO:17):GCAGACAGGGAGCAAGAGAG
Chr21-14-probe(SEQIDNO:18):FAM-CCAACGTGGACCAGTCGGCC-BHQ1
Chr21-15-F(SEQIDNO:19):GTGCTGAGAAGGACACCTCC
Chr21-15-R(SEQIDNO:20):TACAGCCGAGCACTGACAAG
Chr21-15-probe(SEQIDNO:21):FAM-AGAAGGGGACAGCGCCACCT-BHQ1
Chr21-16-F(SEQIDNO:22):GCACTTGGTGAAGACAAGGC
Chr21-16-R(SEQIDNO:23):ACATCCTCAAGAGCTGTGGC
Chr21-16-probe(SEQIDNO:24):FAM-ATCACGCGGCCGAGACATGG-BHQ1
Chr21-18-F(SEQIDNO:25):GTGTCAGCAAGCTGGGTTTG
Chr21-18-R(SEQIDNO:26):CCCGAGAGTCACTGGTTCAC
Chr21-18-probe(SEQIDNO:27):FAM-AGTGTCTTCCAAGCGACGGTGT-BHQ1
Chr21-19-F(SEQIDNO:28):ACAAGGTCGGCAACTCCTTT
Chr21-19-R(SEQIDNO:29):GCCGTCTCCATCATTGTCCA
Chr21-19-probe(SEQIDNO:30):FAM-AGCAGGAGGTTGTGGACAAAGTCA-BHQ1
Chr18-4-F(SEQIDNO:31):CAAACAGACTTGGGCCTCTTA
Chr18-4-R(SEQIDNO:32):CCTTTTTGCTCCCTCTCCAC
Chr18-4-probe(SEQIDNO:33):VIC-CCTGGAGAGAGACTGGTGGCTGG-TAMRA
Chr18-6-F(SEQIDNO:34):AGTCCCAATGCCTCACTTTG
Chr18-6-R(SEQIDNO:35):GCCAAGGTAGAGTTGACCAG
Chr18-6-probe(SEQIDNO:36):VIC-CGGGTGCGTGGTGGGCTTCA-TAMRA
Chr18-8-F(SEQIDNO:37):ATCTTCATCAAGTCCGCCAC
Chr18-8-R(SEQIDNO:38):TCTCGTAGTCGAAAGCCTTG
Chr18-8-probe(SEQIDNO:39):VIC-CTCCAGGACGCTGTTGGTACAC-TAMRA
Chr18-12-F(SEQIDNO:40):AGTGGAGTGAGCAGCAAGAC
Chr18-12-R(SEQIDNO:41):CAGGAGCTCATACGGCAGAG
Chr18-12-probe(SEQIDNO:42):VIC-ACTGGGGGCACCTGCATTCC-TAMRA
Chr18-13-F(SEQIDNO:43):TGGGATGAAGGTCCAACAGC
Chr18-13-R(SEQIDNO:44):AAGCTGGGAATACACCGCAA
Chr18-13-probe(SEQIDNO:45):VIC-CTGGAGCCGGCAGCAGTGAG-TAMRA
Chr18-14-F(SEQIDNO:46):GCACACTTCTCTGCACCTCT
Chr18-14-R(SEQIDNO:47):TCGTAGCACCCTCCATCAGA
Chr18-14-probe(SEQIDNO:48):VIC-TGCACAGCAATGCCAGTGAGTCC-TAMRA
Chr18-16-F(SEQIDNO:49):TTTCGGTGACTTCCGCATCA
Chr18-16-R(SEQIDNO:50):CGGTCTCCTAAAAGCAGGCA
Chr18-16-probe(SEQIDNO:51):VIC-CCAGAGCATCAGGCCGCCAC-TAMRA
Chr18-17-F(SEQIDNO:52):TCCAGACATTCTCGCTTCCC
Chr18-17-R(SEQIDNO:53):TGTGCCCAAGTTTCACTGGA
Chr18-17-probe(SEQIDNO:54):VIC-ACCCACGAAACTGCCCTGGC-TAMRA
Chr18-18-F(SEQIDNO:55):CCCTCCACCCTTGGACTTTC
Chr18-18-R(SEQIDNO:56):TCTGTCTCTGCAGCTGTGTG
Chr18-18-probe(SEQIDNO:57):VIC-CGGCACCCTTGCGCTTTTGC-TAMRA
Chr18-19-F(SEQIDNO:58):ACGTCGCTGATGGAGAAAGG
Chr18-19-R(SEQIDNO:59):GTGAGACACAAGAGACGCGA
Chr18-19-probe(SEQIDNO:60):VIC-CCCGCAGAAGGAACGGCCTG-TAMRA
Chr13-1-F(SEQIDNO:61):AAAACCCAGAAGGTCCGCAT
Chr13-1-R(SEQIDNO:62):GCTTCGAAGATGACCCGGAA
Chr13-1-probe(SEQIDNO:63):VIC-AGGCTCCCTGTGGTGGACCT-TAMRA
Chr13-2-F(SEQIDNO:64):CTCCTCCCCAGCTCTTCTCT
Chr13-2-R(SEQIDNO:65):GCCTTTCCCTTGAGTCCCTC
Chr13-2-probe(SEQIDNO:66):VIC-CTGCAGGCTGCACCTCTGGC-TAMRA
Chr13-7-F(SEQIDNO:67):GTTTAACGACCGCACAGCTC
Chr13-7-R(SEQIDNO:68):TTTCATGGATGCCTTGGGCT
Chr13-7-probe(SEQIDNO:69):VIC-TGGCCTCTATCGTTATGCTGCAGA-TAMRA
Chr13-8-F(SEQIDNO:70):TCAAATCGAAGAGTTGTGAACTG
Chr13-8-R(SEQIDNO:71):GAGAGATAATGGCTTGCGTGC
Chr13-8-probe(SEQIDNO:72):VIC-CCCATTGAAAAGACCGAGCCTTGT-TAMRA
Chr13-10-F(SEQIDNO:73):CAGGGACACACGCATCACTA
Chr13-10-R(SEQIDNO:74):CTGGGGACTCAGCAAGAGTG
Chr13-10-probe(SEQIDNO:75):VIC-CCCTGGCTGCAGTGGGAGGA-TAMRA
Chr13-11-F(SEQIDNO:76):CGAGATACGGGCACATACGG
Chr13-11-R(SEQIDNO:77):ACAGCGGCAAAAGCTTCTCT
Chr13-11-probe(SEQIDNO:78):VIC-TGCTGCCGGAGCGTTACATCA-TAMRA
Chr13-12-F(SEQIDNO:79):CAGGCAGGAGGACTATGCAG
Chr13-12-R(SEQIDNO:80):GGCACCACATCACCACAAAC
Chr13-12-probe(SEQIDNO:81):VIC-AGGCATGCAAGGTGCTGGGC-TAMRA
Chr13-17-F(SEQIDNO:82):AGAATGTGGATGGCCGTGTT
Chr13-17-R(SEQIDNO:83):TGAGCCTGCGGAGAGAGTAG
Chr13-17-probe(SEQIDNO:84):VIC-ACAGGCGACCTTTCAGCAGAGA-TAMRA
Chr13-19-F(SEQIDNO:85):AACTGCATGTGGGCTATGGG
Chr13-19-R(SEQIDNO:86):GCGCTAGATGACACCCTCTC
Chr13-19-probe(SEQIDNO:87):VIC-CCCTGGTCATGTGCCCCTTCGCAGC-TAMRA
Chr13-20-F(SEQIDNO:88):TGGAGAGATGCCAGTGACCT
Chr13-20-R(SEQIDNO:89):GCTACCTGCCCCTTTGTCAT
Chr13-20-probe(SEQIDNO:90):VIC-ACTGCCTGGTGCAGTGTCCAC-TAMRA
Embodiment 2 maternal blood extracts for plasma dna
A, extract pregnant 12-23 week maternal blood 10ml in EDTA anticoagulant tube, mixing 8 times of turning upside down, room temperature preservation；B, separation peripheral blood blood plasma, with 10min centrifugal under 1600g room temperature, after centrifugal, supernatant (blood plasma) is dispensed in 1.5ml centrifuge tube, again remove residual cells with 10min centrifugal under 16000g room temperature, collecting centrifugal obtained supernatant and be dispensed in 1.5ml centrifuge tube, often the volume of pipe subpackage blood plasma is 450 μ L；C, the DNA extracted in the blood plasma that collection obtains also carry out quantitatively。Wherein extract plasma dna and adopt adsorption column method, concretely comprise the following steps and the serum sample of 450ul Refrigerator store is melted in room temperature。16000g, room temperature is centrifuged 5min, takes in the 400-430ul centrifuge tube transferring to new 1.5ml。Add the ProteinaseK solution of 40 μ l, of short duration centrifugal after mixing；Add buffer GB and the 1ulCarrierRNA (can first be made into mixed liquor adding) of 400 μ l, gently reverse mixing, 56 DEG C of temperature bath 10min, and jog sample frequently；Adding dehydrated alcohol (if room temperature is more than 25 DEG C, ethanol is please put pre-cooling on ice) the reverse mixing sample gently of 400 μ l, room temperature places 5min；Being shifted in 700ul to an adsorption column CR2 by previous step gained solution, 12,000rpm (～13,400 × g) are centrifuged 30sec, abandon waste liquid (can repeatedly shift)；In adsorption column CR2, add the centrifugal 30sec of 500 μ l buffer GD, 12,000rpm (～13,400 × g), abandon waste liquid；Adding 600 μ l rinsing liquid PW (having added dehydrated alcohol) in adsorption column CR2,12,000rpm (～13,400 × g) are centrifuged 30sec, abandon waste liquid；Repeat rising operation once, abandon waste liquid；12,000rpm (～13,400 × g) are centrifuged 2min, are transferred to by adsorption column CR2 in new collecting pipe, and room temperature places 2-5min；To the unsettled dropping 50 in adsorbed film centre position μ l eluent, room temperature places the centrifugal 2min in 2-5min, 12,000rpm (～13,400 × g), collects eluent。Qubit quantitative extracting gained sample DNA concentration。
The preparation of embodiment 3 reaction system test template
Utilizing sky root poba gene group DNA extraction kit (DP318-02) to extract complete genome DNA as reaction system test template, use nanodrop that DNA is measured, the OD scope 1.7～2.0 of DNA, dilution final concentration is to 25ng/ μ l, standby。
The determination of the best primer of embodiment 4 and probe
According to human genome the 13rd, No. 18 and No. 21 karyological character sequence, separately design 20 primers and probe combinations according to the thought of the present invention。Utilize No. 13, No. 18 and No. 21 chromosomal special primers pair carry out PCR reaction (the final concentration respectively 1 × PCRBuffer of each component, 0.25mMdNTP, 1U/ reaction dna polymerase, 500nM primer, 250nM probe, 25ng template DNA and surplus are water in 20 μ L systems) with the negative control of probe combinations and preparation for template, PCR reaction condition is: 95 DEG C of denaturations 10 minutes, a circulation；40 amplification cycles, 95 DEG C of degeneration 15 seconds, 60 DEG C of annealing also extend 60 seconds, and PCR primer carries out electrophoresis detection。Primer pair and the probe combinations amplification efficiency of result SEQIDNo:1-SEQIDNo:90 composition are high, and band is special。
The determination of the best primer concentration of embodiment 5
Arrange, with the nucleotides sequence of SEQIDNo:1-SEQIDNo:60, the negative control prepared from probe and embodiment 1 for primer and carry out, for template, the PCR that primer concentration is different, all the other component conditions identical (the final concentration of 1 × PCRBuffer of each composition, 0.25mMdNTP, 1U/ reaction dna polymerase, 100nM probe, 25ng template DNA in 20 μ L systems, surplus is water), primer concentration respectively 20nM, 50nM, 100nM, 200nM and 300nM。PCR reaction condition is identical with embodiment 3, and amplified production carries out electroresis appraisal, and result is as shown in Figure 1。From the figure 3, it may be seen that primer concentration all has purpose band to expand in 20～100nM scope, when primer concentration is 50nM, expanding effect is best。
The determination of the best concentration and probe concentration of embodiment 6
Arrange the negative control for primer and probe and embodiment 1 preparation with the nucleotides sequence of SEQIDNo:1-SEQIDNo:60 and carry out the PCR of different probe concentration for template, all the other component conditions identical (the final concentration of 1 × PCRBuffer of each composition, 0.25mMdNTP, 1U/ reaction dna polymerase, 50nM probe, 25ng template DNA in 20 μ L systems, surplus is water), primer concentration is 50nM, 100nM and 150nM respectively。PCR reaction condition is identical with embodiment 1, and amplified production carries out electroresis appraisal, and result is as shown in Figure 4。As shown in Figure 4, concentration and probe concentration all has purpose band in 50nM～150nM scope, optimum when concentration and probe concentration is 50nM。
The determination of embodiment 7 primer and probe combinations amplification optimal reaction system
Arrange as primer and probe with the nucleotides sequence of SEQIDNo:1-SEQIDNo:60, quantitative fluorescent PCR checking (in 20 μ L systems the final concentration of 1 × PCRBuffer of each composition, 0.25mMdNTP, 1U/ reaction dna polymerase, 50nM primer, 50nM probe, 25ng template DNA, surplus is water) is carried out for template with the negative control of embodiment 1 preparation。Quantitative fluorescent PCR reaction condition: 60 DEG C of annealing also extend 60 seconds, a circulation；95 DEG C of denaturations 10 minutes, a circulation；40 amplification cycles, 95 DEG C of degeneration 15 seconds, 60 DEG C of annealing also extend 60 seconds, and result is as shown in Figure 5。As shown in Figure 5, primer and probe combinations amplification efficiency are best。
The determination of embodiment 8 primer and probe combinations amplification optimal reaction system
Arrange as primer and probe with the nucleotides sequence of SEQIDNo:1-SEQIDNo:30 and SEQIDNo:61-SEQIDNo:90, quantitative fluorescent PCR checking (in 20 μ L systems the final concentration of 1 × PCRBuffer of each composition, 0.25mMdNTP, 1U/ reaction dna polymerase, 50nM primer, 50nM probe, 25ng template DNA, surplus is water) is carried out for template with the negative control of embodiment 1 preparation。Quantitative fluorescent PCR reaction condition: 60 DEG C of annealing also extend 60 seconds, a circulation；95 DEG C of denaturations 10 minutes, a circulation；40 amplification cycles, 95 DEG C of degeneration 15 seconds, 60 DEG C of annealing also extend 60 seconds, and result is as shown in Figure 6。It will be appreciated from fig. 6 that primer and probe combinations amplification efficiency are best。
Embodiment 9 adopts the method that the application test kit carries out Chromosome Analysis
Detecting chromosome number by digital pcr, detection method provided by the invention includes
(1) according to the mankind No. 13, No. 18 and No. 21 chromosomal gene orders, 10 pairs of specific primers and 10 specificity T aqman probes are separately designed。
(2) pregnant woman blood plasma DNA extraction
(3) reaction system of digital pcr amplification gene sequence is prepared。
(4) detect chromosome number by digital pcr and calculate 13,18 and No. 21 chromosome numbers of fetus。Utilize FAM and the VIC fluorescence of each reaction member of digital pcr detection chip, calculate three chromosomal copy numbers respectively。
The reaction system of digital pcr is:
The reaction condition of described digital pcr is 96 DEG C of denaturations 10 minutes；39 circulations 60 DEG C extend 2 minutes, 98 DEG C of degeneration 30 seconds；60 DEG C extend 2 minutes。React after terminating at digital pcr detection of platform FAM and VIC fluorescence signal, and pass through QuantStudioTM3DAnalysisSuiteTMSoftware calculates every chromosomal copy number。13rd, 18, No. 21 chromosomal copy numbers are designated as A1, B1, C1 difference ratio calculated C1/A1 and C1/B1 respectively, are designated as R1 and R2 respectively。
When R1 and R2 is equal to or approximates 1, then show that fetal chromosomal is normal；
When R1 and R2 is all higher than or approximates 1.025, then show that fetus is trisomy 21；
When R1 be at or about 1, R2 be less than or approximately equal to 0.975 time, then show that fetus is 18 3 bodies；
When R2 be at or about 1, R1 be less than or approximately equal to 0.975 time, then show that fetus is 13 3 bodies。
All lists of references quoted in this manual and patent application are herein incorporated by reference, and are specifically and individually incorporated herein by reference the same just as each publication or patent application。Although for the purpose being expressly understood that, already by the mode illustrated with embodiment, describe foregoing invention in detail, but under the teachings of the present invention, for those of ordinary skill in the art, change it is readily apparent that the present invention can be carried out some and revise the spirit or scope without departing from claims。
Claims (10)
1. for detecting a test kit for foetal chromosome aneuploidy, including: according to the mankind No. 13, No. 18 and No. 21 chromosomal gene sequence, the multipair specific primer of design and a plurality of specificity T aqman probe；And for carrying out the reaction system of digital pcr。
2. test kit as claimed in claim 1, the nucleotides sequence of wherein said multipair specific primer and a plurality of specificity T aqman probe is classified as SEQIDNO:1-90。
3. the test kit as described in any one of claim 1-2, wherein said multipair specific primer and a plurality of specificity T aqman probe sequence are divided into two groups: first group of nucleotide sequence including SEQIDNO:1-60, second group of nucleotide sequence including SEQIDNO:1-30,61-90。
4., for detecting specific primer and the Taqman probe of foetal chromosome aneuploidy, its nucleotides sequence is classified as SEQIDNO:1-90。
5. specific primer and the Taqman probe for detecting foetal chromosome aneuploidy as claimed in claim 4, wherein said specific primer and Taqman probe are divided into two groups, first group of nucleotide sequence including SEQIDNO:1-60, second group of nucleotide sequence including SEQIDNO:1-30,61-90。
6. a method for external Noninvasive detection foetal chromosome aneuploidy, comprises the steps:
(1) according to the mankind No. 13, No. 18 and No. 21 chromosomal gene sequence, multipair specific primer and a plurality of specificity T aqman probe are designed；
(2) gather pregnant 12-23 week maternal blood to extract for plasma dna；
(3) reaction system of digital pcr amplification gene sequence is prepared；
(4) detect chromosome number by digital pcr and calculate 13,18 and No. 21 chromosome numbers of fetus, utilize FAM and the VIC fluorescence of each reaction member of digital pcr detection chip, calculate 13,18 and 21 chromosomal copy numbers respectively。
7. method as claimed in claim 6, the sequence of wherein said multipair specific primer and a plurality of specificity T aqman probe is: SEQIDNO:1-90。
8. the method as described in any one of claim 6-7, the extraction step of wherein said maternal plasma DNA is: a, extraction maternal blood 10ml, in EDTA anticoagulant tube, turn upside down and mix 8 times, room temperature preservation；B, separation peripheral blood blood plasma, with 10min centrifugal under 1600g room temperature, after centrifugal, supernatant (blood plasma) is dispensed in 1.5ml centrifuge tube, again remove residual cells with 10min centrifugal under 16000g room temperature, collecting centrifugal obtained supernatant and be dispensed in 1.5ml centrifuge tube, often the volume of pipe subpackage blood plasma is 450 μ L；C, the DNA extracted in the blood plasma that collection obtains also carry out quantitatively。
9. the method as described in any one of claim 6-8, the preparation of the reaction system of wherein said digital pcr amplification gene sequence includes the step that primer and probe sequence SEQIDNO:1-90 are divided into first group and second group。
10. the method as described in any one of claim 6-9, wherein said first group of nucleotide sequence including SEQIDNO:1-60, described second group of nucleotide sequence including SEQIDNO:1-30,61-90, and first group is used for detecting 21 and No. 18 chromosomes, second group is used for detecting 21 and No. 13 chromosomes。
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