CN108531572A - It is a kind of it is antenatal detection progeny genotypes method and application - Google Patents

It is a kind of it is antenatal detection progeny genotypes method and application Download PDF

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CN108531572A
CN108531572A CN201810189906.0A CN201810189906A CN108531572A CN 108531572 A CN108531572 A CN 108531572A CN 201810189906 A CN201810189906 A CN 201810189906A CN 108531572 A CN108531572 A CN 108531572A
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gene loci
dna
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李国栋
冯冬
申童
王玲
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Beijing Aipuyi Medical Testing Center Co Ltd
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Abstract

The present invention provides a kind of method of antenatal detection progeny genotypes and applications, the described method comprises the following steps：(1) it chooses and is no less than 30 gene locis, calculate the minimum gene frequency of each gene loci；(2) set of the minimum gene frequency described to step (1) clusters, and is divided into four classes, determines the cluster result of each gene loci；(3) according to the cluster result of step (2) gene loci, obtain parental generation and filial generation the gene loci genotype combination；(4) according to parental generation the gene loci genotype, obtain filial generation the gene loci genotype.Mathematics Clustering is applied to field of biology by the method for the present invention, by determining the cluster result of minimum gene frequency, realizes the detection to gestational period fetus genotype.

Description

It is a kind of it is antenatal detection progeny genotypes method and application
Technical field
The invention belongs to biotechnology, it is related to method and the application of a kind of antenatal detection progeny genotypes.
Background technology
Deafness is that auditory system and auditory nerve in sense of hearing conduction path and nervous centralis at different levels occur caused by lesion Auditory function obstacle can cause different degrees of dysacousis.Congenital deafness is one of most common inborn defect, and most Common mankind sensory system's disease, incidence 0.1-0.3%.Wherein, 60% deafness is caused by inherent cause, in addition 40% is related with environmental factor.Hereditary hearing impairment is divided into comprehensive deaf and non-comprehensive deafness, is unique symptom with deafness Non- comprehensive deafness accounts for the 70% of all hereditary hearing impairments, and heterogeneous with high genetic, and comprehensive deafness accounts for 30%.
Currently, be reported with the non-comprehensive relevant gene of deafness include 28 autosomal recessive genes, 22 Autosomal dominant gene and 1 X linked gene, wherein autosomal recessive inheritance accounts for 80%, and dominant inheritance accounts for 15%, X companies Lock heredity accounts for 1-3%, and mitochondrial inheritance accounts for 1% hereinafter, GJB2, SLC26A4, GJB3 and mitochondrial DNA (mtDNA) 12SrRNA is the main non-comprehensive deaf gene in China.
Although newborn's deaf gene detection technique is ripe and introduces to the market, detection technique is suitable for birth Baby is a kind of remedial measure to result, still society and new life Long Er families can be given to bring economy and mental burden.It relies on Amniocentesis technology may be implemented to carry out accurate genetic test in foetal period, but amniocentesis may cause infection, miscarriage or Transient respiratory distress of the newborn, there are certain risks.
CN 107190064A disclose the SNaPshot kits of 22 site deaf gene polymorphisms of detection, contribute to It finds to carry the infant that deaf gene is mutated early, however the kit is only applicable to the baby of birth, is one kind to result Remedial measure still can give society and new life Long Er families to bring economy and mental burden, can not achieve antenatal prediction.
Therefore it provides a kind of safe, the high noninvasive antenatal detection progeny genotypes of accuracy method, has important Meaning.
Invention content
In view of the deficiencies of the prior art, the present invention provide it is a kind of it is antenatal detection progeny genotypes method and application, it is described Method is a kind of high throughput, high coverage rate, high-accuracy, the antenatal detection method of noninvasive genotype that at low cost, the period is short, can be used In the antenatal detection of deaf gene genotype, it is advantageously implemented the prevention in advance of hereditary hearing impairment.
For this purpose, the present invention provides following technical scheme：
In a first aspect, the present invention provides a kind of method of antenatal detection progeny genotypes, the described method comprises the following steps：
(1) it chooses and is no less than 30 gene locis, calculate the minimum gene frequency of each gene loci；
(2) set of the minimum gene frequency described to step (1) clusters, and is divided into four classes, determines each gene The cluster result in site；
(3) according to the cluster result of step (2) gene loci, obtain parental generation and filial generation the gene loci base Because type combines；
(4) according to parental generation the gene loci genotype, obtain filial generation the gene loci genotype.
In the present invention, it is assumed that filial generation Circulating DNA (cfDNA) accounting in total cfDNA is α, the gene of parental generation and filial generation Type combines as shown in table 1 with the relationship of minimum gene frequency：
Table 1
It follows that parental generation and the genotype combination of filial generation share 7 kinds, including four kinds of minimum gene frequencies, therefore, The genotype combination of parental generation and filial generation can be determined by minimum gene frequency, then by the genotype of parental generation, determines filial generation Genotype.
In the present invention, since clustering method is all the common methods in digital operation, but it is not used in biology In, and the clustering method in mathematics is applied to field of biology by inventor, finds to be capable of auxiliary judgment filial generation by cluster Genotype judges genotype combination and the filial generation of parental generation and filial generation by determining the cluster result of minimum gene frequency Genotype.
Preferably, step (1) described gene loci includes deaf gene site.
Preferably, the deaf gene site include rs121908362, rs750188782, rs111033220, rs111033204、rs80338939、rs111033313、rs201562855、rs111033305、rs80338943、 rs200455203、rs111033318、rs192366176、rs74315319、rs267606619、rs267606617、 rs146044344、rs17878444、rs28369942、rs2307959、rs2067235、rs2308292、rs1610937、 rs1305056、rs2307652、rs1057516953、rs111033380、rs121908363、rs1611048、rs1611001、 rs2308072、rs3081400、rs8190570、rs201771066、rs150042219、rs773528125、rs17238892、 rs2308163、rs2307433、rs1610905、rs2307581、rs1305047、rs16438、rs8178524、rs16388、 In rs6481 or rs16363 any one or at least two combination.
Preferably, step (2) cluster is using arbitrary in gauss hybrid models, hierarchical clustering or K-means clusters It is a kind of or at least two combination, preferably use gauss hybrid models.
In the present invention, minimum gene frequency is clustered using mathematics clustering method, is divided into four classes, so that it is determined that The genotype combination of each gene loci parental generation and filial generation.
Preferably, step (3) described genotype combination include parental generation and filial generation be homozygote, parental generation be homozygote filial generation It is that heterozygote filial generation is homozygote or parental generation and filial generation is any one in heterozygote for heterozygote, parental generation.
In the present invention, the genotype combination of parental generation and filial generation shares 7 kinds, including parental generation and filial generation are homozygote (AAAA/ Aaaa), parental generation be homozygote filial generation be heterozygote (AAAa/aaAa), parental generation is that heterozygote filial generation is homozygote (AaAA/Aaaa) It is heterozygote (AaAa) with parental generation and filial generation.
Preferably, further include before step (1)：
(1 ') extracts dissociative DNA from pregnant woman's body fluid；
(2 ') it uses the primer sets of gene loci to carry out PCR amplification to the dissociative DNA of extraction, builds DNA library；
(3 ') carry out high-flux sequence to the DNA library and obtain gene loci after sequencing result is compared and analyzes Sequencing result.
Preferably, step (the 1 ') body fluid includes any one in peripheral blood, urine or saliva or at least two Combination, preferably peripheral blood.
Preferably, the quality of step (the 1 ') dissociative DNA be 10-50ng, such as can be 10ng, 11ng, 12ng, 13ng、14ng、15ng、16ng、17ng、18ng、19ng、20ng、21ng、22ng、23ng、24ng、25ng、26ng、27ng、 28ng、29ng、30ng、31ng、32ng、33ng、34ng、35ng、36ng、37ng、38ng、39ng、40ng、41ng、42ng、 43ng, 44ng, 45ng, 46ng, 47ng, 48ng, 49ng or 50ng, preferably 15-20ng.
Preferably, step (the 2 ') primer sets are as shown in SEQ ID NO.1-46.In the present invention, the primer of gene loci Sequence is as shown in table 2：
Table 2
Preferably, the average sequencing depth of step (the 3 ') high-flux sequence be 1000-3000 ×, preferably 2000 ×。
As optimal technical scheme, the present invention provides a kind of method of antenatal detection progeny genotypes, the method includes Following steps：
(1) 10-50g dissociative DNAs are extracted from pregnant woman's body fluid, are carried out using the primer sets as shown in SEQ ID NO.1-46 PCR amplification, structure include the DNA library in 15 deaf gene sites and 31 auxiliary gene sites, and high-flux sequence is average to survey Sequence depth be 1000-3000 ×, sequencing result is compared and is analyzed, sequencing result is obtained；
(2) the minimum gene frequency of each gene loci is calculated；
(3) set of the minimum gene frequency described to step (2) is clustered using gauss hybrid models, is divided into four Class determines the cluster result of each gene loci；
(4) according to the cluster result of step (3) gene loci, obtain parental generation and filial generation the gene loci base Because type combines；
(5) according to parental generation the gene loci genotype, obtain filial generation the gene loci genotype.
Second aspect, a kind of method as described in relation to the first aspect of present invention offer is in non-invasive antenatal detection hereditary hearing impairment base Because of the application of genotype.
In the present invention, only detecting genotype can not directly diagnose whether have hereditary hearing impairment, it is also necessary to and other Means auxiliary diagnosis could finally determine whether the deafness that is hereditary.
Compared with prior art, the present invention has the advantages that：
(1) mathematics Clustering is applied to field of biology by method of the invention, by determining minimum allele frequency The cluster result of rate realizes the detection of the genotype to 15 deaf gene sites of gestational period fetus and 31 auxiliary sites；
(2) testing result after fetal birth is compared, the antenatal Detection accuracy in 15 deaf gene sites is up to 100%, assist the accuracy rate in site to be up to 93.5%, comprehensive accuracy rate is up to 95.7%；
(3) method of the invention is noninvasive, safe, accuracy rate is high, at low cost, the period is short, is carried to hereditary hearing impairment Preceding prevention is of great significance.
Specific implementation mode
The technological means and its effect taken for the present invention is further explained, with reference to embodiments to the present invention make into Illustrate to one step.It is understood that the specific embodiments described herein are used only for explaining the present invention, rather than to this hair Bright restriction.
In the examples where no specific technique or condition is specified, according to technology or condition described in document in the art, Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, be can be by regular channel commercially available from The conventional products of acquisition.
The determination of 1 gene loci of embodiment
15 hot spot Chinese population deaf gene sites of selection and 31 auxiliary gene sites are detected.
Hereditary hearing impairment gene loci：rs121908362、rs750188782、rs111033220、rs111033204、 rs80338939、rs111033313、rs201562855、rs111033305、rs80338943、rs200455203、 rs111033318、rs192366176、rs74315319、rs267606619、rs267606617；
Auxiliary gene site：rs146044344、rs17878444、rs28369942、rs2307959、rs2067235、 rs2308292、rs1610937、rs1305056、rs2307652、rs1057516953、rs111033380、rs121908363、 rs1611048、rs1611001、rs2308072、rs3081400、rs8190570、rs201771066、rs150042219、 rs773528125、rs17238892、rs2308163、rs2307433、rs1610905、rs2307581、rs1305047、 rs16438、rs8178524、rs16388、rs6481、rs16363.
The extraction of 2 maternal blood cfDNA of embodiment
In the case of examinee's informed consent, the peripheral blood 10mL for the pregnant woman that acquisition pregnant week is 12-24 weeks detaches blood Slurry, using ABI companies MagMAXTMCell Free DNA Isolation Kit kits extract dissociative DNA (cfDNA), freeze Be stored in -20 DEG C it is spare.
3 cfDNA specificity databases of embodiment are built
(1) it builds in library specific primers sequence such as table 2 shown in SEQ ID NO.1-46, is closed by Hangzhou connection river biology At；
(2) one-step method PCR is used to build DNA library, reaction system is as shown in table 3, and response procedures are as shown in table 4；
Table 3
Table 4
(3) DNA library of structure is purified using purifying magnetic bead.
4 Illumina of embodiment sequencings and bioinformatic analysis
(1) library is quantified, high-flux sequence, sequencing reading length is carried out using Illumina NextSeq 500 150bp, average sequencing depth 2000 ×；
(2) after obtaining sequencing result, Quality Control, removal joint sequence, primer sequence and low-quality are carried out using FastQC softwares Sequence is measured, result is compared with reference gene group using BWA, the sequencing result of 46 gene locis is obtained, using GATK It is analyzed, obtains the minimum gene frequency (MAF) of 46 gene locis, pass through 46 gene positions of mixed Gauss model pair The minimum gene frequency of point is clustered, and L1 (MAF=0~0.1760), L2 (MAF=0.0563~0.0712) are divided into, L3 (MAF=0.4365~0.4508) and L4 (MAF=0.4608~0.500).
The judgement of 5 fetus deaf gene type of embodiment
According to the cluster result of the minimum gene frequency of 46 gene locis, the genotype group of mother/fetus is predicted It closes；After fetal birth, acquisition Heel blood extracts DNA, carries out high-flux sequence；Quality Control is carried out to sequencing data and is analyzed, as a result As shown in table 5.
Table 5
The results show that the rate of accuracy reached 100% in 15 deaf gene sites, the rate of accuracy reached in 31 auxiliary sites 93.5%, the rate of accuracy reached 95.7% of whole 46 gene locis.
In conclusion mathematics Clustering is applied to field of biology by the present invention, by determining minimum allele frequency The cluster result of rate realizes the detection of the genotype to 15 deaf gene sites of gestational period fetus and 31 auxiliary sites, The antenatal Detection accuracy in 15 deaf gene sites is up to 100%, assists the accuracy rate in site to be up to 93.5%, synthesis is accurate Rate is up to 95.7%；The method of the present invention is noninvasive, safe, accuracy rate is high, at low cost, the period is short, to hereditary hearing impairment Prevent to be of great significance in advance.
Applicant states that the present invention illustrates the method detailed of the present invention, but the present invention not office by above-described embodiment It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implement.Technical field Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention Addition, the selection etc. of concrete mode, all fall within protection scope of the present invention and the open scope.
Sequence table
<110>The general beneficial medical test centered finite company of Beijing love
<120>It is a kind of it is antenatal detection progeny genotypes method and application
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<213>Artificial synthesized ()
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<212> DNA
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<212> DNA
<213>Artificial synthesized ()
<400> 30
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<210> 31
<211> 65
<212> DNA
<213>Artificial synthesized ()
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<212> DNA
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<400> 32
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<211> 65
<212> DNA
<213>Artificial synthesized ()
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<212> DNA
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<211> 65
<212> DNA
<213>Artificial synthesized ()
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<210> 36
<211> 65
<212> DNA
<213>Artificial synthesized ()
<400> 36
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<211> 65
<212> DNA
<213>Artificial synthesized ()
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gccct 65
<210> 38
<211> 65
<212> DNA
<213>Artificial synthesized ()
<400> 38
cttgtgtgct ggtccattct cttgtaacca tgtcaggtga aggaacagcc ccgaggaaag 60
gggcg 65
<210> 39
<211> 65
<212> DNA
<213>Artificial synthesized ()
<400> 39
agggtcaggg ttggctctcg ttggcatggc cctggcccat ccagacctgt atctttgcca 60
ttctg 65
<210> 40
<211> 65
<212> DNA
<213>Artificial synthesized ()
<400> 40
gttcacgaag ccacaagtat ttactatctg gccctttata gaaaaagctc actgaccctg 60
gtcta 65
<210> 41
<211> 65
<212> DNA
<213>Artificial synthesized ()
<400> 41
ggctggattg aagtgcattt gaaagcacaa cgggttgaat cctgttttgt tgtccccatc 60
cctga 65
<210> 42
<211> 65
<212> DNA
<213>Artificial synthesized ()
<400> 42
aaggtggatt tagcagtaaa ctaagagtag agtgcttagt tgaacagggc cctgaagcgc 60
gtaca 65
<210> 43
<211> 65
<212> DNA
<213>Artificial synthesized ()
<400> 43
aaggtggatt tagcagtaaa ctaagagtag agtgcttagt tgaacagggc cctgaagcgc 60
gtaca 65
<210> 44
<211> 64
<212> DNA
<213>Artificial synthesized ()
<400> 44
gcgcctccgc cgtctgcatc gtactcacca tctgtgagct ctgctacctc atctgccaca 60
gggt 64
<210> 45
<211> 64
<212> DNA
<213>Artificial synthesized ()
<400> 45
aggtggccac taggagaggg acaggtggcc actaggagag gggcaggggt actagacgag 60
aacc 64
<210> 46
<211> 64
<212> DNA
<213>Artificial synthesized ()
<400> 46
tgggatttaa aattgttatt agatttatga atgatatgtt ttcttttggg caagtaagtc 60
aggt 64

Claims (10)

1. a kind of method of antenatal detection progeny genotypes, which is characterized in that the described method comprises the following steps：
(1) it chooses and is no less than 30 gene locis, calculate the minimum gene frequency of each gene loci；
(2) set of the minimum gene frequency described to step (1) clusters, and is divided into four classes, determines each gene loci Cluster result；
(3) according to the cluster result of step (2) gene loci, obtain parental generation and filial generation the gene loci genotype Combination；
(4) according to parental generation the gene loci genotype, obtain filial generation the gene loci genotype.
2. according to the method described in claim 1, it is characterized in that, step (1) described gene loci includes deaf gene site；
Preferably, the deaf gene site include rs121908362, rs750188782, rs111033220, rs111033204、rs80338939、rs111033313、rs201562855、rs111033305、rs80338943、 rs200455203、rs111033318、rs192366176、rs74315319、rs267606619、rs267606617、 rs146044344、rs17878444、rs28369942、rs2307959、rs2067235、rs2308292、rs1610937、 rs1305056、rs2307652、rs1057516953、rs111033380、rs121908363、rs1611048、rs1611001、 rs2308072、rs3081400、rs8190570、rs201771066、rs150042219、rs773528125、rs17238892、 rs2308163、rs2307433、rs1610905、rs2307581、rs1305047、rs16438、rs8178524、rs16388、 In rs6481 or rs16363 any one or at least two combination.
3. method according to claim 1 or 2, which is characterized in that step (2) it is described cluster using gauss hybrid models, Hierarchical clustering or K-means cluster in any one or at least two combination, preferably use gauss hybrid models.
4. according to claim 1-3 any one of them methods, which is characterized in that step (3) described genotype combination includes parent Generation and filial generation be homozygote, parental generation be homozygote filial generation be heterozygote, parental generation be heterozygote filial generation be homozygote or parental generation and Filial generation is any one in heterozygote.
5. according to claim 1-4 any one of them methods, which is characterized in that further include before step (1)：
(1 ') extracts dissociative DNA from pregnant woman's body fluid；
(2 ') it uses the primer sets of gene loci to carry out PCR amplification to the dissociative DNA of extraction, builds DNA library；
(3 ') carry out high-flux sequence to the DNA library, after sequencing result is compared and analyzes, obtain the survey of gene loci Sequence result.
6. according to claim 1-5 any one of them methods, which is characterized in that step (the 1 ') body fluid include peripheral blood, In urine or saliva any one or at least two combination, preferably peripheral blood；
Preferably, the quality of step (the 1 ') dissociative DNA is 10-50ng, preferably 15-20ng.
7. according to claim 1-6 any one of them methods, which is characterized in that step (the 2 ') primer sets such as SEQ ID Shown in NO.1-46.
8. according to claim 1-7 any one of them methods, which is characterized in that step (the 3 ') high-flux sequence is averaged Sequencing depth be 1000-3000 ×, preferably 2000 ×.
9. according to claim 1-8 any one of them methods, which is characterized in that the described method comprises the following steps：
(1) 10-50g dissociative DNAs are extracted from pregnant woman's body fluid, and PCR expansions are carried out using the primer sets as shown in SEQ ID NO.1-46 Increase, structure includes the DNA library in 15 deaf gene sites and 31 auxiliary gene sites, and high-flux sequence, average sequencing is deeply Degree be 1000-3000 ×, sequencing result is compared and is analyzed, sequencing result is obtained；
(2) the minimum gene frequency of each gene loci is calculated；
(3) set of the minimum gene frequency described to step (2) is clustered using gauss hybrid models, is divided into four classes, Determine the cluster result of each gene loci；
(4) according to the cluster result of step (3) gene loci, obtain parental generation and filial generation the gene loci genotype Combination；
(5) according to parental generation the gene loci genotype, obtain filial generation the gene loci genotype.
10. a kind of such as any one of claim 1-9 the methods in non-invasive antenatal detection hereditary hearing impairment gene type Using.
CN201810189906.0A 2018-03-08 2018-03-08 It is a kind of it is antenatal detection progeny genotypes method and application Pending CN108531572A (en)

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CN112410420B (en) * 2020-12-19 2022-12-06 中国人民解放军联勤保障部队第九八四医院 Special primer for simultaneously and accurately detecting SNP (single nucleotide polymorphism) loci of related genes of 'one palm induced deafness' and 'one needle induced deafness' and application
CN113481291A (en) * 2021-07-07 2021-10-08 长沙金域医学检验实验室有限公司 Kit for detecting deafness gene 21 locus

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Application publication date: 20180914