CN104120187A - Detection probe for DUOX2 gene mutation and detection method thereof - Google Patents

Abstract

The invention discloses a detection probe for DUOX2 gene mutation and a detection method thereof, particularly relates to a method for detecting DUOX2 gene mutation related to a congenital hypothyroidism (CH) pathogenic gene, and belongs to the technical field of molecular biology. A PCR primer sequence of a DUOX2 gene for detecting the CH is a nucleotide sequence as shown in SEQ ID No.1-SEQ ID No.18 in the sequence table. The detection probe has the advantages that the DUOX2 gene causing the CH is detected by utilizing new generation sequencing, and 24 new mutation sites which are not yet reported home or abroad are discovered. The detection probe applied to clinical gene mutation detection is beneficial to etiological analysis and diagnosis of CH, further recognition of mutation spectrum of the CH gene and further research on pathogenesis of CH.

Description

The detection probes of DUOX2 transgenation and detection method thereof

Technical field

The present invention relates to a kind of detection probes and detection method thereof of DUOX2 transgenation, specially refer to the detection method of gene mutation with congenital hypothyroidism Disease-causing gene DUOX2, belong to technical field of molecular biology.

Background technology

Congenital hypothyroidism (is called for short congenital first low, Congenital hypothyroidism, CH) be that the thyroxine secretion that causes of the disappearance due to relevant enzyme or iodine in thyreoaplasia, dystopy, disappearance or thyroxine building-up process is not enough, thereby cause the paediatrics endocrinopathy of intelligence and anthropometic obstacle.CH hid often in neonatal period, clinical manifestation is not obvious, be difficult to cause the head of a family or even doctor's attention thereby incur loss through delay medical diagnosis on disease and treatment, cause brain development to produce irreversible infringement, the early diagnosis of CH and treatment can prevent patient that mental retardation occurs, and the state of an illness is greatly improved.Therefore, at CH premorbid, it is carried out to early diagnosis and therapy and seem particularly urgent.

When CH be take birth, thyroid hormones level declines, thyrotropic hormone (TSH) raises is feature.CH morbidity and gene and environmental correclation, but concrete mechanism is still not clear at present.Abroad studies have reported that, there is the CH patient of 2-3% can detect transgenation, the CH related genes of having reported is divided into two classes: the gene that (1) is relevant with thyreoaplasia, mainly comprises Pax-8, TSHR, NKX2.1, FOXE1, NKX2.5, TTF1, TTF2 gene etc.; (2) gene relevant with thyroid hormone dyssynthesis, mainly comprises DUOX2, SLC5A5, TG, Dehal1, SLC26A4 and TPO gene.

In the Disease-causing gene research of CH, find that the mutation frequency of gene DUOX2 is relatively high, become gradually the emphasis of research.DUOX2 transgenation induction CH morbidity is autosomal recessive inheritance, and its encoding gene is positioned at No. 15 karyomit(e) 15q15.3 regions of the mankind, comprises 33 exons, and cDNA length is 6.4KB.The DUOX2 albumen of its translation is comprised of 1548 amino acid, is positioned at the teleblem of thyroid cell, a spirane structure that C end comprises six cross-films and NADPH and FAD binding site, and N end comprises peroxidase spline structure and a transmembrane helix structure.DUOX2 is relevant to the generation of Tiroidina hydrogen peroxide (H2O2).H2O2 is as electron acceptor(EA), and in Triiodothyronine building-up process, Tiroidina oxide compound enzyme (TPO) needs the acid iodate of residue of H2O2 catalytic amino and the coupling of amino-acid iodide prepared residue.Once the sudden change of DUOX2 producer, causes its dysfunction, can not normally provide H2O2, and iodine can not be organised, Triiodothyronine is synthetic not enough, thereby causes CH morbidity.Foreign literature report approximately has 30 kinds of DUOX2 transgenations at present, and domestic report is rarely seen.

Nucleic acid sequencing is by goal gene fragment is carried out to DNA sequence dna detection, thereby obtains goal gene DNA sequence dna information, is a widely used important technology in modern molecular biology, has been widely used in the fields such as detection in Gene Mutation and gene type.Generation order-checking is called again " Sanger order-checking ", and because its cost is expensive, flux is too low, and the manual operations time is long, and clinical application is still not extensive.

Summary of the invention

Main purpose of the present invention is to overcome the deficiency of existing CH detection technique, provide one group can to causing, the polygene of congenital hypothyroidism carries out fast, polygene sequencing primer complete detection and that be applicable to clinical patients detection in Gene Mutation and classification of diseases.

Second object of the present invention be to provide that a kind of result is accurate, flux is high, time between short, detection method that cost low energy detects a plurality of candidate genes of congenital hypothyroidism simultaneously.

For achieving the above object, the present invention is by the following technical solutions:

One group of PCR primer sequence that detects the DUOX2 gene of congenital hypothyroidism is the nucleotide sequence shown in sequence table SEQ ID No.1-SEQ ID No.18.

The present invention mainly utilizes oligo6.0 software design DUOX2 gene PCR amplimer, and design of primers is followed following several principle: the length range of primer is suitable at 19-23bp; Primer should be without dimer, the possibility that especially 3 ' end dimer forms; Without hairpin structure; Upstream and downstream primer Tm value differs should not be over 5 ℃; GC content is advisable with 45-55%.After upstream and downstream primer is determined, in ncbi database, utilize Blast to compare to upstream and downstream primer, guarantee specificity and the amplification efficiency of primer.According to exon at a distance of far and near degree, merge part exon Joint Designing primer, 33 exons design 9 pairs of primers altogether, synthetic by Shanghai Sheng Gong biotechnology Services Co., Ltd, the upstream primer P-F of above-mentioned exon and the sequence of downstream primer P-R are as follows, wherein P-F is upstream primer, and P-R is downstream primer, as follows for the concrete design of primers of different exons:

Exon E1-3 upstream primer P-F:5 '-GTGGGCTGCTCTCAACGCTCT-3 ' (SEQ ID No.1);

Downstream primer P-R:5 '-GAAGTGGTTGGGAGTCGGATGG-3 ' (SEQ ID No.2);

Exon E4-8 upstream primer P-F:5 '-CCACTTCCTCTCTACGCAGCAC-3 ' (SEQ ID No.3);

Downstream primer P-R:5 '-CAAATCCTACACCCAGCCACC-3 ' (SEQ ID No.4);

Exon E9-12 upstream primer P-F:5 '-GTAAACGCACATCACCAAATCTC-3 ' (SEQ ID No.5);

Downstream primer P-R:5 '-CTGGGAATCAAGGGCTCATAGG-3 ' (SEQ ID No.6);

Exon E13-17 upstream primer P-F:5 '-TCTCTTTTCTCACCTGGGTCCT-3 ' (SEQ ID No.7);

Downstream primer P-R:5 '-TCACCGAATCCTCACAACAATG-3 ' (SEQ ID No.8);

Exon E18-19 upstream primer P-F:5 '-CTTTCTGATTTGGACTTTGGG-3 ' (SEQ ID No.9);

Downstream primer P-R:5 '-GGCTTTTCTGGCTGGTGTTG-3 ' (SEQ ID No.10);

Exon E20-22 upstream primer P-F:5 '-GGCTGCTTTCTCTGATTGGTC-3 ' (SEQ ID No.11);

Downstream primer P-R:5 '-CTCACTGTCTCCCTGCTACTCC-3 ' (SEQ ID No.12);

Exon E23-25 upstream primer P-F:5 '-AGAAGTAAAGGGTTGGAGGAGG-3 ' (SEQ ID No.13);

Downstream primer P-R:5 '-CACTTTGTTGTTCAGGCTTGTC-3 ' (SEQ ID No.14);

Exon E26-28 upstream primer P-F:5 '-TTGCTGTGTGCCTTGTATTGTTC-3 ' (SEQ ID No.15);

Downstream primer P-R:5 '-TTCCCATCCTCAAACCCCTCTG-3 ' (SEQ ID No.16);

Exon E29-33 upstream primer P-F:5 '-TGGGAAGAGGGAGTAGAGAGGAG-3 ' (SEQ ID No.17);

Downstream primer P-R:5 '-GCCTAAGGTGGATTCTGATGGAG-3 ' (SEQ ID No.18).

The amplified production length of primer sequence of the present invention on average, in 1.5kb left and right, increases by PCR long segment, can effectively catch target area.

A polygene order-checking detection method that detects congenital hypothyroidism, adopts new-generation sequencing method, uses pcr amplification primer, to the detection that suddenlys change of DUOX2 gene.

Described pcr amplification primer is for being the nucleotide sequence shown in sequence table SEQ ID No.1-SEQ ID No.18.

Said method comprising the steps of:

1. design polygene PCR primer sequence: be the nucleotide sequence shown in sequence table SEQ ID No.1-SEQ ID No.18;

2. extract DNA sample: the DNA sample that extracts tester by ordinary method;

Amplification target area: utilize TAKARA high-fidelity enzyme, according to 25 μ l systems, utilize American AB Veriti grads PCR instrument, 30 circulations of increasing, wherein the thermal cycle conditions of each gene extron pcr amplification is as follows:

95 ℃ of denaturation 5min; 95 ℃, 30sec, 64-69 ℃, 30sec, 72 ℃, 60-150sec; 30 circulations; 72 ℃ are extended 10min;

4.PCR product purification: all products are all got 2.5 μ l and carried out electrophoresis evaluation; All residue PCR products all adopt U.S. Omega company to cross post test kit to carry out purifying recovery;

5. the preparation in library: the transposon that carries sequencing primer fragment, interrupt at random the amplified production (amplicon) of above-mentioned steps, and pcr amplification is carried out in the two ends that sequencing primer fragment is connected in the amplicon (length is about 300bp) of fragmentation, sequencing primer is comprised of sequence label, P5/P7 sequence etc., obtains can be used for the DNA fragmentation of order-checking; (test kit that adopts is U.S. illumina company xT DNA builds storehouse test kit, and concrete P5/P7, sequence label are shown in xT DNA Sample Preparation Guide)

6.miseq order-checking: utilize the order-checking of MiSeq Reagent Kit (300cycles PE) test kit, dilution library also makes its sex change, prepare required pre-filled test kit, library mixed solution is installed in the test kit of specified slot, experimental procedure is set and checks each operating parameter, select Sequence to bring into operation, after experiment finishes, check sequencing result;

7. bioinformatic analysis

First new-generation sequencing data are carried out to pre-treatment, comprise wipe out 3' end mass value lower than 20 sequence, remove average quality value lower than 20 sequence; Then use bwa by the sequence alignment after Quality Control to list (hg19 of version number) with reference to genome sequence, utilize GATK instrument interpretation SNP and Indel site; Utilize SNPnexus tool tips SNP, find the functional site of possibility, finally utilize the databases such as dbSNP, thousand human genome databases, HGMD, the pleomorphism site existing in filtration crowd.

The present invention passes through dbSNP, HGMD, the database compare of analysis such as ensemble are got rid of polymorphic site, utilize the method such as 1000genomes database, PubMed retrieval analysis to find novel mutation, result detects altogether 24 kinds of DUOX2 new mutants and has no report, wherein missense mutation (missense) 13 kind in studying at home and abroad, 2 kinds of splice sites (splicing), 3 kinds of nonsense mutations (nosense), 6 kinds of phase shift mutations (frameshift), specific as follows described in:

The new mutant site of the DUOX2 gene that described congenital hypothyroidism is relevant is following one or more:

(1) CCA of the codon in exon Exon3 62 sports TCA;

(2) CCG 76 of the codons of exon Exon3 sports CTG;

(3) CGC 82 of the codons of exon Exon3 sports AGC;

(4) TAT at the codon 185 of exon Exon5 becomes GAT;

(5) at the cDNA of exon Exon5 sequence 596 base C, lack;

(6) TCG at the codon 185 of exon Exon5 becomes CCG;

(7) 301 TGG of codon at exon Exon7 become TGT;

(8) 320 CTA of codon at exon Exon8 become CCA;

(9) AGG 411 of the codons of exon Exon10 becomes AAG;

(10) CGT at the codon 432 of exon Exon11 sports CAT;

(11) 579 CTG of codon at exon Exon14 become CCG;

(12) TGC at the codon 606 of exon Exon14 becomes CGC;

(13) CAA at the codon 570 of exon Exon14 becomes TAA;

(14) bases G in the cDNA of exon Exon15 sequence 1868 lacks;

(15) at the cDNA of exon Exon16 sequence 2102-2104, there is GAG base deletion;

(16) the codon 734TGG at exon Exon17 becomes TGA;

(17) there is the insertion of TCGA base in the codon 2885 at exon Exon21;

(18) CGA at the codon 1084 of exon Exon24 becomes CAA;

(19) the base C in the cDNA of exon Exon24 sequence 3339 lacks;

(20) the base CTG at the cDNA of exon Exon25 sequence 3475-3477 lacks;

(21) GCG 1323 of exon Exon29 codons becomes ACG;

(22) GGA 1521 of the codons of exon Exon33 becomes TGA;

(23) in the 2nd, the downstream of introne 30 base, from T, become G;

(24) in the 1st, the downstream of intron 27 base, from G, become T.

The mutated genes of the DUOX2 gene that congenital hypothyroidism is relevant, contains following one or more mutational sites:

(1) CCA of the codon in exon Exon3 62 sports TCA;

(2) CCG 76 of the codons of exon Exon3 sports CTG;

(3) CGC 82 of the codons of exon Exon3 sports AGC;

(4) TAT at the codon 185 of exon Exon5 becomes GAT;

(5) at the cDNA of exon Exon5 sequence 596 base C, lack;

(6) TCG at the codon 188 of exon Exon5 becomes CCG;

(7) 301 TGG of codon at exon Exon7 become TGT;

(8) 320 CTA of codon at exon Exon8 become CCA;

(9) AGG 411 of the codons of exon Exon10 becomes AAG;

(10) CGT at the codon 432 of exon Exon11 sports CAT;

(11) 579 CTG of codon at exon Exon14 become CCG;

(12) TGC at the codon 606 of exon Exon14 becomes CGC;

(13) CAA at the codon 570 of exon Exon14 becomes TAA;

(14) bases G in the cDNA of exon Exon15 sequence 1868 lacks;

(15) at the cDNA of exon Exon16 sequence 2102-2104, there is GAG base deletion;

(16) the codon 734TGG at exon Exon17 becomes TGA;

(17) there is the insertion of TCGA base in the codon 2885 at exon Exon21;

(18) CGA at the codon 1084 of exon Exon24 becomes CAA;

(19) the base C in the cDNA of exon Exon24 sequence 3339 lacks;

(20) the base CTG at the cDNA of exon Exon25 sequence 3475-3477 lacks;

(21) GCG 1323 of exon Exon29 codons becomes ACG;

(22) GGA 1521 of the codons of exon Exon33 becomes TGA;

(23) in the 2nd, the downstream of introne 30 base, from T, become G;

(24) in the 1st, the downstream of intron 27 base, from G, become T.

The PCR primer sequence of the DUOX2 gene of one group of described detection congenital hypothyroidism is for the preparation of the purposes of the detection reagent of Diagnosis of Congenital Hypothyroidism.

The mutated genes of the DUOX2 gene that described congenital hypothyroidism is relevant is for the preparation of the purposes of the detection reagent of Diagnosis of Congenital Hypothyroidism.

The mutated genes of the DUOX2 gene that described congenital hypothyroidism is relevant is for the preparation of the purposes of the medicine for the treatment of congenital hypothyroidism.

The present invention adopt two generation sequencing technologies, this technology is called again new-generation sequencing (next generation sequencing, NGS), it is the revolutionary progress of sequencing technologies, adopt the theory of " order-checking while synthesizing ", can once to up to a million DNA moleculars, carry out sequencing, make the group of transcribing of species become a reality with the analysis that full genome carries out careful overall picture.Guaranteeing, on the basis of pinpoint accuracy, to make the cost that checks order.New-generation sequencing method is compared and is had following advantage with traditional generation sequencing: 1) cost is low, is only 1% of tradition order-checking cost; 2) flux is high, can to a plurality of samples, check order simultaneously, and carry out once can producing the data of about 600G base; 3) accuracy high (higher than 98.4%), has solved the problem that reads of poly tumor-necrosis factor glycoproteins effectively.On the other hand, the sequence number that high sequencing throughput is checking order is definite, has improved conversely again the order-checking degree of depth (for example, for each sequence, can repeatedly check order) of sequence, thereby has guaranteed the reliability of sequencing result.The birth of new-generation sequencing, makes genomics and functional genomics enter a low cost, extensive, high-throughout order-checking epoch.

New-generation sequencing technology is due to its high-throughput, and low cost is applicable to that polygene causes a disease, Disease-causing gene is huge and the gene diagnosis of the not clear disease of hereditary pattern.At present relevant CH gene paathogenic factor is still not clear, and disease to relate to gene more.By gene sequencing method of new generation, detect research CH associated gene mutation type and feature, due to new-generation sequencing technology tens of up to a hundred genes involveds of examination at short notice, can allow the clinical gene diagnosis of CH become quicker, more comprehensive, greatly improve CH genes involved recall rate, contribute to us to the mutation spectrum of CH gene, to have more deep understanding, understand the role and influence of its genes involved in CH patient's course of disease, deepen us to the pathogenetic research of CH, and lay the first stone for follow-up CH molecular diagnosis and the research and development of diagnostic kit.

PCR primer sequence and s-generation sequence measurement that we use the present invention to design, 24 kinds of DUOX2 gene new mutant types of CH patient have been found, at home and abroad in research, be not reported at present, be respectively: the CCA of the codon 62 in exon Exon3 becomes TCA, make the proline(Pro) (pro) of codon 62 become Serine (ser); The CCG of 76 of the codons of exon Exon3 becomes CTG, makes the proline(Pro) (pro) of 76 of codons become leucine (leu); The CGC of 82 of the codons of exon Exon3 becomes AGC, makes the arginine (Arg) of 82 of codons become Serine (ser); The TAT of the codon 185 of exon Exon5 becomes GAT, makes the tyrosine (Tyr) of 185 of codons become aspartic acid (Asp); C.596delC exon Exon5, makes 596 disappearance base C of cDNA sequence; The codon TCG of exon Exon5 becomes CCG, makes the Serine (Ser) of 188 of codons become proline(Pro) (Pro); 301 TGG of codon of exon Exon7 become TGT, make the tryptophane (Trp) of 301 of codons become halfcystine (Cys); 320 CTA of codon of exon Exon8 become CCA, make the leucine (Leu) of 320 of codons become proline(Pro) (Pro); The AGG of 411 of the codons of exon Exon10 becomes AAG, makes the arginine (Arg) of 411 of codons become Methionin (Lys); The CGT of 432 of the codons of exon Exon11 becomes CAT, makes the arginine (Arg) of 432 of codons become Histidine (His); 579 CTG of codon of exon Exon14 become CCG, make the leucine (Leu) of 579 of codons become proline(Pro) (Pro); The TGC of the codon 606 of exon Exon14 becomes CGC, makes the halfcystine (Cys) of 606 of codons become arginine (Arg); The CAA of the codon 570 of exon Exon14 becomes TAA, makes the L-glutamic acid (CAA) of 570 of codons become terminator codon (TAA); Exon Exon15c.1868delG, makes cDNA sequence 1868 disappearance bases G; Exon Exon16c.2102-2104delGAG, makes cDNA sequence 2102-2104 disappearance bases G AG; The codon 734TGG of exon Exon17 becomes TGA, makes the tryptophane (Trp) of 734 of codons become terminator codon; Exon Exon21c.2885insTCGA, makes cDNA sequence 2885 insert base TCGA; The codon CGA of exon Exon24 becomes CAA, makes the arginine (Arg) of 1084 of codons become glutamine (Gln); Exon Exon24c.3339delC, makes cDNA sequence 3339 disappearance base C; Exon Exon25c.3475-3477delCTG, makes cDNA sequence 3475-3477 disappearance base CTG; The GCG that exon Exon29 codon is 1323 becomes ACG, makes the L-Ala (Ala) of 1323 of codons become Threonine (Thr); The GGA of 1521 of the codons of exon Exon33 becomes TGA, makes the glycine (Gly) of 1521 of codons become terminator codon; The 2nd, the downstream of introne 30 base becomes G (IVS30+2T>G) from T; The 1st, the downstream of intron 27 base becomes T (IVS27+1G>T) from G.

Advantage of the present invention is: the present invention utilizes new-generation sequencing to detect causing the DUOX2 gene of congenital hypothyroidism, has found 24 new mutational sites of not yet reporting both at home and abroad.The method is applicable to large sample and a plurality of exon and detects, have expend time in less, detection efficiency is high, cost is low, the equal higher feature of sensitivity and specific degree, can be fast, show clinical patients detection in Gene Mutation and classification of diseases comprehensively.Be applied to clinical detection in Gene Mutation and contribute to congenital hypothyroidism etiological analysis and diagnosis, contributing to has more deep understanding to the mutation spectrum of Hypothyroidism gene, deepens the pathogenetic research of congenital hypothyroidism.

Below in conjunction with the drawings and specific embodiments, further set forth the present invention.Be interpreted as: these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, for example Sambrook equimolecular is cloned: laboratory manual is shown in the condition described in New York Cold Spring Harbor Laboratory press version in 1989, or the condition of advising according to manufacturer.

Accompanying drawing explanation

Fig. 1 is that sequencer map occurs c.596delC to suddenly change DUOX2 gene Exon5

Fig. 2 is that sequencer map occurs c.1868delG to suddenly change DUOX2 gene Exon15

Fig. 3 is that sequencer map occurs c.2102-2104delGAG to suddenly change DUOX2 gene Exon16

Fig. 4 is that sequencer map occurs c.2885insTCGA to suddenly change DUOX2 gene Exon21

Fig. 5 is that sequencer map occurs c.3339delC to suddenly change DUOX2 gene Exon24

Fig. 6 is that sequencer map occurs c.3475-3477delCTG to suddenly change DUOX2 gene Exon25

Fig. 7 is that the 1323 bit codon GCG of DUOX2 gene Exon29 become ACG sudden change sequencer map

Fig. 8 is that the 606 bit codon TGC of DUOX2 gene Exon14 become CGC sudden change sequencer map

Fig. 9 is that the 1521 bit codon GGA of DUOX2 gene Exon33 become TGA sudden change sequencer map

Figure 10 is that the 1st, the downstream base of DUOX2 gene intron 27 becomes T sudden change sequencer map from G

Figure 11 is that the 2nd, the downstream base of DUOX2 gene intron 30 becomes G sudden change sequencer map from T

Figure 12 is that the 320 bit codon CTA of DUOX2 gene Exon8 become CCA sudden change sequencer map

Figure 13 is that the 579 bit codon CTG of DUOX2 gene Exon14 become CCG sudden change sequencer map

Figure 14 is that the 62 bit codon CCA of DUOX2 gene Exon3 become TCA sudden change sequencer map

Figure 15 is that the 76 bit codon CCG of DUOX2 gene Exon3 become CTG sudden change sequencer map

Figure 16 is that the 570 bit codon CAA of DUOX2 gene Exon14 become TAA sudden change sequencer map

Figure 17 is that the 82 bit codon CGC of DUOX2 gene Exon3 become AGC sudden change sequencer map

Figure 18 is that the 411 bit codon AGG of DUOX2 gene Exon10 become AAG sudden change sequencer map

Figure 19 is that the 432 bit codon CGT of DUOX2 gene Exon11 become CAT sudden change sequencer map

Figure 20 is that the 1084 bit codon CGA of DUOX2 gene Exon24 become CAA sudden change sequencer map

Figure 21 is that the 301 bit codon TGG of DUOX2 gene Exon7 become TGT sudden change sequencer map

Figure 22 is that the 734 bit codon TGG of DUOX2 gene Exon17 become TGA sudden change sequencer map

Figure 23 is that the 185 bit codon TAT of DUOX2 gene Exon5 become GAT sudden change sequencer map

Figure 24 is that the TCG of 188 bit codons of DUOX2 gene Exon5 becomes CCG sudden change sequencer map

Embodiment

Embodiment 1:CH polygene PCR design of primers

Utilize oligo software design DUOX2 gene PCR amplimer, design of primers is followed following several principle: the length range of primer is suitable at 19-23bp; Primer should be without dimer, the possibility that especially 3 ' end dimer forms; Without hairpin structure; Upstream and downstream primer Tm value differs should not be over 5 ℃; GC content is advisable with 45-55%.After upstream and downstream primer is determined, in ncbi database, utilize Blast to compare to upstream and downstream primer, guarantee specificity and the amplification efficiency of primer.According to exon at a distance of far and near degree, merge part exon Joint Designing primer, 33 exons design 9 pairs of primers altogether, synthetic by Shanghai Sheng Gong biotechnology Services Co., Ltd, the upstream primer P-F of above-mentioned exon and the sequence of downstream primer P-R are as follows, wherein P-F is upstream primer, and P-R is downstream primer, as follows for the concrete design of primers of different exons:

Exon E1-3 upstream primer P-F:5 '-GTGGGCTGCTCTCAACGCTCT-3 ' (SEQ ID No.1);

Downstream primer P-R:5 '-GAAGTGGTTGGGAGTCGGATGG-3 ' (SEQ ID No.2);

Exon E4-8 upstream primer P-F:5 '-CCACTTCCTCTCTACGCAGCAC-3 ' (SEQ ID No.3);

Downstream primer P-R:5 '-CAAATCCTACACCCAGCCACC-3 ' (SEQ ID No.4);

Exon E9-12 upstream primer P-F:5 '-GTAAACGCACATCACCAAATCTC-3 ' (SEQ ID No.5);

Downstream primer P-R:5 '-CTGGGAATCAAGGGCTCATAGG-3 ' (SEQ ID No.6);

Exon E13-17 upstream primer P-F:5 '-TCTCTTTTCTCACCTGGGTCCT-3 ' (SEQ ID No.7);

Downstream primer P-R:5 '-TCACCGAATCCTCACAACAATG-3 ' (SEQ ID No.8);

Exon E18-19 upstream primer P-F:5 '-CTTTCTGATTTGGACTTTGGG-3 ' (SEQ ID No.9);

Downstream primer P-R:5 '-GGCTTTTCTGGCTGGTGTTG-3 ' (SEQ ID No.10);

Exon E20-22 upstream primer P-F:5 '-GGCTGCTTTCTCTGATTGGTC-3 ' (SEQ ID No.11);

Downstream primer P-R:5 '-CTCACTGTCTCCCTGCTACTCC-3 ' (SEQ ID No.12);

Exon E23-25 upstream primer P-F:5 '-AGAAGTAAAGGGTTGGAGGAGG-3 ' (SEQ ID No.13);

Downstream primer P-R:5 '-CACTTTGTTGTTCAGGCTTGTC-3 ' (SEQ ID No.14);

Exon E26-28 upstream primer P-F:5 '-TTGCTGTGTGCCTTGTATTGTTC-3 ' (SEQ ID No.15);

Downstream primer P-R:5 '-TTCCCATCCTCAAACCCCTCTG-3 ' (SEQ ID No.16);

Exon E29-33 upstream primer P-F:5 '-TGGGAAGAGGGAGTAGAGAGGAG-3 ' (SEQ ID No.17);

Downstream primer P-R:5 '-GCCTAAGGTGGATTCTGATGGAG-3 ' (SEQ ID No.18).

Embodiment 2: the extraction of blood sample collection and genomic dna:

By the < < of Ministry of Health examination of newborn infant diseases technical specifications > > Case definition MethodsThe cases enrolled, choose altogether CH patient's 192 examples from Guangxi province consanguinity-less relation (age: 9 days-6 years old, the median age 18 days).All persons under inspection or family members all sign written Informed Consent Form, this research obtains agreeing to through the court Ethics Committee, meets the Declaration of Helsinki > > of < < World Medical Association: the ethic principle of human medical research.

192 routine blood samples are prepared sample by the following method, use test kit: TIANGEN Biotech (Beijing) Co., Ltd., poba gene group is extracted test kit (DP318)

1. in 500 μ l anticoagulations, add ligsis buffer1000 μ l, fully mix to limpid on top.With 4000rpm, centrifugal 5min.Abandon supernatant liquor.

2. in precipitation, add ligsis buffer1500 μ l, fully mix.With 6000rpm, centrifugal 5min.

3. thorough supernatant discarded, adds extraction buffer500 μ l (lysing cell), mixes and is placed in 37 ℃, water-soluble 1h.

4. the Proteinase K that adds 8 μ l, top is mixed, 37 ℃ spend the night (or 55 ℃, 3h).

5. every pipe adds the saturated phenol of 450 μ l (getting solution lower floor) slowly to rock 10min, with 5500rpm, and centrifugal 15min.

6. get supernatant, every pipe adds the saturated phenol of 250 μ l and 250 μ l chloroform-primary isoamyl alcohol, shakes up 10min, with 5500rpm, and centrifugal 15min.

7. get supernatant, every pipe adds 500 μ l chloroform-primary isoamyl alcohol, shakes up 10min, with 5500rpm, and centrifugal 15min.

8. get supernatant, every pipe adds the NaAC of the 3M of 50 μ l, and appropriate dehydrated alcohol (precooling) is to full, shake up put into-20 ℃ preserve 2h more than.

9. with 12000rpm, centrifugal 20min.Remove supernatant, add 70% ethanol 500 μ l, with 12000rpm, centrifugal 5min, removes supernatant, and 50-60 ℃ dry.

10. add 50 μ l sterilizing deionized waters, turn bullet, mix.

Embodiment 3: target area amplification

It is synthetic that all primers are all sent to the raw work in Shanghai, and primer is synthetic makes dry powder precipitation by the centrifugal 2min of 12,000g, adds autoclaving distilled water be diluted to final concentration 10uM according to explanation, after it fully dissolves, for PCR, tests.All primer annealing temperature all utilize American AB Veriti grads PCR instrument to grope, and pcr amplification system is 25 μ l.For the target fragment of high GC content, all in system, add DMSO to final concentration be 2%.For the lower primer of amplified band specificity, conventionally consider to change annealing temperature or redesign primer, guarantee that amplification object band is clear, special.

1. primer sequence: be the nucleotide sequence of sequence table SEQ ID No.1-SEQ ID No.18.

2.PCR amplification system: total reaction volume is 25 μ l (the Fermentas dreamTaq Green Buffer of 12.5 μ l, the upstream and downstream primer of 0.5 μ l, the DNA of 1 μ l20ng/ μ l, the high pressure distilled waters of 10.5 μ l)

PCR reaction conditions: wherein the thermal cycle conditions of each exon pcr amplification is as follows:

95 ℃ of denaturation 5min; 95 ℃, 30sec, 64-69 ℃, 30sec, 72 ℃, 60-150sec; 30 circulations; 72 ℃ are extended 10min; (concrete Tm value and extension time are as shown in table 1)

Table 1: best Tm value and extension time (x sec)

Exon	Tm (annealing temperature, ℃)	PCR product length (bp)	The extension time (sec)
				E1-3	69	1996	120
E4-8	64	2242	150
				E9-12	64	2187	150
E13-17	64	2179	150
				E18-19	64	737	60
E20-22	64	1802	120
				E23-25	64	1498	90
E26-28	64	1442	90
				E29-33	64	2256	150

4. gel electrophoresis: after above-mentioned PCR reaction finishes, get 3 μ l products and carry out 1% agarose gel electrophoresis, ethidium bromide staining, electrophoretic voltage is 150mV, after 20min, at Bio-Rad gel imaging system, carry out the specificity of pcr amplification product, as be the single pcr amplification band of corresponding length, think and increase successfully.

The purifying of embodiment 4:PCR product

Use test kit: TIANGEN Biotech (Beijing) Co., Ltd., a large amount of sepharoses reclaim test kit (DP210)

After 1.PCR reaction finishes, from PCR reaction tubes, reaction solution is moved in clean 1.5ml centrifuge tube, add the Binding Solution B of 5 times of volumes, turn upside down and mix.

2. mixed solution is transferred to cover and put in the GenClean post of collection tube, room temperature is placed 2min, in the centrifugal 1min of 8000rpm.

3. take off GenClean Column, outwell waste liquid in collection tube.

Attention: waste liquid is transferred back to GenClean Column repeating step 2 and 3 and can suitably improves organic efficiency.

4. GenClean post is relay and reclaims in collector, add 500 μ lWash Solution,, in the centrifugal 1min of 12000rpm, outwell waste liquid in collection tube.

5. repeating step 4 once.

6. GenClean post is relay and reclaim in collector, the centrifugal 1min of 12,000rpm, thoroughly to remove Wash Solution.

7. GenClean post is put into clean 1.5ml centrifuge tube, in the central authorities of GenClean post adsorption film, carefully added 30～50 μ l Elution Buffer, place 2min for 37 ℃.The centrifugal 1min of 12,000rpm, the liquid in centrifuge tube is the DNA fragmentation that purifying is good.

8. get 2～5 μ l and carry out electrophoresis detection concentration, the DNA that purifying is good can be immediately for subsequent experimental or frozen in-20 ℃.

Embodiment 5: library preparation

Use test kit: U.S. illumina company xT DNA builds storehouse test kit

1.NTA preparation

(1) take out a new NTA plate (Nextera XT Tagment Amplicon Plate), in each hole, add 10 μ l TD buffer successively, 5 μ l0.2ng/ μ l DNA (applied sample amount is 1ng) and 5 μ l ATM.

(2) sealer after the volley of rifle fire mixes 5 times, 20 ℃ of centrifugal 1min of 280g, metal bath 55 degree 5min.

(3) temperature drops to after 10 ℃, tear at once film and add 5 μ l NT buffer, in and NTA, sealer after the volley of rifle fire mixes 5 times.

(4) 20 ℃ of centrifugal 1min of 280g, room temperature is placed 5min.

2.PCR amplification

(1) thaw reagent N PM and index primers, mix.

(2) index 1primers (i7) traverse, guarantees that N701 is at first, and N706 is at the 6th; Index2primers (i5) places vertically, guarantees that S501 is in A position, and S504 is in D position, and records above-mentioned position.

(3) 96 orifice plates are placed on Truseq index plate fixture, add 15 μ l NPM, and utilize the volley of rifle fire to add respectively 5 μ l index2 and 5 μ l index1, for fear of crossed contamination, and the white more renewing and orange lid, the volley of rifle fire mixes 5 times.

(4) sealer, drum rolling, 20 ℃ of centrifugal 1min of 280g.

3.PCR purifying

(1), by 20 ℃ of centrifugal 1min of 280g of 96 orifice plate, prepare a new MIDI plank (CAA), volley of rifle fire transferase 45 0 μ l PCR product (from NTA to CAA).

(2) mix magnetic bead, with each hole of the volley of rifle fire, add 25 μ l AMpure XP magnetic beads, soft piping and druming mixes 10 times, the standing 5min of room temperature.

(3) CAA plate is placed to magnetic frame 2 minutes, remove supernatant, add 200 μ l80% ethanol to place 30 seconds, remove supernatant, repeated washing once, blots ethanol, dries 15min on magnetic frame.

(4) CAA plate is taken away from magnetic frame, added 52.5 μ l RSB, blow and beat gently 10 times, room temperature is placed 2min, prepares a new CAN plate (clean amplified NTA Plate), uses the volley of rifle fire from CAA plate transferase 45 0 μ l supernatant to CAN plate.

4. library stdn

(1) washing LNP, prepares a new MIDI plate LNP, and the volley of rifle fire is from CAN plate transferase 12 0 μ l supernatant to LNP plate.

(2) for 96 samples (192 samples are loading at twice, is 96 samples) at every turn, add 4.4mlLNA1 to 15ml pipe, with rifle, mix LNB1, shift 800 μ l LNB1 and mix to the 15ml pipe that contains LNA1.

(3) the every hole of the volley of rifle fire adds miniature concussion instrument 1800rpm30min after 45 μ lLNA1/LNB1 mixed solution sealers, and magnetic frame is placed 2min, removes supernatant, and LNP is taken away from magnetic frame.

(4) once, every hole adds 45 μ l LNW1 with the volley of rifle fire to repeated washing LNWI, miniature concussion instrument 1800rpm5min after sealer, and magnetic frame is placed 2min, abandons supernatant.

(5) LNP plate is taken away from magnetic frame, added the miniature concussion instrument of 30 μ l0.1N NaOH sealer 1800rpm5min, prepare in the meantime SGP (storaGe Plate) barcode and be attached on 96 new hole PCR plates in SGP plate.

(6) every hole adds after the miniature concussion instrument of 30 μ l LNS1 1800rpm5min, guarantees that liquid fully mixes.Magnetic frame is placed 2min.From LNP plate, shift 30 μ l supernatants to SGP plate, the centrifugal 1min of 1000g after sealer.

Embodiment 6:miseq order-checking

Utilize MiSeq Reagent Kit (300cycles PE) test kit, 96 routine samples (192 samples are loading at twice, is 96 samples at every turn) are divided into two run and check order.

1. prepare the metal bath to 96 ℃ of 1.5ml pipe, the reagent that thaws is to room temperature.

2. mix test kit and sample, from SGP transferase 45 μ l library to be measured, place PCR8 connecting leg, number a new PAL (pooled amplicon library) EP pipe, sample in 8 connecting legs is transferred to PAL and manages and mix.

3. a numbering new DAL (Diluted amplicon library) EP pipe, adds respectively sample in 576 μ l HT1 and 24 μ l PAL pipes to manage to DAL, and piping and druming mixes.

4. concussion instrument mixes DAL, after 96 ℃ of 2min, puts upside down and mixes and be placed in frozen water immediately.

5. ice bath 5min, preserves PAL and SGP plate for-20 ℃.

6. the specified location that DAL is placed on to MISEQ box starts order-checking, from software interface, select Sequence with the setting steps that brings into operation, clean and the mobile rooved face of finish-drying, pack mobile groove into, pack PR2 bottle into, and guarantee that waste water bottle is empty, pack test kit into, check the front check result of operating parameter and operation.

Embodiment 7: bioinformatic analysis

1. data Quality Control, removes inferior quality sequence: first go out average quality value lower than 20 reads, secondly every reads clips mass value lower than 20 base, until a certain base mass value is more than or equal to 20 from 3' end.

2. sequence alignment: use bwa (0.7.5a-r405) sequence alignment software that the data after Quality Control and the mankind are compared with reference to genome sequence (hg19), base mismatch number is less than 3, and allowing to compare is to occur gap.

3. remove by PCR and produce tumor-necrosis factor glycoproteins, first use samtools view to convert the comparison result file of sam form to bam formatted file, then use samtools sort to sort to comparison result, then spend samtools rmdup and remove tumor-necrosis factor glycoproteins.

4. detect single base mutation (SNP) and small segment and insert, lack (indel), first use Realigner Target Creator and the IndelRealigner module of GATK to reset sequence, then detect SNP and Indel by the Select Variants module of GATK.

5.SNP annotation: use SNPnexus tool tips SNP, find possibility to change the site of gene function, comprise missense, be not intended to, phase shift mutation etc.

6. the site that annotation has been reported, utilizes the databases such as dbSNP, thousand human genome databases, HGMD, the pleomorphism site existing in filtration crowd.

The results are shown in Figure shown in 1 to Figure 24, is the CH gene mutation site that the present invention finds first, is below location parameter.

Fig. 1: 596delC Total count:13; A:0; C:0; G:13 (100%, 4+, 9-);

T:0；N:0；DEL:8；INS:0

Fig. 2: 1868delG Total count:55; A:0; C:54 (98%, 24+, 30-);

G:1(2％,1+,0-)；T:0；N:0；DEL:37；INS:0

Figure 32 102-2104delGAG Total count:192; A:0; C:192 (100%, 112+, 80-);

G:0；T:0；N:0；

DEL:101；INS:1

Figure 42 885insTCGA Total count:119; A:118 (99%, 61+, 57-); C:0;

G:0；T:1(1％,1+,0-)；N:0；

DEL:0；INS:32

Figure 53 339delC Total count:68; A:1 (1%, 1+, 0-); C:0;

G:67(99％,26+,41-)；T:0；N:0；

DEL:61；INS:0

Figure 63 475-3477delCTG Total count:179; A:0; C:6 (3%, 1+, 5-);

G:179(97％,95+,78-)；

T:0；N:0；DEL:104；INS:1

Fig. 7 A (GCG) 1323T (ACG) Total count:494; A:242 (49%, 143+, 99-); C:0;

G:252(51％,89+,163-)；T:0；N:0

Fig. 8 C (TGC) 606R (CGC) Total count:360; A:196 (54%, 97+, 99-); C:0;

G:164(46％,91+,73-)；T:0；N:0

Fig. 9 G (GGA) 1521* (TGA) Total count:169; A:54 (32%, 36+, 18-);

C:114(67％,73+,41-)；G:0；T:1(1％,1+,0-)；N:0

Figure 10 IVS27+1G>T Total count:143; A:80 (56%, 34+, 46-);

C:63(44％,37+,26-)；G:0；T:0；N:0

Figure 11 IVS30+2T>G Total count:179; A:85 (47%, 36+, 49-);

C:94(53％,38+,56-)；G:0；T:0；N:0

Figure 12 L (CTA) 320P (CCA) Total count:315; A:167 (53%, 107+, 60-);

C:0；G:148(47％,80+,68-)；T:0；N:0

Figure 13 L (CTG) 579P (CCG) Total count:296; A:169 (57%, 80+, 89-);

C:1(0％,1+,0-)；G:126(43％,48+,78-)；T:0；N:0

Figure 14 P (CCA) 62S (TCA) Total count:86; A:45 (52%, 18+, 27-); C:0;

G:41(48％,15+,26-)；T:0；N:0

Figure 15 P (CCG) 76L (CTG) Total count:252; A:140 (56%, 51+, 89-); C:0;

G:111(44％,34+,77-)；T:1(0％,0+,1-)；N:0

Figure 16 Q (CAA) 570* (TAA) Total count:50; A:24 (48%, 13+, 11-); C:0;

G:26(52％,14+,12-)；T:0；N:0

Figure 17 R (CGC) 82S (AGC) Total count:13; A:2 (15%, 1+, 11-); C:0;

G:0；T:11(85％,5+,6-)；N:0

Figure 18 R (AGG) 411K (AAG) Total count:263; A:0; C:151 (57%, 93+, 58-);

G:0；T:112(43％,57+,55-)；N:0

Figure 19 R (CGT) 432H (CAT) Total count:207; A:0; C:107 (52%, 54+, 53-);

G:0；T:100(48％,38+,62-)；N:0

Figure 20 R (CGA) 1084Q (CAA) Total count:119; A:0; C:58 (49%, 23+, 35-);

G:0；T:61(51％,18+,43-)；N:0

Figure 21 W (TGG) 301C (TGT) Total count:217; A:122 (56%, 40+, 82-);

C:95(44％,28+,67-)；G:0；T:0；N:0

Figure 22 W (TGG) 734* (TGA) Total count:134; A:0; C:70 (52%, 44+, 26-);

G:0；T:64(48％,34+,30-)；N:0

Figure 23 Y (TAT) 185D (GAT) Total count:15; A:12 (80%, 8+, 4-); C:3 (20%, 1+, 2-);

G:0；T:0；N:0

Figure 24 S (TCG) 188P (CCG) Total count:13; A:10 (77%, 7+, 3-);

C:0；G:3(23％,1+,2-)；T:0；N:0

The present embodiment operation steps is practical, and the DUOX2 gene test based on new-generation sequencing detects 24 kinds of new mutant types altogether.Utilize DUOX2 transgenation provided by the invention and detection method, whether the DNA that can detect the low patient of first and family member thereof there is DUOX2 gene said mutation or other mutation type, contribute to understand the mutation spectrum of DUOX2 gene, contribute to understand the role and influence of DUOX2 gene in CH patient's course of disease, intensification is to the pathogenetic research of CH, and lays the first stone for follow-up CH molecular diagnosis and the research and development of diagnostic kit.

Claims (10)

1. one group of PCR primer sequence that detects the DUOX2 gene of congenital hypothyroidism, is characterized in that: be the nucleotide sequence shown in sequence table SEQ ID No.1-SEQ ID No.18.

2. a detection method that detects the DUOX2 gene of congenital hypothyroidism, is characterized in that: adopt new-generation sequencing method, use pcr amplification primer, to the detection that suddenlys change of DUOX2 gene.

3. the detection method of the DUOX2 gene of detection congenital hypothyroidism according to claim 2, is characterized in that: described pcr amplification primer is the nucleotide sequence shown in sequence table SEQ ID No.1-SEQ ID No.18.

4. the detection method of the DUOX2 gene of detection congenital hypothyroidism according to claim 2, is characterized in that: said method comprising the steps of:

(1) design polygene PCR primer sequence: be the nucleotide sequence shown in sequence table SEQ ID No.1-SEQ ID No.18;

(2) extract DNA sample: the DNA sample that extracts tester by ordinary method;

(3) amplification target area: utilize TAKARA high-fidelity enzyme, according to 25 μ l systems, utilize American AB Veriti grads PCR instrument, 30 circulations of increasing, wherein the thermal cycle conditions of each gene extron pcr amplification is as follows:

95 ℃ of denaturation 5min; 95 ℃, 30sec, 64-69 ℃, 30sec, 72 ℃, 60-150sec; 30 circulations; 72 ℃ are extended 10min;

(4) PCR product purification: all products are all got 2.5 μ l and carried out electrophoresis evaluation; All residue PCR products all adopt U.S. Omega company to cross post test kit to carry out purifying recovery;

(5) preparation in library: the transposon that carries sequencing primer fragment, interrupt at random the amplified production (amplicon) of above-mentioned steps, and pcr amplification is carried out in the two ends that sequencing primer fragment is connected in the amplicon (length is about 300bp) of fragmentation, sequencing primer is comprised of sequence label, P5/P7 sequence etc., obtains can be used for the DNA fragmentation of order-checking;

(6) miseq order-checking: utilize the order-checking of MiSeq Reagent Kit (300cycles PE) test kit, dilution library also makes its sex change, prepare required pre-filled test kit, library mixed solution is installed in the test kit of specified slot, experimental procedure is set and checks each operating parameter, select Sequence to bring into operation, after experiment finishes, check sequencing result;

(7) bioinformatic analysis

First new-generation sequencing data are carried out to pre-treatment, comprise wipe out 3' end mass value lower than 20 sequence, remove average quality value lower than 20 sequence; Then use bwa by the sequence alignment after Quality Control to list (hg19 of version number) with reference to genome sequence, utilize GATK instrument interpretation SNP and Indel site; Utilize SNPnexus tool tips SNP, find the functional site of possibility, finally utilize the databases such as dbSNP, thousand human genome databases, HGMD, the pleomorphism site existing in filtration crowd.

5. the new mutant site of the DUOX2 gene that congenital hypothyroidism is relevant, it is characterized in that: be 13 kinds of missense mutation (missense), 2 kinds of splice sites (splicing), 3 kinds of nonsense mutations (nosense), 6 kinds of phase shift mutations (frameshift).

6. the new mutant site of the DUOX2 gene that congenital hypothyroidism according to claim 5 is relevant, is characterized in that, described mutational site is following one or more:

(1) CCA of the codon in exon Exon3 62 sports TCA;

(2) CCG 76 of the codons of exon Exon3 sports CTG;

(3) CGC 82 of the codons of exon Exon3 sports AGC;

(4) TAT at the codon 185 of exon Exon5 becomes GAT;

(5) at the cDNA of exon Exon5 sequence 596 base C, lack;

(6) TCG at the codon 185 of exon Exon5 becomes CCG;

(7) 301 TGG of codon at exon Exon7 become TGT;

(8) 320 CTA of codon at exon Exon8 become CCA;

(9) AGG 411 of the codons of exon Exon10 becomes AAG;

(10) CGT at the codon 432 of exon Exon11 sports CAT;

(11) 579 CTG of codon at exon Exon14 become CCG;

(12) TGC at the codon 606 of exon Exon14 becomes CGC;

(13) CAA at the codon 570 of exon Exon14 becomes TAA;

(14) bases G in the cDNA of exon Exon15 sequence 1868 lacks;

(15) at the cDNA of exon Exon16 sequence 2102-2104, there is GAG base deletion;

(16) the codon 734TGG at exon Exon17 becomes TGA;

(17) there is the insertion of TCGA base in the codon 2885 at exon Exon21;

(18) CGA at the codon 1084 of exon Exon24 becomes CAA;

(19) the base C in the cDNA of exon Exon24 sequence 3339 lacks;

(20) the base CTG at the cDNA of exon Exon25 sequence 3475-3477 lacks;

(21) GCG 1323 of exon Exon29 codons becomes ACG;

(22) GGA 1521 of the codons of exon Exon33 becomes TGA;

(23) in the 2nd, the downstream of introne 30 base, from T, become G;

(24) in the 1st, the downstream of intron 27 base, from G, become T.

7. the mutated genes of the DUOX2 gene that congenital hypothyroidism is relevant, is characterized in that, contains following one or more mutational sites:

(1) CCA of the codon in exon Exon3 62 sports TCA;

(2) CCG 76 of the codons of exon Exon3 sports CTG;

(3) CGC 82 of the codons of exon Exon3 sports AGC;

(4) TAT at the codon 185 of exon Exon5 becomes GAT;

(5) at the cDNA of exon Exon5 sequence 596 base C, lack;

(6) TCG at the codon 185 of exon Exon5 becomes CCG;

(7) 301 TGG of codon at exon Exon7 become TGT;

(8) 320 CTA of codon at exon Exon8 become CCA;

(9) AGG 411 of the codons of exon Exon10 becomes AAG;

(10) CGT at the codon 432 of exon Exon11 sports CAT;

(11) 579 CTG of codon at exon Exon14 become CCG;

(12) TGC at the codon 606 of exon Exon14 becomes CGC;

(13) CAA at the codon 570 of exon Exon14 becomes TAA;

(14) bases G in the cDNA of exon Exon15 sequence 1868 lacks;

(15) at the cDNA of exon Exon16 sequence 2102-2104, there is GAG base deletion;

(16) the codon 734TGG at exon Exon17 becomes TGA;

(17) there is the insertion of TCGA base in the codon 2885 at exon Exon21;

(18) CGA at the codon 1084 of exon Exon24 becomes CAA;

(19) the base C in the cDNA of exon Exon24 sequence 3339 lacks;

(20) the base CTG at the cDNA of exon Exon25 sequence 3475-3477 lacks;

(21) GCG 1323 of exon Exon29 codons becomes ACG;

(22) GGA 1521 of the codons of exon Exon33 becomes TGA;

(23) in the 2nd, the downstream of introne 30 base, from T, become G;

(24) in the 1st, the downstream of intron 27 base, from G, become T.

8. the PCR primer sequence of the DUOX2 gene of one group of detection congenital hypothyroidism claimed in claim 1 is for the preparation of the purposes of the detection reagent of Diagnosis of Congenital Hypothyroidism.

9. the mutated genes of the DUOX2 gene that congenital hypothyroidism claimed in claim 7 is relevant is for the preparation of the purposes of the detection reagent of Diagnosis of Congenital Hypothyroidism.

10. the mutated genes of the DUOX2 gene that congenital hypothyroidism claimed in claim 7 is relevant is for the preparation of the purposes of the medicine for the treatment of congenital hypothyroidism.