CN103352086A

CN103352086A - PCR-LDR kit for detecting 11 deaf susceptibility genes

Info

Publication number: CN103352086A
Application number: CN2013103214474A
Authority: CN
Inventors: 步迅; 张全芳; 刘艳艳; 夏子芳
Original assignee: High Tech Research Center Of Shandong Academy Of Agricultural Sciences
Current assignee: High Tech Research Center Of Shandong Academy Of Agricultural Sciences
Priority date: 2013-07-29
Filing date: 2013-07-29
Publication date: 2013-10-16

Abstract

The invention provides a PCR-LDR (Polymerase Chain Reaction-Ligase Detection Reaction) kit for simultaneously detecting 11 deaf susceptibility genes. The PCR-LDR kit comprises a packaging box body (1) as well as PCR amplification agents and LDR reaction agents (2)-(5); the PCR-LDR kit further comprises 6 pairs of PCR primers, 12 groups of probes, 1 group of general probes and 11 groups of detection probes. By adopting the technical scheme, the PCR-LDR kit has high sensitivity, and is clear and accurate in interpretation; compared with the traditional sequencing technology, the kit can complete high-throughput screening of diagnosis results of the obtained 11 key deaf susceptibility genes in one attempt, and has the advantages of economy, high efficiency and high accuracy rate.

Description

A kind of PCR-LDR detection kit of 11 deaf tumor susceptibility genes

Technical field

The present invention relates to technical field of biological, specifically a kind of PCR-LDR detection kit of 11 deaf tumor susceptibility genes.

Background technology

Deafness is a kind of human life quality's of having a strong impact on common congenital disorders, it can cause by term single gene sudden change or heterogeneic complex mutation, also can by environmental factors (such as medical factor, environmental exposure, wound, medicine etc.) or gene and environment acting in conjunction and cause.China has hearing loss crowd 2,780 ten thousand now, occupies (33%) first of all kinds of deformity.There is research to find that GJB2, SLC26A4, chondriogen are 3 modal Disease-causing genes that cause most of China non-syndrome induced deafness.

The GJB2 gene is first hereditary hearing impairment Disease-causing gene that is cloned and identifies, also is to cause the deaf modal Disease-causing gene of non-comprehensive.In the non-syndrome induced deafness, approximately have 20% to be that the GJB2 transgenation causes.The Cx26 albumen of GJB2 genes encoding is high level expression in cochlea, form a complete Gap junctions with the inserted by connexin of flanking cell.These passages play an important role in information transmission and exchange of substance, are that ionogen, second messenger and meta-bolites are in the important channel of iuntercellular conversion.After the GJB2 transgenation, make the potassium ion backflow enter endolymphatic circulation and be affected, cause phonosensitive nerve deafness.The modal mutation type of GJB2 gene is 235delC, secondly is 299-230delA＞T and 176-191del16, and its gene frequency is respectively 11.90%, 2.22% and 0.65%.Pathologic 235delC sudden change causes phase shift mutation, produces non-functional protein, the slit is connected damaged, thereby affects the normal switching of ionic channel.The 299-300delAT sudden change causes the most of disappearance in the CL district of Cx26 polypeptide, and TM3, EC2 and TM4 district lack fully, may lose the regulation and control to Gap junctions pH value, reduces the slit connection to the resolving ability of paraprotein.16 bases were lost after 176-191del16 referred to 176, since No. 59 codon frameshit, so that termination codon is advanced to No. 75, produced non-functional protein.

The SLC26A4 gene belongs to (the solute carder family26A of ion transport body 26A family.SLC26A), coding ion transport associated protein.In the keeping of body ion component balance, play a significant role.The SLC26A4 transgenation can cause the deaf DFNB4 of autosomal recessive and Pendred syndrome (aqueductus vestibuli enlarges or companion's inner ear malformations, nerve deafness and thyrocele).The common mutations type of SLC26A4 gene is IVS7-2A＞G, comprises in addition 2168A＞G, 1226G＞A, 1174A＞T, 1975G＞C, the hot spot mutations such as 2027T＞A.

Mitochondrial DNA Mutation is one of major reason that causes hearing loss.Wherein, plastosome 12S rRNA transgenation is deaf relevant with syndromic deafness and nonsyndromic, and the 1555A＞G and the 1494C＞T sudden change that are positioned at 12S rRNA area decoder are to cause aminoglycosides antibiotics ototoxicity and the deaf common molecular mechanism of nonsyndromic.These sudden changes may cause the change of 12S rRNA secondary structure, have destroyed the synthetic of mitochondrial protein, reduce the generation of ATP in the cell, are that the inside and outside ionic concn of cell is unbalance, the cochlear hair cell apoptosis, and the mitochondria dysfunction that causes thus causes deafness.

Gene pleiomorphism detection technique commonly used mainly comprises at present: direct Sequencing technology, restriction fragment length polymorphism (RFLP), DNA chip technology (Micro array), sex change high performance liquid chromatography (DHPLC), single strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) etc.The problems such as these methods exist respectively that this cost is high, accuracy rate is low, complex operation and poor repeatability.This test kit adopts polymerase chain reaction-Ligase detection reaction (polymerase chain reaction-ligase detection reaction, PCR-LDR) detect 11 deaf tumor susceptibility genes in conjunction with capillary electrophoresis technique, the principle of LDR is to utilize the realization of high temperature conjunction enzyme to the identification of gene polymorphism sites, if in a single day the high temperature conjunction enzyme detects the mispairing that two oligonucleotide joint corresponding positions of DNA and complementation exist the single core thuja acid, namely can stop the connection of hybridization probe.

Summary of the invention

The purpose of this invention is to provide a kind of 11 deaf tumor susceptibility gene GJB2:235delC, 299-230delA＞T, 176-191del16 of detecting simultaneously, SLC26A4:IVS7-2A＞G, 2168A＞G, 1226G＞A, 1174A＞T, 1975G＞C, 2027T＞A, the PCR-LDR detection kit of plastosome 12SrDNA:1555A＞G and 1494C＞T.

To achieve these goals, this test kit comprises pcr amplification and LDR reaction reagent; Comprise 6 pairs of PCR primers, 12 groups of probes, 1 group of general probe and 11 groups of detection probes.

The present invention is to GJB2:235delC, 299-230delA＞T, 176-191de116, SLC26A4:IVS7-2A＞G, 2168A＞G, 1226G＞A, 1174A＞T, 1975G＞C, 2027T＞A, it is detected object that plastosome 12SrDNA:1555A＞G and 1494C＞T amount to 11 deaf sick tumor susceptibility genes, by amplification and the capillary electrophoresis to above-mentioned deaf susceptibility loci, with the contrast of genotyping standard thing, can filter out the individuality that contains above-mentioned site mutation in 3 hours.This test kit adopts compound LDR Ligase detection reaction first, simultaneously to 6JB2:235delC, 299-230delA＞T, 176-191del16, SLC26A4:IVS7-2A＞G, 2168A＞G, 1226G＞A, 1174A＞T, 1975G＞C, 2027T＞A, plastosome 12SrDNA:1555A＞G and 1494C＞T carry out the PCR-LDR amplification, and by capillary electrophoresis post analysis clip size, detect when can realize 11 deaf sites.The method is highly sensitive, and interpretation is clear accurately; Relatively traditional sequencing technologies, this test kit can be finished the high-throughput examination of the crucial deaf genes sudden change of 11 of disposable acquisitions diagnostic results, have the advantages such as economy, efficient and high-accuracy.

Description of drawings

Fig. 1 is test kit of the present invention for the GJB2 gene: result behind the dna sequencing somatotype of 235de1C mutated individual blood cake source;

Fig. 2 is test kit of the present invention for result behind GJB2 gene 299-230delA＞T mutated individual blood cake source dna sequencing somatotype;

Fig. 3 is test kit of the present invention for the GJB2 gene: result behind the dna sequencing somatotype of 176-191del16 mutated individual blood cake source;

Fig. 4 is test kit of the present invention for result behind SLC26A4:IVS7-2A＞G mutated individual blood cake source dna sequencing somatotype;

Fig. 5 is test kit of the present invention for result behind SLC26A4:2168A＞G mutated individual blood cake source dna sequencing somatotype;

Fig. 6 is test kit of the present invention for result behind SLC26A4:1226G＞A mutated individual blood cake source dna sequencing somatotype;

Fig. 7 is test kit of the present invention for result behind SLC26A4:1174A＞T mutated individual blood cake source dna sequencing somatotype;

Fig. 8 is test kit of the present invention for result behind SLC26A4:1975G＞C mutated individual blood cake source dna sequencing somatotype;

Fig. 9 is test kit of the present invention for tying behind SLC26A4:2027T＞A mutated individual blood cake source dna sequencing somatotype;

Figure 10 is test kit of the present invention for result behind plastosome 12SrDNA:1555A＞G mutated individual blood cake source dna sequencing somatotype;

Figure 11 is test kit of the present invention for result behind plastosome 12SrDNA:1494C＞T mutated individual blood cake source dna sequencing somatotype.

Figure 12 STR fluoroscopic examination result

Figure 13 PCR-LDR detected result

Figure 14 sensitivity test result

The composition structure iron of Figure 15 test kit

Embodiment

Embodiment 1

1. genomic dna is prepared

Sample to be checked selects magnetic bead or Chelex-100 method to extract genomic dna, and the sample gene group DNA of extraction is quantitatively at about 1ng/ul-10ng/ul.

2.PCR reaction:

According to the following procedure experiment:

System: 25ul

Each experiment all requires to do simultaneously negative control and positive control

The PCR loop parameter:

Remarks: different because of various places PCR instrument performance, the annealing temperature in the loop parameter can improve or reduce by 1 ℃.

3, ligase enzyme reaction system:

Reaction conditions:

94℃ 2min

94℃ 30S； 60℃ 2min，30 Cycles

20℃ pause

4 electrophoresis detection

Get 1ul connection product and mix 3 minutes ice baths of 95 ℃ of sex change, electrophoresis detection with 0.5ul Marker ROX-300 and 10ul deionized formamide.

Test required template and primer as follows:

LDR common template: CCCATTAGTCTTGCGAGTACAGGTTTGCG

General fluorescent probe: P-GAATCAATCAGACTG-FAM 15

The primer sequence that wherein is used for three sites of GJB2 gene is as follows:

GJB2_176_M：P-TGGCTGCAGGGTGTTGCATTTTCGCAAACCTGTACTC

GJB2_176_del：TTTGCTAACTTCCCCTCTGACCCA

GJB2_176_NOdel：TTTATCGTAGCACACGTTCTTGCAGCC

GJB2_235_M：P-GCTCGATAATGGGAAGTAGTTTTTTCGCAAACCTGTACTC

GJB2_235_delC：TTTTTGCGTGGACACGAAGATCAGCTGCA

GJB2_235_C：TTTTTTTGCGTGGACACGAAGATCAGCTGCAG

GJB2_299_M：P-ATGTGCGGTAGGAAACGTGTTTTTTTTTTCGTAAACGGTACTC

GJB2_299_delAT：TTTTTTTTTTTGGTGCCTTTCCTCTTCTTCTC

GJB2_299_AT：TTTTTTTTTTTTGACTATAGTCCTCTTCTTCTCAT

6 detection site primer sequences of SLC26A4 gene are as follows:

1975_M：P-AAGGCGTTGCTTTGAAACTTTTCGCAAACGAGTTGTC

1975_C：TAAAGATCATGCTCCAGTCTCACCAAG

1975_G：TTTAAAGATCATCCTCCACCTTTCAGCTACAC

2027_M：P-GAGTTCTCACTAAGAACAACTTTTTTTCGCAAACCTCTAATC

2027_T：TTTTTTAAAACCCTCAACACTTAGCCCCGCA

2027_A：TTTTTTTTTAAAACTCAAAGGCTAACGGCT

1174_M：P-GCCGAACCCACTACGCATGAATTTTTTTTTCGCAAAATCAGCC

1174_A：TTTTTTTCGGCAAAGAACTGAGCTCATT

1174_T：TTTTTTTTTTCACCAGAATGATCCCTCAGAATCTG

1226_M：P-CCATCAAGAGCAGTACGTGACACTTTTTTTTTTTTTTCGCCTATGTGC

1226_G：TTTTTTTTTCCTGTCTCCTGCACCTGGAGTTAC

1226_A：TTTTTTTTTTTTCCGATGCGCTAGGCTGAACCTCT

IVS7-2_M：P-GAAATCCGAACTTAATCATGTGAATTTTTTTTTTTTTTTTTTCGCAAATGTACTC

56

IVS7-2_A：TTTTTTTTTTTGAAATCCCAGTAGCAATAATTGTCT

IVS7-2_G：TTTTTTTTTTTTTTGAAAGCAGTAGCCCTTATCCGACC

2168_M：P-GGCTGTCTAAACAATGTGTTTTTTTTTTTTTTTTTTTTTTCCTAAACCAGGACAC

2168_A：TTTTTTTTTTTTTTTGGTTCCCATAGGTAGACTATAGGTCAT 2168_G：TTTTTTTTTTTTTTTTTTGGTTCTGTCGAATAATCTACCTCACTAC

The primer sequence information of 2 detection site of 12s rRNA gene is as follows:

1494_M：P-CGATCGGGCGTCAGTGTCCCCGCTTTTTTTTTTTTCGCACCTCTGTACTC

1494_C：TTTTTTTTTTTCCTTTGAGATCTATACTTCTTGAGG

1494_T：TTTTTTTTTTTTTCCTTTGATCCTTATACAATGAGCCAACAA

1555_M：P-ATCATCCCTATAATCTGCGTATTTTTTTTTTTTTTTCGAATTCATCACTC

1555_G：TTTTTTTTTTTTTTAGTTAACTTACCCAGTTACTGACTTAG

1555_A：TTTTTTTTTTTTTTTTTAGCATCACGGTATAGATGTGCAACGACCCTA

Embodiment 2

One, 1555 site templates are prepared

The PCR system:

Two, specificity experiment (STR fluoroscopic examination)

The mixture that the dilution that marks take above-mentioned yellow respectively is 5 times, 50 times, 500 times carries out PCR and carries out capillary electrophoresis by original reagent and corresponding program as template.

1, response procedures:

The pcr amplification program:

2.1STR fluoroscopic examination result (Figure 12)

With Z-0-5, namely contain the 1555 site plasmid mixtures that mutant plasmid concentration and wild plasmid concentration are 1ng/ul, wherein mutant plasmid content is 0, dilutes 5 times, detects 1555 and is false positive.

Take Z-0-50 as template, namely contain the 1555 site plasmid mixtures that mutant plasmid concentration and wild plasmid concentration are 1ng/ul, wherein mutant plasmid content is 0, dilutes 50 times, detects 1555 and is false positive.Z-0-500 namely contains the 1555 site plasmid mixtures that mutant plasmid concentration and wild plasmid concentration are 1ng/ul, and wherein mutant plasmid content is 0, dilutes 500 times, and without the purpose peak, false positive disappears.

Conclusion: with respect to the STR experiment, PCR-LDR test kit specificity is stronger.

2.2PCR-LDR detected result (Figure 13)

Take Z-0-50 as template, namely containing mutant plasmid concentration is zero, and wild plasmid concentration is the 1555 site plasmid mixtures of 20ng/ul, dilutes 50 times, does not detect the positive, and is consistent with sequencing result.

Three, sensitivity experiment

Take Z-1-5X as template, namely contain the 1555 site plasmid mixtures that mutant plasmid concentration and wild plasmid concentration are 1ng/ul, wherein mutant plasmid content is 1%, dilutes 5 times mixture and tests through PCR-LDR, detects the positive (Figure 14).

Conclusion:

1. with respect to direct sequencing, the sensitivity of PCR-LDR test kit is higher, and data read more convenient, and is quick.

2. experimental period relatively: direct sequencing needs 10 hours, and the PCR-LDR test kit needs 4 hours, has greatly saved the time.

3. saving cost.Compare with direct sequencing, PCR-LDR has saved a large amount of reagent, and can set up multiple system, has further saved cost.

Claims

1. a PCR-LDR detection kit that detects simultaneously 11 deaf tumor susceptibility genes is characterized in that, described test kit comprises Packaging box body (1), pcr amplification and LDR reaction reagent;

Described pcr amplification reagent is respectively pcr amplification Reaction Mix (2) and (3); Described LDR reaction reagent is LDR reaction Probe Mix (4) and (5);

Described 11 deaf tumor susceptibility genes are GJB2:235delC, 299-230delA＞T, 176-191del16, SLC26A4:IVS7-2A＞G, 2168A＞G, 1226G＞A, 1174A＞T, 1975G＞C, 2027T＞A, plastosome 12SrDNA:1555A＞G and 1494C＞T;