CN101135665B - Reagent kit detecting elevated plain pneumochysis susceptibility based on mitochondria DNA T6680C mononucleotide polymorphism - Google Patents

Abstract

The reagent box comprises the following separately-packed reagents: 1) primer mixed liquor; 2) PCR reaction mixed liquor; 3) mitochondrion DNA 6680 site mononucleotide polymorphism T contrast DNA; 4)mitochondrion DNA 6680 site mononucleotide site mononucleotide polymorphism C contrast DNA; 5) probe mixed liquor; 6)coupled reaction mixed liquor; electrophoresis mixed liquor.

Description

Detect the kit of elevated plain pneumochysis susceptibility based on mitochondrial DNA T6680C single nucleotide polymorphism

Technical field

The present invention relates to the kit that a kind of disease detection is used, be specifically related to detect the kit of elevated plain pneumochysis susceptibility based on mitochondrial DNA T6680C single nucleotide polymorphism.

Background technology

(high altitude pulmonary edema is that a kind of spy sends out the pulmonary edema in environment of low oxygen plateau HAPE) to plateau pneumochysis, is the acute severe altitude sickness that serious threat enters plateau population health and life.This disease onset is anxious, and progress is fast, if give treatment to untimelyly, can be developed to respiratory failure in the short period of time, even dead.Bibliographical information, in the crowd of plateau Han nationality who enters more than 3600 meter of height above sea level, the incidence of disease of plateau pneumochysis is up to 0.4%-2%.The have tangible family and the individual susceptible of HAPE morbidity are inclined to, and this points out our inherent causes playing an important role in the HAPE pathogenesis [Zhang Xuefeng, the acute heavy altitude sickness prevalence survey of plateau construction crowd, " plateau medicine magazine ", 2005; 15 (3): 7-8; Old have, plateau pneumochysis pathogenesis progress, " plateau medicine magazine ", 2005,15 (2): 62-64; Gao Yuqi, the pathogenesis of plateau pneumochysis and control, " the medical officer people ", 2005; 482 (2): 108-111].

Because mitochondria is to play an important role in hypoxic acclimatization and the adaptation, HAPE is the bad acute high altitude sickness of a kind of acclimatization, thereby important effect [Gao Wenxiang is taking place in mitochondria in the generation of HAPE, the research of hypoxemia rat brain mitochondria in-vitro transcription activity, " Chinese applied physiology magazine ", 2001; 17:323-326; Moudgil R, Hypoxic pulmonary vasoconstriction, " J ApplPhysiol ", 2005; 98:390-403].Single nucleotide polymorphism (SNP) is a kind of novel molecular genetic marker, is widely used in the neurological susceptibility prediction [Kato of complex disease, Mitochondrial DNApolymorphisms in bipolar disorder, " J Affect Disord ", 2001,62:151-164].LDR (ligase detection reaction) is a kind of economy, detects SNP method commonly used fast, it detects principle is to utilize the identification of high temperature ligase realization to gene polymorphism sites, in a single day the high temperature ligase detects the base mispairing that DNA and two complementary oligonucleotide joint corresponding positions exist the point mutation type, and then coupled reaction just can not be carried out.The sequencing and typing principle is as follows:

(1) the technical program obtains to contain the gene segment in mutational site to be detected earlier by PCR (polymerase chain reaction), carries out LDR then, reads testing result by the sequenator electrophoresis at last;

(2) testing result shows, site, the left side, and promptly mutational site one is the A/C heterozygote, site, the right, promptly mutational site two is a T homozygote (see figure 1).

LDR compares with traditional SNP somatotype means, and the LDR technology has multinomial advantage:

(1) accuracy height: compare with the false negative false positive of technology such as PCR, the LDR technology can be carried out somatotype to SNP more accurately;

(2) simple to operate: similar with present round pcr, the LDR operation is simple, and the technician is not had special professional requirement;

(3) cost is low: use the LDR detection system only to need existing P CR instrument equimolecular Laboratory Instruments to get final product, need not to drop into a large amount of fund purchasing equipments again;

(4) highly versatile: ripe LDR detection system is applicable to the somatotype in various SNP site;

(5) high flux: the LDR detection system can be carried out somatotype to reaching up to a hundred SNP sites simultaneously, and the detection efficiency height satisfies the diagnostic requirements of complex disease and pharmacogenomics.

Summary of the invention

The purpose of this invention is to provide a kind of kit, can be used for plateau pneumochysis (being called for short HAPE) susceptible person's screening, instruct the prevention of HAPE based on mitochondrial DNA T6680C single nucleotide polymorphism detection elevated plain pneumochysis susceptibility.

The inventor is analyzed by the 6680th site SNP to the mitochondrial DNA (mtDNA) of 206 routine HAPE patients of Han nationality and 144 routine Han nationality normal healthy controls, the SNP of research mtDNA and the correlativity of HAPE have been sought out responsive believable HAPE susceptible biological heredity mark.Step is the total length amplification of passing through earlier the mtDNA of the irrelevant individuality of 26 routine Han nationality, order-checking, and 6680 sites of tentatively pointing out mtDNA are the high frequency site of the Hans mtDNA.Utilize 10398 loci polymorphisms of mtDNA then, all samples are divided into single doubly group M and N by RFLP (restriction fragment length polymorphism).Be reflected in the large sample correlativity that (single doubly group M and N) detects mtDNA T6680CSNP and HAPE by (PCR polymerase chain reaction)-LDR (ligase detection reaction) again.In single doubly group M, the 6680C of mtDNA is 12.2% in HAPE case class frequency, and the frequency in Han nationality's control group is 1.7% (P=0.028).Conclusion: single nucleotide polymorphism 6680C is the hazards of HAPE morbidity, can be used as one of genetic marker of HAPE.Therefore,, can design the primer and the probe of suitable length, be used for the neurological susceptibility that PCR-LDR detects plateau pneumochysis based on this SNP site.

Kit based on mitochondrial DNA T6680C single nucleotide polymorphism detection elevated plain pneumochysis susceptibility of the present invention is characterized in that this kit comprises the reagent of following separation packing:

Reagent one, the primer mixed liquor, sequence is as follows:

Upstream primer HP2-F:5 '-ATCCTACCAGGCTTCGGAAT-3 ';

Downstream primer HP2-R:5 '-GCCACCTACGGTGAAAAGAA-3 ';

Reagent two, the PCR reaction mixture;

Reagent three, mitochondrial DNA 6680 site single nucleotide polymorphism T contrast DNA;

Reagent four, mitochondrial DNA 6680 site single nucleotide polymorphism C contrast DNA;

Reagent five, the probe mixed liquor, three kinds of probe sequences are as follows:

①P2-F：

5 '-GTTACAATATGGGAGATTATTCCGATTTTTTTTTTTTTTTT-3 ', fluorescence FAM mark;

2. P2-D1: single nucleotide polymorphism T:

5’-TTTTTTTTTTTTTTTTTGGTTCTTTTTTTCCGGAGAAGTAA-3’；

3. P2-D2: single nucleotide polymorphism C:

5’-TTTTTTTTTTTTTTTTTTTTGGTTCTTTTTTTCCGGAGAAGTAG-3’；

Reagent six, the coupled reaction mixed liquor;

Reagent seven, the electrophoresis mixed liquor.

Described kit based on mitochondrial DNA T6680C single nucleotide polymorphism detection elevated plain pneumochysis susceptibility is characterized in that:

Reagent one, primer mixed liquor 100 μ l; Each 50 μ l of upstream primer and downstream primer mix, and concentration is 10pmol/ μ l;

Reagent two, PCR reaction mixture 1000 μ l; Composition has 1 * PCR buffer, 2.0mMMgCl ₂, 200mM dNTPs, 1U Taq enzyme, surplus is a distilled water;

Reagent three, 6680 site single nucleotide polymorphism T contrast DNA 50 μ l, concentration is 50ng/ μ l;

Reagent four, 6680 site single nucleotide polymorphism C contrast DNA 50 μ l, concentration is 50ng/ μ l;

Reagent five, probe mixed liquor 120 μ l, three kinds of each 40 μ l of probe mix, and these three kinds of concentration and probe concentration are 12.5pmol/ul.

Reagent six, coupled reaction mixed liquor 100 μ l; Composition has the 20mM Tris-HCl of pH 7.6, the 25mM potassium acetate, and the 10mM magnesium acetate, the 10mM dithiothreitol (DTT), the 1mM NAD, 0.1% TritonX-100,0.5 μ l Taq dna ligase, surplus is a distilled water;

Reagent seven, electrophoresis mixed liquor 100 μ l; Fluorescent material 1 * ROX and sample-loading buffer 6 * loadingbuffer respectively are 50 μ l.

The present invention can be used for people from Plain and enter before the plateau plateau pneumochysis susceptible person's screening, instructs prevention and the treatment of HAPE, and the threat that alleviates acute severe altitude sickness helps entering the plateau population health; This kit uses simple, and is easy to operate, detects fast.

Description of drawings

Fig. 1 LDR somatotype principle.

Fig. 2 mitochondrial DNA 6680 sites are single nucleotide polymorphism C.

Fig. 3 mitochondrial DNA 6680 sites are single nucleotide polymorphism T.

Embodiment

Kit based on mitochondrial DNA T6680C single nucleotide polymorphism detection elevated plain pneumochysis susceptibility of the present invention comprises the reagent that following separation is packed:

Reagent one, primer mixed liquor 100 μ l; Each 50 μ l of upstream primer and downstream primer mix, and concentration is 10pmol/ μ l, and sequence is as follows:

Upstream primer HP2-F:5 '-ATCCTACCAGGCTTCGGAAT-3 ';

Downstream primer HP2-R:5 '-GCCACCTACGGTGAAAAGAA-3 ';

Reagent two, PCR reaction mixture 1000 μ l; Composition has 1 * PCR buffer, 2.0mM MgCl ₂, 200mM dNTPs, 1U Taq enzyme, surplus is a distilled water;

Reagent three, mitochondrial DNA 6680 site single nucleotide polymorphism T contrast DNA 50 μ l, concentration is 50ng/ μ l;

Reagent four, mitochondrial DNA 6680 site single nucleotide polymorphism C contrast DNA 50 μ l, concentration is 50ng/ μ l;

Reagent five, probe mixed liquor 120 μ l; Three kinds of each 40 μ l of probe mix, and these three kinds of concentration and probe concentration are 12.5pmol/ul, and probe sequence is as follows:

①2-F：

5 '-GTTACAATATGGGAGATTATTCCGATTTTTTTTTTTTTTTT-3 ', fluorescence FAM mark;

2. P2-D1: 6680 sites of mitochondrial DNA are single nucleotide polymorphism T:

5’-TTTTTTTTTTTTTTTTTGGTTCTTTTTTTCCGGAGAAGTAA-3’；

3. P2-D2: 6680 sites of mitochondrial DNA are single nucleotide polymorphism C:

5’-TTTTTTTTTTTTTTTTTTTTGGTTCTTTTTTTCCGGAGAAGTAG-3’；

Reagent six, coupled reaction mixed liquor 100 μ l; Composition has the 20mM Tris-HCl of pH 7.6, the 25mM potassium acetate, and the 10mM magnesium acetate, the 10mM dithiothreitol (DTT), the 1mM NAD, 0.1%TritonX-100,0.5 μ l Taq dna ligase, surplus is a distilled water;

Reagent seven, electrophoresis mixed liquor 100 μ l; Fluorescent material 1 * ROX and sample-loading buffer 6 * loadingbuffer respectively are 50 μ l.

All reagent of this kit and consumptive material all can be given birth to worker's biotechnology Services Co., Ltd in Shanghai, and internal reagent companies such as precious bioengineering (Dalian) company limited buy, and primer and probe also can be synthetic in above-mentioned biotech firm according to above sequence.

The operation instruction of this kit:

The first step, adopt Shanghai to give birth to the worker's biotechnology UNIQ-10 of Services Co., Ltd pillar clinical sample genome extraction agent box (article No. SK1342) and extract leucocyte genome DNA in the individual venous blood to be measured, with the uv-spectrophotometric instrument DNA is carried out quantitatively then;

In second step, three pipes are prepared in the preparation of PCR reaction system altogether;

First pipe is got DNA of individual 0.5 μ l to be measured, reagent one 1 μ l, reagent 2 18.5 μ l, mixing for sample to be tested;

Reagent 3 0.5 μ l are got, reagent one 1 μ l, reagent 2 18.5 μ l, mixing for single nucleotide polymorphism T contrast in 6680 sites of the second pipeline mitochondrial DNA;

Reagent 4 0.5 μ l are got, reagent one 1 μ l, reagent 2 18.5 μ l, mixing for single nucleotide polymorphism C contrast in 6680 sites of the 3rd pipeline mitochondrial DNA;

The 3rd step, three of second step is managed the material of mixing put into the PCR instrument respectively, all carry out the PCR reaction by following condition: 30 seconds → 56 ℃ annealing of 15 minutes → 94 ℃ sex change of 94 ℃ of sex change are extended 30 seconds → 56 ℃ annealing of 1 minute → 94 ℃ sex change of 1 minute → 72 ℃ extensions of 30 seconds → 56 ℃ annealing of 1 minute → 94 ℃ sex change for 1 minute → 72 ℃ and were extended 1 minute for 1 minute → 72 ℃ ..., wherein 30 seconds → 56 ℃ annealing of 94 ℃ of sex change are extended circulation in 1 minute 25 times for 1 minute → 72 ℃, last 72 ℃ of last eventually 7min that extend obtain three pipe PCR products;

The 4th step, check that with 3% agarose gel electrophoresis (voltage 80V, electrophoresis time 40 minutes) the 3rd goes on foot three pipe PCR products of gained, fragment length is 318bp;

The 5th step, get three PCR pipes again, every pipe all adds reagent 51 μ l, and reagent 6 1.15 μ l, every then pipe add three each 7.85 μ l of pipe PCR product of the 3rd step gained, mixing respectively; Probe length 41bp when mtDNA 6680T, when being 6680C, probe length 44bp;

The 6th step, the 5th steps three pipe homomixture is put into the PCR instrument respectively, carry out the LDR coupled reaction, the LDR response procedures is, 95 ℃ 2 minutes → 94 ℃ 30 seconds → 60 ℃ 2 minutes → 94 ℃ 30 seconds → 60 ℃ 2 minutes → 94 ℃ 30 seconds → 60 ℃ 2 minutes ..., wherein 94 ℃ 30 seconds → 60 ℃ circulated 15 times in 2 minutes altogether, obtained 3 pipes and connected products;

The 7th step connected the products from the 6th step gained 3 pipe respectively and respectively gets 2 μ l, all each and 2 μ l mixings of reagent seven; The electrophoresis that on 3730 sequenators, checks order, voltage is 3KV during electrophoresis, electrophoresis time 1.5 hours;

In the 8th step, the result carries out data analysis by Genemapper software, is single nucleotide polymorphism C when connecting product length for 82bp, referring to Fig. 2; When connecting product length for 85bp is single nucleotide polymorphism T, referring to Fig. 3.As result as shown in Figure 2 the time, be the HAPE susceptible individual.

Claims (1)

1. detect the kit of elevated plain pneumochysis susceptibility based on mitochondrial DNA T6680C single nucleotide polymorphism, it is characterized in that this kit comprises the reagent of following separation packing:

Reagent one, the primer mixed liquor, sequence is as follows:

Upstream primer HP2-F:5 '-ATCCTACCAGGCTTCGGAAT-3 ';

Downstream primer HP2-R:5 '-GCCACCTACGGTGAAAAGAA-3 ';

Primer mixed liquor 100 μ l; Each 50 μ l of upstream primer HP2-F and downstream primer HP2-R mix, and concentration is 10pmol/ μ l;

Reagent two, PCR reaction mixture 1000 μ l; Composition has 1 * PCR buffer, 2.0mMMgCl ₂, 200mM dNTPs, 1 U Taq enzyme, surplus is a distilled water;

Reagent three, 6680 site single nucleotide polymorphism T contrast DNA, the 50 μ l of mitochondrial DNA, concentration is 50ng/ μ l;

Reagent four, 6680 site single nucleotide polymorphism C contrast DNA, the 50 μ l of mitochondrial DNA, concentration is 50ng/ μ l;

Reagent five, probe mixed liquor 120 μ l, three kinds of each 40 μ l of probe mix, and these three kinds of concentration and probe concentration are 12.5pmol/ μ l; Three kinds of probe sequences are as follows:

①P2-F：

5 '-GTTACAATATGGGAGATTATTCCGATTTTTTTTTTTTTTTT-3 ', fluorescence FAM mark;

2. P2-D1: 6680 sites of mitochondrial DNA are single nucleotide polymorphism T:

5’-TTTTTTTTTTTTTTTTTGGTTCTTTTTTTCCGGAGAAGTAA-3’；

3. P2-D2: 6680 sites of mitochondrial DNA are single nucleotide polymorphism C:

5’-TTTTTTTTTTTTTTTTTTTTGGTTCTTTTTTTCCGGAGAAGTAG-3’；

Reagent six, coupled reaction mixed liquor 100 μ l; Composition has the 20mM Tris-HCl of pH 7.6, the 25mM potassium acetate, and the 10mM magnesium acetate, the 10mM dithiothreitol (DTT), the 1mM NAD, 0.1% TritonX-100,0.5 μ l Taq dna ligase, surplus is a distilled water;

Reagent seven, electrophoresis mixed liquor 100 μ l; Fluorescent material 1 * ROX and sample-loading buffer 6 * loadingbuffer respectively are 50 μ l.

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