CN104293958B - A kind of test kit predicting susceptibility of ankylosing spondylitis and method - Google Patents

Abstract

The invention discloses a kind of test kit predicting susceptibility of ankylosing spondylitis and method, belong to medical biotechnology field.The present invention, by extracting the genomic DNA of host cell, measures the genotype of the TNXB gene extron district 32063600 position pleomorphism site of experimenter, it was predicted that experimenter's susceptibility to ankylosing spondylitis.The invention have the advantage that the dependency elaborating TNXB gene extron district 32063600 position pleomorphism site first with ankylosing spondylitis, provide a kind of method predicting susceptibility of ankylosing spondylitis, the method can be used for the prevention of ankylosing spondylitis, auxiliary diagnosis and treatment, it is also possible to for new drug development.

Description

A kind of test kit predicting susceptibility of ankylosing spondylitis and method

Technical field

The present invention relates to a kind of test kit predicting susceptibility of ankylosing spondylitis and method, be by surveying in particular Fixed and AS related gene TNXB polymorphism predicts experimenter's susceptibility for ankylosing spondylitis, and the method can Auxiliary for disease diagnoses, treats and new drug development, belongs to biological technical field.

Background technology

Ankylosing spondylitis (Ankylosing spondylitis, AS) is a kind of autoimmune disease, is mainly in 16-40 The person between twenty and fifty in year, the ill ratio of men and women is about 4～10:1.Pathological changes Chang Shouxian betides sacroiliac joint, minority patient with severe symptoms Show as whole rigid spine.Additionally, some patients is with hip joint in various degree, eye, lung, cardiovascular, kidney Deng the outer pathological changes of spinal column.AS sickness rate in white race crowd is about 1%～3%, and China's AS prevalence is about 0.2-0.6%, Wherein the patient of more than 60% gets involved with hip joint, causes more than 20% AS patient disabilities, and inflammation mainly involves pass The bone attachment point of condyle, tendon and ligament, causes local joint accretion tetanic, limitation of activity.The most scarce The weary medicine that can obviously relieve and control disease development.AS belongs to multigenic disease, has obvious genetic predisposition, though So it has been generally acknowledged that heredity plays a leading role with immune factor in AS falls ill, but definite cause and onset of disease mechanism is the most not Clear.

Carrying out AS inherited pathogenic factor research at present, many employing SNP, as the association analysis method of genomic marker, are to have Effect, it is proven.SNP refers to the DNA sequence that in chromogene group level, single nucleotide diversity causes Polymorphism, the frequency in crowd needs > 1%, SNPs is biallelic marker, has 70.1% in the change of this single base Conversion between homotype base: such as G/A or T/C, 29.1% is that the transversion between purine and pyrimidine occurs.C (cytosine) is the most labile site in human genome, because great majority are methylated cytosines, it is possible to Spontaneous deaminizating is converted to T (thymus pyrimidine), and SNP contains the 80-90% of known polymorphism, is modal something lost The change of disease is different.

Owing to the selection pressure of existence causes SNP distribution in term single gene and whole genome to be inhomogeneities. The SNPs quantity in gene noncoding region is 4 times of coding region, and sum is up to 3,000,000.SNP is high with its density (average every 1kb just has 1), representativeness are strong, and (SNP being positioned at gene internal may directly affect protein structure Or expression), hereditary stability good (with microsatellite polymorphism comparatively speaking), be prone to automated analysis (because of SNP In crowd, mostly be biallelic marker, can be simply with " +/-or 1/0 " directly typing) etc. feature become fine Genetic marker.

1973, first Brewerton etc. was found that and the Human leukocyte antigen-B27 of AS strong correlation (HLA-B27).Along with progress of research, other tumor susceptibility genes such as tumor necrosis factor-alpha (TNF-relevant to AS α), il-1 (IL-1) is identified successively.

TNXB (tenascin XB, tenascin XB) gene is positioned at 6q21.3, total length 68220bp.This base Because being positioned human leukocyte antigen III region, closely related with immunoreation and self property immunological diseases generation development etc..

The research that TNXB gene is associated with AS there is no report in recent years.

Summary of the invention

The main object of the present invention is to provide a kind of test kit predicting susceptibility of ankylosing spondylitis, draws including PCR Thing and the test kit containing this primer.

Second object of the present invention is to provide a kind of method detecting susceptibility of ankylosing spondylitis gene.

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

A kind of nucleotide sequence detecting susceptibility of ankylosing spondylitis, has the base shown in sequence table SEQ ID No.1 Sequence, its 32063600 position is i.e. variant sites, indicates with letter " R ".This nucleotide sequence is TNXB Gene Partial sequence.Fig. 1 is TNXB gene structure and the schematic diagram in polymorphic variation site thereof, includes in accompanying drawing Multiple exons, 32063600 are marked on the relevant position in TNXB gene extron district.

A kind of method detecting susceptibility of ankylosing spondylitis, by extracting the genomic DNA of host cell, measures The genotype of the TNXB gene extron district 32063600 position pleomorphism site of experimenter, it was predicted that experimenter is to by force The susceptibility of straightforward spondylitis: when the genotype of position, TNXB gene extron district 32063600 is AA, tested The susceptibility of person is minimum；When carrying G allele, the susceptibility of experimenter raises.

The primer of one group of detection susceptibility of ankylosing spondylitis, the nucleotide sequence of primer is respectively sequence table SEQ ID Shown in No.2 and sequence table SEQ ID No.3, length is respectively 23bp and 25bp, and can specifically expand Go out to include the product of 32063600 positions in sequence shown in SEQ ID No.1.

The invention provides a kind of diagnostic kit detecting susceptibility of ankylosing spondylitis, wherein contain the present invention special Property amplification position, TNXB gene extron district 32063600 primer to the test kit for PCR augmentation detection General components, reagent, buffer etc., those skilled in the art know these general components and detection method.The present invention Whole components, content, source and using method in test kit are as follows:

A kind of test kit detecting ankylosing spondylitis tumor susceptibility gene, is made up of following reagent:

(1) 10 μ L 10 × PCR buffer；

(2) 2 μ L 10mMdNTP mixed liquors；

(3) 2 μ L Taq DNA polymerase, 2unit/ μ L；

(4) 1 μ L F1 primers, for the nucleotide sequence shown in SEQ ID NO.2, concentration is 10pmol/ μ L；

(5) 1 μ L R1 primers, for the nucleotide sequence shown in SEQ ID NO.3, concentration is 10pmol/ μ L；

(6) 8 μ L 10 × LC-Green Plus saturated fluorescence dyestuffs；

(7) 2 μ L oligonucleotide internal references, are made up of 4 kinds of each 0.5 μ L of internal reference primer, and concentration is 10pmol/ μ L, its Middle low temperature oligonucleotide internal reference forward primer F is the nucleotide sequence shown in SEQ ID NO.4, in low temperature oligonucleotide Ginseng downstream primer R is the nucleotide sequence shown in SEQ ID NO.5, and high temperature oligonucleotide internal reference forward primer F is Nucleotide sequence shown in SEQ ID NO.6, high temperature oligonucleotide internal reference downstream primer R is SEQ ID NO.7 institute The nucleotide sequence shown；

(8) 64 μ L pure water.

Using method:

(1) PCR amplification: by PCR amplification TNXB gene extron district Partial Fragment, prepares mixed liquor: 10 × PCR reaction buffer 1 μ L, 10mM/LdNTP0.2 μ L, Taq DNA polymerase 0.2 μ L, 10pM/L Forward primer 0.1 μ L, 10pM/L downstream primer 0.1 μ L, 10 × LC-Green Plus saturated fluorescence dyestuff 0.8 μ L, Oligonucleotide internal reference 0.2 μ L (the high and low temperature oligonucleotide internal reference upstream and downstream each 0.05 μ L of primer) (sequence is shown in Table 1), Genomic DNA 1 μ L, adds pure water to 10 μ L.PCR reaction condition is 95 DEG C of denaturations 3min, 95 DEG C of degeneration 30s, 69 DEG C of annealing 30s, 72 DEG C extend 4s, altogether 45 circulations, 72 DEG C of overall elongation 2min.Carrying out high-resolution Before melting curve analysis, carry out degeneration and renaturation processes: 95 DEG C of 30s, 25 DEG C of 2min, 94 DEG C of 30s, 24 DEG C of 2min. In each system, the paraffin oil of 20 μ L is added, to prevent from evaporating before PCR.

(2) genotype judges: PCR primer moved in special 96 orifice plates of HRM, at Light Scanner TMHR-I Carry out HRM analysis on 96, with Light Scanner Call IT software, the curve after gathering is analyzed, according to The difference of melting curve judges genotype.

TNXB gene extron district 32063600 position pleomorphism site is in the reagent preparing diagnosing ankylosing spondylitis Purposes.

TNXB gene extron district 32063600 position pleomorphism site is in the medicine of preparation treatment ankylosing spondylitis Purposes.

The assay method of the present invention determines the genomic DNA deriving from people, and sample source is unrestricted, such as: body fluid (blood, ascites and urine etc.), histiocyte (such as hepatic tissue) etc..Can make with these samples of purification by extracting Standby genomic DNA.Adjust the concentration of genomic DNA so that it is the most consistent.With genomic DNA as mould Plate, amplifiable go out nucleic acid fragment containing TNXB gene mutation site, to obtain the great amount of samples measured.This pass through The sample that the amplification DNA fragmentation containing TNXB genovariation point obtains, is particularly suited for use as measuring material.

When carrying out gene auxiliary diagnosis, the present invention is preferably applied in the mutation type measured according to TNXB gene and exists Auxiliary diagnostic, auxiliary diagnostic includes the particular agent as neccessary composition, and it is corresponding to being used for measuring The method of TNXB gene mutation type.Suitable particular agent is selected, such as DNA sheet by the assay method used Section and/or the primer for PCR amplification step.

The invention have the advantage that the present invention illustrates TNXB gene extron district 32063600 position polymorphism first The dependency of site and AS, it is provided that a kind of method predicting AS susceptibility, the method can be used for AS prevention, Auxiliary diagnosis and treatment, it is also possible to for new drug development.

With detailed description of the invention, the present invention is further described below in conjunction with the accompanying drawings, in order to summary of the invention is had more by the public Deep understanding, not limitation of the present invention, the equivalent of all any this areas done according to the disclosure of invention Replace, belong to protection scope of the present invention.

Accompanying drawing explanation

Fig. 1 is TNXB gene structure and the schematic diagram in polymorphic variation site thereof

Fig. 2 is the TNXB genovariation site gene type figure through HRM method

Fig. 3 is the sequencer map in TNXB genovariation site

Detailed description of the invention

In the following example, represent that the english abbreviation of reagent is as follows:

10 × PCR buffer: 10mM Tris-HCI (pH=8.3), 500mM potassium chloride (KCl), 10mM chlorine Change magnesium (MgCl₂), 0.01% (W/V) gelatin

DNTP: deoxynucleoside triphosphate

EDTA: disodiumedetate

TE:10mM Tris-HCl (pH=7.5), 1mM EDTA (pH=8.0)

Embodiment 1: blood sample collection and the extraction of genomic DNA:

One. case is selected in:

By the New York Revised diagnostic criteria MethodsThe cases enrolled of 1984, choose the AS from Ningxia, China consanguinity-less relation altogether Patient 273 example (age: 8-71 year, average 27 years old), with area normal healthy controls volunteer 240 example (age: 18-21 year, average 19 years old).All persons under inspection are Han nationality and signature Written informed consent, and this research obtains Beijing Hospital, the accreditation of ethics audit committee of Ministry of Public Health Gerontological Research Center institute, meet " world medicine association Meeting Declaration of Helsinki ": the ethic principle of human medical research.

Two. according to following method, prepare human gene group DNA.

1. in the 1.5mLEP pipe of label, add 1000 μ L erythrocyte cracked liquids, rear addition 400 μ LEDTA anticoagulants Blood (anticoagulation is reverse mixing 3-5 time before adding), reverse mixing, room temperature stands 10 minutes；

After 2.13000rpm is centrifuged 30 seconds, remove supernatant；

3. in gained precipitates, add 480 μ L nucleic acid cleavage liquid, after attack tube wall, fully mixing, add 20 μ L albumen Enzyme K (with splitting karyolymph 20 times of diluted protein enzyme K of dilution), reverse mixing, 65 DEG C of incubators 10 minutes, (period is frequently Mix up and down, it is ensured that without grumeleuse)；

4. it is down to room temperature after taking out, adds 300 μ L albumen precipitation liquid, the most reverse mixing, stand 10 minutes, 13000rpm Centrifugal 2 minutes；

5. supernatant is moved in new EP pipe, add the isopropanol of 670 μ L pre-coolings, the most reverse mixing (10 times Above), it is seen that linear DNA gradually forms little agglomerate, 13000rpm is centrifuged 2 minutes；

6. abandon supernatant and guarantee that precipitation is stayed in EP pipe, adding 670 μ L70% ethanol, mixing of turning upside down, 13000rpm is centrifuged 2 minutes；

7. abandoning supernatant, in making pipe, ethanol volatilization is clean；

8. add TE solution (400 μ L), fully dissolve, the genomic DNA extracted is carried out concentration and purity point Analysis, draw part DNA solution as working solution, concentration correction to 20ng/ μ L, be placed in 4 DEG C standby, remain gene Group DNA puts-20 DEG C of Refrigerator stores.

The identification of embodiment 2:SNP is identified

The present invention uses PCR-high-resolution melting curve (HRM) analytic process and PCR sequencing technologies simultaneously to TNXB The genotype of position, gene extron district 32063600 (its loci is A/G) detects.Fig. 2 is TNXB Genovariation site is through sequencer map that the gene type figure of HRM method, Fig. 3 are TNXB genovariation site.

The determination of one .PCR-HRM primer

Looking into the DNA base sequence (Seq ID № 1) taken near 32063600 positions from Genebank, primer sets Meter completes under Oligo7.0 software.Purpose fragment is positioned at TNXBB gene extron district, and total length 52bp is special Property primer sequence is as follows:

F1:5 '-TTTTACGTGGGCTATGGCGGTGA-3 ' (SEQ ID No.2)

R1:5 '-TTTTTCGAGGCTCTTCCTTCCCGCA-3 ' (SEQ ID No.3)

Two .PCR reaction system and reaction conditions

By PCR amplification TNXB gene extron district Partial Fragment, PCR reaction system is: 10 × PCR reacts Buffer 1 μ L, 10mM/LdNTP 0.2 μ L, Taq DNA polymerase 0.2 μ L, 10pM/L forward primer 0.1 μ L, 10pM/L downstream primer 0.1 μ L, 10 × LC-Green Plus saturated fluorescence dyestuff 0.8 μ L, oligonucleotide Internal reference 0.2 μ L (the high and low temperature oligonucleotide internal reference upstream and downstream each 0.05 μ L of primer) (sequence is shown in Table 1), gene Group DNA1 μ L, adds deionized water to 10 μ L.In each system, add 20 μ L paraffin oil during PCR, prevent Evaporate.PCR reaction condition is 95 DEG C of denaturations 3min, 95 DEG C of degeneration 30s, and 69 DEG C of annealing 30s, 72 DEG C are prolonged Stretch 4s, altogether 45 circulations, 72 DEG C of overall elongation 2min.Before carrying out high-resolution fusion curve analysis, become Property and renaturation process: 95 DEG C of 30s, 25 DEG C of 2min, 94 DEG C of 30s, 24 DEG C of 2min.

Table 1: high and low temperature oligonucleotide internal reference primer sequence, annealing temperature and product sheet segment length

Three .HRM judge genotype

PCR primer is moved in special 96 orifice plates of HRM, Light Scanner TMHR-I 96 carries out HRM and divides Analysis: from the beginning of 40 DEG C, gathers melting curve, to 98 DEG C of end, with Light Scanner Call IT with the slope of 0.3 DEG C/s Curve after gathering is analyzed by software, it is determined that genotype.

Four. order-checking judges genotype

From the individuality of the different genotype of gained, randomly draw 10 example samples respectively carry out sequence verification.Order-checking sample Re-starting PCR amplification, sequencing primer sequence is: F2:5 '-GTGCATCTGTTGGGAAGGCTAC-3 ' (SEQ ID No.8), R2:5 '-GGCATGTCTGGATGGCACAGTC-3 ' (SEQ ID No.9), expands Increase sheet segment length 347bp.It is 30 μ L that PCR reacts total system, comprises: genomic DNA 2 μ L, 10 × PCRBuffer3 μ L, 10mMdNTP0.5 μ L, Taq DNA polymerase (5U/ μ L) 0.5 μ L, upstream and downstream primer (10pM/ μ L) each 0.5 μ L, pure water is supplemented to cumulative volume 30 μ L.PCR reaction condition is: after 95 DEG C of denaturations 3min Entering major cycle, 95 DEG C of degeneration 30s, 64 DEG C of annealing 30s, 72 DEG C extend 20s, 45 circulations, and 72 DEG C extend 2min. PCR primer detects through 8% polyacrylamide gel electrophoresis, send Hua Da gene sequencing after gel imaging system observation is qualified Portion carries out sequence verification.(Fig. 3)

Embodiment 3: gene SNP and the dependency of ankylosing spondylitis (AS)

One. statistical method:

Statistical method: use the populational representation of Hardy-Weinberg balance test research sample.Utilize SPSS11.0 In software, Pearson X 2 test calculates the allele of position, TNXB gene extron district 32063600, genotype exists Distribution frequency between AS case group and Normal group, the risk OR value of AS and 95%CI credibility interval thereof, with P < 0.05 is significance of difference standard.

Two. result: be positioned at genotype and the equipotential of the position, TNXB gene extron district 32063600 in 6p21.3 region Gene frequency distribution between case and matched group refers to table 2.

The genotype of position, table 2TNXB gene extron district 32063600 and gene frequency distribution between case-control group

Note: OR: odds ratio；CI: credibility interval.* G allele is the risk allele being susceptible to suffer from AS.Experimenter is divided into AS's Risk allele AG carrier, and non-risk allele AA carrier.The chi-square value of genotype is by relatively non-wind Danger allelotype AA draws with risk allelotype AG.

From table 2, the G allele of TNXB gene 32063600 position, it is i.e. C on its DNA complementary strand Allele, the distribution frequency in PATIENT POPULATION is significantly higher than its allele distributions frequency in healthy normal population Rate (4.9%vs.0.4%), has significant difference (P=1.3 × 10^-5), and the OR value in A site is 0.08, 95%CI:0.02-0.34；In the risk allele AG carrier and non-risk allele AA carrier of AS, (the P ＜ 0.05) that risk genotype distribution frequency in case group is significantly higher than in matched group, all shows TNXB base It is proportionate because position, exon 1 32063600 is ill with AS, may have the risk increasing AS morbidity.

Enforcement 4: detection kit

Preparation detection AS relevant risk test kit, include amplifiable go out TNXB gene 32063600 position primer Right, and other PCR-HRM corresponding reagent.Test kit of the present invention, for 10 person-portion detection application, keeps in Dark Place in-20 DEG C, Its component, content and source include:

10 μ L 10 × PCR buffer (Pharmacia),

2 μ L 10mMdNTP mixed liquor (Pharmacia),

2 μ L Taq DNA polymerase (2unit/ μ L) (Takara)

1μL F1(SEQ ID No.2)(10pM/μL)

1 μ L R1 (SEQ ID No.3) (10pM/ μ L) primer,

8 μ L 10 × LC-Green Plus saturated fluorescence dyestuff (American I daho company),

2 μ L oligonucleotide internal references, are made up of 4 kinds of each 0.5 μ L of internal reference primer, and concentration is 10pmol/ μ L, and wherein low temperature is few Nucleotide internal reference forward primer F is the nucleotide sequence shown in SEQ ID NO.4, low temperature oligonucleotide internal reference downstream primer R is the nucleotide sequence shown in SEQ ID NO.5, and high temperature oligonucleotide internal reference forward primer F is SEQ ID NO.6 Shown nucleotide sequence, high temperature oligonucleotide internal reference downstream primer R is the nucleotide sequence shown in SEQ ID NO.7；

64 μ L pure water.

After PCR-HRM detects, TNXB gene extron district 32063600 position polymorphism can be detected easily.

Use kit measurement AS patient of the present invention and position, normal person's TNXB gene extron district 32063600 polymorphic Property, randomly draw AS patient gene and organize DNA and each 10 examples of normal person's genomic DNA.

One. method:

1.PCR expands: by PCR amplification TNXB gene extron district Partial Fragment, PCR reaction system is: 10 × PCR reaction buffer 1 μ L, 10mM/LdNTP 0.2 μ L, Taq DNA polymerase 0.2 μ L, 10pM/L Forward primer 0.1 μ L, 10pM/L downstream primer 0.1 μ L, 10 × LC-Green Plus saturated fluorescence dyestuff 0.8 μ L, Oligonucleotide internal reference 0.2 μ L (the high and low temperature oligonucleotide internal reference upstream and downstream each 0.05 μ L of primer) (sequence is shown in Table 1), Genomic DNA 1 μ L, adds deionized water to 10 μ L.In each system, 20 μ L paraffin oil are added during PCR, Prevent from evaporating.PCR reaction condition is 95 DEG C of denaturations 3min, 95 DEG C of degeneration 30s, 69 DEG C of annealing 30s, 72 DEG C extend 4s, altogether 45 circulations, 72 DEG C of overall elongation 2min.Before carrying out high-resolution fusion curve analysis, enter Row degeneration and renaturation process: 95 DEG C of 30s, 25 DEG C of 2min, 94 DEG C of 30s, 24 DEG C of 2min.

2. genotype judges: PCR primer moved in special 96 orifice plates of HRM, at Light Scanner TMHR-I Carry out HRM analysis on 96: from the beginning of 40 DEG C, gather melting curve with the slope of 0.3 DEG C/s, to 98 DEG C of end, With Light Scanner Call IT software, the curve after gathering is analyzed, it is determined that genotype.

Two. result:

Result shows, genotype is AG the 3 of AS patient's TNXB gene extron district 32063600 position polymorphism Example, 7 examples of AA；10 example matched group genotype are AA.This method can effectively determine TNXB gene extron District 32063600 position polymorphism, when the genotype of position, TNXB gene extron district 32063600 is AA, experimenter Susceptibility minimum；When carrying G allele, the susceptibility of experimenter raises.

The present invention has an illustration of practicality:

The detection method of the TNXB gene pleiomorphism of the present invention can be used for analyzing the TNXB in huamn autosomal 6p21.3 district The G allele of the rare variant sites on gene, is i.e. C allele on its DNA complementary strand, and it is right to be applied to How are the complementary diagnosis of AS and the individual AS risk of assessment, are beneficial to carry out the early intervention of AS and treatment.

The present invention is utilized to illustrate the nucleotide variation of position, TNXB gene extron district 32063600, as biomarker One of, can be used as the screening of the molecular target of drug design, divide with the activity helping searching to have regulation TNXB expression Son, promotes AS new drug development.

The nucleotide sequence of detection TNXB gene pleiomorphism that the present invention sets up and AS related locus, can high sensitivity, spy The test kit being applied to the auxiliary diagnosis of AS gene of the opposite sex.

As mentioned above, it was therefore concluded that, the polymorphism of position, TNXB gene extron district 32063600 has notable phase with AS Guan Xing.Therefore, measure this polymorphism according to the present invention, can be used for the gene auxiliary diagnosis of AS.

Invention describes the new mutation site that TNXB Gene A S-phase is closed, and it is many to provide a kind of mensuration TNXB gene The method of state property, and, according to the present invention, it is only necessary to a small amount of DNA sample just be enough to measure gene polynorphisms.Knot Really, the invention provides a kind of gene aided diagnosis method measuring AS related gene polymorphism.

Claims (2)

1. the primer of one group of detection susceptibility of ankylosing spondylitis, it is characterised in that: the nucleotide sequence of primer is respectively Shown in sequence table SEQ ID No.2 and sequence table SEQ ID No.3.

2. the test kit detecting ankylosing spondylitis tumor susceptibility gene, it is characterised in that be made up of following reagent:

(1) 10 μ L 10 × PCR buffer；

(2) 2 μ L 10mMdNTP mixed liquors；

(3) 2 μ L Taq DNA polymerase, 2unit/ μ L；

(4) 1 μ L F1 primers, for the nucleotide sequence shown in SEQ ID NO.2, concentration is 10pmol/ μ L；

(5) 1 μ L R1 primers, for the nucleotide sequence shown in SEQ ID NO.3, concentration is 10pmol/ μ L；

(6) 8 μ L 10 × LC-Green Plus saturated fluorescence dyestuffs；

(7) 2 μ L oligonucleotide internal reference primers, are made up of 4 kinds of each 0.5 μ L of internal reference primer, and concentration is 10pmol/ μ L, Wherein low temperature oligonucleotide internal reference forward primer F is the nucleotide sequence shown in SEQ ID NO.4, low temperature oligonucleotide Internal reference downstream primer R is the nucleotide sequence shown in SEQ ID NO.5, high temperature oligonucleotide internal reference forward primer F For the nucleotide sequence shown in SEQ ID NO.6, high temperature oligonucleotide internal reference downstream primer R is SEQ ID NO.7 Shown nucleotide sequence；

(8) 64 μ L pure water.