CN1180093C - Detection kit for autosomal dominant inheritance polycystic kidney disease - Google Patents

Abstract

The present invention relates to the technical field of the medical molecular biology detection, which is a kit for detecting autosomal dominant inherited polycystic kidney diseases. In the kit, a corresponding primer is synthesized according to the structure characteristics of patients with I and II type disease-causing genes. The molecular biology technology combined with a single-chain conformation polymorphism analytical method or a high-pressure liquid chromatograph analytical method for detection can directly detect the mutation site and the mutation type of the disease-causing gene of autosomal dominant inherited polycystic kidney diseases. The kit provides an efficient path to early prevent and cure the disease and get rid of the transfer between the involvement family generations of the disease-causing gene.

Description

The autosomal dominant polycystic kidney disease detection kit

Technical field

The present invention relates to Medical Molecular Biology detection technique field, is a kind of test kit that is used to detect autosomal dominant polycystic kidney disease.

Background technology

Autosomal dominant polycystic kidney disease is a kind of very common, relevant with heredity kidney disease, the concealment of onset process, the many just morbidities after 30 years old of patient can cause body bilateral kidney multiple cyst, and unusual with other multi-organ function, final most of patients is because of renal failure death.To the clinical diagnosis of this disease, mainly utilize iconography technology and familial inheritance medical history and make, though this diagnostic method is easy, direct, be only applicable to just can make a definite diagnosis when the existing multiple kidney tumour of body occurs, be difficult to diagnosis ahead of time, and early treatment.Molecular diagnosis to this disease, mainly be to utilize little satellite linkage analysis technology, by detecting several and the closely linked genetic marker of autosomal dominant polycystic kidney disease Disease-causing gene changes from generation to generation in family and makes indirect diagnosis, whether this detection technique can carry Disease-causing gene by indirect reflection body from gene level is done sth. in advance diagnosis, but can't directly detect the particular location and the type of Disease-causing gene sudden change, be subject to the influence of family dna homolog reorganization between the generation and reduce accuracy rate of diagnosis, seldom adopt at present.

Autosomal dominant polycystic kidney disease is because due to the sudden change of I type Disease-causing gene and II type Disease-causing gene, and wherein 85% patient suddenlys change relevantly with the former, and only accounts for 15% with the latter's relevant ratio of suddenling change.I type Disease-causing gene is positioned at human chromosomal 16p13.1, and long 53kb comprises 46 exons, and wherein the 1-33 exon is positioned at the tumor-necrosis factor glycoproteins district, and the 34-46 exon is positioned at the non repetitive sequence district.II type Disease-causing gene is positioned at human chromosomal 4q13, and long 68kb comprises 15 exons.The nucleotide sequence of I, II type Disease-causing gene sees Genbank for details, and accession number is respectively L39891 and U50928.Because autosomal dominant polycystic kidney disease Disease-causing gene complex structure, wherein there are a plurality of homologous sequences in I type Disease-causing gene, has directly reduced the specificity that detects.Influenced by it, present most of Disease-causing gene mutation researches only limit to the subregion of I type Disease-causing gene, though external indivedual research group tentatively carried out its full detection in Gene Mutation ( Rossetti S., Strmecki L., Gamble V., et al.Mutation analysis of the entire PKD1 gene:genetic and iagnostic implications.Am J Hum Genet, 2001,68:46-63), but the Protocols in Molecular Biology that adopts is of a great variety, lacks unity and repeatability.Thereby development autosomal dominant polycystic kidney disease detection kit directly detects particular location and mutation type that its I type Disease-causing gene and II type Disease-causing gene complete sequence suddenly change, is the problem of needing solution badly.

Summary of the invention

The invention provides a kind of test kit that detects autosomal dominant polycystic kidney disease I type Disease-causing gene and II type Disease-causing gene mutational site and mutation type that is directly used in.

According to the constructional feature of autosomal dominant polycystic kidney disease gene with at I type Disease-causing gene and II type Disease-causing gene complete sequence order, have tens ofly in the test kit of the present invention to primer, its reason is:

1.I type Disease-causing gene and II type Disease-causing gene sequence are long, do not find that so far there is mutantional hotspot in Disease-causing gene, for realizing the Mutation Screening in the full gene scope, the corresponding increase of design primer quantity.

2.I the type Disease-causing gene is made up of tumor-necrosis factor glycoproteins district and non repetitive sequence district, also has homologous sequence in the tumor-necrosis factor glycoproteins district.For overcoming the interference of homologous sequence, the tumor-necrosis factor glycoproteins district has been designed long-chain PCR primer, this primer can go out I type Disease-causing gene sequence by specific amplification, the amplified production mean length is 4.2kb (and general pcr amplified fragment length is many below 1.6kb), so the introducing of long-chain PCR primer is another reason that quantity increases.

3. when adopting the single-strand conformation polymorphism analysis method or adopting the high pressure liquid chromatograph analytical procedure that pcr amplification product is carried out preliminary screening, both are all restricted to the length of screening product, the former is optimum range with 160-400bp, and the latter then is no more than 600bp with fragment and is advisable.And I type Disease-causing gene adopts long-chain PCR specific amplification products therefrom to be not suitable for the requirement of above-mentioned analytical procedure, so must be template with long-chain PCR specific amplification products again, carry out the amplification second time by nest-type PRC, the long segment that the long-chain pcr amplification is gone out is divided into the short-movie section by the nest-type PRC amplification, for use in single-strand conformation polymorphism analysis method or the analysis of employing high pressure liquid chromatograph.The introducing of nest-type PRC primer is the another major reason that primer quantity increases.

4. according to the constructional feature of I, II type Disease-causing gene, pcr amplification product both can adopt the screening of single-strand conformation polymorphism analysis method also can pass through the high pressure liquid chromatograph screening, the present invention has designed the corresponding PCR primer of two covers respectively, it is that two kinds of analytical procedures can be shared that a considerable amount of primers are wherein arranged, but for easy to use, prevent mistake, thus two cover primers in the shared test kit of two kinds of screening methods, disposed simultaneously for you to choose, thus test kit primer quantity is obviously increased.

The nucleotide sequence of primer of the present invention sees Table 1 respectively, table 2 and table 3.Wherein I type Disease-causing gene adopt the screening of single-strand conformation polymorphism analysis method the forward and reverse primer of pcr amplification referring to sequence table 1, by the forward and reverse primer of the pcr amplification of high pressure liquid chromatograph analytical procedure referring to sequence table 2.II type Disease-causing gene adopts the pcr amplification product of single-strand conformation polymorphism analysis method screening to be suitable for the high pressure liquid chromatograph analytical procedure equally, and the forward and reverse primer of its pcr amplification is referring to sequence table 3.The primer synthetic method preparation routinely of above-mentioned each primer.

For ease of narration, introduce the primer mixture code name in the test kit earlier:

1. be used for the I type primer mixture of single-strand conformation polymorphism analysis, represent with I-S-n, wherein I represents I type Disease-causing gene, and S represents the single-strand conformation polymorphism analysis method, and n represents the primer numbering of I type Disease-causing gene.

2. be used for the I type primer mixture that high pressure liquid chromatograph is analyzed, represent that with I-H-n wherein H represents the high pressure liquid chromatograph analytical procedure, I and n are the same.

3.II the type primer mixture is represented with II-SH-n, wherein II represents II type Disease-causing gene, and SH represents that this serial primer all is suitable for single-strand conformation polymorphism analysis method and high pressure liquid chromatograph analytical procedure, and n represents the primer numbering of II type Disease-causing gene.

The code name of other reagent is in the test kit:

1. be exclusively used in the I type negative control thing of single-strand conformation polymorphism analysis method, represent with-I-S, the normal control thing is represented with+I-S; Be exclusively used in the I type negative control thing of high pressure liquid chromatograph analytical method, represent with-I-H, the normal control thing is represented with+I-H.

2. in single-strand conformation polymorphism analysis method and high pressure liquid chromatograph analytical method, II type negative control thing is all represented with-II-SH, and the normal control thing is all represented with+II-SH.

3. the liquid code name develops the color:

Represent with colour developing liquid A, colour developing liquid B, colour developing liquid C, colour developing liquid D, colour developing liquid E, colour developing liquid F respectively.

Test kit of the present invention comprises following reagent:

1. adopt I type primer mixture 82 pipes of single-strand conformation polymorphism analysis method screening, respectively with I-S-1, I-S-2, I-S-3 ..., I-S-82 represents.Wherein I-S-1 to I-S-8 is a long-chain PCR primer, and totally 8 pairs, I-S-9 to I-S-65 is the nest-type PRC primer, and totally 57 pairs, all the other are non-iteron PCR primer, totally 17 pairs, are respectively I-S-66 to I-S-82.

2. adopt high pressure liquid chromatograph to analyze I type primer mixture 72 pipes of screening, respectively with I-H-1, I-H-2, I-H-3 ..., I-H-72 represents.Wherein I-H-1 to I-H-8 is a long-chain PCR primer, and totally 8 pairs, I-H-9 to I-H-59 is the nest-type PRC primer, totally 51 pairs.All the other are non-iteron PCR primer, totally 13 pairs, are respectively I-H-60 to I-H-72.

3.II type primer mixture 17 pipe, use respectively II-SH-1, II-SH-2, II-SH-3 ..., II-SH-17 represents.

4.I each 1 pipe of type negative control thing is represented with-I-S and-I-H respectively; Each 1 pipe of I type normal control thing is represented with+I-S and+I-H respectively; II type negative control thing 1 pipe is represented with-II-SH; II type normal control thing 1 pipe is represented with+II-SH.

5. sample-loading buffer 1 is managed.

6. colour developing liquid A, B, C, D, E, F are respectively 1 bottle.

Wherein identical in two of the I type Disease-causing gene cover primers have:

1. long-chain PCR primer: I-S-1 to I-S-8 is corresponding identical respectively with I-H-1 to I-H-8;

2. what the nest-type PRC primer was identical has 31 pairs, is respectively:

The same I-H-13 of I-S-14, the same I-H-14 of I-S-15, the same I-H-15 of I-S-16, the same I-H-16 of I-S-17, the same I-H-17 of I-S-18, the same I-H-18 of I-S-19, the same I-H-19 of I-S-20, I-S-22 to I-S-28 be respectively with I-H-22 to I-H-28, the same I-H-42 of I-S-47, the same I-H-43 of I-S-48, the same I-H-44 of I-S-49, the same I-H-45 of I-S-50, the same I-H-46 of I-S-51, the same I-H-47 of I-S-52, the same I-H-48 of I-S-53, the same I-H-49 of I-S-54, the same I-H-50 of I-S-55, the same I-H-51 of I-S-56, the same I-H-52 of I-S-57, the same I-H-53 of I-S-58, the same I-H-54 of I-S-59, the same I-H-55 of I-S-60, the same I-H-56 of I-S-61, the same I-H-58 of I-S-64, the same I-H-59 of I-S-65.

3. what non repetitive sequence district primer was identical has 2 pairs, i.e. the same I-H-60 of I-S-66, the same I-H-61 of I-S-67.

Above-mentioned each reagent is respectively:

1. primer mixture: each primer mixture is 200 μ l/ pipe.

I type primer mixture: contain Tutofusin tris 80mmol/L, potassium acetate 0-170mmol/L, methyl-sulphoxide 0-10%, Octylphenoxy gathers ethoxy ethanol 0.2%, deoxynucleoside triphosphate 500-1200 μ mol/L, magnesium acetate 0-2.5mmol/L or magnesium chloride 0-2mmol/L, thermostable type deoxynucleotide polysaccharase 16-80U, each 2-6 μ mol/L of forward and reverse primer.

II type primer mixture: the poly-ethoxy alcohol concn of its Tutofusin tris, methyl-sulphoxide, Octylphenoxy is identical with I type primer mixture, deoxynucleoside triphosphate 1200 μ mol/L, magnesium chloride 2mmol/L, thermostable type deoxynucleotide polysaccharase 20U, each 2 μ mol/L of forward and reverse primer.

2. contrast

(1) I type and II type negative control thing: sterilization deionization ultrapure water, each 0.4ml/ pipe.

(2) I type and II type normal control thing: contain normal gene 100ng/ μ l, each 0.4ml/ pipe.

3. sample-loading buffer: 10ml/ manages

Wherein contain sucrose 10%, bromjophenol blue 0.01%, dimethylbenzene green grass or young crops 0.01%.

4. colour developing liquid:

(1) colour developing liquid A: dehydrated alcohol, 60ml/ bottle.

(2) colour developing liquid B: concentrated nitric acid, 10ml/ bottle.

(3) colour developing liquid C:30% silver nitrate solution, the 10ml/ bottle.

(4) colour developing liquid D:25% sodium carbonate solution, the 60ml/ bottle.

(5) colour developing liquid E: acetate, 60ml/ bottle.

(6) colour developing liquid F:37% formaldehyde solution, the 6ml/ bottle.

Description of drawings:

Fig. 1 for the primer of test kit of the present invention in the relative position synoptic diagram of autosomal dominant polycystic kidney disease I type Disease-causing gene structure;

Fig. 2 is that test kit primer of the present invention is in the relative position synoptic diagram of autosomal dominant polycystic kidney disease II type Disease-causing gene structure;

Fig. 3 is embodiment 5 electrophoretogram synoptic diagram;

Fig. 4 is embodiment 6 electrophoretogram synoptic diagram;

Fig. 5 is that embodiment 7 high pressure liquid chromatographs are analyzed peak figure.

Embodiment

Concrete preparation method is as follows for above-mentioned each reagent:

1. the preparation of each solution of primer mixture

(1) 0.8mol/L tris solution

Adding 96.9 gram Tutofusin triss in 800ml deionization ultrapure water, is 8.9 with the concentrated hydrochloric acid adjust pH, is settled to 1L with the deionization ultrapure water.

(2) 1.7mol/L potassium acetate solution

In 800ml deionization ultrapure water, add 166.8 gram potassium acetates, fully after the dissolving, be settled to 1L with the deionization ultrapure water.

(3) dimethyl sulfoxide solution

It is standby that concentration is pressed the packing of 10ml/ bottle greater than 99% dimethyl sulfoxide solution.

(4) 20% Octylphenoxies gather ethoxy ethanol

Getting concentration is poly-ethoxy ethanol (the magnificent biotechnology in the Shanghai company limited) 1.88ml of commercialization Octylphenoxy of 106.5% (w/v), is settled to 10ml with the deionization ultrapure water.

(5) deoxynucleoside triphosphate solution

It is standby that concentration is that the commercialization deoxynucleoside triphosphate solution (Japanese TaKaRa company) of 10mmol/L is pressed the packing of 1.5ml/ pipe.

(6) 250mmol/L magnesium acetate solution

In 800ml deionization ultrapure water, add 35.61 gram potassium acetates, fully after the dissolving, be settled to 1L with the deionization ultrapure water.

(7) 200mmol/L magnesium chloride solution

In 800ml deionization ultrapure water, add 19.06 gram potassium acetates, fully after the dissolving, be settled to 1L with the deionization ultrapure water.

(8) thermostable type deoxynucleotide polysaccharase solution

It is standby that enzymic activity is that the commercial hot stable form deoxynucleotide polysaccharase (U.S. Applied Biosystems company) of 4U/ μ l is pressed the packing of 1.5ml/ pipe.

(9) each primer solution, concentration are 50 μ mol/L.

Each primer powder that will synthetic gained, being diluted to concentration with sterilization deionization ultrapure water respectively is 50 μ mol/L, pressing 0.5ml/, to manage packing standby.

2. the preparation of each primer mixture (by 8 person-portion usage quantitys, the 200ul/ pipe)

Embodiment 1: preparation primer mixture I-S-1

Getting concentration is 0.8mol/L tris solution 20 μ l, 1.7mol/L potassium acetate solution 20 μ l, dimethyl sulfoxide solution 20 μ l, 20% Octylphenoxy gathers ethoxy ethanol 2 μ l, 10mmol/L deoxynucleoside triphosphate solution 24 μ l, 250mmol/L magnesium acetate solution 2 μ l, enzymic activity is 4U/ μ l thermostable type deoxynucleotide polysaccharase 20 μ l, each 8 μ l of the forward and reverse primer of 50 μ mol/LI-S-1, fully behind the mixing, be settled to 200 μ l with sterilization deionization ultrapure water, put in the 0.5ml thin-walled tube standby in-20 ℃.This moment, final concentration was respectively: Tutofusin tris 80mmol/L, potassium acetate 170mmol/L, methyl-sulphoxide 10%, Octylphenoxy gathers ethoxy ethanol 0.2%, deoxynucleoside triphosphate 1200 μ mol/L, magnesium acetate 2.5mmol/L, thermostable type deoxynucleotide polysaccharase 80U, each 2 μ mol/L of forward and reverse primer.

Primer mixture I-H-1 preparation method is with embodiment 1.

Embodiment 2: preparation primer mixture I-S-2

Get 10mmol/L deoxynucleoside triphosphate solution 10 μ l, enzymic activity be 4U/ μ l thermostable type deoxynucleotide polysaccharase 4 μ l other respectively become amount that branch gets with embodiment 1.Fully behind the mixing, be settled to 200 μ l, put in the 0.5ml thin-walled tube standby in-20 ℃ with sterilization deionization ultrapure water.

Primer mixture I-S-3 to I-S-8 and I-H-2 and I-H-8 preparation method are with embodiment 2.

Embodiment 3: preparation I-S-9 primer mixture

Getting concentration is 0.8mol/L tris solution 20 μ l, 20% Octylphenoxy gathers ethoxy ethanol 2 μ l, 10mmol/L deoxynucleoside triphosphate solution 24 μ l, 200mmol/L magnesium chloride solution 2 μ l, enzymic activity is 4U/ μ l thermostable type deoxynucleotide polysaccharase 5 μ l, and each 8 μ l of the forward and reverse primer of 50 μ mol/L I-S-9 are fully behind the mixing, be settled to 200 μ l with sterilization deionization ultrapure water, put in the 0.5ml thin-walled tube standby in-20 ℃.This moment, final concentration was respectively: Tutofusin tris 80mmol/L, Octylphenoxy gathers ethoxy ethanol 0.2%, deoxynucleoside triphosphate 1200 μ mol/L, magnesium chloride 2.5mmol/L, thermostable type deoxynucleotide polysaccharase 20U, each 2 μ mol/L of forward and reverse primer.

Primer mixture I-S-10 to I-S-65, I-S-67 to I-S-73, I-S-78 to I-S-80 and I-H-9 to I-H-59, I-H-61 to I-H-65 and I-H-70 preparation method are with embodiment 3.

Embodiment 4: preparation primer mixture I-S-66

Get dimethyl sulfoxide solution 20 μ l, other respectively becomes the branch amount of getting with embodiment 3.This moment, final concentration was respectively: Tutofusin tris 80mmol/L, and Octylphenoxy gathers ethoxy ethanol 0.2%, methyl-sulphoxide 10%, deoxynucleoside triphosphate 1200 μ mol/L, magnesium chloride 2.5mmol/L, thermostable type deoxynucleotide polysaccharase 20U, each 2 μ mol/L of forward and reverse primer.

Primer mixture I-S-74 to I-S-77, I-S-8 1, I-S-82 and I-H-60, I-H-66 to I-H-69, I-H-71, I-H-72 preparation method are with embodiment 4.

3.I, the preparation of II type feminine gender and normal control thing

(1) negative control thing-I-S ,-preparation of I-H and-II-SH

Be sub-packed in the 0.5ml thin-walled tube by the 0.4ml/ pipe with sterilization deionization ultrapure water, put-20 ℃ standby.

(2) normal control thing+I-S ,+preparation of I-H and+II-SH

With 10 milliliters of normal people's peripheral bloods, phenol chloroform method (" molecular cloning " experiment guide routinely, the Jin Dongyan compiling, the 465-467 page or leaf) its genome of difference extracting, being diluted to concentration with sterilization deionization ultrapure water is 100ng/ μ l,, be sub-packed in the 0.5ml thin-walled tube by the 0.4ml/ pipe as normal I, II type Disease-causing gene template solution with this, it is standby to put 4 ℃ of refrigerators.During use, be the working concentration of 50ng/ μ l with the dilution of sterilization deionization ultrapure water.

4. the preparation of sample-loading buffer

In 80ml deionization ultrapure water, add 10 gram sucrose, 0.01 gram bromjophenol blue and 0.01 gram dimethylbenzene green grass or young crops respectively, fully after the mixing dissolving, be settled to 100ml with the deionization ultrapure water, press the packing of 10ml/ pipe, room temperature is standby.Its final concentration is respectively sucrose 10%, bromjophenol blue 0.01%, dimethylbenzene green grass or young crops 0.01%.

5. the preparation of colour developing liquid

(1) colour developing liquid A

It is standby that dehydrated alcohol is pressed the packing of 60ml/ bottle.

(2) colour developing liquid B

It is standby that concentrated nitric acid is pressed the packing of 10ml/ bottle.

(3) colour developing liquid C

In 80ml deionization ultrapure water, add 30 gram Silver Nitrates, fully after the mixing dissolving, be settled to 100ml with the deionization ultrapure water, the packing of 10ml/ bottle is put 4 ℃ of refrigerators and is kept in Dark Place.It is 30% silver nitrate solution.

(4) colour developing liquid D

In 80ml deionization ultrapure water, add 25 gram anhydrous sodium carbonates, fully after the mixing dissolving, be settled to 100ml with the deionization ultrapure water, it is standby to press the packing of 60ml/ bottle.It is 25% sodium carbonate solution.

(5) colour developing liquid E

It is standby that acetate is pressed the packing of 60ml/ bottle.

(6) colour developing liquid F

It is standby that 37% formaldehyde solution is pressed the packing of 6ml/ bottle.

6. assembling test kit

(1) assembling single-strand conformation polymorphism analysis method detection kit: the primer mixture I-S-1 to I-S-82 that gets above-mentioned preparation respectively, II-SH-1 to II-SH-17, contrast-I-S ,+I-S ,-II-SH and+II-SH each 1 the pipe, sample-loading buffer 1 pipe and colour developing liquid A, B, C, D, E and F place in the test kit for each 1 bottle, are the present invention and are exclusively used in the test kit that detects sudden change by the single-strand conformation polymorphism analysis approach.

(2) assembling high pressure liquid chromatograph analytical method detection kit: primer mixture I-H-1 to I-H-72 and each 1 pipe of II-SH-1 to II-SH-17 of getting above-mentioned preparation respectively, contrast-I-H ,+I-H ,-II-SH and+each 1 pipe of II-SH places in the test kit, is the present invention and is exclusively used in the test kit that detects sudden change by the high pressure liquid chromatograph analysis approach.

(3) detection kit of two kinds of analytical method dual-purposes of assembling: primer mixture I-S-1 to I-S-82, I-H-1 to I-H-72 and each 1 pipe of II-SH-1 to II-SH-17 of getting above-mentioned preparation respectively, contrast-I-S ,+I-S ,-I-H ,+I-H ,-II-SH and+II-SH each 1 the pipe, sample-loading buffer 1 pipe and colour developing liquid A, B, C, D, E and F place in the test kit for each 1 bottle, are the detection kit of two kinds of analytical method dual-purposes.

Certainly, can also be assembled into all ingredients boxes such as special survey I type or the sudden change of II type Disease-causing gene as required.

The use of test kit

As everyone knows, autosomal dominant polycystic kidney disease is because I, due to the sudden change of II type Disease-causing gene, that sudden change has taken place which Nucleotide actually? when adopting this test kit to detect, can be undertaken by following program: sudden change accounts for this patient's 85% according to I type Disease-causing gene, the non repetitive sequence district is than tumor-necrosis factor glycoproteins district this characteristics that increase easily, the I type that can increase earlier Disease-causing gene non repetitive sequence district, through single-strand conformation polymorphism analysis method or the screening of high pressure liquid chromatograph analytical method, if screening goes out unusual nucleotide fragments, then this sample and corresponding normal control are further made nucleotide sequence analysis, by comparing, find out the mutational site with normal sequence.If this zone is unscreened and is detected unusual nucleotide fragments, then needn't make nucleotide sequence analysis, but further make the pcr amplification of II type Disease-causing gene sequence area, because this sequence area more easily increases than I type Disease-causing gene tumor-necrosis factor glycoproteins district, amplified fragments is screening by the same method, when II type Disease-causing gene sequence area also screening detect the tumor-necrosis factor glycoproteins district of I type Disease-causing gene again when the unusual nucleotide fragments.This trace routine can reduce blindness, improves detection efficiency.

The step that detects is as follows:

1. prepare genes of individuals group solution to be measured;

2. be template with prepared genome, carry out pcr amplification with the primer mixture in the test kit;

3. with pcr amplification gained nucleotide fragments, carry out screening with single strand conformation polymorphism method or high pressure liquid chromatograph, by comparing with normal control thing pcr amplification result, screening goes out unusual nucleotide fragments;

4. unusual nucleotide fragments and corresponding normal oligodeoxynucleotide fragment are carried out nucleotide sequencing,, determine mutational site and mutation type by comparing with normal sequence.

Above said single-strand conformation polymorphism analysis method, be to detect one of dna mutation method commonly used.Its principle is: the DNA heat denatured forms two strands, if wherein a strand has coding mutation, the formed three-dimensional structure of this strand is different with corresponding normal strand, when carrying out native polyacrylamide gel electrophoresis simultaneously, cause travelling speed difference to occur so with the normal control thing, electrophoresis band is at same position, thereby determines that it is unusual nucleotide fragments.

As for the high pressure liquid chromatograph analytical method is a kind of novel method that detects dna mutation, and it uses ion chromatography post, and the different of residence time are analyzed to utilize between mutator gene and normal gene trickle base difference to cause them in chromatography column.This analytical procedure has realized the semi-automation of screening process, and detection speed is accelerated greatly.But employed instrumentation costs an arm and a leg, and the corresponding testing cost that increased also is difficult to popularize at present.

Advantage of the present invention and positively effect:

Use test kit of the present invention to detect autosomal dominant polycystic kidney disease, easy and simple to handle, good reproducibility, surveyed area is contained wide, can directly detect the mutational site and the mutation type of Disease-causing gene.And, improved the accuracy rate that detects because the interference of homologous sequence in the I type Disease-causing gene has been avoided in the introducing of long-chain primer and nested primer.Use the high pressure liquid chromatograph screening can obviously improve detection sensitivity, and shorten sense cycle greatly.Test kit of the present invention can be used for the gene diagnosis and the antenatal diagnosis of autosomal dominant polycystic kidney disease family, provides effective way for preventing and treating, break away from Disease-causing gene early in the transmission of family between the generation of getting involved.

Now in conjunction with following 2 embodiment, test kit of the present invention suddenlyd change to detect with the single-strand conformation polymorphism analysis method to elaborate.

Embodiment 5: the Wang, and the male sex, 32 years old, health check-up B ultrasonic diagnosis " bilateral polycystic kidney companion polycystic liver ", its mother and uncle have the polycystic kidney medical history for many years, and father is healthy.

1. the genome solution for preparing personnel to be measured

Collect Wang and its mother's peripheral blood 2ml, the phenol chloroform method prepares its genome respectively routinely, is the solution of 50ng/ μ l with sterilization deionization ultrapure water with its dilution, and it is standby to put 4 ℃ of refrigerators.

2.I the sudden change in type Disease-causing gene non repetitive sequence district detects

(1) pcr amplification: get 17 pcr amplification pipes, be numbered A I-S-66, A I-S-67, A I-S-82, add above-mentioned Wang's genome solution 2 μ l respectively as amplification template, add 23 μ l sterilization deionization ultrapure water more successively, behind the mixing, 95 ℃ 5 minutes, in frozen water, left standstill immediately 3 minutes, add primer mixture I-S-66 successively, I-S-67, each 25 μ l of I-S-82, behind the mixing, 95 ℃ 1 minute, then 95 ℃ 30 seconds, anneal 40 seconds (recommend annealing temperature, down with) referring to table 1,72 ℃ 40 seconds, circulate 35 times, last 72 ℃ 10 minutes, 17 pcr amplification nucleotide fragments, put 4 ℃ standby.By same quadrat method, Wang's maternal gene group, negative control thing and normal control thing are carried out pcr amplification, wherein mother's Wang reaction tubes numbering be respectively B I-S-66, B I-S-67 ..., B I-S-82, the reaction tubes of negative control thing and normal control thing numbering is respectively-I-S-66 ,-I-S-67 ... ,-I-S-82 and+I-S-66 ,+I-S-67 ... ,+I-S-82.

(2) single-strand conformation polymorphism analysis

With above-mentioned amplification gained nucleotide fragments by corresponding numbered packets, promptly sample A I-S-66, B I-S-66 and contrast-I-S-66 and+the I-S-66 volume is one group, weaves into 17 groups successively altogether.With first group is that example is operated as follows:

1. with the nucleotide fragments thermally denature

Get A I-S-66, B I-S-66 ,+I-S-66 and-each 2 μ l of I-S-66, respectively with 10 μ l sample-loading buffer mixings rearmounted 95 ℃ 6 minutes, make the sex change of double chain nucleotide fragment become two strand nucleotide fragments, it is standby to put frozen water;

2. electrophoresis

Prepare 8% non-denaturing polyacrylamide gel according to a conventional method, electrophoretic buffer is the TBE (0.045mol/L Tutofusin tris, 0.045mol/L boric acid and 0.001mol/L ethylenediamine tetraacetic acid (EDTA)) of 0.5 times of dilution, with voltage 300V elder generation prerunning 15 minutes, get each the 6 μ l of sample after the above-mentioned sex change again, point sample respectively, just shifted out gel with the bromjophenol blue dyestuff till.

3. colour developing

Colour developing liquid is done following preparation respectively:

A liquid: get colour developing liquid A 10ml,, standby with distilled water diluting to 100ml.

B liquid: get colour developing liquid B 1ml,, standby with distilled water diluting to 100ml.

C liquid: get colour developing liquid C 0.5ml, to 100ml, it is standby to add colour developing liquid F 120u1 mixing with distilled water diluting.

D liquid: get colour developing liquid D 10ml,, standby with distilled water diluting to 100ml.

E liquid: get colour developing liquid E 10ml,, standby with distilled water diluting to 100ml.

Gel behind the above-mentioned electrophoresis is made following color development treatment in the colour developing liquid that respectively prepares:

Successively gel was soaked 10 minutes in a liquid; In b liquid, soaked 5 minutes, with distilled water rinsing 2 times, each 1 minute; In c liquid, soaked 60 minutes, with distilled water rinsing 2 times, each 1 minute; In d liquid, soaked 5 minutes, treat that the colour developing of tawny band is clear; In e liquid, soaked 5 minutes; Used the distilled water immersion rinsing at last 30 minutes, and took a picture again.Each is organized The selection result and finds, A I-S-77 respectively has the different (see figure 4)s with the migration position of normal control thing+I-S-77 of electrophoresis band with B I-S-77, illustrate that nucleotide fragments is unusual, so these two samples and normal control thing+I-S-77 thereof are carried out nucleotide sequencing.

(3) nucleotide sequencing: the nucleotide sequencing result is: normal control thing+I-S-77 nucleotides sequence is classified as-GGGTAGCCTACGCCC AGCTGGCCAT-, and A I-S-77 and B I-S-77 nucleotides sequence are classified as-GGGTAGCCTACGCCC GGCTGGCCAT-, replacement mutation all takes place for both to this shows the back: promptly the VITAMIN B4 A of the 50906th of I type Disease-causing gene group sports guanine G, thereby the autosomal dominant polycystic kidney disease of determining Wang and mother thereof is because the sudden change of I type Disease-causing gene causes, the mutational site is in its 44th exon, and mutation type is the caused missense mutation of base substitution.

Embodiment 6: Zhao, women, 28 years old, conceived 3 months.Health check-up B ultrasonic diagnosis " bilateral polycystic kidney companion polycystic liver ", the affine uncle of his father has the polycystic kidney medical history for many years, mother is healthy, now utilize test kit of the present invention to detect its autosomal dominant polycystic kidney disease Disease-causing gene mutational site and mutation type, and conceived fetus carried out antenatal diagnosis, whether suffer from the possibility of genotype multicystic kidney disease.

1. the preparation of personnel's genome solution to be measured

Zhao is identical with embodiment 5 with the genomic preparation method of its father.Detection to fetus prepares its genome with the fetus cast-off cells in the amniotic fluid.

Collect fetus amniotic fluid 1.5ml at least by the amniocentesis approach, abandon supernatant after centrifugal 5 minutes, be precipitated as the cast-off cells of fetus.With 10mmol/L tris solution (containing the 1mmol/L disodium ethylene diamine tetraacetate) washing precipitation, add 10 μ l 0.1mol/L sodium hydroxide and 2mol/L sodium-chlor mixing solutions, and shake up, 100 ℃ of boiling water boiled 2 minutes, centrifugal 10 minutes of strong again vibration back, get supernatant liquor, quantitatively and with sterilization deionization ultrapure water it is diluted to the genome solution that concentration is 50ng/ μ l, put in 4 ℃ of refrigerators standby through ultraviolet spectrophotometer.

2.I the detection in type Disease-causing gene non repetitive sequence district

Detection method is with embodiment 5, and detected result three there is no unusually, so carry out the detection of II type Disease-causing gene.

3.II the sudden change of type Disease-causing gene detects

(1) pcr amplification: get 17 pcr amplification pipes, be numbered A II-SH-1, A II-SH-2 ..., A II-SH-17, add above-mentioned Zhao's genome solution 2 μ l respectively as amplification template, add 23 μ l sterilization deionization ultrapure water more successively, behind the mixing, 95 ℃ 5 minutes, in frozen water, left standstill immediately 3 minutes.Add successively primer mixture II-SH-1, II-SH-2 ..., each 25 μ l of II-SH-17, behind the mixing, 95 ℃ 1 minute, 95 ℃ 30 seconds, anneal and (recommended annealing temperature in 40 seconds referring to table 3, down together), 72 ℃ 40 seconds, circulate 35 times, last 72 ℃ 10 minutes, pcr amplification nucleotide fragments 17 pipe, put 4 ℃ standby.Same quadrat method, carry out the pcr amplification of father's Zhao genome, Zhao's fetus genome, negative control thing and normal control thing, wherein father Zhao PCR product be numbered B II-SH-1, B II-SH-2 ..., B II-SH-17, Zhao fetus PCR product be numbered C II-SH-1, C II-SH-2 ..., C II-SH-17, the numbering of negative control thing and normal control thing is respectively-II-SH-1 ,-II-SH-2 ... ,-II-SH-17 and+II-SH-1 ,+II-SH-2 ... ,+II-SH-17.

(2) single-strand conformation polymorphism analysis

Analytical procedure is with embodiment 5, and the result is the no abnormality seen nucleotide fragments also, so at last I type Disease-causing gene tumor-necrosis factor glycoproteins district is detected.

4.I the sudden change in type Disease-causing gene tumor-necrosis factor glycoproteins district (comprising that exons 1 is to exon 3 3) detects

(1) long-chain pcr amplification:

1. the long-chain pcr amplification of exons 1: get the pcr amplification pipe of 1 sterilization, be numbered A I-S-1, add Zhao's genome solution 16 μ l, replenish volume to 25 μ l with sterilization deionization ultrapure water, behind the mixing, 98 ℃ 5 minutes, in frozen water, left standstill 3 minutes immediately.Add primer mixture I-S-1 25 μ l, behind the mixing, 95 ℃ 1 minute, 95 ℃ 30 seconds, annealed 40 seconds, 72 ℃ 40 seconds, circulate 35 times, last 72 ℃ 10 minutes, long-chain pcr amplification product A I-S-1 put 4 ℃ standby.

Get father's Zhao pcr amplification product B I-S-1 by the same method respectively, the pcr amplification product C I-S-1 of fetus, the pcr amplification product+I-S-1 of the pcr amplification product-I-S-1 of negative control thing and normal control thing.

2. exon 2 is to the long-chain pcr amplification of exon 8: the pcr amplification pipe of getting 7 sterilizations, numbering is respectively AI-S-2 to A I-S-8, each adds Zhao's genomic templates solution 10 μ l, replenish volume to 40 μ l with sterilization deionization ultrapure water, behind the mixing, 98 ℃ 5 minutes, in frozen water, left standstill immediately 3 minutes.Add each 50 μ l of primer mixture I-S-2 to I-S-8 respectively to each pipe successively, behind the mixing, 95 ℃ 1 minute, 95 ℃ 30 seconds, annealed 40 seconds, 72 ℃ 40 seconds, circulate 35 times, last 72 ℃ 10 minutes, pcr amplification product A I-S-2 to A I-S-8, put 4 ℃ standby.

By the same method, respectively father's Zhao pcr amplification product B I-S-2 to B I-S-8, pcr amplification product C I-S-2 to the C I-S-8 of fetus, the pcr amplification product-I-S-2 of negative control thing is to the pcr amplification product+I-S-2 of-I-S-8 and normal control thing to+I-S-8.

(2) nest-type PRC amplification:

1. the nest-type PRC of exons 1 amplification: the pcr amplification pipe of getting 3 sterilizations, be numbered A I-S-9, A I-S-10, A I-S-11, add Zhao A I-S-1 long-chain pcr amplification product 1 μ l respectively, replenish volume to 25 μ l with sterilization deionization ultrapure water, behind the mixing, 95 ℃ 5 minutes, in frozen water, left standstill immediately 3 minutes.Add each 25 μ l of primer mixture I-S-9, I-S-10, I-S-11 successively, behind the mixing, 95 ℃ 1 minute, 95 ℃ 30 seconds, annealed 40 seconds, 72 ℃ 40 seconds, circulate 30 times, last 72 ℃ 10 minutes, respectively pcr amplification product A I-S-9, A I-S-10, A I-S-11, put 4 ℃ standby.

By the same method, get father's Zhao pcr amplification product B I-S-9, B I-S-10, B I-S-11 respectively, pcr amplification product C I-S-9, the C I-S-10 of fetus, C I-S-11, the pcr amplification product-I-S-9 of negative control thing ,-I-S-10 ,-pcr amplification product+I-S-9 of I-S-11 and normal control thing ,+I-S-10 ,+I-S-11

The operation steps of the nest-type PRC amplification of other exon of tumor-necrosis factor glycoproteins district is that different templates need be selected corresponding PCR primer for use with above-mentioned exons 1.Its corresponding relation is as follows:

I-S-12 to I-S-19 is selected in the I-S-2 amplification for use, and I-S-20 to I-S-26 is selected in the I-S-3 amplification for use,

I-S-27 to I-S-44 is selected in the I-S-4 amplification for use, and I-S-45 to I-S-52 is selected in the I-S-5 amplification for use,

I-S-53 to I-S-59 is selected in the I-S-7 amplification for use, and I-S-60 to I-S-65 is selected in the I-S-8 amplification for use.

The numbering of nest-type PRC amplified production is respectively: Zhao for A I-S-12, A I-S-13 ..., A I-S-65, father Zhao be B I-S-12, B I-S-13 ..., B I-S-65, Zhao fetus be C I-S-12, C I-S-13 ..., C I-S-65, negative control thing-I-S-12 ,-I-S-13 ... ,-I-S-65, normal control thing+I-S-12 ,+I-S-13 ... ,+I-S-65.

If adopt high pressure liquid chromatograph to detect, then corresponding nest-type PRC amplimer is:

I-H-9 to I-H-11 is selected in the I-H-1 amplification for use, and I-H-12 to I-H-18 is selected in the I-H-2 amplification for use,

I-H-19 to I-H-26 is selected in the I-H-3 amplification for use, and I-H-27 to I-H-39 is selected in the I-H-4 amplification for use,

I-H-40 to I-H-47 is selected in the I-H-5 amplification for use, and I-H-48 to I-H-54 is selected in the I-H-7 amplification for use,

I-H-55 to I-H-59 is selected in the I-H-8 amplification for use.

Because the pcr amplification product fragment of I-S-6 or I-H-6 is shorter, only is 300bp, therefore needn't carry out the amplification of next round nest-type PRC, can carry out single-strand conformation polymorphism analysis or high pressure liquid chromatograph analysis with the above-mentioned nest-type PRC product that obtains.

(3) single-strand conformation polymorphism analysis

With above-mentioned nest-type PRC product by corresponding numbered packets, for example A I-S-9, B I-S-9, C I-S-9 ,+I-S-9 and-it is one group that I-S-9 compiles, and is divided into into 57 groups, carries out single-strand conformation polymorphism analysis respectively, working method is with embodiment 5.Found that wherein there are unusual banding pattern in Zhao's A I-S-30, father's Zhao B I-S-30, so and Zhao's the normal (see figure 5) of fetus C I-S-30 banding pattern to A I-S-30, B I-S-30, C I-S-30 and+sample of I-S-30 carries out nucleotide sequencing respectively.

(4) nucleotide sequencing: the nucleotide sequencing result is: the nucleotides sequence of+I-S-30 and C I-S-30 is classified as-CTACCACGTGC GCCTGGAGGTCAA-, and A I-S-30, B I-S-30 are-CTACCACGTGCCTGGAGGTCAA-, comparison result, begin to lack two bases G C the 27678th of autosomal dominant polycystic kidney disease I type Disease-causing gene group, phase shift mutation all takes place in both to this shows the back, thereby the autosomal dominant polycystic kidney disease of determining Zhao and father thereof is because the sudden change of I type Disease-causing gene causes, the mutational site is at its 15th exon, and mutation type is the caused phase shift mutation of base deletion.And C I-S-30 sequence is normal, illustrates that fetus do not carry mother Zhao's autosomal dominant polycystic kidney disease sudden change Disease-causing gene.

Below by an embodiment, test kit of the present invention suddenlyd change to detect with high pressure liquid chromatograph to be described in detail.

Embodiment 7: Lee, and the male sex, 62 years old, health check-up B ultrasonic diagnosis " bilateral polycystic kidney " had multicystic kidney disease history family history before 2 years.

1. the preparation of the preparation of genome solution, long-chain and nest-type PRC amplified production is selected primer difference with embodiment 5, the numbering of its amplified production be respectively A I-H-60, A I-H-61 ..., A I-H-72.The numbering of normal control thing pcr amplification product is respectively+I-H-60 ,+I-H-61 ... ,+I-H-72.

2. high pressure liquid chromatograph analysis

1. get the pcr amplification pipe of 26 sterilizations, numbering be respectively A I-H-60 to A I-H-72 and+I-H-60 is to+I-H-72, after adding each 10 μ l of pcr amplification product of above-mentioned identical numbering successively, follow these steps to carry out denatured pretreatment: 95 ℃ 3 minutes, slowly cool to 65 ℃, the temperature lowering speed is controlled at 0.1 ℃/second; 65 ℃ 30 minutes; Continue slowly to be cooled to 37 ℃, left standstill 10 minutes, and then sample and contrast organized into groups one to one by corresponding numbering, for example A I-H-60 and+I-H-60 is one group, all go up sample respectively to high pressure liquid chromatograph (U.S. Transgenomic company, WAVE nucleotide analysis system).Have and shut the operation of sample-wash-out-equilibrated and undertaken by its process specifications that provides.The all no abnormal peak of result type, thus the autosomal dominant polycystic kidney disease that can determine Lee is not because I type Disease-causing gene non repetitive sequence region mutation causes.

3.II the sudden change of type Disease-causing gene detects

(1) with II-SH-1 to II-SH-17 primer II type Disease-causing gene is carried out pcr amplification, its process is with embodiment 6, the amplified production of gained Lee and normal control thing numbering be respectively A II-SH-1, A II-SH-2 ..., A II-SH-17 and+II-SH-1 ,+II-SH-2 ... ,+II-SH-17.

(2) it is then the same to carry out the high pressure liquid chromatograph analysis.The peak type that detects found that referring to Fig. 6 the anomaly peak type appears in A II-SH-8, then A II-SH-8 and normal control thing+II-SH-8 is carried out nucleotide sequencing.

(3) nucleotide sequencing.The nucleotide sequencing result is: the nucleotides sequence of normal control thing+II-SH-8 is classified as-GGAAATT CGCATTCACAAACTA-, and A II-SH-8 is-GGAAATTGCATTCACAAACTA-, comparison result, base C of the 202nd disappearance in autosomal dominant polycystic kidney disease II type Disease-causing gene group exon 6, this shows that phase shift mutation takes place the latter, thereby determine that Lee's autosomal dominant polycystic kidney disease is that mutation type is the caused phase shift mutation of base deletion because the sudden change of II type Disease-causing gene causes, and the mutational site is at its 6th exon.

Table 1 single strand conformation polymorphism detects the nucleotide sequence of autosomal dominant polycystic kidney disease I type Disease-causing gene mutant primer

The primer mixture code name	The primer numbering	Nucleotide sequence		Annealing temperature	The exon numbering
						Forward primer	Reverse primer
		I-S-1	1					5’-AGCGCAACTACTTGGAGGCCC-3’	5’-CCACCTCATCGCCCCTTCCTAAGCAT-3’	69℃	1
I-S-2	2			5’-ATTTTTTGAGATGGAGCTTCACTCTTGCAGG-3’	5’-CGCTCGGCAGGCCCCTAACC-3’	68℃	2-7
		I-S-3	3					5’-CCGCCCCCAGGAGCCTAGACG-3’	5’-CATCCTGTTCATCCGCTCCACGGTTAC-3’	68℃	8-12
I-S-4	4			5’-TGGAGGGAGGGACGCCAATC-3’	5’-GTCAACGTGGGCCTCCAAGT-3’	68℃	13-15
		I-S-5	5					5’-AGCGCAACTACTTGGAGGCCC-3’	5’-GCAGGGTGAGCAGGTGGGGCCATCCTA3’	70℃	15-21
I-S-6	6			5’-GAGGCTGTGGGGGTCCAGTCAAGTGG-3’	5’-AGGGAGGCAGAGGAAAGGGCCGAAC-3’	72℃	22
		I-S-7	7					5’-CCCCGTCCTCCCCGTCCTTTTGTC-3’	5’-AAGCGCAAAAGGGCTGCGTCG-3’	69℃	23-28
I-S-8	8			5’-GGCCCTCCCTGCCTTCTAGGCG-3’	5’-GTTGCAGCCAAGCCCATGTTA-3’	68℃	29-34
		I-S-9	9					5’-GGTCGCGCTGTGGCGAAGG-3’	5’-CGGCGGGCGGCATCGT-3’	68℃	1
I-S-10	10			5’-CCTGAGCTGCGGCCTCCG-3’	5’-CAGTTGACGCGGCAGGCG-3’	66℃	1
		I-S-11	11					5’-TGCGAGCCCCCCTGCCTC-3’	5’-AACCCGCCCACGCCCGCCCGTCC3’	66℃	1
I-S-12	12			5’-CTTGGGGATGCTGGCAATG-3’	5’-GGGATTCGGCAAAGCTGATG-3’	62℃	2
		I-S-13	13					5’-CCATCAGCTTTGCCGAATCC-3’	5’-AACTGGGAGGGCAGAAGGG-3’	62℃	3
I-S-14	14			5’-AGTGGGGGGCTGGCATAGAC-3’	5’-TGAGCCCTGCCCAGTGTCT-3’	62℃	4
		I-S-15	15					5’-GAGCCAGGAGGAGCAGAACCC-3’	5’-AGAGGGACAGGCAGGCAAAGG-3’	65℃	5
I-S-16	16			5’-CCCAGCCCTCCAGTGCCT-3’	5’-CCCAGGCAGCACATAGCGAT-3’	65℃	5
		I-S-17	17					5’-CCGAGGTGGATGCCGCTG-3’	5’-AACGAGGGTGTCAACGGTCAG-3’	65℃	5
I-S-18	18			5’-ACCGTTGACACCCTCGTTCC-3’	5’-TCTCTGCCCCAGTGCTTCAG-3’	64℃	6
		I-S-19	19					5’-CTGTGAGGGTGGGAGGATGG-3’	5’-CGCTCGGCAGGCCCCTAACC-3’	56℃	7
I-S-20	20			5’-TCTGTTCGTCCTGGTGTCCTG-3’	5’-GGAGGGCAGGTTGTAGAACGTG-3’	64℃	8
		I-S-21	21					5’-GGTAGGGGGAGTCTGGGCTT-3’	5’-GAGGCCACCCCGAGTCC-3’	64℃	9
I-S-22	22			5’-GTTGGGCATCTCTGACGGTG-3’	5’-GGAAGGTGGCCTGAGGAGAT-3’	64℃	10
		I-S-23	23					5’-GGGGTCCACGGGCCATG-3’	5’-AAGCCCAGCAGCACGGTGAG-3’	64℃	11
I-S-24	24			5’-GCTTGCAGCCACGGAAC-3’	5’-GCAGTGCTACCACTGAGAAC-3’	64℃	11
		I-S-25	25					5’-TGCCCCTGGGAGACCAACGATAC-3’	5’-GGCTGCTGCCCTCACTGGGAAG-3’	67℃	11
I-S-26	26			5’-GAGGCGACAGGCTAAGGG-3’	5’-CATGAAGCAGAGCAGAAGGC-3’	64℃	12
		I-S-27	27					5’-TGGAGGGAGGGACGCCAATC-3’	5’-GAGGCTGGGGCTGGGACAAG-3’	67℃	13
I-S-28	28			5’-CCCGGTTCACTCACTGCG-3’	5’-CCGTGCTCAGAGCCTGAAAG-3’	64℃	14
		I-S-29	29					5’-CGGGTGGGGAGCAGGTGG-3’	5’-GCTCTGGGTCAGGACAGGGGA-3’	67℃	15
I-S-30	30			5’-CGCCTGGGGGTGTTCTTT-3’	5’-CACATGCTCCACTGTTGCCTCC-3’	64℃	15
		I-S-31	31					5’GCCCCCGTGGTGGTCAGC-3’	5’-CAGGCTGCGTGGGGATGC-3’	67℃	15
I-S-32	32			5’-CTGGAGGTGCTGCGCGTT-3’	5’-CTGGCTCCACGCAGATGC-3’	67℃	15
		I-S-33	33					5’-CGTGAACAGGGCGCATTA-3’	5’-GCAGCAGAGATGTTGTTGGAC-3’	65℃	15
I-S-34	34			5’-CCAGGCTCCTATCTTGTGACA-3’	5’-TGAAGTCACCTGTGCTGTTGT-3’	60℃	15
		I-S-35	35					5’-CTACCTGTGGGATCTGGGG-3’	5’-TGCTGAAGCTCACGCTCC-3’	67℃	15
I-S-36	36			F：5’-GGGCTCGTCGTCAATGCAAG-3’	5’-CACCACCTGCAGCCCCTCTA-3’	67℃	15
		I-S-37	37					5’-CCGCCCAGGACAGCATCTTC-3’	5’-CGCTGCCCAGCATGTTGG-3’	64℃	15
I-S-38	38			5’-CGGCAAAGGCTTCTCGCTC-3’	5’-CCGGGTGTGGGGAAGCTATG-3’	64℃	15
		I-S-39	39					5’-CGAGCCATTTACCACCCATAG-3’	5’-GCCCAGCACCAGCTCACAT-3’	65℃	15

I-S-40	40	5’-CCACGGGCACCAATGTGAG-3’	5’-GGCAGCCAGCAGGATCTGAA-3’	64℃	15
						I-S-41	41	5’-CAGCAGCAAGGTGGTGGC-3’	5’-GCGTAGGCGACCCGAGAG-3’	67℃	15
I-S-42	42	5’-ACGGGCACTGAGAGGAACTTC-3’	5’-ACCAGCGTGCGGTTCTCACT-3’	64℃	15
						I-S-43	43	5’-GCCGCGACGTCACCTACAC-3’	5’-TCGGCCCTGGGCTCATCT-3’	67℃	15
I-S-44	44	5’-GTCGCCAGGGCAGGACACAG-3’	5’-GTCAACGTGGGCCTCCAAGT-3’	68℃	15
						I-S-45	45	5’-AGCGCAACTACTTGGAGGCCC-3’	5’-TGATGGGCACCAGGCGCTC-3’	69℃	15
I-S-46	46	5’-CATCCAGGCCAATGTGACGGT-3’	5’-CCTGGTGGCAAGCTGGGTGTT-3’	64℃	15
						I-S-47	47	5’-TAAAACTGGATGGGGCTCTC-3’	5’-GGCCTCCACCAGCACTAA-3’	56℃	16
I-S-48	48	5’-GGGTCCCCCAGTCCTTCCAG-3’	5’-TCCCCAGCCCGCCCACA-3’	67℃	17
						I-S-49	49	5’-GCCCCCTCACCACCCCTTCT-3’	5’-TCCCGCTGCTCCCCCCAC-3’	67℃	18
I-S-50	50	5’-GATGCCGTGGGGACCGTC-3’	5’-GTGAGCAGGTGGCAGTCTCG-3’	67℃	19
						I-S-51	51	5’-CCACCCCCTCTGCTCGTAGGT-3’	5’-GGTCCCAAGCACGCATGCA-3’	64℃	20
I-S-52	52	5’-TGCCGGCCTCCTGCGCTGCTGA-3’	5’-GCAGGGTGAGCAGGTGGGGCCATCCTA-3’	67℃	21
						I-S-53	53	5’-CTGCACTGACCTCACGCATGT-3’	5’-GCCAAAGGGAAAGGGATTGGA-3’	62℃	23
I-S-54	54	5’-GCTCATCTTTCTGGTGG-3’	5’-CCCCCAAGAACAAGGC-3’	56℃	23
						I-S-55	55	5’-TATGCTTTCAGGCCCGTGGCA-3’	5’-AGAGCCCATACCCGGTCCAGTCC-3’	62℃	24
I-S-56	56	5’-GGACTGGACCGGGTATGGGCTCT-3’	5’-CACCCAGGCCCTCCTCGACTC-3’	62℃	25
						I-S-57	57	5’-CTCTATCCTGAGAAGGC-3’	5’-TGAGAGCAGGGGAGGC-3’	62℃	26
I-S-58	58	5’-CAGGCCAAAGCTGAGATGACTTG-3’	5’-AGAGGCGCAGGAGGGAGGTC-3’	62℃	27
						I-S-59	59	5’-CCCTCTGCCCCCGCATTG-3’	5’-AAGCGCAAAAGGGCTGCGTCG-3’	62℃	28
I-S-60	60	5’-TCCGTGGGAGGTTGGG-3’	5’-GCCACACAGGTGAGGC-3’	64℃	29
						I-S-61	61	5’-CCTCTTCCTGCCCAGCCCTTC-3’	5’-CTTCCCGAGCAGCCTTTGGTG-3’	62℃	30
I-S-62	62	5’-GTCCCATATATCCAGCATTCT-3’	5’-ACAGTGTCTTGAGTCCAAGC-3’	56℃	31
						I-S-63	63	5’-GCCTTGGCGCAGCTTGGACT-3’	5’-ACACCCAGCAAGGACACGCA-3’	65℃	32
I-S-64	64	5’-GGTTTGCTCGGAAGCCC-3’	5’-TGAGCTTCAGAGCCCCCTCCTC-3’	61℃	33
						I-S-65	65	5’-TGCAGCTGGGCCCACCCT-3’	5’-CTGGGGATCCCATGAGGC-3’	60℃	34
I-S-66	66	5’-TCAAGAAACTGCCCGCC-3’	5’-GGGGCTACGCAAGCAC-3’	64℃	35
						I-S-67	67	5’-GACAGGTGTGCTTGCGTAGCC-3’	5’-GATGGAGGCCTGTAGCCTACCCC-3’	66℃	36
I-S-68	68	5’-GCTGTGGCTGTGGCTGTCTC-3’	5’-CGGGCTCTCTACCAGGGTGTC-3’	66℃	36-37
						I-S-69	69	5’-GGTCTTGCTGGAAGCCCTGTAC-3’	5’-CATGCCATGTAGCCTCTTGACC-3’	64℃	37
I-S-70	70	5’-CACCCCACGGCTTTGCAC-3’	5’-CTTTGCAGACGGTAGGCGTG-3’	66℃	37-38
						I-S-71	71	5’-CATGCTTTTTCTGCTGGTGACC3’	5’-GCTCTGGGCTGGACTGGTTC-3’	66℃	38-39
I-S-72	72	5’-CGTGCTGCTGCCCTACGTC-3’	5’-CGTCCCCGAGCCATTGTG-3’	66℃	39-40
						I-S-73	73	5’-TTCAGCACCAGCGATTACGAC-3’	5’-CCTGTTGTCCAGCCAGTTGTG-3’	60℃	40-41
I-S-74	74	5’-AGCTGCACAACTGGCTGGAC3’	5’-CCGAGGTGAGCAGAGGCAG-3’	64℃	41-42
						I-S-75	75	5’-AGGTGTGCCTGCTGCTGTT-3’	5’-CCACCTGGTGAAGCTAGTG-3’	60℃	43
I-S-76	76	5’-CTGACCGCCAGTGGACCC-3’	5’-TCGGCATAATGTCTTGCCAAAG-3’	60℃	43-44
						I-S-77	77	5’-CCAGTGGTCCGTCTTTGG-3’	5’-GGGGTGACAGGTGCCAGGAC-3’	64℃	44-45
I-S-78	78	5’-TCTACCCTGTGGTCCTGCCGAG-3’	5’-AGGAACAACTCCACCATCTCGTAG-3’	60℃	45
						I-S-79	79	5’-GGCTGGGGGCTGTTATTCTC-3’	5’-TGGAGGAGCGAGAGGGCAG-3’	66℃	45-46
I-S-80	80	5’-TCCGCTTTGAAGGGATGGAG-3’	5’-GGGAGGGCTCAGGCTCACAC-3’	66℃	46
						I-S-81	81	5’-GGGACAAGGTGTGAGCCTGAG-3’	5’-CAAGGCGGCTGGGCAGTG-3’	68℃	46
I-S-82	82	5’-GATCTTCCCGTGGCCCAT-3’	5’-GTGTCCACTCCGACTCCA-3’	58℃	46

Table 2 high pressure liquid chromatograph detects the nucleotide sequence of autosomal dominant polycystic kidney disease I type Disease-causing gene mutant primer

The primer mixture code name	The primer numbering	Nucleotide sequence		Annealing temperature	The exon numbering
						Forward primer	Reverse primer
		I-H-1	?1					5’-AGCGCAACTACTTGGAGGCCC-3’	5’-CCACCTCATCGCCCCTTCCTAAGCAT-3’	69℃	1
I-H-2	?2			5’-ATTTTTTGAGATGGAGCTTCACTCTTGCAGG-3’	5’-CGCTCGGCAGGCCCCTAACC-3’	68℃	2-7
		I-H-3	?3					5’-CCGCCCCCAGGAGCCTAGACG-3’	5’-CATCCTGTTCATCCGCTCCACGGTTAC-3’	68℃	8-12
I-H-4	?4			5’-TGGAGGGAGGGACGCCAATC-3’	5’-GTCAACGTGGGCCTCCAAGT-3’	68℃	13-15
		I-H-5	?5					5’-AGCGCAACTACTTGGAGGCCC-3’	5’-GCAGGGTGAGCAGGTGGGGCCATCCTA-3’	70℃	15-21
I-H-6	?6			5’-GAGGCTGTGGGGGTCCAGTCAAGTGG-3’	5’-AGGGAGGCAGAGGAAAGGGCCGAAC-3’	72℃	22
		I-H-7	?7					5’-CCCCGTCCTCCCCGTCCTTTTGTC-3’	5’-AAGCGCAAAAGGGCTGCGTCG-3’	69℃	23-28
I-H-8	?8			5’-GGCCCTCCCTGCCTTCTAGGCG-3’	5’-GTTGCAGCCAAGCCCATGTTA-3’	68℃	29-34
		I-H-9	?9					5’-GGTCGCGCTGTGGCGAAGG-3’	5’-CGGCGGGCGGCATCGT-3’	68℃	1
I-H-10	?10			5’-CCTGAGCTGCGGCCTCCG-3’	5’-CAGTTGACGCGGCAGGCG-3’	66℃	1
		I-H-11	?11					5’-TGCGAGCCCCCCTGCCTC-3’	5’-AACCCGCCCACGCCCGCCCGTCC3’	66℃	1
I-H-12	?12			5’-TAGGGGCTCTGGCCCTGAC-3’	5’-CCAGCCAGGACCCCACCCAAAG-3’	55℃	2-3
		I-H-13	?13					5’-AGTGGGGGGCTGGCATAGAC-3’	5’-TGAGCCCTGCCCAGTGTCT-3’	62℃	4
I-H-14	?14			5’-GAGCCAGGAGGAGCAGAACCC-3’	5’-AGAGGGACAGGCAGGCAAAGG-3’	65℃	5
		I-H-15	?15					5’-CCCAGCCCTCCAGTGCCT-3’	5’-CCCAGGCAGCACATAGCGAT-3’	65℃	5
I-H-16	?16			5’-CCGAGGTGGATGCCGCTG-3’	5’-AACGAGGGTGTCAACGGTCAG-3’	65℃	5
		I-H-17	?17					5’-ACCGTTGACACCCTCGTTCC-3’	5’-TCTCTGCCCCAGTGCTTCAG-3’	64℃	6
I-H-18	?18			5’-CTGTGAGGGTGGGAGGATGG-3’	5’-CGCTCGGCAGGCCCCTAACC-3’	56℃	7
		I-H-19	?19					5’-TCTGTTCGTCCTGGTGTCCTG-3’	5’-GGAGGGCAGGTTGTAGAACGTG-3’	64℃	8
I-H-20	?20			5’-GGTAGGGGGAGTCTGGGCTT-3’	5’-CTGGGAACCACTCTGGTGGC-3’	64℃	9
		I-H-21	?21					5’-GGTAGGGGGAGTCTGGGCTT-3’	5’-CACCCACCACCCAGAGTCCC-3’	64℃	9
I-H-22	?22			5’-GTTGGGCATCTCTGACGGTG-3’	5’-GGAAGGTGGCCTGAGGAGAT-3’	64℃	10
		I-H-23	?23					5’-GGGGTCCACGGGCCATG-3’	5’-AAGCCCAGCAGCACGGTGAG-3’	64℃	11
I-H-24	?24			5’-GCTTGCAGCCACGGAAC-3’	5’-GCAGTGCTACCACTGAGAAC-3’	64℃	11
		I-H-25	?25					5’-TGCCCCTGGGAGACCAACGATAC-3’	5’-GGCTGCTGCCCTCACTGGGAAG-3’	67℃	11
I-H-26	?26			5’-GAGGCGACAGGCTAAGGG-3’	5’-CATGAAGCAGAGCAGAAGGC-3’	64℃	12
		I-H-27	?27					5’-TGGAGGGAGGGACGCCAATC-3’	5’-GAGGCTGGGGCTGGGACAAG-3’	67℃	13
I-H-28	?28			5’-CCCGGTTCACTCACTGCG-3’	5’-CCGTGCTCAGAGCCTGAAAG-3’	64℃	14
		I-H-29	?29					5’-TGGGGAGCAGGTGGGGGTGC-3’	5’-AGACGCGCACATCCGCCTGGGCCG-3’	64℃	15
I-H-30	?30			5’-CGTGCGCCTGGAGGTCAAC-3’	5’-GGCTGCGTGGGGATGCAG-3’	68℃	15
		I-H-31	?31					5’-CGTGCTGGTCTTCGTCCTGG-3’	5’-TGTAGCGGTAGGGGAACGG-3’	64℃	15
I-H-32	?32			5’-GTTTGTGCAGCTCGGGGAC-3’	5’-AAGCGTGGGTGACCTCCG-3’	65℃	15
		I-H-33	?33					5’-CCCGCCAGCTACCTGTGG-3’	5’-GCGGAGCCCACCTCGTTC-3’	66℃	15
I-H-34	?34			5’-CTTCCGCTCCGTGGGCAC-3’	5’-GGAGGCGGCCACCATCAG-3’	65℃	15
		I-H-35	?35					5’-AGCGCCTGGGCCGACTGCAC-3’	5’-AGCTGCCCCCAAAAGGGC-3	65℃	15
I-H-36	?36			5’-GAGCCCGGAGGCAGCTTC-3’	5’-GGGAGCACCTCGGGGTTG-3’	66℃	15
		I-H-37	?37					5’-AGCTGTCACCTTCCGCCTG-3’	5’-GCACCTGGATCTCCAACAGCC-3’	67℃	15
I-H-38	?38			5’-GCTGGTCATCCTGTCGGGC-3’	5’-CACCAGGTTGGAGGCGTTC-3’	66℃	15
		I-H-39	?39					5’-CCAGGGCCGAGCACTCCTAC-3’	5’-GTCAACGTGGGCCTCCAAGT-3’	67℃	15
I-H-40	?40			5’-AGCGCAACTACTTGGAGGCCC-3’	5’-TGGGGTCGTAGGACTCGCTC-3’	66℃	15

I-H-41	41	5’-CGCCTGGTGCCCATCATTG-3’	5’-GGACGGGTGAGGGGCATG-3’	66℃	15
						I-H-42	42	5’-TAAAACTGGATGGGGCTCTC-3’	5’-GGCCTCCACCAGCACTAA-3’	56℃	16
I-H-43	43	5’-GGGTCCCCCAGTCCTTCCAG-3’	5’-TCCCCAGCCCGCCCACA-3’	67℃	17
						I-H-44	44	5’-GCCCCCTCACCACCCCTTCT-3’	5’-TCCCGCTGCTCCCCCCAC-3’	67℃	18
I-H-45	45	5’-GATGCCGTGGGGACCGTC-3’	5’-GTGAGCAGGTGGCAGTCTCG-3’	67℃	19
						I-H-46	46	5’-CCACCCCCTCTGCTCGTAGGT-3’	5’-GGTCCCAAGCACGCATGCA-3’	64℃	20
I-H-47	47	5’-TGCCGGCCTCCTGCGCTGCTGA-3’	5’-GCAGGTGAGCAGGTGGGGCCATCCTA-3’	67℃	21
						I-H-48	48	5’-CTGCACTGACCTCACGCATGT-3’	5’-GCCAAAGGGAAAGGGATTGGA-3’	62℃	23
I-H-49	49	5’-GCTCATCTTTCTGGTGG-3’	5’-CCCCCAAGAACAAGGC-3’	56℃	23
						I-H-50	50	5’-TATGCTTTCAGGCCCGTGGCA-3’	5’-AGAGCCCATACCCGGTCCAGTCC-3’	62℃	24
I-H-51	51	5’-GGACTGGACCGGGTATGGGCTCT-3’	5’-CACCCAGGCCCTCCTCGACTC-3’	62℃	25
						I-H-52	52	5’-CTCTATCCTGAGAAGGC-3’	5’-TGAGAGCAGGGGAGGC-3’	62℃	26
I-H-53	53	5’-CAGGCCAAAGCTGAGATGACTTG-3’	5’-AGAGGCGCAGGAGGGAGGTC-3’	62℃	27
						I-H-54	54	5’-CCCTCTGCCCCCGCATTG-3’	5’-AAGCGCAAAAGGGCTGCGTCG-3’	62℃	28
I-H-55	55	5’-TCCGTGGGAGGTTGGG-3’	5’-GCCACACAGGTGAGGC-3’	64℃	29
						I-H-56	56	5’-CCTCTTCCTGCCCAGCCCTTC-3’	5’-CTTCCCGAGCAGCCTTTGGTG-3’	62℃	30
I-H-57	57	5’-GAGCAGGTCTGAGCTGCCG-3’	5’-GCACCAGGGCTCGAGGTTTC-3’	62℃	31-32
						I-H-58	58	5’-GGTTTGCTCGGAAGCCC-3’	5’-TGAGCTTCAGAGCCCCCTCCTC-3’	61℃	33
I-H-59	59	5’-TGCAGCTGGGCCCACCCT-3’	5’-CTGGGGATCCCATGAGGC-3’	60℃	34
						I-H-60	60	5’-TCAAGAAACTGCCCGCC-3’	5’-GGGGCTACGCAAGCAC-3’	64℃	35
I-H-61	61	5’-GACAGGTGTGCTTGCGTAGCC-3’	5’-GATGGAGGCCTGTAGCCTACCCC-3’	66℃	36
						I-H-62	62	5’-TCCATCACGGGGGACCCCTCT-3’	5’-AAAGGGGGACAGGAGTGGTCCT-3’	65℃	37
I-H-63	63	5’-AAAGCCCTGCTGTCACTGTGG-3’	5’-TAGGGTCTGGCTGGACTAAAG-3’	65℃	38
						I-H-64	64	5’-GGGTCTCTGGTGGCCGCTCA-3’	5’-ATGCCAGAGCTCCGCTAAAGG-3’	66℃	39
I-H-65	65	5’-CACTCCTGTTCCCTTTTGATG-3’	5’-CGGCACTCCTGGAGAACTACT-3’	65℃	40
						I-H-66	66	5’-CGGCCTCCTGACCAGCCTGGCTC-3’	5’-TAGGCCAGCGGGGGCCGGAGGAGTG-3’	66℃	41
I-H-67	67	5’-TGCCACCCGCTCCTACTGA-3’	5’-TGGAGGCGCGGGGTCT-3’	70℃	42
						I-H-68	68	5’-CGTCCCTCCCGCCCTCCTGA3’	5’-TCTCTCTGCTTGCAGCCCTGGGGTGTG-3’	68℃	43
I-H-69	69	5’-GCCTCGCTGCTCTTCCTG-3’	5’-GCTGAGCTGAGCTAAGACGCCCTCC-3’	66℃	44
						I-H-70	70	5’-AGCTCAGCTGTACGCCCTCA-3’	5’-TCTCCCTCTCCCCCCCACTG-3’	65℃	45
I-H-71	71	5’-GAGAGGGACACGCCCTGGGCTCTGC-3’	5’-GGCAAGGCGGCTGGGCAGTGCTGG-3’	66℃	46
						I-H-72	72	5’-CCCGTGGCCCATCCCCGGGCCTGCGG-3’	5’-TACGTGCAGCCATTCTGCCTGGCCC-3’	66℃	46

The sudden change of table 3 autosomal dominant polycystic kidney disease II type Disease-causing gene detects the nucleotide sequence of primer

The primer mixture code name	The primer numbering	Nucleotide sequence		Annealing temperature	The exon numbering
						Forward primer	Reverse primer
		II-SH-1	?1					?5’CCGCCCCCGCCGCGCGCCGGACGCCAGTG ?ACC3’	5’CCTGCCGGGAGCACGACGAG3’	70℃	1
II-SH-2	?2			?5’GCCCCCGCCGCCGCGGCCTCCCCTTCTCCT3’	5’CTGGGCTGGGGCACGGCGGG3’	72℃	1
		II-SH-3	?3					?5’GGGGGCTACCACGGCGCGGGC3’	5’CGGCCCGCCGCCCCCGCCCGCGGC CGTTCTGGTTCGTGCATCTG3’	70℃	1
II-SH-4	?4			?5’AAATGATATCTTTTCTTTTCTTCA3’	5’AACTTTCCCATTAGTGCAAG3’	56℃	2
		II-SH-5	?5					?5’GGCGTTCATTTGGATCTTTC3’	5’TGTGATAGAGAGGTACTTTCA3’	56℃	3
II-SH-6	?6			?5’CTTTTTCAAAGATGTTTCCTTTGC3’	5’CCGAGTGCCAATGAGTCACA3’	56℃	4
		II-SH-7	?7					?5’GCCTCAAGTGTTCCACTGAT3’	5’ACCACACAGAAATAGGAGGG3’	56℃	5
II-SH-8	?8			?5’TTGTTATTGTTTTAATTGTTCTTA3’	5’TTGTAGAATAGAATAGGAAATTTGG3’	56℃	6
		II-SH-9	?9					?5’TTGGTGAAGAAAAATATACTAGTCA3’	5’TGGAACTCATTTTTTTTAAAGA3’	56℃	7
II-SH-10	?10			?5’TTTTATTATACACAGTCACACC3’	5’CTACTCTGACTAAATTTTTCTTCTT3’	56℃	8
		II-SH-11	?11					?5’TTTGGTTTTGTATTGTGGTG3’	5’AAGGATTTACGAAGTTTAAATTG3’	56℃	9
II-SH-12	?12			?5’TTTTTGCCCTCCTTTCATTTA3’	5’GAAACAATGCTCATTTTATGTC3’	56℃	10
		II-SH-13	?13					?5’AAACCAAGTCTTTTATTTTTTCTC3’	5’AGAACCTCAGGAAGCATGATT3’	56℃	11
II-SH-14	?14			?5’GATGAATGTTATCTGTATCCTCTC?3’	5’TAGGTACCAAATCAAATCCG3’	56℃	12
		II-SH-15	?15					?5’GTCTCAGTGTTCTGCTCCTC3’	5’CAAATTCTGCCAATTCCTTTA3’	56℃	13
II-SH-16	?16			?5’TTTGTCCCTCTGTACTGTGTTT3’	5’AAATACAACTGTCAGCAACATA3’	56℃	14
		II-SH-17	?17					?5’TGACCCCCAACACCAGTTTC3’	5’GGACAGCCACTTCCTCACTT3’	56℃	15

Claims (14)

1. an autosomal dominant polycystic kidney disease detection kit comprises Disease-causing gene primer mixture, negative control thing, normal control thing, it is characterized in that the composition of Disease-causing gene primer mixture is:

Tutofusin tris 80mmol/L, potassium acetate 0-170mmol/L, methyl-sulphoxide 0-10%, Octylphenoxy gathers ethoxy ethanol 0.2%, deoxynucleoside triphosphate 500-1200 μ mol/L, magnesium acetate 0-2.5mmol/L or magnesium chloride 0-2mmol/L, each 2-6 μ mol/L of thermostable type deoxynucleotide polysaccharase 16-80U and the forward and reverse primer of Disease-causing gene, said forward and reverse primer is I-H-1 to I-H-72, II-SH-1 to II-SH-17 and/or I-S-1 to I-S-82.

2. by the described autosomal dominant polycystic kidney disease detection kit of claim 1, it is characterized in that the contained primer of said Disease-causing gene primer mixture is I-H-1 to I-H-72.

3. by the described autosomal dominant polycystic kidney disease detection kit of claim 1, it is characterized in that the said contained primer of Disease-causing gene primer mixture is II-SH-1 to II-SH-17.

4. by the described autosomal dominant polycystic kidney disease detection kit of claim 3, it is characterized in that the contained primer of said Disease-causing gene primer mixture also comprises I-H-1 to I-H-72.

5. by the described autosomal dominant polycystic kidney disease detection kit of claim 1, also comprise sample-loading buffer and colour developing liquid, it is characterized in that the contained primer of said Disease-causing gene primer mixture is I-S-1 to I-S-82.

6. by the described autosomal dominant polycystic kidney disease detection kit of claim 1, also comprise sample-loading buffer and colour developing liquid, it is characterized in that the contained primer of said Disease-causing gene primer mixture is H-SH-1 to II-SH-17.

7. by the described autosomal dominant polycystic kidney disease detection kit of claim 5, it is characterized in that the contained primer of said Disease-causing gene primer mixture also comprises II-SH-1 to II-SH-17.

8. by claim 5 or 6 or 7 described autosomal dominant polycystic kidney disease detection kit, it is characterized in that said sample-loading buffer composition is: sucrose 10%, bromjophenol blue 0.01% and dimethylbenzene green grass or young crops 0.01%.

9. by claim 5 or 6 or 7 described autosomal dominant polycystic kidney disease detection kit, it is characterized in that said colour developing liquid composition is respectively: colour developing liquid A---dehydrated alcohol; Colour developing liquid B---concentrated nitric acid; Colour developing liquid C---30% silver nitrate solution; Colour developing liquid D---25% sodium carbonate solution; Colour developing liquid E---acetate; Colour developing liquid F---37% formaldehyde solution.

10. by the described autosomal dominant polycystic kidney disease detection kit of claim 8, it is characterized in that said colour developing liquid composition is respectively: colour developing liquid A---dehydrated alcohol; Colour developing liquid B---concentrated nitric acid; Colour developing liquid C---30% silver nitrate solution; Colour developing liquid D---25% sodium carbonate solution; Colour developing liquid E---acetate; Colour developing liquid F---37% formaldehyde solution.

11., it is characterized in that the contained primer of said Disease-causing gene primer mixture also comprises I-H-1 to I-H-72 by the described autosomal dominant polycystic kidney disease detection kit of claim 7.

12., it is characterized in that the contained primer of said primer mixture also comprises I-H-1 to I-H-72 by the described autosomal dominant polycystic kidney disease detection kit of claim 8.

13., it is characterized in that the contained primer of said Disease-causing gene primer mixture also comprises I-H-1 to I-H-72 by the described autosomal dominant polycystic kidney disease detection kit of claim 9.

14., it is characterized in that the contained primer of Disease-causing gene primer mixture also comprises I-H-1 to I-H-72 by the described autosomal dominant polycystic kidney disease detection kit of claim 10.

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