CN102816846A - Kit for detecting mutation hotspot of GSTP1 gene - Google Patents

Abstract

The kit for detecting mutation hotspot of GSTP1 gene comprises a red blood cell lysate, a whole blood DNA extraction reagent, absolute ethyl alcohol, a PCR amplification reaction liquid, a positive reference substance, a negative reference substance and a pyrosequencing reaction liquid. The kit is characterized by comprising target gene upstream and downstream primers F and R-Biotin for detecting an exon 5, and a sequencing primer FCX. The kit can detect polymorphism of a mutation hotspot (I105V) of the GSTP1 gene, and has good specificity and high accuracy.

Description

Be used to detect the test kit of GSTP1 gene hot mutant site

Technical field

The invention belongs to life science and biological technical field; Be particularly related to a kind of glutathione-S-transferase P1 (glutathione-S-transferase P1 that is used to detect; GSTP1) test kit of gene hot mutant site (I105V) can detect the GSTP1 gene mutation site, and specificity is good; Accuracy is high, can improve the sudden change recall rate.

Background technology

Glutathione s-transferase (GSTs) is one group of multi-functional drug metabolism enzyme; Wherein GSTP1 extensively is present in the body tumor tissue; Expression rate is higher in the malignant tumour of human body epithelial origin, like gastrointestinal cancer, the esophageal carcinoma in digestive tube source, and the lung cancer in respiratory tract source.Normal discovery GSTP1Vall05 genotype over-expresses in precancerous lesion and tumour patient.

In the research of the relation of GSTP1 gene pleiomorphism and population health, find; The SNP in the Ile105Val site of GSTP1 gene causes changing in GSTP1 albumen codon 105 amino acids; Have influence on the catalysis of GSTP1; Enzymic activity reduces, and makes its anti-oxidant function descend the existence of favourable patient in chemotherapy.Be the favourable chemotherapy of polymorphum in GSTP1Ile105Val site, prognosis is better.

At present, GSTP1 transgenation conventional detection comprises enzyme cutting method, common PCR sequencing PCR and tetra-sodium PCR sequencing PCR etc.In practical application, the method that is used to detect colorectal cancer PIK3CA transgenation is mainly direct sequencing, although this method is a gold standard; If but mutation rate is lower than 15%; The phenomenon of omission can take place, and process of the test is too loaded down with trivial details, the reagent type that needs is various; Waste time and energy, thus to a certain degree limit the application of this method.

Tetra-sodium order-checking (Pyrosequencing) technology is a kind of new sequential analysis technology, is by the enzyme cascade chemiluminescence reaction in 4 kinds of enzymatic same reaction systems.In real work, a lot of situation need be carried out the sequence checking to the dna fragmentation of known array, and this analysis is often surveyed tens bp and just can be satisfied the demand.In this case, the Sanger method may not be only dna sequence analysis technology.And the tetra-sodium sequencing technologies is the dna sequence analysis technology of the most suitable these application at present.Be characterized in easy and simple to handle, big flux, robotization, be fit to the rapid detection of great amount of samples, interpretation is carried out in the mutational site of sequence, thereby the fixed point that realizes the GSTP1 gene mutation site detects according to peak value.

Summary of the invention

The objective of the invention is to overcome the defective of prior art, provide with a kind of quick, accurately, high-throughout GSTP1 gene hot sudden change (Ile105Val) detection kit.

Be used to detect the test kit of GSTP1 gene hot mutant site; Comprise erythrocyte cracked liquid, whole blood DNA extraction agent, absolute ethyl alcohol, pcr amplification reaction liquid, positive reference substance, negative control article and tetra-sodium sequencing reaction liquid; It is characterized in that: said test kit comprises detection GSTP1 the 5th exon goal gene upstream and downstream primers F, R-Biotin, sequencing primer FCX, and its sequence is respectively:

F：TGGTGGACATGGTGAATGA，

R-Biotin：GGTCAGCCCAAGCCAC，

FCX：CCTCCGCTGCAAATA。

Further, the method for use of said test kit comprises the steps:

(1) gathers blood sample to be measured, extract DNA;

(2) be template with this DNA, with the said corresponding PCR primer that is used for amplified sample DNA GSTP1 gene the 5th exon of claim 1: F, R increase, and obtain the PCR reaction product;

(3) the PCR reaction product is carried out the tetra-sodium order-checking with sequencing primer FCX, determine whether to exist sudden change.

Preferably, the PCR of step (2) reaction is increased by following condition: 94 ℃ of 5min; 98 ℃ of 30s, 58 ℃ of 30s, 68 ℃ of 30s, 35 circulations; Last 68 ℃ of 5min.

The present invention has designed exon 5PCR amplimer and tetra-sodium sequencing primer according to GSTP1 gene hot sudden change position.

Table 1. exon 5 primer sequences

F, R are the amplimer of No. 5 exons, corresponding fragment in the biological sample to be checked that can increase, and wherein reverse primer is done biotin labeling; FCX is a tetra-sodium order-checking forward sequencing primer, and aforementioned amplification gained fragment is checked order.

The present invention is applied to the detection of GSTP1 gene hot mutant site with sequencing technologies, designs exon 5PCR amplimer and tetra-sodium sequencing primer, carries out pcr amplification and carries out the tetra-sodium sequencing analysis.Can detect GSTP1 gene hot mutant site (Ile105Val) polymorphum, specificity is good, and accuracy is high, and it all is being significant aspect primary prevention of instructing tumour and medicinal design and the clinical rational drug use.

Description of drawings

After Fig. 1 is the amplification of GSTP1 the 5th exon genes, the electrophorogram behind 1.5% agarose gel electrophoresis.As shown in the figure, the purpose fragment is about 176bp.Wherein, M is the Marker of TAKARA2000, and 1-5 is examined samples.

Fig. 2 A be the wild-type sample behind the Exon5 primer amplification, the tetra-sodium sequencing result.Because reverse mark vitamin H, so forward order-checking standard sequence should be C

TCCCTCAT.

Fig. 2 B be the mutant sample behind the Exon5 primer amplification, the tetra-sodium sequencing result.Can know by figure; Complete A → G conversion appears in this sample; C

TCCCTCAT is the homozygous mutation sample.

Fig. 2 C be the mutant sample behind the Exon5 primer amplification, the tetra-sodium sequencing result.Can know by figure; Part A → G conversion appears in this sample; C

TCCCTCAT is the heterozygous mutant sample.

Embodiment

The present invention is used to detect the test kit of colorectal cancer PIK3CA gene hot mutant site; Comprise erythrocyte cracked liquid, whole blood DNA extraction agent, absolute ethyl alcohol, pcr amplification reaction liquid, positive reference substance, negative control article and tetra-sodium sequencing reaction liquid; It is characterized in that: said test kit comprises detection 5 exon goal gene upstream and downstream primers F, R-Biotin, sequencing primer FCX, and its sequence is:

F：TGGTGGACATGGTGAATGA，

R-Biotin：GGTCAGCCCAAGCCAC，

FCX：CCTCCGCTGCAAATA。

Embodiment 1

(1) sample extracting:

1.1 extract 300 μ l blood, add 900 μ L erythrocyte cracked liquids, put upside down mixing, room temperature was placed 5 minutes, during put upside down mixing more several times.Centrifugal 1 minute of 10000rpm (if the whizzer maximum speed does not allow, but the centrifugal 5min of 3000rpm) inhales and removes supernatant, stays the white corpuscle deposition, adds 200 μ L damping fluid GA, vibration to thorough mixing.

1.2 add 20 μ l Proteinase K solution, mixing.

1.3 add 200 μ L damping fluid GB, fully put upside down mixing, to place 10 minutes for 70 ℃, the solution strain is limpid, and is brief centrifugal to remove the globule of cap wall.

1.4 add people's 200 μ L absolute ethyl alcohols, the mixing 15 seconds of fully vibrating, flocks may appear in this moment, and is brief centrifugal to remove the globule of cap wall.

All add among the adsorption column CB3 (adsorption column is put into collection tube) 1.5 will go up step gained solution and a flocks, 12,000rpm (13,400 * g) centrifugal 30 seconds, outwell waste liquid, CB3 puts back in the collection tube with adsorption column.

1.6 in adsorption column CB3, add 500 μ L damping fluid GD (please checking to have added absolute ethyl alcohol whether earlier before using), 12,000rpm (13,400 * g) centrifugal 30 seconds, outwell waste liquid, CB3 puts into collection tube with adsorption column.

1.7 in adsorption column CB3, add 700 μ L rinsing liquid PW (please checking to have added absolute ethyl alcohol whether earlier before using), 12,000rpm (13,400 * g) centrifugal 30 seconds, outwell waste liquid, CB3 puts into collection tube with adsorption column.

1.8 in adsorption column CB3, add 500 μ L rinsing liquid PW, 12,000rpm (13,400 * g) centrifugal 30 seconds, outwell waste liquid.

1.9 adsorption column CB3 is put back in the collection tube, 12,000rpm (13,400 * g) centrifugal 2 minutes, outwell waste liquid.Place room temperature to place several minutes adsorption column CB3, thoroughly to dry rinsing liquid remaining in the sorbing material.

1.10 adsorption column CB3 is changed in the clean centrifuge tube, and to the unsettled dropping 100 μ L elution buffer TE in the middle part of adsorption film, room temperature was placed 2-5 minute, and 12,000rpm (13,400 * g) centrifugal 2 minutes, solution is collected in the centrifuge tube.

(2) amplification of target gene fragment

2.1 getting the every pipe 47 μ L of pre-mixed PCR reaction solution by sample number n (sample number=number of awaiting test sample+1+positive control of negative control 1) is sub-packed in the reaction tubes.

2.2 the above-mentioned sample to be tested of handling well and negative, positive control are respectively got 3 μ L and added respectively in the reaction tubes, mixing, the low-speed centrifugal several seconds, carry out pcr amplification, concrete reaction system and cyclic amplification system are following:

Table 2. exon 5PCR reaction system

Table 3. exon 5PCR circulating system

Embodiment 2

Electrophoresis is identified: 1.5% agarose gel electrophoresis, and 140V, 20min, gel imaging system is observed.The 9th exon purpose fragment is respectively 176bp, and Marker is DL2000.

Embodiment 3

The preparation of single-stranded template: with aforementioned amplification gained PCR product, every sample adds the magnetic bead that 3 μ L Streptavidins encapsulate according to 50 μ L and at room temperature hatched 10 minutes.With PCR product after magnetic bead combines respectively at 75% ethanol, respectively hatched 10 seconds in the NaOH solution of 0.2mol/L and the Tris elutriant, fully reaction back sex change obtains single stranded DNA.To discharge with the strand after magnetic bead combines and be suspended in (100mM Tris-acetate pH 7.75,20mM Mg-acetate) (containing the 10pmol sequencing primer) in the 45 μ L annealing buffers.Under 80 ℃, hatched 2 minutes, at room temperature placed then 5 minutes.

Embodiment 4

Tetra-sodium order-checking: sequencing reaction detects under the SQA of PYROMARKID instrument pattern down at 28 ℃ automatically, and the dNTP application of sample be A → T → C → G in proper order, and along with the carrying out of the reaction of enzymatic, the ccd video camera detection is the collection optical signal also, finally obtains detecting sequence.

Embodiment 5

The tetra-sodium sequencing result is analyzed: the PCR reaction product is carried out the tetra-sodium order-checking, compare with the wild type gene sequence, determine whether to exist sudden change.The mutation type of the 5th exon is mainly replacement mutation, occurs A → G conversion in the 105th amino acids, causes that the Isoleucine in this site changes Xie Ansuan (I105V) in the albumen.

SEQUENCE?LISTING

 

< 110>the limited company of Jilin Ai Dikang medical test

 

< 120>be used to detect the test kit of GSTP1 gene hot mutant site

 

<130>

 

<160> 3

 

<170> PatentIn?version?3.3

 

<210> 1

<211> 19

<212> DNA

< 213>artificial sequence

 

<400> 1

tggtggacat?ggtgaatga 19

 

 

<210> 2

<211> 16

<212> DNA

< 213>artificial sequence

 

<400> 2

ggtcagccca?agccac 16

 

 

<210> 3

<211> 15

<212> DNA

< 213>artificial sequence

 

<400> 3

cctccgctgc?aaata 15

 

 

 

Claims (3)

1. be used to detect the test kit of GSTP1 gene hot mutant site; Comprise erythrocyte cracked liquid, whole blood DNA extraction agent, absolute ethyl alcohol, pcr amplification reaction liquid, positive reference substance, negative control article and tetra-sodium sequencing reaction liquid; It is characterized in that: said test kit comprises detection 5 exon goal gene upstream and downstream primers F, R-Biotin, sequencing primer FCX, and its sequence is:

F：TGGTGGACATGGTGAATGA，

R：Biotin-GGTCAGCCCAAGCCAC，

FCX：CCTCCGCTGCAAATA。

2. test kit as claimed in claim 1 is characterized in that the method for use of said test kit comprises the steps:

(1) gathers blood sample to be measured, extract DNA;

(2) be template with this DNA, with the said corresponding PCR primer that is used for amplified sample DNA GSTP1 gene of claim 1: F, R increase, and obtain the PCR reaction product;

(3) the PCR reaction product is carried out the tetra-sodium order-checking with sequencing primer FCX, determine whether to exist sudden change.

3. test kit as claimed in claim 2 is characterized in that, the PCR reaction of step (2) is increased by following condition: 94 ℃ of 5min; 98 ℃ of 30s, 58 ℃ of 30s, 68 ℃ of 30s, 35 circulations; Last 68 ℃ of 5min.

Images