CN202519251U

CN202519251U - Microsatellite instability detection kit

Info

Publication number: CN202519251U
Application number: CN2012201647068U
Authority: CN
Inventors: 唐金海; 曹海霞; 井昶雯; 吴建中; 王卓
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-04-17
Filing date: 2012-04-17
Publication date: 2012-11-07
Anticipated expiration: 2022-04-17

Abstract

The utility model discloses a microsatellite instability detection kit, which comprises a kit body 1, a kit cover 2, a primer reagent bottle 3, an enzyme mixture reagent bottle 4, a de-ionized water pipe 5, a PCR (Polymerase Chain Reaction) pipe 6 and a baffle 7. The kit has the characteristic of simple structure, rational design, convenience in use, easy manufacturing and low probability of pollution in a detection process, and has the advantages of high sensitivity, high specificity, short consumed time and the like during microsatellite instability detection.

Description

A kind of microsatellite instability detection kit

Technical field

The utility model relates to a kind of detection kit, especially relates to a kind of microsatellite instability detection kit.

Background technology

Little satellite (microsatellites) is to be dispersed throughout the fragment that same deoxy-oligonucleotide repeats to be in series in the human genome; Repetitive sequence is 1～6bp; Multiplicity is no more than 60 times; Fragment length shows the individual specificity of height usually less than 350bp in the crowd, and genetic stability.Comprise tens thousand of microsatellite locus in the human genome,, in the crowd, present the height polymorphum because they generally are in the noncoding DNA zone that can accumulate neutral mutation.Microsatellite polymorphism is microsatellite instability (microsatellite instability, performance MSI).Microsatellite instability finds expression in same microsatellite locus between the Different Individual and between the healthy tissues of same individuality and some abnormal structure, and the number of the repeating unit of microsatellite locus is different.。The generation reason of microsatellite instability possibly be " chain the is sliding " phenomenon in the dna replication dna process.In the dna replication dna process, after duplicating mixture and duplicating a repeating unit (repeat unit), subchain is separated with template strand, with next or several repeating units recombine down, one or several repeating unit is formed then " ring is protruding ".This structure can be proofreaied and correct by mismatch repair system (mismatch repair system) under the normal circumstances, but corrective system is when not normal, and subchain DNA gets final product mutagenesis as continuing to extend.

Generally acknowledge that research index (working group of the U.S. state-run cancer association provided in 1997) shows, directly BAT-25, BAT-26, D5S346, D2S123 and D17S250 are detected in 5 sites totally, if wherein there are 2 sudden changes then to be MSI-H (High frequency MSI) state; Sensitivity and specific degree all can significantly improve; MSI tumour multidigit is in being positioned at proximal colonic simultaneously, and gross tumor volume is big, poor differentiation; Mucinous adenocarcinoma sees that lymphocytic infiltration is often arranged more.Say that further MSI-H is common in most hereditary nonpolyposis property colorectal cancer (HNPCC) and few part sporadic colorectal cancer, both totals account for 30% [7] of intestinal cancer.Generally speaking, compare with MSI-H, the prognosis of MSI-L (Low frequency MSI) MSS (microsatellite stable) tumour patient is relatively poor, and transfer and relapse is common.

Therefore, microsatellite instability detects significant for colorectal cancer prognostic evaluation and chemotherapy side effect property.And detect MSI method commonly used denaturing gel electrophoresis, dhplc analysis etc. are arranged, but these method susceptibility are lower.And method is very unstable, is difficult in wide clinical application.So explore a kind of hot issue that microsatellite instability rapid and reliable method has become clinical and experimental study that detects.The utility model content

In order to solve the problems of the technologies described above, the utility model provides a kind of microsatellite instability detection kit, comprises box body 1, lid 2, primer reagent bottle 3, enzyme mixture reagent bottle 4, de-ionized water pipe 5, PCR pipe 6 and is arranged on the baffle plate 7 on the box body 1.Said baffle plate is used for blocking primer reagent bottle 3, enzyme mixture reagent bottle 4, de-ionized water pipe 5, PCR pipe 6, and it is fixed.

Said primer is the primer in these 5 sites of specific amplification BAT-25, BAT-26, D5S346, D2S123 and D17S250.The upstream primer in amplification BAT-25 site is: 5 '-TCG CCT CCA AGA ATG TAAGT-3 ', downstream primer is: 5 '-TCT GCA TTT TAA CTA TGG CTC-3 ', the primer amount is 1pmol; The upstream primer in amplification BAT-26 site is: 5 '-TGA CTA CTT TTG ACT TCA GCC-3 ', downstream primer is: 5 '-AAC CA T TCA ACA TTT TTA ACC C-3 ', the primer amount is 1pmol; The upstream primer in amplification D2S123 site is: 5 '-AAA CAG GAT GCC TGC CTT TA-3 ', downstream primer is: 5 '-GGACTT TCC ACC TAT GGG AC-3 ', the primer amount is 1pmol; Amplification D17S250 upstream primer is: 5 '-GGAAGA ATC AAA TAG ACA AT-3 ', downstream primer is: 5 '-GCT GGC CAT ATA TAT ATT TAAACC-3 ', the primer amount is 10pmol; Amplification D5S346 upstream primer is: 5 '-ACT CAC TCT AGT GATAAA TCGGG-3 ', downstream primer is: 5 '-AGC AGA TAA GAC AGT ATT ACT AGTT-3 ', the primer amount is 1pmol.

Said enzyme mixture comprises that concentration is the dNTPs of 0.4mmol/L, the Mg of 3mmol/L ²⁺, 2 * damping fluid (2 * buffer), the rTaq of 1.25U/25 μ l.

The utility model is compared with currently available products, has following actively useful effect:

This test kit is simple in structure, reasonable in design, easy to use, easy to manufacture, testing process pollution not; This test kit has advantages such as susceptibility height, high specificity, weak point consuming time when detecting microsatellite instability.

Description of drawings

Fig. 1 is the one-piece construction synoptic diagram of the utility model.

Embodiment

Below in conjunction with accompanying drawing and embodiment the utility model is further specified.

Table 1 is the utility model PCR reaction system.

As shown in Figure 1, an embodiment of the utility model is a kind of microsatellite instability detection kit, comprises box body 1, lid 2, primer reagent bottle 3, enzyme mixture reagent bottle 4, de-ionized water pipe 5, PCR pipe 6 and is arranged on the baffle plate 7 on the box body 1.Said baffle plate is used for blocking primer reagent bottle 3, enzyme mixture reagent bottle 4, de-ionized water pipe 5, PCR pipe 6, and it is fixed.

Said enzyme mixture comprises that concentration is the dNTPs of 0.4mmol/L, the Mg of 3mmol/L ²⁺, 2xbuffer, the rTaq of 1.25U/25 μ l.

Working method:

1) gets template DNA

Get patients blood 1ml and focus paraffin section, the conventional DNA that extracts gets 1ulDNA as template.

2) reaction system

With each RM according to carrying out proportioning like reaction system in the following table 1:

Table 1

3) reaction

Each material in the reaction system in middle mixing, is reacted according to following program:

4) nucleic acid electrophoresis

The pcr amplification result is reflected by the nucleic acid electrophoresis result.If electrophoretic band is correctly clear, then can carry out next step operation.Otherwise then amplification again.

5) interpretation of result

Carry out fragment analysis through the ABI3500 sequenator, whether change according to clip size whether stable detect little satellite.Five sites have two or more than then be judged as the high unsteady state of little satellite (MSI-H), have only a site to change and then be the low unsteady state (MSI-L) of little satellite, do not have the site to change and then be stable (MSS) state of little satellite.

Five site clip size are as shown in table 2:

The site	Clip size (bp)
		BAT-25	120
BAT-26	116
		D2S123	197-227
D17S250	151-169
		D5S346	96-122

In sum; The described embodiment of the utility model only provides a kind of embodiment of the best; The technology contents and the technical characterstic of the utility model disclose as above, yet the personage who is familiar with this technology still maybe be based on content that the utility model disclosed and done various replacement and the modifications that do not deviate from creation spirit of the present invention; Therefore, the protection domain of the utility model is not limited to the technology contents that embodiment discloses, so the equivalence that all shapes according to the utility model, structure and principle are done changes, all is encompassed in the protection domain of the utility model.

Claims

1. a microsatellite instability detection kit is characterized in that, comprises box body (1), lid (2), primer reagent bottle (3), enzyme mixture reagent bottle (4), de-ionized water pipe (5), PCR pipe (6) and is arranged on the baffle plate (7) on the box body (1).