CN107217103A - Detect the multiple fluorescence PCR amplifing reagent and kit of microsatellite instability - Google Patents
Detect the multiple fluorescence PCR amplifing reagent and kit of microsatellite instability Download PDFInfo
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Abstract
The invention discloses a kind of multiple fluorescence PCR amplifing reagent and kit for detecting microsatellite instability.The PCR amplifing reagents include the sense primer and anti-sense primer designed respectively for microsatellite locus BAT25, BAT26, D2S123, D5S346 and D17S250, can expand multiple microsatellite locus simultaneously in same PCR system and carry out multiple fluorescence PCR detection microsatellite instability.The fluorescence peak that the amplified production of each microsatellite locus is produced after electrophoresis detection does not have the phenomenon of juxtaposition, and detection efficiency is high, and sensitivity is good.
Description
Technical field
The present invention relates to field of biological detection, more particularly to a kind of multiple fluorescence PCR for detecting microsatellite instability
Amplifing reagent and kit.
Background technology
Microsatellite is also known as STR (Short tandem repeats, STRs) or simple repeated sequence
(Simple sequences repeats, SSRs), is distributed widely in human genome.Repetitive sequence is general by 1~6 core
Thuja acid is constituted, and with dinucleotides repetitive sequence is particularly that (CA/GT) n is most commonly seen, n is number of repetition, and usually 15~60
It is secondary.The conjugated protein or direct coding protein of microsatellite energy autospecific, what is had may then participate in the folding of chromatid
Formation of folded or telomere etc..Microsatellite is by changing DNA structure or playing gene regulation by being combined with specific protein
Effect, is the molecular labeling that a class is new, polymorphism information capacity is high.
Microsatellite instability (Microsatellite Instability, MSI) refers to due to mispairing reparation or duplication
The increase of simple repeated sequence or loss caused by mistake etc., cause the length of microsatellite to change.The main original that MSI occurs
Because being that defect occurs for the gene function for participating in pairing errors repair, thus can not normal correct and copy mistake, cause microsatellite
DNA change, prevent it from normally playing regulating and controlling effect.MSI is easily caused cell propagation and disdifferentiation, or even promotees
Hair malignant tumour is formed.Compared with normal structure, the microsatellite in tumour is easier to cause microsatellite length to change.Grind
Study carefully that to show that the tumours such as MSI and colorectal cancer, stomach cancer, carcinoma of endometrium occur closely related.Wherein about 15% sporadic knot
The carcinoma of the rectum is related to MSI, more than 90% hereditary nonpolyposis colorectal cancer (HNPCC, also referred to as Lynch syndromes) with
MSI is related, therefore detects clinically significant to MSI.
The method for detecting microsatellite instability is general by selected special primer, then micro- in PCR amplified samples
Satellite site, the product after amplification is analyzed in the display system after electrophoresis detection (such as Capillary Electrophoresis), and and normal structure
Compare, judge sequence length of microsatellite locus etc. whether there is change.NCI (National Cancer Institute) recommends
The standard post that 5 microsatellite locus are detected as MSI (microsatellite instability), respectively BAT25, BAT26,
D2S123, D5S346 and D17S250.Wherein BAT25 and BAT26 have a mononucleotide repeat sequence, D2S123, D5S346 and
D17S250 has dinucleotide repetitive sequence.The testing result of comparison of tumor and normal sample after detection, if the above 5 is micro-
There are 2 in satellite site or more than 2 microsatellite locus change, referred to as high-frequency type (High-frequency MSI, MSI-
H)；Only 1 microsatellite locus is changed, referred to as low frequency type (low-frequency MSI, MSI-L), and 5 are not sent out
Raw change is referred to as microsatellite stable type (Microsatellite stable, MSS).
However, traditional PCR amplifing reagents to microsatellite locus (BAT25, BAT26, D2S123, D5S346 and
D17S250) enter after performing PCR amplification, the amplified production that the amplification of each microsatellite locus is obtained fluorescence peak after electrophoresis detection is easy
Juxtaposition, thus it is more difficult in same PCR system simultaneously detect microsatellite locus BAT25, BAT26, D2S123, D5S346 and
Whether D17S250 changes.
The content of the invention
Based on this, it is necessary to which the amplified production for providing each microsatellite locus after a kind of PCR amplifications is produced after electrophoresis detection
Raw fluorescence peak will not juxtaposition, can simultaneously be detected in same PCR system microsatellite locus BAT25, BAT26,
D2S123, D5S346 and D17S250 whether change detection microsatellite instability multiple fluorescence PCR amplifing reagent and
Detection kit.
A kind of multiple fluorescence PCR amplifing reagent for detecting microsatellite instability, including：
The sense primer BAT25-F1 and anti-sense primer BAT25-R1 designed for microsatellite locus BAT25, the upstream
Primer BAT25-F1 base sequence is as shown in SEQ ID No.1, the base sequence such as SEQ of the anti-sense primer BAT25-R1
Shown in ID No.2；
The sense primer BAT26-F1 and anti-sense primer BAT26-R1 designed for microsatellite locus BAT26, the upstream
Primer BAT26-F1 base sequence is as shown in SEQ ID No.3, the base sequence such as SEQ of the anti-sense primer BAT26-R1
Shown in ID No.4；
For the microsatellite locus D2S123 sense primer D2S123-F1 designed and anti-sense primer D2S123-R1, it is described on
Primer D2S123-F1 base sequence is swum as shown in SEQ ID No.5, the base sequence of the anti-sense primer D2S123-R1 is such as
Shown in SEQ ID No.6；
For the microsatellite locus D5S346 sense primer D5S346-F1 designed and anti-sense primer D5S346-R1, it is described on
Primer D5S346-F1 base sequence is swum as shown in SEQ ID No.7, the base sequence of the anti-sense primer D5S346-R1 is such as
Shown in SEQ ID No.8；And for the sense primer D17S250-F1 and anti-sense primer of microsatellite locus D17S250 designs
D17S250-R1, the sense primer D17S250-F1 base sequence are as shown in SEQ ID No.9, the anti-sense primer
D17S250-R1 base sequence is as shown in SEQ ID No.10.
In one embodiment, the amplifing reagent also includes：
The sense primer Penta C-F1 and anti-sense primer Penta C-R1 designed for microsatellite locus Penta C, institute
Sense primer Penta C-F1 base sequence is stated as shown in SEQ ID No.11, the base of the anti-sense primer Penta C-R1
Sequence is as shown in SEQ ID No.12.
In one embodiment, the amplifing reagent also includes：
The sense primer Penta D-F1 and anti-sense primer Penta D-R1 designed for microsatellite locus Penta D, institute
Sense primer Penta D-F1 base sequence is stated as shown in SEQ ID No.13, the base of the anti-sense primer Penta D-R1
Sequence is as shown in SEQ ID No.14.
In one embodiment, in the amplifing reagent,
The concentration of the sense primer BAT25-F1 is 50nmol/L~200nmol/L, the anti-sense primer BAT25-R1
Concentration be 50nmol/L~200nmol/L；And/or,
The concentration of the sense primer BAT26-F1 is 50nmol/L~200nmol/L, the anti-sense primer BAT26-R1
Concentration be 50nmol/L~200nmol/L；And/or,
The concentration of the sense primer D2S123-F1 is 50nmol/L~200nmol/L, the anti-sense primer D2S123-
R1 concentration is 50nmol/L~200nmol/L；And/or,
The concentration of the sense primer D5S346-F1 is 50nmol/L~200nmol/L, the anti-sense primer D5S346-
R1 concentration is 50nmol/L~200nmol/L；And/or,
The concentration of the sense primer D17S250-F1 is 50nmol/L~200nmol/L, the anti-sense primer
D17S250-R1 concentration is 50nmol/L~200nmol/L.
In one embodiment, at least one in the sense primer BAT25-F1 and the anti-sense primer BAT25-R1
Individual mark has at least one mark in fluorophor, the sense primer BAT26-F1 and the anti-sense primer BAT26-R1
Note has at least one mark in the second fluorophor, the sense primer D2S123-F1 and the anti-sense primer D2S123-R1
At least one for having in the 3rd fluorophor, the sense primer D5S346-F1 and the anti-sense primer D5S346-R1 is marked with
At least one in 4th fluorophor, the sense primer D17S250-F1 and the anti-sense primer D17S250-R1 is marked with
5th fluorophor, and the 3rd fluorophor is selected from two kinds of fluorophors differed with the 4th fluorophor.
In one embodiment, first fluorophor is marked on the anti-sense primer BAT25-R1, and described
Two fluorophors are marked on the sense primer BAT26-F1, and the 3rd fluorophor is marked at the sense primer
On D2S123-F1, the 4th fluorophor is marked on the sense primer D5S346-F1, the 5th fluorophor mark
Note is on the anti-sense primer D17S250-R1.
In one embodiment, first fluorophor, second fluorophor, the 3rd fluorophor,
4th fluorophor and the 5th fluorophor are selected from FAM fluorophors, TAMRA fluorophors, ROX fluorophors
With one kind in JOE fluorophors.
In one embodiment, first fluorophor is FAM fluorophors, and second fluorophor is
TAMRA fluorophors, the 3rd fluorophor is FAM fluorophors, and the 4th fluorophor is JOE fluorophors, institute
The 5th fluorophor is stated for JOE fluorophors.
A kind of multiple fluorescence PCR detection reagent box for detecting microsatellite instability, including above-mentioned amplifing reagent.
In one embodiment, Taq archaeal dna polymerases and PCR reaction buffers are also included in the kit.
The multiple fluorescence PCR amplifing reagent of above-mentioned detection microsatellite instability, including respectively for 5 microsatellite locus
The sense primer and anti-sense primer of (BAT25, BAT26, D2S123, D5S346 and D17S250) design.Result of the test shows, on
The corresponding microsatellite locus of specific amplification, the amplified production band of each microsatellite locus can be distinguished by stating the primer of particular sequence
Unicity is good.Simultaneously 5 microsatellite locus (BAT25, BAT26, D2S123, D5S346 and D17S250) are entered with performing PCR amplification
Afterwards, the fluorescence peak that each amplified production is produced after electrophoresis detection does not have the phenomenon of juxtaposition, so as in same PCR system
In detect microsatellite locus BAT25, BAT26, D2S123, D5S346 and D17S250 microsatellite instability, detection effect simultaneously
Rate is high, and sensitivity is good.
Brief description of the drawings
The expansion that the DNA that Fig. 1 is extracted in primer pair people's saliva for each microsatellite locus in embodiment 1 enters after performing PCR amplification
Increase production the electrophoresis comparison diagram of thing；
Fig. 2 is to carry out list using the microsatellite locus BAT25 designed in embodiment 1 primer pair wild type BAT25 plasmids
The CE electrophoretograms of product after weight fluorescent PCR amplification；
Fig. 3 is to carry out list using the microsatellite locus BAT26 designed in embodiment 1 primer pair wild type BAT26 plasmids
The CE electrophoretograms of product after weight fluorescent PCR amplification；
Fig. 4 is to be carried out using the microsatellite locus D2S123 designed in embodiment 1 primer pair wild type D2S123 plasmids
The CE electrophoretograms of product after the amplification of substance fluorescent PCR；
Fig. 5 is to be carried out using the microsatellite locus D5S346 designed in embodiment 1 primer pair wild type D5S346 plasmids
The CE electrophoretograms of product after the amplification of substance fluorescent PCR；
Fig. 6 is to be entered using the microsatellite locus D17S250 designed in embodiment 1 primer pair wild type D17S250 plasmids
The CE electrophoretograms of product after the amplification of row substance fluorescent PCR；
Fig. 7 is to be entered using the microsatellite locus Penta C designed in embodiment 2 primer pair wild type Penta C plasmids
The CE electrophoretograms of product after the amplification of row substance fluorescent PCR；
Fig. 8 is that wild plasmid is carried out after sixfold fluorescent PCR amplification using the PCR amplifing reagents designed in embodiment 2
The CE electrophoretograms of product；
Fig. 9 is to wild plasmid and the mixing plasmid of mutant plasmids using the PCR amplifing reagents designed in embodiment 2
Carry out the CE electrophoretograms of product after sixfold fluorescent PCR amplification；
Figure 10 is using normal structure and tumour of the PCR amplifing reagents designed in embodiment 2 to clinically sample 1701215
The genomic DNA of tissue carries out the CE electrophoresis comparison diagrams of product after sixfold fluorescent PCR amplification；
Figure 11 is using normal structure and tumour of the PCR amplifing reagents designed in embodiment 2 to clinically sample 1717924
The genomic DNA of tissue carries out the CE electrophoresis comparison diagrams of product after sixfold fluorescent PCR amplification；
Figure 12 is using normal structure and tumour of the PCR amplifing reagents designed in embodiment 2 to clinically sample 1700021
The genomic DNA of tissue carries out the CE electrophoresis comparison diagrams of product after sixfold fluorescent PCR amplification.
Embodiment
In order to facilitate the understanding of the purposes, features and advantages of the present invention, with reference to specific embodiment and
Accompanying drawing is described in detail to the embodiment of the present invention.Elaborate in the following description many details in order to
Fully understand the present invention.But the invention can be embodied in many other ways as described herein, art technology
Personnel can do similar improvement in the case of without prejudice to intension of the present invention, therefore the present invention is not by following public specific implementation
Limitation.
The multiple fluorescence PCR amplifing reagent of the detection microsatellite instability of one embodiment, including：
The sense primer BAT25-F1 and anti-sense primer BAT25-R1 designed for microsatellite locus BAT25, sense primer
BAT25-F1 base sequence is as shown in SEQ ID No.1, anti-sense primer BAT25-R1 base sequence such as SEQ ID No.2 institutes
Show.The sense primer BAT26-F1 and anti-sense primer BAT26-R1 designed for microsatellite locus BAT26, sense primer BAT26-
F1 base sequence is as shown in SEQ ID No.3, and anti-sense primer BAT26-R1 base sequence is as shown in SEQ ID No.4.Pin
The sense primer D2S123-F1 and anti-sense primer D2S123-R1 designed microsatellite locus D2S123, sense primer D2S123-
F1 base sequence is as shown in SEQ ID No.5, and anti-sense primer D2S123-R1 base sequence is as shown in SEQ ID No.6.Pin
The sense primer D5S346-F1 and anti-sense primer D5S346-R1 designed microsatellite locus D5S346, sense primer D5S346-
F1 base sequence is as shown in SEQ ID No.7, and anti-sense primer D5S346-R1 base sequence is as shown in SEQ ID No.8.With
And for sense primer D17S250-F1 and anti-sense primer D17S250-R1 that microsatellite locus D17S250 is designed, sense primer
D17S250-F1 base sequence is as shown in SEQ ID No.9, anti-sense primer D17S250-R1 base sequence such as SEQ ID
Shown in No.10.
Inventor has found, traditional PCR amplifing reagents to microsatellite locus (BAT25, BAT26, D2S123, D5S346 and
D17S250) enter after performing PCR amplification, the sequence length for the amplified production that the amplification of each microsatellite locus is obtained is relatively.Through electricity
The easy juxtaposition of fluorescence peak after swimming detection, it is more difficult to detect whether multiple microsatellite locus occur simultaneously in same PCR system
Change.Inventor has carried out substantial amounts of research to microsatellite locus BAT25, BAT26, D2S123, D5S346 and D17S250, weight
New design primer, be found surprisingly that the sense primer BAT25-F1 with specific base sequence, anti-sense primer BAT25-R1, on
Swim primer BAT26-F1, anti-sense primer BAT26-R1, sense primer D2S123-F1, anti-sense primer D2S123-R1, sense primer
D5S346-F1, anti-sense primer D5S346-R1, sense primer D17S250-F1 and anti-sense primer D17S250-R1 combine to be formed
PCR amplifing reagents can distinguish the corresponding microsatellite locus of specific amplification, and each amplified production is produced after electrophoresis detection
Raw fluorescence peak does not have the phenomenon of juxtaposition, so as to detected simultaneously in same PCR system microsatellite locus BAT25,
Whether BAT26, D2S123, D5S346 and D17S250 change, and detection efficiency is high, and sensitivity is good.
Further, the multiple fluorescence PCR amplifing reagent of detection microsatellite instability can be directed to from saliva or paraffin
The DNA of the extractions such as investing tissue's genome is expanded, and requires relatively low to amplification template, sensitivity is good.
Specifically, it has also been found that entering to the microsatellite locus D17S250 with dinucleotide repetitive sequence in research process
During row amplification, there is nonspecific amplified production more than comparison, obtained amplified production band unicity is poor, and inventor passes through
Microsatellite locus D17S250 primer is redesigned, is found surprisingly that base sequence is upper as shown in SEQ ID No.9
Swimming primer D17S250-F1, the anti-sense primer D17S250-R1 as shown in SEQ ID No.10 coordinates with base sequence, being capable of specificity
Amplification microsatellite locus D17S250, the amplified production band of generation is single.
In one embodiment, amplifing reagent also includes the sense primer designed for microsatellite locus Penta C
Penta C-F1 and anti-sense primer Penta C-R1, sense primer Penta C-F1 base sequence such as SEQ ID No.11 institutes
Show, anti-sense primer Penta C-R1 base sequence is as shown in SEQ ID No.12.
Microsatellite locus Penta C have fermentation by five tubes, control microsatellite locus during frequently as PCR.So
And, microsatellite locus Penta C are similar to D17S250, and amplified production band unicity is poor, both amplifications after PCR amplifications
The easy juxtaposition of product fluorescence peak after electrophoresis detection, is unfavorable for analysing and comparing.And by being redesigned to primer,
Sense primer Penta C-F1 and base sequence such as SEQ of the base sequence as shown in SEQ ID No.11 are added in amplifing reagent
Anti-sense primer Penta C-R1 shown in ID No.12, being capable of specific amplification microsatellite locus Penta C, and Penta C
Amplified production electrophoresis detection after the fluorescence peak that produces and microsatellite locus BAT25, BAT26, D2S123, D5S346 and
The fluorescence peak produced after D17S250 amplified production electrophoresis detection will not juxtaposition.Therefore, it is possible in same PCR system
Microsatellite locus (BAT25, BAT26, D2S123, D5S346 and D17S250) to be measured and the microsatellite compareed are detected simultaneously
Whether site Penta C change, and detection efficiency is high, and is capable of deciding whether the testing result for false positive, the standard of detection
True property is good.
In one embodiment, amplifing reagent also includes the sense primer designed for microsatellite locus Penta D
Penta D-F1 and anti-sense primer Penta D-R1, sense primer Penta D-F1 base sequence such as SEQ ID No.13 institutes
Show, anti-sense primer Penta D-R1 base sequence is as shown in SEQ ID No.14.
Microsatellite locus Penta D also have fermentation by five tubes, control microsatellite locus during frequently as PCR.
Sense primer Penta C-F1 and base sequence such as SEQ of the base sequence as shown in SEQ ID No.11 are added in amplifing reagent
The ID No.13 sense primer Penta D-F1 and anti-sense primer Penta D-R1 as shown in SEQ ID No.14, can be specific
Expand microsatellite locus Penta D, and the fluorescence peak and microsatellite locus produced after Penta D amplified production electrophoresis detection
The fluorescence peak produced after BAT25, BAT26, D2S123, D5S346 and D17S250 amplified production electrophoresis detection will not intersect weight
It is folded.Therefore, it is possible to detect microsatellite locus (BAT25, BAT26, D2S123, D5S346 to be measured simultaneously in same PCR system
And D17S250) and the microsatellite locus Penta D of control whether change, detection efficiency is high, and be capable of deciding whether for
The testing result of false positive, the accuracy of detection is good.
Further, amplifing reagent includes sense primer BAT25-F1, anti-sense primer BAT25-R1, sense primer BAT26-
F1, anti-sense primer BAT26-R1, sense primer D2S123-F1, anti-sense primer D2S123-R1, sense primer D5S346-F1, under
Swim primer D5S346-R1, sense primer D17S250-F1, anti-sense primer D17S250-R1, sense primer Penta C-F1, downstream
Primer Penta C-R1, sense primer Penta D-F1 and anti-sense primer Penta D-R1.Microsatellite locus BAT25, BAT26,
The fluorescence peak produced after D2S123, D5S346, D17S250, Penta C and Penta D amplified production electrophoresis detection is not
Can juxtaposition.Therefore, it is possible to detected simultaneously in same PCR system microsatellite locus to be measured (BAT25, BAT26,
D2S123, D5S346 and D17S250) and control microsatellite locus (Penta C and Penta D) whether change, examine
Efficiency high is surveyed, and can more accurately determine whether the testing result of false positive, the accuracy of detection is good.
In one embodiment, amplifing reagent middle and upper reaches primer BAT25-F1 concentration for 50nmol/L~
200nmol/L, anti-sense primer BAT25-R1 concentration are 50nmol/L~200nmol/L.
In one embodiment, sense primer BAT26-F1 concentration is 50nmol/L~200nmol/L, anti-sense primer
BAT26-R1 concentration is 50nmol/L~200nmol/L.
In one embodiment, sense primer D2S123-F1 concentration is 50nmol/L~200nmol/L, and downstream is drawn
Thing D2S123-R1 concentration is 50nmol/L~200nmol/L.
In one embodiment, sense primer D5S346-F1 concentration be 50nmol/L~200nmol/L, it is described under
The concentration for swimming primer D5S346-R1 is 50nmol/L~200nmol/L.
In one embodiment, sense primer D17S250-F1 concentration is 50nmol/L~200nmol/L, and downstream is drawn
Thing D17S250-R1 concentration is 50nmol/L~200nmol/L.
In detection, the primer concentration in amplifing reagent has certain to the intensity of fluorescence peak after amplified production electrophoresis detection
The sensitivity of influence, considering cost and detection, sense primer BAT25-F1, anti-sense primer BAT25-R1, sense primer
BAT26-F1, anti-sense primer BAT26-R1, sense primer D2S123-F1, anti-sense primer D2S123-R1, sense primer D5S346-
F1, anti-sense primer D5S346-R1, sense primer D17S250-F1 and anti-sense primer D17S250-R1 concentration are 50nmol/L
~200nmol/L, such as 70nmol/L, 100nmol/L, 150nmol/L or 190nmol/L.
Preferably, amplifing reagent middle and upper reaches primer BAT25-F1, anti-sense primer BAT25-R1, sense primer BAT26-F1,
Anti-sense primer BAT26-R1, sense primer D2S123-F1, anti-sense primer D2S123-R1, sense primer D5S346-F1, downstream are drawn
Thing D5S346-R1, sense primer D17S250-F1 and anti-sense primer D17S250-R1 concentration are 100nmol/L.
Further, amplifing reagent also includes the sense primer Penta C-F1 designed for microsatellite locus Penta C
During with anti-sense primer Penta C-R1, sense primer Penta C-F1 and anti-sense primer Penta C-R1 concentration are 50nmol/L
~200nmol/L, such as 70nmol/L, 100nmol/L, 150nmol/L or 190nmol/L.
Further, amplifing reagent also includes the sense primer Penta D-F1 designed for microsatellite locus Penta D
During with anti-sense primer Penta D-R1, sense primer Penta D-F1 and anti-sense primer Penta D-R1 concentration are 50nmol/L
~200nmol/L, such as 70nmol/L, 100nmol/L, 150nmol/L or 190nmol/L.
In one embodiment, at least one in sense primer BAT25-F1 and anti-sense primer BAT25-R1 is marked with
First fluorophor.At least one in sense primer BAT26-F1 and anti-sense primer BAT26-R1 is marked with the second fluorescent base
Group.At least one in sense primer D2S123-F1 and anti-sense primer D2S123-R1 is marked with the 3rd fluorophor.Draw upstream
At least one in thing D5S346-F1 and anti-sense primer D5S346-R1 is marked with the 4th fluorophor.Sense primer D17S250-
At least one in F1 and anti-sense primer D17S250-R1 is marked with the 5th fluorophor.And the 3rd fluorophor and the 4th fluorescence
Group is selected from two kinds of fluorophors differed.
By mark fluorescent group, it is easy to fluorescent value to detect the amount of corresponding amplified production.3rd fluorophor and the 4th
Fluorophor is selected from two kinds of fluorophors differed, is easy to distinguish microsatellite locus D2S123 and D5S346 amplified production
Fluorescence peak.Inventor has found in research process, the sequence length of microsatellite locus D2S123 and D5S346 amplified production compared with
It is fluorophor that is close, being differed to microsatellite locus D2S123 and D5S346 two kinds of amplimer mark, is conducive to area
The fluorescence peak produced after the amplified production electrophoresis for dividing microsatellite locus D2S123 and D5S346, improves the sensitivity of detection.
It will be appreciated, of course, that in other embodiments or add respectively detection microsatellite locus BAT25,
BAT26, D2S123, D5S346 and D17S250 probe, and mark on probe fluorophor.Now, it can not be marked on primer
Remember fluorophor.
In one embodiment, the first fluorophor is marked on anti-sense primer BAT25-R1, the second fluorophor mark
Note is on sense primer BAT26-F1, and the 3rd fluorophor is marked on sense primer D2S123-F1, the 4th fluorophor mark
On sense primer D5S346-F1, the 5th fluorophor is marked on anti-sense primer D17S250-R1.Inventor is in research process
Middle to find, fluorophor is marked on sense primer or anti-sense primer, the PCR primer electrophoresis detection produced after being expanded to PCR
Fluorescence peak has a certain impact, the mark position of the fluorophor of present embodiment, the corresponding amplification production produced after PCR amplifications
The fluorescence peak of thing electrophoresis detection is single, and sensitivity is high.
In one embodiment, the first fluorophor, the second fluorophor, the 3rd fluorophor, the 4th fluorophor
One in FAM fluorophors, TAMRA fluorophors, ROX fluorophors and JOE fluorophors is selected from the 5th fluorophor
Kind.
In one embodiment, the first fluorophor is FAM fluorophors, and the second fluorophor is TAMRA fluorescent bases
Group, the 3rd fluorophor is FAM fluorophors, and the 4th fluorophor is JOE fluorophors, and the 5th fluorophor is JOE fluorescence
Group.
Further, amplifing reagent also includes the sense primer Penta C-F1 designed for microsatellite locus Penta C
During with anti-sense primer Penta C-R1, at least one mark in sense primer Penta C-F1 and anti-sense primer Penta C-R1
There is the 6th fluorophor.6th fluorophor is selected from FAM fluorophors, TAMRA fluorophors, ROX fluorophors and JOE fluorescence
One kind in group.
Further, amplifing reagent also includes the sense primer Penta D-F1 designed for microsatellite locus Penta D
During with anti-sense primer Penta D-R1, at least one mark in sense primer Penta D-F1 and anti-sense primer Penta D-R1
There is the 7th fluorophor.7th fluorophor is selected from FAM fluorophors, TAMRA fluorophors, ROX fluorophors and JOE fluorescence
One kind in group.
Result of the test shows, the multiple fluorescence PCR amplifing reagent of above-mentioned detection microsatellite instability, and particular sequence draws
Thing can distinguish the corresponding microsatellite locus of specific amplification, and the amplified production band unicity of each microsatellite locus is good.Simultaneously
5 microsatellite locus (BAT25, BAT26, D2S123, D5S346 and D17S250) are entered after performing PCR amplification, each amplified production warp
The fluorescence peak produced after electrophoresis detection does not have the phenomenon of juxtaposition, so as to detect micro- defend simultaneously in same PCR system
Whether championship point BAT25, BAT26, D2S123, D5S346 and D17S250 change, and detection efficiency is high, and sensitivity is good.
Further, when the multiple fluorescence PCR amplifing reagent of above-mentioned detection microsatellite instability also includes defending for micro-
The sense primer Penta C-F1 and anti-sense primer Penta C-R1 of championship point Penta C designs, and/or PCR amplifing reagents are also
During including for Penta D the sense primer Penta D-F1 designed and anti-sense primer Penta D-R1.Being capable of same PCR system
In detect that microsatellite locus (BAT25, BAT26, D2S123, D5S346 and D17S250) to be measured and the micro- of control defend simultaneously
Whether championship point Penta C and/or Penta D change, and detection efficiency is high, and is capable of deciding whether the detection for false positive
As a result, the accuracy of detection is good.
The multiple fluorescence PCR detection reagent box of the detection microsatellite instability of one embodiment, including above-mentioned amplification
Reagent.
Further, Taq archaeal dna polymerases and PCR reaction buffers are also included in above-mentioned detection kit, kit.
The detection kit can simultaneously be detected in same PCR system microsatellite locus BAT25, BAT26, D2S123,
Whether D5S346 and D17S250 changes, and detection efficiency is high, and sensitivity is good.
It is specific embodiment part below.
In following examples, unless otherwise instructed, the experimental method of unreceipted actual conditions, generally according to normal condition,
For example, see Pehanorm Brooker, EF, not the written molecular cloning of Ritchie, T Mannies A Disi etc. (Jin Dongyan, Li Mengfeng etc. are translated) is real
Test guide [M] (Beijing：Science Press, 1992) described in condition or kit manufacturer recommend method realize.
Reagent used in embodiment is commercially available.
Embodiment 1
The gene numbering where microsatellite locus to be measured is consulted in Gene Bank, it is determined that the master of each microsatellite locus
Want the base sequence of repetitive sequence and main repetitive sequence upstream and downstream 1000bp or so, the relevant information such as table of microsatellite locus
Shown in 1.
Table 1：The essential information of microsatellite locus
| Microsatellite locus | Gene is numbered | Main repetitive sequence |
| BAT25 | L04143 | (A)25 |
| BAT26 | U41210 | (A)26 |
| D2S123 | Z16551.1 | (CA)15 |
| D5S346 | NM_005669 | (CA)20 |
| D17S250 | NR_033753.2 | (CA)24 |
Sense primer and anti-sense primer, and corresponding mark fluorescent base are targetedly designed each microsatellite locus
Group, it is specific as shown in table 2.
Table 2:The sense primer and anti-sense primer of microsatellite locus
The primer of design by raw work biosynthesis, by the sense primer BAT25-F1 of synthesis, anti-sense primer BAT25-R1,
Sense primer BAT26-F1, anti-sense primer BAT26-R1, sense primer D2S123-F1, anti-sense primer D2S123-R1, upstream are drawn
Thing D5S346-F1, anti-sense primer D5S346-R1, sense primer D17S250-F1 and anti-sense primer D17S250-R1 mixing, shape
Into the multiple fluorescence PCR amplifing reagent of detection microsatellite instability, wherein, the concentration of each primer is 100nmol/L.This reality
Apply example PCR amplifing reagents can in same PCR system and meanwhile 5 microsatellite locus BAT25 of amplification, BAT26, D2S123,
D5S346 and D17S250, the amplified production that the amplification of each microsatellite locus is obtained fluorescence peak after electrophoresis detection will not intersect weight
It is folded.
Embodiment 2
Sense primer BAT25- in the multiple fluorescence PCR amplifing reagent of the detection microsatellite instability of the present embodiment
F1, anti-sense primer BAT25-R1, sense primer BAT26-F1, anti-sense primer BAT26-R1, sense primer D2S123-F1, downstream
Primer D2S123-R1, sense primer D5S346-F1, anti-sense primer D5S346-R1, sense primer D17S250-F1 and downstream
Primer D17S250-R1 is same as Example 1, unlike, the microsatellite locus for control is also added into the present embodiment
The sense primer Penta C-F1 and anti-sense primer Penta C-R1 of Penta C designs.Microsatellite locus Penta C related letter
Breath is as shown in table 3.
Table 3：Microsatellite locus Penta C essential information
| Microsatellite locus | Gene is numbered | Main repetitive sequence |
| Penta C | AL138752 | (AAAAG)3 |
Sense primer Penta C-F1 and the downstream of Penta C design is targetedly designed microsatellite locus Penta C
Primer Penta C-R1, and corresponding mark fluorescent group, it is specific as shown in table 4.
Table 4:The sense primer and anti-sense primer of microsatellite locus
The primer of design is by raw work biosynthesis, by the sense primer Penta C-F1 and anti-sense primer Penta of synthesis
C-R1 is mixed with the PCR amplifing reagents of embodiment 1, forms the multiple fluorescence PCR amplifing reagent of detection microsatellite instability.Its
In, sense primer Penta C-F1 and anti-sense primer Penta C-R1 concentration are 100nmol/L.The PCR of the present embodiment expands
Increase reagent can in same PCR system simultaneously 6 microsatellite locus BAT25 of amplification, BAT26, D2S123, D5S346,
D17S250 and Penta C, the amplified production that the amplification of each microsatellite locus is obtained fluorescence peak after electrophoresis detection will not intersect weight
It is folded.
Embodiment 3
Sense primer BAT25- in the multiple fluorescence PCR amplifing reagent of the detection microsatellite instability of the present embodiment
F1, anti-sense primer BAT25-R1, sense primer BAT26-F1, anti-sense primer BAT26-R1, sense primer D2S123-F1, downstream
Primer D2S123-R1, sense primer D5S346-F1, anti-sense primer D5S346-R1, sense primer D17S250-F1 and downstream
Primer D17S250-R1 is same as Example 1, unlike, the microsatellite locus for control is also added into the present embodiment
The sense primer Penta D-F1 and anti-sense primer Penta D-R1 of Penta D designs.Microsatellite locus Penta C related letter
Breath is as shown in table 5.
Table 5：Microsatellite locus Penta D essential information
| Microsatellite locus | Gene is numbered | Main repetitive sequence |
| Penta D | Ac000014 | (AAAAG)8 |
Sense primer Penta D-F1 and the downstream of Penta D design is targetedly designed microsatellite locus Penta D
Primer Penta D-R1, and corresponding mark fluorescent group, it is specific as shown in table 6.
Table 6:The sense primer and anti-sense primer of microsatellite locus
The primer of design is by raw work biosynthesis, by the sense primer Penta D-F1 and anti-sense primer Penta of synthesis
D-R1 is mixed with the PCR amplifing reagents of embodiment 1, forms the multiple fluorescence PCR amplifing reagent of detection microsatellite instability.Its
In, sense primer Penta D-F1 and anti-sense primer Penta D-R1 concentration are 100nmol/L.The PCR of the present embodiment expands
Increase reagent can in same PCR system simultaneously 6 microsatellite locus BAT25 of amplification, BAT26, D2S123, D5S346,
D17S250 and Penta D, the amplified production that the amplification of each microsatellite locus is obtained fluorescence peak after electrophoresis detection will not intersect weight
It is folded.
Embodiment 4
The multiple fluorescence PCR amplifing reagent of the detection microsatellite instability of the present embodiment includes sense primer BAT25-
F1, anti-sense primer BAT25-R1, sense primer BAT26-F1, anti-sense primer BAT26-R1, sense primer D2S123-F1, downstream
Primer D2S123-R1, sense primer D5S346-F1, anti-sense primer D5S346-R1, sense primer D17S250-F1, anti-sense primer
D17S250-R1, sense primer Penta C-F1 and anti-sense primer Penta C-R1, sense primer Penta D-F1 and downstream are drawn
Thing Penta D-R1.The sequence of specific primer is referring to upper table 2, table 4 and table 5.The concentration of each primer is 100nmol/L.This reality
Apply example PCR amplifing reagents can in same PCR system and meanwhile 7 microsatellite locus BAT25 of amplification, BAT26, D2S123,
D5S346, D17S250, Penta C and Penta D, the amplified production that the amplification of each microsatellite locus is obtained is after electrophoresis detection
Fluorescence peak will not juxtaposition.
Test one
Detect the band of amplified production
The saliva sample of people is gathered, (raw work is biological, production code member with kit：B518266 genomic DNA) is extracted, with
The saliva genomic DNA of people be template, be separately added into the microsatellite locus BAT25 designed as shown in table 1, BAT26, D2S123,
D5S346, D17S250 sense primer and anti-sense primer enter performing PCR amplification, and amplified production carries out gel electrophoresis, as a result referring to Fig. 1
It is shown.Far Left swimming lane is that (raw work is biological, production code member by electrophoresis Marker：B600303), Marker stripe sizes are followed successively by
500bp, 400bp, 300bp, 200bp, 150bp, 100bp, 75bp, 50bp and 25bp.BAT25、BAT26、D2S123、
D5S346, D17S250 each at least four parallel laboratory test of amplified production.It will be seen from figure 1 that being drawn using design table 1 Suo Shi
After thing amplification BAT25, BAT26, D2S123, D5S346, D17S250, the band of amplified production is single, illustrates the special of each primer
Property it is good, being capable of the corresponding microsatellite locus of specific amplification.
Test two
Substance fluoroscopic examination amplified production
Respectively with 1pg/ μ L wild type BAT25 plasmids, wild type BAT26 plasmids, wild type D2S123 plasmids, wild type
D5S346 plasmids, wild type D17S250 plasmids and wild type P enta C plasmids are used as amplification template.Using microsatellite locus
BAT25, BAT26, D2S123, D5S346, D17S250 and Penta C each self-corresponding sense primer and anti-sense primer carry out single
Weight fluorescent PCR amplification, and amplified production is subjected to CE electrophoresis, as a result respectively as shown in Figure 2 to 7.Abscissa is amplification in figure
The base numerical value of the genetic fragment of product, ordinate is fluorescence values.It can be seen that microsatellite locus BAT25,
Fluorescence peak after BAT26, D2S123, D5S346, D17S250 and Penta C amplified production CE electrophoresis compares concentration, in phase
The base value bit answered is equipped with obvious specific peak value.
Test three
The amplified production of sixfold fluoroscopic examination wild type microsatellite locus
By wild type BAT25 plasmids, wild type BAT26 plasmids, wild type D2S123 plasmids, wild type D5S346 plasmids,
Wild type D17S250 plasmids and the mixing of wild type Penta C plasmids are used as amplification template.The concentration of wherein each plasmid is 1pg/
μL.Fluorescent PCR amplification is carried out using the multiple fluorescence PCR amplifing reagent amplification of embodiment 2, and amplified production is subjected to CE electricity
Swimming, as a result as shown in Figure 8.Abscissa is the base numerical value of the genetic fragment of amplified production in figure, and ordinate is fluorescence values.From
It can be seen from the figure that, microsatellite locus BAT25, BAT26, D2S123, D5S346, D17S250 and Penta C amplified production CE
Fluorescence peak after electrophoresis does not have the phenomenon of juxtaposition, so as to detect microsatellite locus simultaneously in same PCR system
Whether BAT25, BAT26, D2S123, D5S346, D17S250 and Penta C change, and detection efficiency is high, and sensitivity is good.
Test four
Sixfold fluoroscopic examination contains the amplified production of the microsatellite locus of saltant type
By wild type BAT25 plasmids, wild type BAT26 plasmids, wild type D2S123 plasmids, wild type D5S346 matter
Grain, wild type D17S250 plasmids and the mixing of wild type Penta C plasmids, then add saltant type BAT25 plasmids (5 weights of missing
Multiple base), the saltant type BAT26 plasmids base of 4 repetitions (missing), saltant type D2S123 plasmids (27 repetitions of missing
Base) and saltant type D17S250 plasmids (base of 10 repetitions of missing) mixing.The concentration of wherein each plasmid is 1pg/ μ L.
Fluorescent PCR amplification is carried out using the multiple fluorescence PCR amplifing reagent amplification of embodiment 2, and amplified production is subjected to CE electrophoresis, knot
Fruit is as shown in Figure 9.Abscissa is the base numerical value of the genetic fragment of amplified production in figure, and ordinate is fluorescence values.Compare Fig. 8
Understood with Fig. 9, add saltant type BAT25 plasmids, saltant type BAT26 plasmids, saltant type D2S123 plasmids and saltant type D17S250
After plasmid, the peak of the amplified production of corresponding four microsatellite locus is moved to left or moved to right, and detection sensitivity is high.
Test five
The genomic DNA using the normal structure sample of clinically same patient and tumor tissues sample is adopted as template respectively
MSI detections are carried out with the multiple fluorescence PCR amplifing reagent of embodiment 2.Sample 1701215, sample 1717924 and sample
1700021 MSI detections CE peak figures are as follows according to the MSI types that MSI testing results collect sample respectively as shown in Figure 10~12
Table 7.
Table 7:MSI testing results
| Sample number into spectrum | Testing result | MSI types |
| 1701215 | Five sites are unchanged | MSS types |
| 1717924 | Five sites are unchanged | MSS types |
| 1700021 | BAT-25, BAT-26 and D2S123 occur bimodal | MSH types |
Embodiment described above only expresses one or more of embodiments of the present invention, and it describes more specific and detailed
Carefully, but can not therefore and be interpreted as the limitation to the scope of the claims of the present invention.It should be pointed out that for the common skill of this area
For art personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to this hair
Bright protection domain.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
SEQUENCE LISTING
<110>Changzhou tung oil tree bio tech ltd
<120>Detect the multiple fluorescence PCR amplifing reagent and kit of microsatellite instability
<160> 14
<170> PatentIn version 3.3
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Claims (10)
1. a kind of multiple fluorescence PCR amplifing reagent for detecting microsatellite instability, it is characterised in that including：
The sense primer BAT25-F1 and anti-sense primer BAT25-R1 designed for microsatellite locus BAT25, the sense primer
BAT25-F1 base sequence is as shown in SEQ ID No.1, the base sequence such as SEQ ID of the anti-sense primer BAT25-R1
Shown in No.2；
The sense primer BAT26-F1 and anti-sense primer BAT26-R1 designed for microsatellite locus BAT26, the sense primer
BAT26-F1 base sequence is as shown in SEQ ID No.3, the base sequence such as SEQ ID of the anti-sense primer BAT26-R1
Shown in No.4；
The sense primer D2S123-F1 and anti-sense primer D2S123-R1 designed for microsatellite locus D2S123, the upstream is drawn
Thing D2S123-F1 base sequence is as shown in SEQ ID No.5, the base sequence such as SEQ of the anti-sense primer D2S123-R1
Shown in ID No.6；
The sense primer D5S346-F1 and anti-sense primer D5S346-R1 designed for microsatellite locus D5S346, the upstream is drawn
Thing D5S346-F1 base sequence is as shown in SEQ ID No.7, the base sequence such as SEQ of the anti-sense primer D5S346-R1
Shown in ID No.8；And for the sense primer D17S250-F1 and anti-sense primer of microsatellite locus D17S250 designs
D17S250-R1, the sense primer D17S250-F1 base sequence are as shown in SEQ ID No.9, the anti-sense primer
D17S250-R1 base sequence is as shown in SEQ ID No.10.
2. the multiple fluorescence PCR amplifing reagent of detection microsatellite instability according to claim 1, it is characterised in that
The amplifing reagent also includes：
For the microsatellite locus Penta C sense primer Penta C-F1 designed and anti-sense primer Penta C-R1, it is described on
Primer Penta C-F1 base sequence is swum as shown in SEQ ID No.11, the base sequence of the anti-sense primer Penta C-R1
As shown in SEQ ID No.12.
3. the multiple fluorescence PCR amplifing reagent of detection microsatellite instability according to claim 1, it is characterised in that
The amplifing reagent also includes：
For the microsatellite locus Penta D sense primer Penta D-F1 designed and anti-sense primer Penta D-R1, it is described on
Primer Penta D-F1 base sequence is swum as shown in SEQ ID No.13, the base sequence of the anti-sense primer Penta D-R1
As shown in SEQ ID No.14.
4. the multiple fluorescence PCR amplifing reagent of detection microsatellite instability according to claim 1, it is characterised in that
In the amplifing reagent,
The concentration of the sense primer BAT25-F1 is 50nmol/L~200nmol/L, and the anti-sense primer BAT25-R1's is dense
Spend for 50nmol/L~200nmol/L；And/or,
The concentration of the sense primer BAT26-F1 is 50nmol/L~200nmol/L, and the anti-sense primer BAT26-R1's is dense
Spend for 50nmol/L~200nmol/L；And/or,
The concentration of the sense primer D2S123-F1 is 50nmol/L~200nmol/L, the anti-sense primer D2S123-R1's
Concentration is 50nmol/L~200nmol/L；And/or,
The concentration of the sense primer D5S346-F1 is 50nmol/L~200nmol/L, the anti-sense primer D5S346-R1's
Concentration is 50nmol/L~200nmol/L；And/or,
The concentration of the sense primer D17S250-F1 is 50nmol/L~200nmol/L, the anti-sense primer D17S250-R1
Concentration be 50nmol/L~200nmol/L.
5. the multiple fluorescence PCR amplifing reagent of detection microsatellite instability according to claim 1, it is characterised in that
At least one in the sense primer BAT25-F1 and the anti-sense primer BAT25-R1 is marked with the first fluorophor, described
At least one in sense primer BAT26-F1 and the anti-sense primer BAT26-R1 is marked with the second fluorophor, the upstream
At least one in primer D2S123-F1 and the anti-sense primer D2S123-R1 is marked with the 3rd fluorophor, and the upstream is drawn
At least one in thing D5S346-F1 and the anti-sense primer D5S346-R1 is marked with the 4th fluorophor, the sense primer
At least one in D17S250-F1 and the anti-sense primer D17S250-R1 is marked with the 5th fluorophor, and described 3rd glimmering
Light group is selected from two kinds of fluorophors differed with the 4th fluorophor.
6. the multiple fluorescence PCR amplifing reagent of detection microsatellite instability according to claim 5, it is characterised in that
First fluorophor is marked on the anti-sense primer BAT25-R1, and second fluorophor is marked at the upstream and drawn
On thing BAT26-F1, the 3rd fluorophor is marked on the sense primer D2S123-F1, the 4th fluorophor mark
Note is on the sense primer D5S346-F1, and the 5th fluorophor is marked on the anti-sense primer D17S250-R1.
7. the multiple fluorescence PCR amplifing reagent of detection microsatellite instability according to claim 5, it is characterised in that
First fluorophor, second fluorophor, the 3rd fluorophor, the 4th fluorophor and the described 5th
Fluorophor is selected from one kind in FAM fluorophors, TAMRA fluorophors, ROX fluorophors and JOE fluorophors.
8. the multiple fluorescence PCR amplifing reagent of the detection microsatellite instability according to claim 5 or 7, its feature exists
In first fluorophor is FAM fluorophors, and second fluorophor is TAMRA fluorophors, the 3rd fluorescence
Group is FAM fluorophors, and the 4th fluorophor is JOE fluorophors, and the 5th fluorophor is JOE fluorescent bases
Group.
9. a kind of multiple fluorescence PCR detection reagent box for detecting microsatellite instability, it is characterised in that including such as claim
Amplifing reagent described in 1~8 any one.
10. the multiple fluorescence PCR detection reagent box of microsatellite instability is detected as claimed in claim 9, it is characterised in that
Also include Taq archaeal dna polymerases and PCR reaction buffers in the kit.
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| CN110904236A (en) * | 2019-12-23 | 2020-03-24 | 武汉百泰基因工程有限公司 | Detection kit for microsatellite unstable state and detection method thereof |
| CN111370063A (en) * | 2020-03-23 | 2020-07-03 | 上海欧易生物医学科技有限公司 | MSI (MSI-based micro satellite instability) detection method and system based on Pacbio data |
| WO2022100766A1 (en) * | 2020-11-16 | 2022-05-19 | 江萤 | Method for detecting human microsatellite instability site, and use thereof |
| CN114107453A (en) * | 2021-09-17 | 2022-03-01 | 艾普拜生物科技(苏州)有限公司 | Kit for detecting instability of microsatellite |
| CN113981046A (en) * | 2021-11-05 | 2022-01-28 | 朱运峰 | DNA methylation detection method based on quantitative PCR technology and kit thereof |
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