CN107217103B - 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 reagents and kit for detecting microsatellite instability.The PCR amplification reagent includes that can expand multiple microsatellite locus simultaneously in same PCR system for the upstream primer and downstream primer of microsatellite locus BAT25, BAT26, D2S123, D5S346 and D17S250 design respectively and carry out multiple fluorescence PCR detection microsatellite instability.The fluorescence peak that the amplified production of each microsatellite locus generates after electrophoresis detection does not have the phenomenon that 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 technique
Microsatellite is also known as short tandem repeat (Short tandem repeats, STRs) or simple repeated sequence
(Simple sequences repeats, SSRs), is distributed widely in human genome.Repetitive sequence is generally by 1~6 core
Thuja acid composition is especially that (CA/GT) n is most commonly seen with dinucleotides repetitive sequence, and n is number of repetition, usually 15~60
It is secondary.The conjugated protein or direct coding protein of microsatellite energy autospecific, some may then participate in the folding of chromatid
Folded or formation of telomere etc..Microsatellite is by changing DNA structure or by playing gene regulation in conjunction with specific protein
Effect, is molecular labeling a new class of, that polymorphism information capacity is high.
Microsatellite instability (Microsatellite Instability, MSI) refers to due to mispairing reparation or duplication
The increase or loss of simple repeated sequence caused by mistake etc., cause the length of microsatellite to change.The main original that MSI occurs
Participate in the gene function of pairing errors repair to occur defect because being, thus cannot normal correct and copy mistake, cause microsatellite
DNA change, prevent it from normally playing regulating and controlling effect.MSI is easy to cause cell Proliferation and disdifferentiation, or even promotees
Hair malignant tumour is formed.Compared with normal tissue, the microsatellite in tumour is easier that microsatellite length is caused to change.It grinds
Study carefully and shows that the generation of the tumours such as MSI and colorectal cancer, gastric cancer, carcinoma of endometrium is closely related.Wherein about 15% sporadic knot
The carcinoma of the rectum is related to MSI, 90% or more hereditary nonpolyposis colorectal cancer (HNPCC, also referred to as Lynch syndrome) with
MSI is related, therefore is clinically of great significance to MSI detection.
The method of detection microsatellite instability is then micro- in PCR amplification sample generally by selecting special primer
Satellite site, the product after amplification is analyzed in the display system after electrophoresis detection (such as Capillary Electrophoresis), and and normal tissue
It compares, judges that whether there is or not changes for sequence length of microsatellite locus etc..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 mononucleotide repeat sequence, D2S123, D5S346 and
D17S250 has dinucleotide repetitive sequence.The testing result of comparison of tumor and normal sample after detection, if above 5 micro-
There are 2 or 2 or more microsatellite locus to change in satellite site, referred to as high-frequency type (High-frequency MSI, MSI-
H)；Only 1 microsatellite locus changes, referred to as low frequency type (low-frequency MSI, MSI-L), and 5 are not sent out
Raw change is known as microsatellite stable type (Microsatellite stable, MSS).
However, traditional PCR amplification reagent to microsatellite locus (BAT25, BAT26, D2S123, D5S346 and
D17S250 after) carrying out PCR amplification, the amplified production that each microsatellite locus expands 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.
Summary of the invention
Based on this, it is necessary to which the amplified production for providing each microsatellite locus after a kind of PCR amplification produces after electrophoresis detection
Raw fluorescence peak will not juxtaposition, can in same PCR system simultaneously detect microsatellite locus BAT25, BAT26,
D2S123, D5S346 and D17S250 it is whether changed detection microsatellite instability multiple fluorescence PCR amplifing reagent and
Detection kit.
A kind of multiple fluorescence PCR amplifing reagent detecting microsatellite instability, comprising:
For the upstream primer BAT25-F1 and downstream primer BAT25-R1 of microsatellite locus BAT25 design, the upstream
The base sequence of primer BAT25-F1 is as shown in SEQ ID No.1, the base sequence such as SEQ of the downstream primer BAT25-R1
Shown in ID No.2；
For the upstream primer BAT26-F1 and downstream primer BAT26-R1 of microsatellite locus BAT26 design, the upstream
The base sequence of primer BAT26-F1 is as shown in SEQ ID No.3, the base sequence such as SEQ of the downstream primer BAT26-R1
Shown in ID No.4；
For the upstream primer D2S123-F1 and downstream primer D2S123-R1 of microsatellite locus D2S123 design, it is described on
The base sequence of primer D2S123-F1 is swum as shown in SEQ ID No.5, the base sequence of the downstream primer D2S123-R1 is such as
Shown in SEQ ID No.6；
For the upstream primer D5S346-F1 and downstream primer D5S346-R1 of microsatellite locus D5S346 design, it is described on
The base sequence of primer D5S346-F1 is swum as shown in SEQ ID No.7, the base sequence of the downstream primer D5S346-R1 is such as
Shown in SEQ ID No.8；And upstream primer D17S250-F1 and downstream primer for microsatellite locus D17S250 design
The base sequence of D17S250-R1, the upstream primer D17S250-F1 are as shown in SEQ ID No.9, the downstream primer
The base sequence of D17S250-R1 is as shown in SEQ ID No.10.
In one embodiment, the amplifing reagent further include:
For the upstream primer Penta C-F1 and downstream primer Penta C-R1 of microsatellite locus Penta C design, institute
The base sequence of upstream primer Penta C-F1 is stated as shown in SEQ ID No.11, the base of the downstream primer Penta C-R1
Sequence is as shown in SEQ ID No.12.
In one embodiment, the amplifing reagent further include:
For the upstream primer Penta D-F1 and downstream primer Penta D-R1 of microsatellite locus Penta D design, institute
The base sequence of upstream primer Penta D-F1 is stated as shown in SEQ ID No.13, the base of the downstream primer Penta D-R1
Sequence is as shown in SEQ ID No.14.
In one embodiment, in the amplifing reagent,
The concentration of the upstream primer BAT25-F1 is 50nmol/L~200nmol/L, the downstream primer BAT25-R1
Concentration be 50nmol/L~200nmol/L；And/or
The concentration of the upstream primer BAT26-F1 is 50nmol/L~200nmol/L, the downstream primer BAT26-R1
Concentration be 50nmol/L~200nmol/L；And/or
The concentration of the upstream primer D2S123-F1 is 50nmol/L~200nmol/L, the downstream primer D2S123-
The concentration of R1 is 50nmol/L~200nmol/L；And/or
The concentration of the upstream primer D5S346-F1 is 50nmol/L~200nmol/L, the downstream primer D5S346-
The concentration of R1 is 50nmol/L~200nmol/L；And/or
The concentration of the upstream primer D17S250-F1 is 50nmol/L~200nmol/L, the downstream primer
The concentration of D17S250-R1 is 50nmol/L~200nmol/L.
In one embodiment, at least one in the upstream primer BAT25-F1 and the downstream primer BAT25-R1
A label has fluorophor, at least one of the upstream primer BAT26-F1 and the downstream primer BAT26-R1 mark
Note has the second fluorophor, at least one of the upstream primer D2S123-F1 and the downstream primer D2S123-R1 label
There is third fluorophor, at least one of the upstream primer D5S346-F1 and the downstream primer D5S346-R1 are marked with
4th fluorophor, at least one of the upstream primer D17S250-F1 and the downstream primer D17S250-R1 are marked with
5th fluorophor, and the third fluorophor and the 4th fluorophor are selected from two kinds of different fluorophors.
In one embodiment, first fluorophor label is on the downstream primer BAT25-R1, and described the
Two fluorophors mark on the upstream primer BAT26-F1, and the third fluorophor label is in the upstream primer
On D2S123-F1, the 4th fluorophor label is on the upstream primer D5S346-F1, the 5th fluorophor mark
Note is on the downstream primer D17S250-R1.
In one embodiment, first fluorophor, second fluorophor, the third fluorophor,
4th fluorophor and the 5th fluorophor are selected from FAM fluorophor, TAMRA fluorophor, ROX fluorophor
One of with JOE fluorophor.
In one embodiment, first fluorophor is FAM fluorophor, and second fluorophor is
TAMRA fluorophor, the third fluorophor are FAM fluorophor, and the 4th fluorophor is JOE fluorophor, institute
Stating the 5th fluorophor is JOE fluorophor.
A kind of multiple fluorescence PCR detection reagent box detecting microsatellite instability, including above-mentioned amplifing reagent.
It in one embodiment, further include Taq archaeal dna polymerase and PCR reaction buffer in the kit.
The multiple fluorescence PCR amplifing reagent of above-mentioned detection microsatellite instability, including it is directed to 5 microsatellite locus respectively
The upstream primer and downstream primer of (BAT25, BAT26, D2S123, D5S346 and D17S250) design.On test result shows
The primer for stating particular sequence can distinguish the corresponding microsatellite locus of specific amplification, the amplified product band of each microsatellite locus
Unicity is good.PCR amplification is carried out to 5 microsatellite locus (BAT25, BAT26, D2S123, D5S346 and D17S250) simultaneously
Afterwards, the fluorescence peak that each amplified production generates after electrophoresis detection does not have the phenomenon that juxtaposition, so as in same PCR system
In detect the microsatellite instability of microsatellite locus BAT25, BAT26, D2S123, D5S346 and D17S250, detection effect simultaneously
Rate is high, and sensitivity is good.
Detailed description of the invention
Fig. 1 is that the DNA extracted in primer pair people's saliva of each microsatellite locus in embodiment 1 carries out the expansion after PCR amplification
Increase production the electrophoresis comparison diagram of object；
Fig. 2 is to carry out list using the primer pair wild type BAT25 plasmid of the microsatellite locus BAT25 designed in embodiment 1
The CE electrophoretogram of product after weight fluorescent PCR amplification；
Fig. 3 is to carry out list using the primer pair wild type BAT26 plasmid of the microsatellite locus BAT26 designed in embodiment 1
The CE electrophoretogram of product after weight fluorescent PCR amplification；
Fig. 4 is to be carried out using the primer pair wild type D2S123 plasmid of the microsatellite locus D2S123 designed in embodiment 1
The CE electrophoretogram of product after the amplification of substance fluorescent PCR；
Fig. 5 is to be carried out using the primer pair wild type D5S346 plasmid of the microsatellite locus D5S346 designed in embodiment 1
The CE electrophoretogram of product after the amplification of substance fluorescent PCR；
Fig. 6 be using the microsatellite locus D17S250 designed in embodiment 1 primer pair wild type D17S250 plasmid into
The CE electrophoretogram of product after the amplification of row substance fluorescent PCR；
Fig. 7 be using the microsatellite locus Penta C designed in embodiment 2 primer pair wild type Penta C plasmid into
The CE electrophoretogram of product after the amplification of row substance fluorescent PCR；
Fig. 8 is after carrying out the amplification of sixfold fluorescent PCR to wild plasmid using the PCR amplification reagent designed in embodiment 2
The CE electrophoretogram of product；
Fig. 9 is using the PCR amplification reagent designed in embodiment 2 to the mixing plasmid of wild plasmid and mutant plasmids
The CE electrophoretogram of product after progress sixfold fluorescent PCR amplification；
Figure 10 is using the PCR amplification reagent designed in embodiment 2 to the normal tissue and tumour of clinically sample 1701215
The genomic DNA of tissue carries out the CE electrophoresis comparison diagram of product after the amplification of sixfold fluorescent PCR；
Figure 11 is using the PCR amplification reagent designed in embodiment 2 to the normal tissue and tumour of clinically sample 1717924
The genomic DNA of tissue carries out the CE electrophoresis comparison diagram of product after the amplification of sixfold fluorescent PCR；
Figure 12 is using the PCR amplification reagent designed in embodiment 2 to the normal tissue and tumour of clinically sample 1700021
The genomic DNA of tissue carries out the CE electrophoresis comparison diagram of product after the amplification of sixfold fluorescent PCR.
Specific embodiment
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, combined with specific embodiments below and
Specific embodiments of the present invention will be described in detail for attached drawing.Be explained 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 without violating the connotation 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, comprising:
For the upstream primer BAT25-F1 and downstream primer BAT25-R1 of microsatellite locus BAT25 design, upstream primer
The base sequence of BAT25-F1 is as shown in SEQ ID No.1, the base sequence of downstream primer BAT25-R1 such as SEQ ID No.2 institute
Show.For the upstream primer BAT26-F1 and downstream primer BAT26-R1 of microsatellite locus BAT26 design, upstream primer BAT26-
The base sequence of F1 is as shown in SEQ ID No.3, and the base sequence of downstream primer BAT26-R1 is as shown in SEQ ID No.4.Needle
To the upstream primer D2S123-F1 and downstream primer D2S123-R1 of microsatellite locus D2S123 design, upstream primer D2S123-
The base sequence of F1 is as shown in SEQ ID No.5, and the base sequence of downstream primer D2S123-R1 is as shown in SEQ ID No.6.Needle
To the upstream primer D5S346-F1 and downstream primer D5S346-R1 of microsatellite locus D5S346 design, upstream primer D5S346-
The base sequence of F1 is as shown in SEQ ID No.7, and the base sequence of downstream primer D5S346-R1 is as shown in SEQ ID No.8.With
And upstream primer D17S250-F1 and downstream primer D17S250-R1 for microsatellite locus D17S250 design, upstream primer
The base sequence of D17S250-F1 is as shown in SEQ ID No.9, the base sequence of downstream primer D17S250-R1 such as SEQ ID
Shown in No.10.
Inventors have found that traditional PCR amplification reagent to microsatellite locus (BAT25, BAT26, D2S123, D5S346 and
D17S250 after) carrying out PCR amplification, the sequence length for the amplified production that each microsatellite locus expands is relatively.Through electricity
Fluorescence peak is easy juxtaposition after swimming detection, it is more difficult to detect whether multiple microsatellite locus occur simultaneously in same PCR system
Variation.Inventor has carried out a large amount of research to microsatellite locus BAT25, BAT26, D2S123, D5S346 and D17S250, weight
New design primer, be found surprisingly that the upstream primer BAT25-F1 with specific base sequence, downstream primer BAT25-R1, on
Swim primer BAT26-F1, downstream primer BAT26-R1, upstream primer D2S123-F1, downstream primer D2S123-R1, upstream primer
D5S346-F1, downstream primer D5S346-R1, upstream primer D17S250-F1 and downstream primer D17S250-R1 combine to be formed
PCR amplification reagent be capable of the corresponding microsatellite locus of amplification of specificity respectively, and each amplified production produces after electrophoresis detection
Raw fluorescence peak does not have the phenomenon that juxtaposition, so as in same PCR system simultaneously detect 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 for detecting microsatellite instability can be directed to from saliva or paraffin
The DNA of the extractions such as investing tissue's genome is expanded, lower to amplification template requirement, and sensitivity is good.
Specifically, in research process it has also been found that the microsatellite locus D17S250 with dinucleotide repetitive sequence into
When row amplification, there is more nonspecific amplified production, obtained amplified product band unicity is poor, and inventor passes through
The primer of microsatellite locus D17S250 is redesigned, be found surprisingly that base sequence as shown in SEQ ID No.9 on
Primer D17S250-F1 and base sequence the downstream primer D17S250-R1 as shown in SEQ ID No.10 cooperation are swum, it being capable of specificity
Amplification microsatellite locus D17S250, the amplified product band of generation is single.
In one embodiment, amplifing reagent further includes the upstream primer for microsatellite locus Penta C design
The base sequence such as SEQ ID No.11 institute of Penta C-F1 and downstream primer Penta C-R1, upstream primer Penta C-F1
Show, the base sequence of downstream primer Penta C-R1 is as shown in SEQ ID No.12.
Microsatellite locus Penta C has fermentation by five tubes, control microsatellite locus when frequently as PCR.So
And microsatellite locus Penta C is similar to D17S250, amplified product band unicity is poor, the amplification both after PCR amplification
Product fluorescence peak after electrophoresis detection is easy juxtaposition, is unfavorable for analysing and comparing.And by being redesigned to primer,
Base sequence the upstream primer Penta C-F1 as shown in SEQ ID No.11 and base sequence such as SEQ are added in amplifing reagent
Downstream primer Penta C-R1 shown in ID No.12 is capable of the amplification microsatellite locus Penta C of specificity, and Penta C
Amplified production electrophoresis detection after the fluorescence peak that generates and microsatellite locus BAT25, BAT26, D2S123, D5S346 and
The fluorescence peak generated after the amplified production electrophoresis detection of D17S250 will not juxtaposition.It therefore, can be in same PCR system
The microsatellite of microsatellite locus to be measured (BAT25, BAT26, D2S123, D5S346 and D17S250) and control is detected simultaneously
Whether site Penta C changes, and detection efficiency is high, and the testing result being capable of deciding whether as false positive, the standard of detection
True property is good.
In one embodiment, amplifing reagent further includes the upstream primer for microsatellite locus Penta D design
The base sequence such as SEQ ID No.13 institute of Penta D-F1 and downstream primer Penta D-R1, upstream primer Penta D-F1
Show, the base sequence of downstream primer Penta D-R1 is as shown in SEQ ID No.14.
Microsatellite locus Penta D also has fermentation by five tubes, control microsatellite locus when frequently as PCR.
Base sequence the upstream primer Penta C-F1 as shown in SEQ ID No.11 and base sequence such as SEQ are added in amplifing reagent
The ID No.13 upstream primer Penta D-F1 and downstream primer Penta D-R1 as shown in SEQ ID No.14 is capable of specificity
Expand microsatellite locus Penta D, and the fluorescence peak and microsatellite locus generated after the amplified production electrophoresis detection of Penta D
The fluorescence peak generated after the amplified production electrophoresis detection of BAT25, BAT26, D2S123, D5S346 and D17S250 will not intersect weight
It is folded.Therefore, microsatellite locus (BAT25, BAT26, D2S123, D5S346 to be measured can be detected 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 accuracy of the testing result of false positive, detection is good.
Further, amplifing reagent includes upstream primer BAT25-F1, downstream primer BAT25-R1, upstream primer BAT26-
F1, downstream primer BAT26-R1, upstream primer D2S123-F1, downstream primer D2S123-R1, upstream primer D5S346-F1, under
Swim primer D5S346-R1, upstream primer D17S250-F1, downstream primer D17S250-R1, upstream primer Penta C-F1, downstream
Primer Penta C-R1, upstream primer Penta D-F1 and downstream primer Penta D-R1.Microsatellite locus BAT25, BAT26,
The fluorescence peak generated after the amplified production electrophoresis detection of D2S123, D5S346, D17S250, Penta C and Penta D is not
It can juxtaposition.Therefore, can in same PCR system simultaneously detect microsatellite locus to be measured (BAT25, BAT26,
D2S123, D5S346 and D17S250) and control microsatellite locus (Penta C and Penta D) whether change, examine
Testing result that is high-efficient, and can more accurately judging whether it is false positive is surveyed, the accuracy of detection is good.
In one embodiment, the concentration of amplifing reagent middle and upper reaches primer BAT25-F1 be 50nmol/L~
The concentration of 200nmol/L, downstream primer BAT25-R1 are 50nmol/L~200nmol/L.
In one embodiment, the concentration of upstream primer BAT26-F1 is 50nmol/L~200nmol/L, downstream primer
The concentration of BAT26-R1 is 50nmol/L~200nmol/L.
In one embodiment, the concentration of upstream primer D2S123-F1 is 50nmol/L~200nmol/L, and downstream is drawn
The concentration of object D2S123-R1 is 50nmol/L~200nmol/L.
In one embodiment, the concentration of upstream primer D5S346-F1 is 50nmol/L~200nmol/L, under described
The concentration for swimming primer D5S346-R1 is 50nmol/L~200nmol/L.
In one embodiment, the concentration of upstream primer D17S250-F1 is 50nmol/L~200nmol/L, and downstream is drawn
The concentration of object D17S250-R1 is 50nmol/L~200nmol/L.
When detecting, the primer concentration in amplifing reagent has the intensity of fluorescence peak after amplified production electrophoresis detection certain
It influences, the sensitivity of considering cost and detection, upstream primer BAT25-F1, downstream primer BAT25-R1, upstream primer
BAT26-F1, downstream primer BAT26-R1, upstream primer D2S123-F1, downstream primer D2S123-R1, upstream primer D5S346-
F1, downstream primer D5S346-R1, upstream primer D17S250-F1 and downstream primer D17S250-R1 concentration be 50nmol/L
~200nmol/L, such as 70nmol/L, 100nmol/L, 150nmol/L or 190nmol/L etc..
Preferably, amplifing reagent middle and upper reaches primer BAT25-F1, downstream primer BAT25-R1, upstream primer BAT26-F1,
Downstream primer BAT26-R1, upstream primer D2S123-F1, downstream primer D2S123-R1, upstream primer D5S346-F1, downstream are drawn
The concentration of object D5S346-R1, upstream primer D17S250-F1 and downstream primer D17S250-R1 are 100nmol/L.
Further, amplifing reagent further includes the upstream primer Penta C-F1 for microsatellite locus Penta C design
When with downstream primer Penta C-R1, upstream primer Penta C-F1 and downstream primer Penta C-R1 concentration are 50nmol/L
~200nmol/L, such as 70nmol/L, 100nmol/L, 150nmol/L or 190nmol/L etc..
Further, amplifing reagent further includes the upstream primer Penta D-F1 for microsatellite locus Penta D design
When with downstream primer Penta D-R1, upstream primer Penta D-F1 and downstream primer Penta D-R1 concentration are 50nmol/L
~200nmol/L, such as 70nmol/L, 100nmol/L, 150nmol/L or 190nmol/L etc..
In one embodiment, at least one of upstream primer BAT25-F1 and downstream primer BAT25-R1 are marked with
First fluorophor.At least one of upstream primer BAT26-F1 and downstream primer BAT26-R1 are marked with the second fluorescent base
Group.At least one of upstream primer D2S123-F1 and downstream primer D2S123-R1 are marked with third fluorophor.Draw upstream
At least one of object D5S346-F1 and downstream primer D5S346-R1 are marked with the 4th fluorophor.Upstream primer D17S250-
At least one of F1 and downstream primer D17S250-R1 are marked with the 5th fluorophor.And third fluorophor and the 4th fluorescence
Group is selected from two kinds of different fluorophors.
By mark fluorescent group, the amount of corresponding amplified production is detected convenient for fluorescent value.Third fluorophor and the 4th
Fluorophor is selected from two kinds of different fluorophors, convenient for distinguishing the amplified production of microsatellite locus D2S123 and D5S346
Fluorescence peak.Inventor has found in the course of the research, the sequence length of the amplified production of microsatellite locus D2S123 and D5S346 compared with
Be it is close, to two kinds of different fluorophors of amplimer label of microsatellite locus D2S123 and D5S346, be conducive to area
Divide the fluorescence peak generated after the amplified production electrophoresis of microsatellite locus D2S123 and D5S346, improves the sensitivity of detection.
It will be appreciated, of course, that in other embodiments, be also possible to be added detect respectively microsatellite locus BAT25,
The probe of BAT26, D2S123, D5S346 and D17S250, and fluorophor is marked on probe.At this point, can not be marked on primer
Remember fluorophor.
In one embodiment, the first fluorophor label is on downstream primer BAT25-R1, the second fluorophor mark
On upstream primer BAT26-F1, third fluorophor marks on upstream primer D2S123-F1 note, the 4th fluorophor label
On upstream primer D5S346-F1, the 5th fluorophor is marked on downstream primer D17S250-R1.Inventor is in research process
Middle discovery, fluorophor marks in upstream primer or downstream primer, to the PCR product electrophoresis detection generated after PCR amplification
Fluorescence peak has a certain impact, the mark position of the fluorophor of present embodiment, and the corresponding amplification generated after PCR amplification produces
The fluorescence peak of object electrophoresis detection is single, high sensitivity.
In one embodiment, the first fluorophor, the second fluorophor, third fluorophor, the 4th fluorophor
One in FAM fluorophor, TAMRA fluorophor, ROX fluorophor and JOE fluorophor is selected from the 5th fluorophor
Kind.
In one embodiment, the first fluorophor is FAM fluorophor, and the second fluorophor is TAMRA fluorescent base
Group, third fluorophor are FAM fluorophor, and the 4th fluorophor is JOE fluorophor, and the 5th fluorophor is JOE fluorescence
Group.
Further, amplifing reagent further includes the upstream primer Penta C-F1 for microsatellite locus Penta C design
When with downstream primer Penta C-R1, at least one of upstream primer Penta C-F1 and downstream primer Penta C-R1 label
There is the 6th fluorophor.6th fluorophor is selected from FAM fluorophor, TAMRA fluorophor, ROX fluorophor and JOE fluorescence
One of group.
Further, amplifing reagent further includes the upstream primer Penta D-F1 for microsatellite locus Penta D design
When with downstream primer Penta D-R1, at least one of upstream primer Penta D-F1 and downstream primer Penta D-R1 label
There is the 7th fluorophor.7th fluorophor is selected from FAM fluorophor, TAMRA fluorophor, ROX fluorophor and JOE fluorescence
One of group.
Test result shows that the multiple fluorescence PCR amplifing reagent of above-mentioned detection microsatellite instability, particular sequence draw
Object can distinguish the corresponding microsatellite locus of specific amplification, and the amplified product band unicity of each microsatellite locus is good.Simultaneously
After carrying out PCR amplification to 5 microsatellite locus (BAT25, BAT26, D2S123, D5S346 and D17S250), each amplified production warp
The fluorescence peak generated after electrophoresis detection does not have the phenomenon that 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 further includes defending for micro-
The upstream primer Penta C-F1 and downstream primer Penta C-R1 and/or PCR amplification reagent of championship point Penta C design are also
Including for Penta D design upstream primer Penta D-F1 and downstream primer Penta D-R1 when.It being capable of same PCR system
In detect micro- defending for microsatellite locus to be measured (BAT25, BAT26, D2S123, D5S346 and D17S250) and control simultaneously
Whether championship point Penta C and/or Penta D change, and detection efficiency is high, and the detection being capable of deciding whether as 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, above-mentioned detection kit further includes Taq archaeal dna polymerase and PCR reaction buffer in kit.
The detection kit can in same PCR system simultaneously detect microsatellite locus BAT25, BAT26, D2S123,
Whether D5S346 and D17S250 changes, and detection efficiency is high, and sensitivity is good.
It is below specific embodiment part.
In following embodiment, unless otherwise instructed, test method without specific conditions, usually according to normal condition,
For example, see Pehanorm Brooker, EF, not the written molecular cloning of Ritchie, T Manny A Disi etc. (Jin Dongyan, Li Mengfeng etc. are translated) is real
It tests condition described in guide [M] (Beijing: Science Press, 1992) or method that kit manufacturer is recommended is realized.
Reagent used in embodiment is commercially available.
Embodiment 1
The gene number where microsatellite locus to be measured is consulted in Gene Bank, determines 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 of microsatellite locus such as table
Shown in 1.
Table 1: the essential information of microsatellite locus
| Microsatellite locus | Gene number | 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 |
Upstream primer and downstream primer, and corresponding mark fluorescent base targetedly are designed to each microsatellite locus
Group, it is specific as shown in table 2.
Table 2: the upstream primer and downstream primer of microsatellite locus
The primer of design passes through raw work biosynthesis, by the upstream primer BAT25-F1 of synthesis, downstream primer BAT25-R1,
Upstream primer BAT26-F1, downstream primer BAT26-R1, upstream primer D2S123-F1, downstream primer D2S123-R1, upstream are drawn
Object D5S346-F1, downstream primer D5S346-R1, upstream primer D17S250-F1 and downstream primer D17S250-R1 mixing, shape
At the multiple fluorescence PCR amplifing reagent of detection microsatellite instability, wherein the concentration of each primer is 100nmol/L.This reality
Apply example PCR amplification reagent can in same PCR system and meanwhile expand 5 microsatellite locus BAT25, BAT26, D2S123,
D5S346 and D17S250, the amplified production that each microsatellite locus expands fluorescence peak after electrophoresis detection will not intersect weight
It is folded.
Embodiment 2
Upstream primer BAT25- in the multiple fluorescence PCR amplifing reagent of the detection microsatellite instability of the present embodiment
F1, downstream primer BAT25-R1, upstream primer BAT26-F1, downstream primer BAT26-R1, upstream primer D2S123-F1, downstream
Primer D2S123-R1, upstream primer D5S346-F1, downstream primer D5S346-R1, upstream 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 upstream primer Penta C-F1 and downstream primer Penta C-R1 of Penta C design.The related letter of microsatellite locus Penta C
Breath is as shown in table 3.
Table 3: the essential information of microsatellite locus Penta C
| Microsatellite locus | Gene number | Main repetitive sequence |
| Penta C | AL138752 | (AAAAG)3 |
Targetedly the upstream primer Penta C-F1 to microsatellite locus Penta C design Penta C design and downstream
Primer Penta C-R1, and corresponding mark fluorescent group, it is specific as shown in table 4.
Table 4: the upstream primer and downstream primer of microsatellite locus
The primer of design passes through raw work biosynthesis, by the upstream primer Penta C-F1 and downstream primer Penta of synthesis
C-R1 is mixed with the PCR amplification reagent of embodiment 1, forms the multiple fluorescence PCR amplifing reagent of detection microsatellite instability.Its
In, the concentration of upstream primer Penta C-F1 and downstream primer Penta C-R1 are 100nmol/L.The PCR of the present embodiment expands
Increase reagent can in same PCR system simultaneously expand 6 microsatellite locus BAT25, BAT26, D2S123, D5S346,
D17S250 and Penta C, the amplified production that each microsatellite locus expands fluorescence peak after electrophoresis detection will not intersect weight
It is folded.
Embodiment 3
Upstream primer BAT25- in the multiple fluorescence PCR amplifing reagent of the detection microsatellite instability of the present embodiment
F1, downstream primer BAT25-R1, upstream primer BAT26-F1, downstream primer BAT26-R1, upstream primer D2S123-F1, downstream
Primer D2S123-R1, upstream primer D5S346-F1, downstream primer D5S346-R1, upstream 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 upstream primer Penta D-F1 and downstream primer Penta D-R1 of Penta D design.The related letter of microsatellite locus Penta C
Breath is as shown in table 5.
Table 5: the essential information of microsatellite locus Penta D
| Microsatellite locus | Gene number | Main repetitive sequence |
| Penta D | Ac000014 | (AAAAG)8 |
Targetedly the upstream primer Penta D-F1 to microsatellite locus Penta D design Penta D design and downstream
Primer Penta D-R1, and corresponding mark fluorescent group, it is specific as shown in table 6.
Table 6: the upstream primer and downstream primer of microsatellite locus
The primer of design passes through raw work biosynthesis, by the upstream primer Penta D-F1 and downstream primer Penta of synthesis
D-R1 is mixed with the PCR amplification reagent of embodiment 1, forms the multiple fluorescence PCR amplifing reagent of detection microsatellite instability.Its
In, the concentration of upstream primer Penta D-F1 and downstream primer Penta D-R1 are 100nmol/L.The PCR of the present embodiment expands
Increase reagent can in same PCR system simultaneously expand 6 microsatellite locus BAT25, BAT26, D2S123, D5S346,
D17S250 and Penta D, the amplified production that each microsatellite locus expands 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 upstream primer BAT25-
F1, downstream primer BAT25-R1, upstream primer BAT26-F1, downstream primer BAT26-R1, upstream primer D2S123-F1, downstream
Primer D2S123-R1, upstream primer D5S346-F1, downstream primer D5S346-R1, upstream primer D17S250-F1, downstream primer
D17S250-R1, upstream primer Penta C-F1 and downstream primer Penta C-R1, upstream primer Penta D-F1 and downstream are drawn
Object Penta D-R1.The sequence ginseng of specific primer sees the above table 2, table 4 and table 5.The concentration of each primer is 100nmol/L.This reality
Apply example PCR amplification reagent can in same PCR system and meanwhile expand 7 microsatellite locus BAT25, BAT26, D2S123,
D5S346, D17S250, Penta C and Penta D, the amplified production that each microsatellite locus expands is after electrophoresis detection
Fluorescence peak will not juxtaposition.
Test one
Detect the band of amplified production
The saliva sample for acquiring people extracts genomic DNA with kit (raw work biology, product number: B518266), with
The saliva genomic DNA of people be template, be separately added into the microsatellite locus BAT25, BAT26 designed as shown in table 1, D2S123,
D5S346, D17S250 upstream primer and downstream primer carry out PCR amplification, and amplified production carries out gel electrophoresis, as a result referring to Fig. 1
It is shown.Far Left swimming lane is electrophoresis Marker (raw work biology, product number: B600303), and Marker stripe size is followed successively by
500bp, 400bp, 300bp, 200bp, 150bp, 100bp, 75bp, 50bp and 25bp.BAT25,BAT26,D2S123,
Each at least four parallel laboratory test of the amplified production of D5S346, D17S250.It will be seen from figure 1 that being drawn using design shown in table 1
After object expands BAT25, BAT26, D2S123, D5S346, D17S250, the band of amplified production is single, illustrates the special of each primer
Property it is good, be capable of specificity the corresponding microsatellite locus of amplification.
Test two
Substance fluorescence detection amplified production
Respectively with the wild type BAT25 plasmid of 1pg/ μ L, wild type BAT26 plasmid, wild type D2S123 plasmid, wild type
D5S346 plasmid, wild type D17S250 plasmid and wild type P enta C plasmid are as amplification template.Using microsatellite locus
The corresponding upstream primer of BAT25, BAT26, D2S123, D5S346, D17S250 and Penta C and downstream primer carry out single
Weight fluorescent PCR amplification, and amplified production is subjected to CE electrophoresis, it is as a result as shown in Figure 2 to 7 respectively.Abscissa is amplification in figure
The base numerical value of the genetic fragment of product, ordinate are fluorescence values.It can be seen from the figure that microsatellite locus BAT25,
Fluorescence peak after the amplified production CE electrophoresis of BAT26, D2S123, D5S346, D17S250 and Penta C compares concentration, in phase
The base value bit answered is equipped with apparent specific peak value.
Test three
The amplified production of sixfold fluorescence detection wild type microsatellite locus
By wild type BAT25 plasmid, wild type BAT26 plasmid, wild type D2S123 plasmid, wild type D5S346 plasmid,
Wild type D17S250 plasmid and the mixing of wild type Penta C plasmid are as amplification template.Wherein the concentration of 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
As can be seen that the amplified production CE of microsatellite locus BAT25, BAT26, D2S123, D5S346, D17S250 and Penta C in figure
Fluorescence peak after electrophoresis does not have the phenomenon that 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 fluorescence detection contains the amplified production of the microsatellite locus of saltant type
By wild type BAT25 plasmid, wild type BAT26 plasmid, wild type D2S123 plasmid, wild type D5S346 matter
Then saltant type BAT25 plasmid (5 weights of missing are added in grain, wild type D17S250 plasmid and the mixing of wild type Penta C plasmid
Multiple base), saltant type BAT26 plasmid (missing 4 duplicate bases), (missing 27 is duplicate for saltant type D2S123 plasmid
Base) and saltant type D17S250 plasmid (10 duplicate bases of missing) mixing.Wherein the concentration of 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
With Fig. 9 it is found that saltant type BAT25 plasmid, saltant type BAT26 plasmid, saltant type D2S123 plasmid and saltant type D17S250 is added
After plasmid, the peak of the amplified production of corresponding four microsatellite locus is moved to left or is moved to right, and detection sensitivity is high.
Test five
Respectively using the genomic DNA of the normal tissue sample of clinically same patient and tumor tissues sample as template, adopt
MSI detection is carried out with the multiple fluorescence PCR amplifing reagent of embodiment 2.Sample 1701215, sample 1717924 and sample
For 1700021 MSI detection CE peak figure respectively as shown in Figure 10~12, the MSI type for summarizing sample according to MSI testing result is as follows
Table 7.
Table 7:MSI testing result
| Sample number into spectrum | Testing result | MSI type |
| 1701215 | Five sites are unchanged | MSS type |
| 1717924 | Five sites are unchanged | MSS type |
| 1700021 | BAT-25, BAT-26 and D2S123 occur bimodal | MSH type |
One or more of embodiments of the invention above described embodiment only expresses, description are more specific and detailed
Carefully, but it cannot be understood as limitations on the scope of the patent of the present invention.It should be pointed out that for the common skill of this field
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 scope.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
SEQUENCE LISTING
<110>Changzhou tung oil tree Biotechnology Co., Ltd
<120>the multiple fluorescence PCR amplifing reagent and kit of microsatellite instability are detected
<160> 14
<170> PatentIn version 3.3
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gcctgccttt aacactgcta ttca 24
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Claims (5)
1. a kind of multiple fluorescence PCR amplifing reagent for detecting microsatellite instability characterized by comprising
For the upstream primer BAT25-F1 and downstream primer BAT25-R1 of microsatellite locus BAT25 design, the upstream primer
The base sequence of BAT25-F1 is as shown in SEQ ID No.1, the base sequence of the downstream primer BAT25-R1 such as SEQ ID
Shown in No.2；
For the upstream primer BAT26-F1 and downstream primer BAT26-R1 of microsatellite locus BAT26 design, the upstream primer
The base sequence of BAT26-F1 is as shown in SEQ ID No.3, the base sequence of the downstream primer BAT26-R1 such as SEQ ID
Shown in No.4；
Draw for the upstream primer D2S123-F1 and downstream primer D2S123-R1, the upstream of microsatellite locus D2S123 design
The base sequence of object D2S123-F1 is as shown in SEQ ID No.5, the base sequence such as SEQ of the downstream primer D2S123-R1
Shown in ID No.6；
Draw for the upstream primer D5S346-F1 and downstream primer D5S346-R1, the upstream of microsatellite locus D5S346 design
The base sequence of object D5S346-F1 is as shown in SEQ ID No.7, the base sequence such as SEQ of the downstream primer D5S346-R1
Shown in ID No.8；And upstream primer D17S250-F1 and downstream primer for microsatellite locus D17S250 design
The base sequence of D17S250-R1, the upstream primer D17S250-F1 are as shown in SEQ ID No.9, the downstream primer
The base sequence of D17S250-R1 is as shown in SEQ ID No.10；And
For the upstream primer Penta C-F1 and downstream primer Penta C-R1 of microsatellite locus Penta C design, it is described on
The base sequence of primer Penta C-F1 is swum as shown in SEQ ID No.11, the base sequence of the downstream primer Penta C-R1
As shown in SEQ ID No.12；
The downstream primer BAT25-R1 is marked with the first fluorophor, and the upstream primer BAT26-F1 is marked with the second fluorescence
Group, the upstream primer D2S123-F1 are marked with third fluorophor, and the upstream primer D5S346-F1 is marked with the 4th
Fluorophor, the downstream primer D17S250-R1 are marked with the 5th fluorophor, and the upstream primer Penta C-F1 is under
At least one of trip primer Penta C-R1 is marked with the 6th fluorophor；
It is first fluorophor, second fluorophor, the third fluorophor, the 4th fluorophor, described
5th fluorophor and the 6th fluorophor be selected from FAM fluorophor, TAMRA fluorophor, ROX fluorophor and
One of JOE fluorophor；
The third fluorophor and the 4th fluorophor are selected from two kinds of different fluorophors；
The concentration of the upstream primer BAT25-F1 is 50nmol/L~200nmol/L, and the downstream primer BAT25-R1's is dense
Degree is 50nmol/L~200nmol/L；And/or
The concentration of the upstream primer BAT26-F1 is 50nmol/L~200nmol/L, and the downstream primer BAT26-R1's is dense
Degree is 50nmol/L~200nmol/L；And/or
The concentration of the upstream primer D2S123-F1 is 50nmol/L~200nmol/L, the downstream primer D2S123-R1's
Concentration is 50nmol/L~200nmol/L；And/or
The concentration of the upstream primer D5S346-F1 is 50nmol/L~200nmol/L, the downstream primer D5S346-R1's
Concentration is 50nmol/L~200nmol/L；And/or
The concentration of the upstream primer D17S250-F1 is 50nmol/L~200nmol/L, the downstream primer D17S250-R1
Concentration be 50nmol/L~200nmol/L；
The concentration of the upstream primer Penta C-F1 is 50nmol/L~200nmol/L, the downstream primer Penta C-R1
Concentration be 50nmol/L~200nmol/L.
2. the multiple fluorescence PCR amplifing reagent of detection microsatellite instability according to claim 1, which is characterized in that
The amplifing reagent further include:
For the upstream primer Penta D-F1 and downstream primer Penta D-R1 of microsatellite locus Penta D design, it is described on
The base sequence of primer Penta D-F1 is swum as shown in SEQ ID No.13, the base sequence of the downstream primer Penta D-R1
As shown in SEQ ID No.14.
3. the multiple fluorescence PCR amplifing reagent of detection microsatellite instability according to claim 1, which is characterized in that
First fluorophor is FAM fluorophor, and second fluorophor is TAMRA fluorophor, the third fluorescent base
Group is FAM fluorophor, and the 4th fluorophor is JOE fluorophor, and the 5th fluorophor is JOE fluorophor.
4. a kind of multiple fluorescence PCR detection reagent box for detecting microsatellite instability, which is characterized in that including such as claim
1~3 described in any item amplifing reagents.
5. the multiple fluorescence PCR detection reagent box of detection microsatellite instability as claimed in claim 4, which is characterized in that
It further include Taq archaeal dna polymerase and PCR reaction buffer in the kit.
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| CN107475442A (en) * | 2017-10-17 | 2017-12-15 | 生工生物工程（上海）股份有限公司 | A kind of method of microsatellite instability detection |
| WO2019162240A1 (en) * | 2018-02-20 | 2019-08-29 | F. Hoffmann-La Roche Ag | Improved detection of microsatellite instability |
| CN110305958A (en) * | 2018-11-23 | 2019-10-08 | 郑州海普生物医药科技有限公司 | A kind of MSI detection method |
| CN109825590B (en) * | 2019-04-01 | 2022-08-05 | 常州桐树生物科技有限公司 | Method, kit and primer for joint detection of tumor-driven gene mutation and microsatellite instability |
| CN110699455A (en) * | 2019-10-29 | 2020-01-17 | 苏州绘真医学检验有限公司 | Human circulating tumor cell MSI detection primer group, kit and detection method |
| 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 |
| CN111370063B (en) * | 2020-03-23 | 2021-09-10 | 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 |
| CN114480632B (en) * | 2020-11-16 | 2023-05-16 | 江萤 | Detection method for human microsatellite unstable sites and application 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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| CN106834479A (en) * | 2017-02-16 | 2017-06-13 | 凯杰（苏州）转化医学研究有限公司 | Microsatellite instability state analysis system in immunotherapy of tumors |
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