CN110358675A - A kind of DNA detection PCR system of fluorescence analysis - Google Patents
A kind of DNA detection PCR system of fluorescence analysis Download PDFInfo
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- CN110358675A CN110358675A CN201910685049.8A CN201910685049A CN110358675A CN 110358675 A CN110358675 A CN 110358675A CN 201910685049 A CN201910685049 A CN 201910685049A CN 110358675 A CN110358675 A CN 110358675A
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
A kind of DNA detection PCR system of fluorescence analysis, fluorophor is added in PCR reaction system, it is in the same closed container that PCR amplification is synchronous in conjunction with detection process to carry out, amplified production is detected after each PCR cycle, obtain the amplification curve of reactant, the method of last relative quantification carries out quantitative analysis to unknown template, determines that unknown template reaches the cycle-index Ct value of specified fluorescence intensity or fluorescence intensity percentage, the concentration of unknown template is determined according to Ct value.Wherein, the measurement cycle-index Ct value of the amplifing reagent of different batches is modified, is made with uniformity by the DNA testing result of different batches amplifing reagent.
Description
Technical field
The present invention relates to the technical fields of DNA detection, more particularly, to a kind of polymerase chain reaction of DNA detection
(PCR) system of fluorescence analysis.
Background technique
DNA nucleic acid carries various information as basic inhereditary material, to its structure, the understanding of function, is conducive to grind
Study carefully the heredity, evolution and medical diagnosis on disease etc. of species.Polymerase chain reaction (PCR) technology is that one kind simulates nature in vitro
The nucleic acid amplification technologies of DNA replication dna process, also referred to as cell-free amplification technique can be by minim DNA heredity object with this technology
Matter simply increases rapidly 1,000,000 times or more.Due to technique high sensitivity, accuracy is strong, therefore is widely used to biological doctor
Field, such as medical diagnosis, food safety, animal quarantine and genetics research.
Fluorescent quantitative PCR technique is to migrate FRET based on fluorescence resonance energy in Standard PCR technical foundation
(Fluorescent Resonance Energy Transfer) principle, is added fluorophor in PCR reaction system, same
It is in one closed container that PCR amplification is synchronous in conjunction with detection process to carry out, amplified production is detected after each PCR cycle,
A fluorescence intensity signals are collected, with the progress that PCR reacts, PCR reaction product is constantly accumulated, and fluorescence signal intensity also waits ratios
Example increases, and when after reaction, the amplification curve of available reactant, the method for last relative quantification or absolute quantitation is not to
Know that template carries out quantitative analysis, determines that unknown template reaches the cycle-index Ct of specified fluorescence intensity or fluorescence intensity percentage
Value, the concentration of unknown template is determined according to Ct value.
The extension of progress primer under Taq DNA polymerase acts in reaction reagent in pcr amplification reaction, and different batches
Preparation process has differences in reaction reagent, causes the pcr amplification reaction result of different batches reaction reagent distinct.It is existing
In technology, Hitachi High-Technologies Corporation proposes a kind of correction different batches examination in the invention of CN201480063430.6
The method of agent testing result, the benchmark percentage of the response curve by changing detection reagent and standard DNA sample, will test
Reagent is adapted to its expected value for the Ct detected value of standard DNA sample.However, being by changing intermediate parameters in the invention
Method amendment Ct detected value, rather than the direct amendment for Ct detected value；Since the datum mark of response curve is usually located at reaction
The biggish part of the slope of curve, lesser curve difference also results in datum mark and has greatly changed, if detection reagent exists
When error is larger at the expected value of standard reagent, it will lead to modified datum error and increase, to influence testing result.
Summary of the invention
The present invention provides a kind of PCR system of fluorescence analysis of DNA detection, can directly repair to cycle-index Ct value
Just, while modified error can be reduced.
As one aspect of the present invention, a kind of DNA detection PCR system of fluorescence analysis is provided, comprising: DNA sample addition
Portion, amplifing reagent addition portion, reacting part, fluorescence measurement portion, analysis portion, amplifing reagent storage unit, standard DNA sample storage unit with
And information storage part；DNA sample addition portion, for the addition detection DNA sample into reacting part；Amplifing reagent storage unit, is used for
Store the amplification reaction reagent of different batches；Amplifing reagent addition portion, for by the particular batch in amplifing reagent storage unit
Amplification reaction reagent is added in reacting part；Reacting part carries out polymerase chain reaction for DNA sample and amplification reaction reagent
It answers；Fluorescence measurement portion is measured by the fluorescence that optical instrument generates reacting part；Analysis portion is based on fluorescence measurement
The measurement data in portion determines the Ct value of DNA sample；Standard DNA sample storage unit stores the standard DNA sample of known Ct value；
The standard DNA sample and the polymerase chain reaction curve with reference to amplifing reagent, the ginseng are stored in the information storage part
Examining amplifing reagent keeps the Ct measured value of the standard DNA sample special in the fluorescence intensity percentage of polymerase chain reaction curve
Determine to be equal to its expected value when percentage；It further include correction value determining section, based on the standard DNA sample and the particular batch
The polymerase chain of the polymerase chain reaction curve of amplification reaction reagent and the standard DNA sample and reference amplifing reagent
Response curve determines the correction value of the particular batch amplification reaction reagent；Measurement number of the analysis portion based on fluorescence measurement portion
Accordingly and the correction value, the Ct value of DNA sample is determined.
Preferably, the particular percentile is 10% ~ 30%.
Preferably, the correction value determining section is based on the first intensity percent value and the second intensity percent value, determines institute
State the polymerase chain reaction curve and the standard DNA sample of standard DNA sample and the particular batch amplification reaction reagent
After the interval region of the polymerase chain reaction curve of reference amplifing reagent, the area and the mark of the interval region are calculated
The polymerase chain reaction curve and the standard DNA sample and ginseng of quasi- DNA sample and the particular batch amplification reaction reagent
Length of the polymerase chain reaction curve in the interval region for examining amplifing reagent is determined according to the area and the length
This is averagely expanded number as correction value by the average amplification number of the interval region.
Preferably, first intensity percent is set as the 2/3 of the particular percentile, second intensity percent
It is set as the 4/3 of the particular percentile.
Preferably, the analysis portion is according to measuring fluorescence measurement portion and the amplification reaction reagent of the particular batch
Time in polymerase chain reaction curve corresponding to the particular percentile position is determined as the Ct value before amendment, repairs described
Ct value before just adds or subtracts the correction value, determines the Ct value of the DNA sample.
Preferably, standard DNA sample described in the interval region and the particular batch amplification reaction reagent polymerize
When enzyme chain reaction curve is located at the standard DNA sample with reference to the polymerase chain reaction curve left side of amplifing reagent, institute
Analysis portion is stated by the Ct value before amendment plus the correction value；Standard DNA sample described in the interval region with it is described specific
The polymerase chain reaction curve of batch amplification reaction reagent is located at the standard DNA sample and the polymerase with reference to amplifing reagent
When on the right of chain reaction curve, the Ct value before amendment is subtracted the correction value by the analysis portion.
Detailed description of the invention
Fig. 1 is the polymerase chain reaction song of standard DNA of embodiment of the present invention sample and particular batch amplification reaction reagent
The schematic diagram of line and standard DNA sample and the polymerase chain reaction curve with reference to amplifing reagent.
Specific embodiment
In order to illustrate more clearly of technical solution of the present invention, embodiment will be used simply to be situated between the present invention below
Continue, it should be apparent that, be described below in be only one embodiment of the present of invention, for those of ordinary skill in the art come
It says, without any creative labor, other technical solutions can also be obtained according to these embodiments, also belonged to
Disclosure of the invention range.
The DNA of the embodiment of the present invention detects PCR system of fluorescence analysis, comprising: DNA sample addition portion, amplifing reagent addition
Portion, reacting part, fluorescence measurement portion, analysis portion, amplifing reagent storage unit, standard DNA sample storage unit and information storage part.
DNA sample addition portion, for adding DNA sample into reacting part.Amplifing reagent storage unit, for storing different batches
Secondary amplification reaction reagent, includes Taq DNA polymerase in amplification reaction reagent reaction reagent, carries out polymerase for DNA sample
Chain reaction.Amplifing reagent addition portion, for the amplification reaction reagent of the particular batch in amplifing reagent storage unit to be added to
In reacting part.
Reacting part carries out polymerase chain reaction for DNA sample and amplification reaction reagent, passes through denaturation, annealing, extension
Circulation makes DNA cloning.Fluorescence measurement portion is measured by the fluorescence that fluorescence intensity sensor generates reacting part, for
Each amplification procedure detects amplified production, collects fluorescence intensity signals, and measurement result is transmitted to analysis portion.
Standard DNA sample storage unit, stores the standard DNA sample of known Ct value, which is standard DNA sample and ginseng
Corresponding amplification cycles number when fluorescence intensity percentage is particular percentile in the polymerase chain reaction curve of examination agent,
The particular percentile is the datum mark of response curve, can be set to 10% ~ 30%, being preferable to provide is 20%.Information storage part,
It stores standard DNA sample and this refers to the polymerase chain reaction curve of reagent, curve C1 as shown in figure 1.Referring to Fig. 1, by
Difference between different batches reagent examines standard DNA sample using the reagent of particular batch in reagent storage unit
When survey, polymerase chain reaction curve C2 is different from standard DNA sample and the polymerase chain reaction curve C1 with reference to reagent.
Correction value determining section, the polymerase chain reaction based on standard DNA sample Yu particular batch amplification reaction reagent
The polymerase chain reaction curve C1 of curve C2 and standard DNA sample and reference amplifing reagent determines that the particular batch expands
The correction value of reaction reagent.Correction value determining section determines the first intensity percent value W1 lower than specific percentage value and is higher than spy
Determine the second intensity percent value W2 of percent value, the 2/3 of datum mark can be set by the first intensity percent W1, the last the second
Degree percentage W2 is set as the 4/3 of datum mark.Correction value determining section is according to the first intensity percent value W1 and the second intensity percentage
Ratio W2 determines polymerase chain reaction curve C2 and the standard DNA examination of standard DNA sample and particular batch amplification reaction reagent
The interval region 10 of sample and the polymerase chain reaction curve C1 with reference to reagent, determines the area S, Yi Jiju of the interval region 10
Polymerase chain response curve C1 is located at length of curve L1 in interval region 10 and polymerase chain reaction curve C2 and is located at interval
Length of curve L2 in region 10.Wherein area S, length of curve L1 and L2 can be determining by conventional image processing method,
Such as area and length are determined according to image pixel number.Correction value determining section S and length of curve L1 and L2 according to area,
Mean breadth L=the 2S/(L1+L2 in counting period region 10), using the corresponding averagely amplification number difference of mean breadth L as the spy
Determine the correction value of batch amplification reaction reagent.
Before PCR system of fluorescence analysis is detected by the amplification reaction reagent corresponding DNA sample of particular batch, first will
The particular batch amplification reaction reagent is reacted with the standard DNA sample of known Ct value and is detected, and correction value determining section is passed through
Determine the particular batch amplification reaction reagent for the correction value of the standard DNA sample of known Ct value；Applying the particular batch
When amplification reaction reagent detects DNA sample, DNA sample is measured by fluorescence measurement portion and the particular batch amplification reaction reagent is anti-
The time for corresponding to reference point location in polymerase chain reaction curve is determined as correcting by the polymerase chain reaction curve answered
Preceding Ct value；Then, the Ct value before amendment is added or is subtracted the detected value that the correction value obtains Ct value by analysis portion.Specifically
, the polymerase chain reaction curve C2 of 10 Plays DNA sample of interval region and particular batch amplification reaction reagent is located at mark
When the polymerase chain reaction curve left side C1 of quasi- DNA sample and reference reagent, the Ct before amendment is added correction value by analysis portion
Obtain detected value；The polymerase chain reaction curve of interval region 10 Plays DNA sample and particular batch amplification reaction reagent
C2 be located at standard DNA sample and with reference to reagent polymerase chain reaction curve C1 on the right of when, analysis portion is by the Ct value before amendment
It subtracts correction value and obtains detected value.
All references mentioned in the present invention all incorporated by reference in this application, are individually recited just as each document
As with reference to such.In addition, it should also be understood that, the above embodiments are merely illustrative of the technical scheme of the present invention and are not intended to be limiting thereof, although
Referring to above-described embodiment, invention is explained in detail, and those of ordinary skill in the art still can be to the present invention
Specific embodiment be modified or replaced equivalently, and any modification without departing from spirit and scope of the invention or equivalent
Replacement, is intended to be within the scope of the claims of the invention.
Claims (4)
1. a kind of DNA detects PCR system of fluorescence analysis, comprising: DNA sample addition portion, amplifing reagent addition portion, reacting part are glimmering
Light measurement portion, analysis portion, amplifing reagent storage unit, standard DNA sample storage unit and information storage part；DNA sample addition portion,
For the addition detection DNA sample into reacting part；Amplifing reagent storage unit, for storing the amplification reaction reagent of different batches；
Amplifing reagent addition portion, for the amplification reaction reagent of the particular batch in amplifing reagent storage unit to be added in reacting part；
Reacting part carries out polymerase chain reaction for DNA sample and amplification reaction reagent；Fluorescence measurement portion, passes through optical instrument pair
It is measured in the fluorescence that reacting part generates；Analysis portion determines the Ct of DNA sample based on the measurement data in fluorescence measurement portion
Value；Standard DNA sample storage unit stores the standard DNA sample of known Ct value；It is characterized by: in the information storage part
It stores the standard DNA sample and refers to the polymerase chain reaction curve of amplifing reagent, the reference amplifing reagent makes described
The Ct measured value of standard DNA sample is equal to it when the fluorescence intensity percentage of polymerase chain reaction curve is particular percentile
Expected value；It further include correction value determining section, it is poly- based on the standard DNA sample and the particular batch amplification reaction reagent
The polymerase chain reaction curve of polymerase chain response curve and the standard DNA sample and reference amplifing reagent, determining should
The correction value of particular batch amplification reaction reagent；Measurement data and the amendment of the analysis portion based on fluorescence measurement portion
Value, determines the Ct value of DNA sample.
2. DNA according to claim 1 detects PCR system of fluorescence analysis, it is characterised in that: the particular percentile is
10%~30%。
3. DNA according to claim 2 detects PCR system of fluorescence analysis, it is characterised in that: the particular percentile is about
20%。
4. DNA according to claim 2 detects PCR system of fluorescence analysis, it is characterised in that: the fluorescence intensity measurement portion
It is measured by the fluorescence that fluorescence intensity sensor generates reacting part, for each amplification procedure, detects amplified production,
Fluorescence intensity signals are collected, and measurement result is transmitted to analysis portion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910685049.8A CN110358675A (en) | 2019-07-27 | 2019-07-27 | A kind of DNA detection PCR system of fluorescence analysis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910685049.8A CN110358675A (en) | 2019-07-27 | 2019-07-27 | A kind of DNA detection PCR system of fluorescence analysis |
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| Publication Number | Publication Date |
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| CN110358675A true CN110358675A (en) | 2019-10-22 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201910685049.8A Withdrawn CN110358675A (en) | 2019-07-27 | 2019-07-27 | A kind of DNA detection PCR system of fluorescence analysis |
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2019
- 2019-07-27 CN CN201910685049.8A patent/CN110358675A/en not_active Withdrawn
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Application publication date: 20191022 |