CN101979544A - Standard sample-based real-time fluorescence PCR relative calibration method - Google Patents

Abstract

The invention relates to standard sample-based real-time fluorescence polymerase chain reaction (PCR) relative calibration method. In the prior art, no real-time fluorescence PCR calibration method is provided. The method of the invention comprises the following steps of: preparing a standard sample, calculating a calibration standard value, detecting data of an instrument to be calibrated, and determining a calibration result. In the method, fluorescent powder which is divided equally according to a measurement range is adopted so that the fluorescence intensity generated by a fluorescent sample can cover the overall measurement range.

Description

A kind of real-time fluorescence PCR relative Calibration method based on standard model

Technical field

The invention belongs to optics and biotechnology crossing domain, relate to a kind of scaling method of real-time fluorescence PCR, especially a kind of real-time fluorescence PCR relative Calibration method based on standard model.

Background technology

Polymerase chain reaction (Polymerase Chain Reaction is called for short PCR) technology is a kind of nucleic acid amplification technologies in in-vitro simulated natural dna replication dna process, also claim the cell-free molecular cloning technology, its principle is similar to duplicating of n DNA, is a kind of method that external enzymatic reaction preference ground synthesizes specific DNA.The real-time fluorescence PCR technology was released by U.S. Applied biosystems (ABI) company in 1996, it is a kind ofly to add fluorophor in the PCR reaction system, utilization detects whole PCR process to the real-time detection of fluorescent signal accumulation, by calibration curve unknown template is carried out quantitative analysis at last.This technology is used fluorescence labeling probe on conventional PCR basis, detect the PCR product in real time.Improve sensitivity on the one hand, utilized the fluorescent signal accumulation whole PCR process of monitoring in real time on the other hand, set up real-time amplification curve, by typical curve unknown template has been carried out quantitative analysis at last.Real time pcr has good advantage, easy and simple to handle, rapidly and efficiently, hypersensitivity, repeatability, specificity and multiplex amplification.

At present, adopt CCD as detector in the real-time fluorescence PCR instrument mostly, its biggest advantage is to scan a plurality of fluorescent signals simultaneously, speed is very fast, but sensitivity is low, and there is interference in the fluorescent signal between test sample simultaneously, makes detected result be subjected to very big influence.Find after having compared the document of a large amount of fluorescent PCR instrument, less to report based on the demarcation aspect of the fluorescent PCR instrument of CCD, concentrate on mostly in the research that how to detect fluorescent signal.This directly has influence on the social recognition and the industrialization quality Control during Production of reliability that the biological PCR product uses, generalization, experimental result.

Summary of the invention

The present invention is directed to the deficiencies in the prior art, propose a kind of real-time fluorescence PCR relative Calibration method based on standard model.The sample that this method utilization is demarcated provides the fluorescent signal of standard for the real-time fluorescence PCR instrument, realizes the demarcation of real-time fluorescence PCR instrument fluoroscopic examination precision.Because of the real-time fluorescence PCR instrument is a kind of method of relative measurement, so the present invention also adopts the method for relative Calibration.The present invention realizes by following technical scheme:

Need a fluorescence detector of having demarcated, real-time fluorescence PCR instrument to be calibrated, a n sample tube, fluorescent powder and an epoxy resin glue in the methods of the invention.Put into the fluorescence detector of having demarcated solidifying the sample tube that is made, obtain the normal data of fluorescence light intensity by fluorescent powder and glue mixing.The fluorescence detector of having demarcated connects integrating sphere as excitation light source by monochromatic LED, photomultiplier is as photodetector, and is that the enlargement ratio and the RS232 serial ports of instrument power supply, modulation photomultiplier outputs to the result on the computer by instrumentation control system.

The concrete steps of the inventive method are:

Step 1: production standard sample

1-1 carries out the n five equilibrium with real-time fluorescence PCR instrument maximum range to be calibrated, gets n identical transparent sample test tube then, and number consecutively is 1 to n, wherein n 〉=10.

1-2 gets a beaker, adds the fluorescent powder of e mg and the epoxy resin glue of f ml in beaker, stirs, and fluorescent powder is dissolved in the epoxy resin glue fully, forms glue mixture; Get a syringe then, (glue mixture of 0＜s＜f) is injected the n test tube, then this invisible spectro fluorescent powder concentration δ with s ml _nFor:

${\mathrm{\δ}}_n=\frac{e}{f}$

Strengthen for the fluorescence intensity that makes this n test tube is linear successively, then i number in vitro the concentration of fluorescent powder be:

${\mathrm{\δ}}_i=\frac{i}{n}{\mathrm{\δ}}_n=\frac{\mathrm{ie}}{\mathrm{nf}},(1\≤i\≤n)$

After then finishing the i test tube, the glue volume delta v that should add in the beaker _I-1For:

$\mathrm{\Δ}{v}_{i-1}=\frac{\mathrm{nf}-[i+(i+1)+...+n]s}{i-1}$

Thereby obtain the linear successively enhanced n test tube of fluorescence intensity;

1-3 leaves standstill n test tube until glue and solidifies fully, thereby finishes the making of the 1st to n standard samples.

Step 2: calculate the calibration criterion value

2-1 opens the fluorescence detector of having demarcated, adjusts multiplier gain, makes instrument stabilizer;

2-2 puts into the fluorescence detector of having demarcated successively to the n that obtains in a step 1 standard samples, obtains one group of standard fluorescence light intensity measurement a ₁, a ₂,, a _nCalculate the calibration criterion value A of one group of correspondence again ₁, A ₂, A _n, A wherein ₁=a ₁/ a _n, A ₂=a ₂/ a _n, A _n=a _n/ a _n

Step 3: instrument data to be calibrated detects

3-1 opens real-time fluorescence PCR instrument to be calibrated, waits for stabilizer instrument.

3-2 puts into real-time fluorescence PCR instrument to be calibrated to the n that obtains in a step 1 standard samples, obtains one group of observed value b ₁, b ₂,, b _nCalculate the observed value B to be calibrated of one group of correspondence again ₁, B ₂, B _n, B wherein ₁=b ₁/ b _n, B ₂=b ₂/ b _n, B _n=b _n/ b _n

Step 4: draw calibration result

Obtain the each point deviation D of real-time fluorescence PCR instrument to be calibrated by calibration criterion Value Data group and observed value data set to be calibrated comparison _iWith average deviation d, thereby finish the real-time fluorescence PCR relative Calibration.

D _i＝|A _i-B _i|

$d=\frac{1}{n}\underset{1\≤i\≤n}{\mathrm{\Σ}}{D}_i$

The present invention compared with prior art, the useful effect that has is:

1, adopt fluorescent powder as standard model, this powder is metastable material, and to temperature and the not influence of light application time length, the fluorescence light intensity that sample is produced is maintained fixed.

2, adopt the fluorescent powder of pressing the useful range five equilibrium, the fluorescence light intensity that the fluorescence sample is produced covers whole useful range, the stepped in theory distribution of the light intensity value that obtains in fluorescence detector and the PCR instrument, clear more easy resolution.

3, adopt sol-gel technique, make fluorescent powder in vitro with the colloid uniform mixing, thereby after making excitation light source shine test tube, produce stable fluorescence, be convenient to the detection of photodetector.

4, adopt the fluorescence detector demarcated, can make experimental data have traceability, and ratio of precision real-time fluorescence PCR instrument height, the result that the data analysis of real-time fluorescence PCR instrument is obtained has reliability.

Embodiment

The required hardware of using comprises in the inventive method: a fluorescence detector of having demarcated, a computer, a real-time fluorescence PCR instrument to be calibrated and a printer.The instrument that the fluorescence detector of having demarcated is demarcated for the national quantitative study of process institute, the real-time fluorescence PCR instrument height that its ratio of precision is general can be used as normal data; Its excitation light source adopts monochromatic LED and integrating sphere, and photodetector is a photomultiplier, can detect the standard fluorescence light intensity that obtains fluorescent substance, and the data that its detection obtains can be kept in the computer by the RS232 serial ports of instrument controlling box.

The photodetector of real-time fluorescence PCR instrument to be calibrated can be CCD or photomultiplier, after sample tube is put into instrument, normally starts instrument, makes its operation full cycle process, detects the data that obtain and is kept in the computer as detected value.

The concrete steps of the inventive method are:

Step 1: production standard sample

1-1 carries out n (n 〉=10) five equilibrium with the maximum range of instrument to be calibrated, gets n identical transparent sample test tube then, and number consecutively is 1 to n.

1-2 gets a 50ml beaker, adds the fluorescent powder of 5mg and the epoxy resin glue of 20ml in beaker, stirs, and fluorescent powder is dissolved in the epoxy resin glue fully, forms glue mixture.Get a syringe then, the glue mixture of 0.01ml is injected the n test tube, add Δ v again _iThe glue of volume is diluted to next concentration with fluorescent powder, to make the n-1 standard model.N number invisible spectro fluorescent powder concentration δ _nFor

${\mathrm{\δ}}_n=\frac{5}{20}=0.25$

Strengthen for the fluorescence intensity that makes this n test tube is linear successively, then i number in vitro the concentration of fluorescent powder be

${\mathrm{\δ}}_i=\frac{i}{n}{\mathrm{\δ}}_n=0.25\frac{i}{n},(1\≤i\≤n)$

After finishing the i+1 standard model, make the i standard model before, remaining fluorescent powder quality m (mg) is in the beaker

$m=5-0.25\×0.01-\frac{n-1}{n}0.25\×0.01-...-\frac{i+1}{n}0.25\×0.01=5-\frac{(i+1)+...+(n-1)+n}{n}0.0025$

The volume v of glue in the beaker then _i(ml) be

$v_i=\frac{m}{{\mathrm{\δ}}_i}=\frac{5-\frac{(i+1)+...+(n-1)+n}{n}0.0025}{0.25\frac{i}{n}}=20\frac{n}{i}-\frac{(i+1)+...+(n-1)+n}{i}0.01$

Finish after the i test tube sample remaining glue volume v in the beaker _i' (ml) be

${v_i}^{\′}=v_i-0.01\mathrm{ml}=20\frac{n}{i}-\frac{i+(i+1)+...+n}{i}0.01$

Then finish the i test tube, prepare before the i-1 test tube glue volume delta v that should add in the beaker _I-1For

$\mathrm{\Δ}{v}_{i-1}=v_{i-1}-{v_i}^{\′}=20\frac{n}{i-1}-0.01\frac{i+...+n}{i-1}-[20\frac{n}{i}-\frac{i+(i+1)+...+n}{i}0.01]$

$=20\frac{n}{i(i-1)}-0.01\frac{i+...+n}{i(i-1)}$

Thereby obtain the linear successively enhanced n test tube of fluorescence intensity.

1-3 leaves standstill n test tube until glue and solidifies fully, thereby finishes the making of the 1st to n standard samples.

Step 2: calculate the calibration criterion value

2-1 opens the fluorescence detector of having demarcated, adjusts multiplier gain, makes instrument stabilizer;

2-2 puts into the fluorescence detector of having demarcated successively to the n that obtains in a step 1 standard samples, obtains one group of standard fluorescence light intensity measurement a ₁, a ₂,, a _n, because of the gain difference of each instrument, the fluorescence light intensity value that has caused measuring is relative, and nisi, in the present invention should be with the maximum value a that measures _nBe benchmark, calculate the calibration criterion value A of one group of correspondence ₁, A ₂, A _n, A wherein ₁=a ₁/ a _n, A ₂=a ₂/ a _n, A _n=a _n/ a _n

Step 3: instrument data to be calibrated detects

3-1 opens real-time fluorescence PCR instrument to be calibrated, waits for stabilizer instrument.

3-2 puts into real-time fluorescence PCR instrument to be calibrated to the n that obtains in a step 1 standard samples, obtains one group of observed value b ₁, b ₂,, b _nWith b _nBe benchmark, calculate the observed value B to be calibrated of one group of correspondence ₁, B ₂, B _n, B wherein ₁=b ₁/ b _n, B ₂=b ₂/ b _n, B _n=b _n/ b _n

Step 4: draw calibration result

Obtain the each point deviation D of real-time fluorescence PCR instrument to be calibrated by calibration criterion Value Data group and observed value data set to be calibrated comparison _iWith average deviation d, thereby finish the real-time fluorescence PCR relative Calibration.

D _i＝|A _i-B _i|

$d=\frac{1}{n}\underset{1\≤i\≤n}{\mathrm{\Σ}}{D}_i$

Claims (1)

1. the real-time fluorescence PCR relative Calibration method based on standard model is characterized in that this method comprises the steps:

Step 1, production standard sample

1-1 carries out the n five equilibrium with real-time fluorescence PCR instrument maximum range to be calibrated, gets n identical transparent sample test tube then, and number consecutively is 1 to n, wherein n 〉=10;

1-2 gets a beaker, adds the fluorescent powder of e mg and the epoxy resin glue of f ml in beaker, stirs, and fluorescent powder is dissolved in the epoxy resin glue fully, forms glue mixture; Get a syringe then, the glue mixture of s ml is injected the n test tube, then this invisible spectro fluorescent powder concentration δ _nFor:

${\mathrm{\δ}}_n=\frac{e}{f}$

Strengthen for the fluorescence intensity that makes this n test tube is linear successively, then i number in vitro the concentration of fluorescent powder be:

${\mathrm{\δ}}_i=\frac{i}{n}{\mathrm{\δ}}_n=\frac{\mathrm{ie}}{\mathrm{nf}},$ 1≤i≤n wherein

After finishing the i test tube thus, the glue volume delta v that should add in the beaker _I-1For:

$\mathrm{\Δ}{v}_{i-1}=\frac{\mathrm{nf}-[i+(i+1)+...+n]s}{i-1}$

Thereby obtain the linear successively enhanced n test tube of fluorescence intensity;

1-3 leaves standstill n test tube until glue and solidifies fully, thereby finishes the making of the 1st to n standard samples;

Step 2, calculating calibration criterion value

2-1 opens the fluorescence detector of having demarcated, adjusts multiplier gain, makes instrument stabilizer;

2-2 puts into the fluorescence detector of having demarcated successively to the n that obtains in a step 1 standard samples, obtains one group of standard fluorescence light intensity measurement a ₁, a ₂,, a _nCalculate the calibration criterion value A of one group of correspondence again ₁, A ₂, A _n, A wherein ₁=a ₁/ a _n, A ₂=a ₂/ a _n, A _n=a _n/ a _n

Step 3, instrument data to be calibrated detect

3-1 opens real-time fluorescence PCR instrument to be calibrated, waits for stabilizer instrument;

3-2 puts into real-time fluorescence PCR instrument to be calibrated to the n that obtains in a step 1 standard samples, obtains one group of observed value b ₁, b ₂,, b _nCalculate the observed value B to be calibrated of one group of correspondence again ₁, B ₂, B _n, B wherein ₁=b ₁/ b _n, B ₂=b ₂/ b _n, B _n=b _n/ b _n

Step 4, draw calibration result

Obtain the each point deviation D of real-time fluorescence PCR instrument to be calibrated by calibration criterion Value Data group and observed value data set to be calibrated comparison _iWith average deviation d, thereby finish the real-time fluorescence PCR relative Calibration; D wherein _i=| A _i-B _i|,