阅读说明：本技术 一种qpcr实时荧光数据定量分析的方法 (QPCR real-time fluorescence data quantitative analysis method ) 是由 王寄竹 石瑞 于 2020-05-29 设计创作，主要内容包括：本发明公开了一种QPCR实时荧光数据定量分析的方法,涉及PCR反应领域,包括S1、采集每个样品在PCR反应过程中多个循环的荧光数据,得到每个样品的循环数—荧光亮度值的曲线A；S2、处理S1中的所有荧光数据,使每个样品的曲线A变得平滑得到曲线B；S3、获取每个样品的曲线B的基线并进行基线校准；S4、根据校准后的基线得到每个样品的Ct值；该方法从数据层面入手,通过一系列算法步骤,明显消除反应、加温、信号采集等对原始数据的影响,从而完成最终的分析,计算出待测样品的Ct值和浓度,定量分析的结果准确度。(The invention discloses a quantitative analysis method of QPCR real-time fluorescence data, which relates to the field of PCR reaction, and comprises S1, collecting fluorescence data of multiple cycles of each sample in the PCR reaction process to obtain a curve A of the cycle number-fluorescence brightness value of each sample; s2, processing all the fluorescence data in S1 to smooth the curve A of each sample to obtain a curve B; s3, acquiring a base line of the curve B of each sample and calibrating the base line; s4, obtaining the Ct value of each sample according to the calibrated baseline; the method starts from a data aspect, and obviously eliminates the influence of reaction, heating, signal acquisition and the like on original data through a series of algorithm steps, thereby completing final analysis, calculating the Ct value and concentration of a sample to be detected, and quantitatively analyzing the result accuracy.)

1. A method for QPCR real-time fluorescence data quantitative analysis is characterized by comprising the following steps:

s1, collecting fluorescence data of each sample in a plurality of cycles in the PCR reaction process to obtain a curve A of the cycle number-fluorescence brightness value of each sample;

s2, processing all the fluorescence data in S1 to smooth the curve A of each sample to obtain a curve B;

s3, acquiring a base line of the curve B of each sample and calibrating the base line;

and S4, obtaining the Ct value of each sample according to the calibrated baseline.

2. The method according to claim 1, wherein S2 comprises:

s21, determining a smooth window, wherein the smooth window is 3 or 5;

s22, starting the first cycle, taking the number of points of the sliding window, and replacing the corresponding collection value with the average value, so that the curve a of each sample becomes a smooth curve B.

3. The method according to claim 2, wherein S3 comprises:

s31, differentiating two continuous points of data in the curve B of each sample, and marking a point m with the maximum differential value;

s32, calculating the difference between the point m and the previous point from the point m to the previous point;

s33, if the difference is larger than 0.1, moving the point m forward until the first point;

s34, taking the point after stopping as m as a baseline end point, and marking the baseline period from the first cycle to the mth cycle as a curve B;

s35, taking the 1 st point to the m th point as a baseline data set to perform linear fitting, and calculating a baseline analytical formula y as ax + b

S36, subtracting the ordinate of the base line after calibration from the ordinate of each point on the curve B to obtain deltaRn.

4. The method according to claim 2, wherein S4 comprises:

s41, taking the fluorescence value deltaRn of each line calibration and the corresponding cycle number as a new coordinate pair of the curve, and calculating 10-time standard deviation of all points in the baseline period as a threshold;

s42, taking the minimum value of the threshold values of all samples as an automatic threshold value;

and S43, calculating the Ct value according to the automatic threshold value and the base line after each sample is calibrated.

Technical Field

The invention relates to the field of PCR (polymerase chain reaction), in particular to a quantitative QPCR (quantitative polymerase chain reaction) real-time fluorescence data analysis method.

Background

QPCR real-time fluorescence data, captured by various types of sensors, requires a series of analyses of the data to determine the concentration of the sample to be measured. In practice, due to errors in the PCR reaction itself, temperature control, and collection process, the final collection result will deviate to different degrees, so that the shape of the S-curve becomes irregular, and the accuracy of the Ct value of the result of quantitative analysis is affected.

Disclosure of Invention

The invention aims to solve the problems and designs a method for QPCR real-time fluorescence data quantitative analysis.

The invention realizes the purpose through the following technical scheme:

a method for QPCR real-time fluorescence data quantitative analysis comprises the following steps:

s1, collecting fluorescence data of each sample in a plurality of cycles in the PCR reaction process to obtain a curve A of the cycle number-fluorescence brightness value of each sample;

s2, processing all the fluorescence data in S1 to smooth the curve A of each sample to obtain a curve B;

s3, acquiring a base line of the curve B of each sample and calibrating the base line;

and S4, obtaining the Ct value of each sample according to the calibrated baseline.

Further, in S2, the method includes:

s21, determining a smooth window, wherein the smooth window is 3 or 5;

s22, starting the first cycle, taking the number of points of the sliding window, and replacing the corresponding collection value with the average value, so that the curve a of each sample becomes a smooth curve B.

Further, in S3, the method includes:

s31, differentiating two continuous points of data in the curve B of each sample, and marking a point m with the maximum differential value;

s32, calculating the difference between the point m and the previous point from the point m to the previous point;

s33, if the difference is larger than 0.1, moving the point m forward until the first point;

s34, taking the point after stopping as m as a baseline end point, and marking the baseline period from the first cycle to the mth cycle as a curve B;

s35, taking the 1 st point to the m th point as a baseline data set to perform linear fitting, and calculating a baseline analytical formula y as ax + b

S36, subtracting the ordinate of the base line after calibration from the ordinate of each point on the curve B to obtain deltaRn.

Further, in S4, the method includes:

s41, taking the fluorescence value deltaRn of each line calibration and the corresponding cycle number as a new coordinate pair of the curve, and calculating 10-time standard deviation of all points in the baseline period as a threshold;

s42, taking the minimum value of the threshold values of all samples as an automatic threshold value;

and S43, calculating the Ct value according to the automatic threshold value and the base line after each sample is calibrated.

The invention has the beneficial effects that: the invention realizes a QPCR data processing method, which starts from a data layer and obviously eliminates the influence of reaction, heating, signal acquisition and the like on original data through a series of algorithm steps so as to finish final analysis, calculate the Ct value and concentration of a sample to be detected and quantify the accuracy of the analysis result.

Drawings

FIG. 1 is a graph of curve A in a QPCR real-time fluorescence data quantitative analysis method of the present invention;

FIG. 2 is a graph of the QPCR real-time fluorescence data quantitative analysis method after baseline calibration.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.

In the description of the present invention, it is to be understood that the terms "upper", "lower", "inside", "outside", "left", "right", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, or the orientations or positional relationships that the products of the present invention are conventionally placed in use, or the orientations or positional relationships that are conventionally understood by those skilled in the art, and are used for convenience of describing the present invention and simplifying the description, but do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention.

Furthermore, the terms "first," "second," and the like are used merely to distinguish one description from another, and are not to be construed as indicating or implying relative importance.

In the description of the present invention, it is also to be noted that, unless otherwise explicitly stated or limited, the terms "disposed" and "connected" are to be interpreted broadly, and for example, "connected" may be a fixed connection, a detachable connection, or an integral connection; can be mechanically or electrically connected; the connection may be direct or indirect via an intermediate medium, and may be a communication between the two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

The following detailed description of embodiments of the invention refers to the accompanying drawings.

A method for QPCR real-time fluorescence data quantitative analysis comprises the following steps:

s1, collecting fluorescence data of each sample in a plurality of cycles in the PCR reaction process to obtain a curve A of the cycle number of each sample to the fluorescence brightness value, as shown in figure 1.

S2, processing all the fluorescence data in S1 to smooth the curve A of each sample to obtain a curve B;

s21, determining a smooth window, wherein the smooth window is 3 or 5;

s22, starting the first cycle, taking the number of points of the sliding window, and replacing the corresponding collection value with the average value, so that the curve a of each sample becomes a smooth curve B.

S3, acquiring a base line of the curve B of each sample and calibrating the base line, wherein the curve after limit calibration is shown in figure 2;

s31, differentiating two continuous points of data in the curve B of each sample, and marking a point m with the maximum differential value;

s32, calculating the difference between the point m and the previous point from the point m to the previous point;

s33, if the difference is larger than 0.1, moving the point m forward until the first point;

s34, taking the point after stopping as m as a baseline end point, and marking the baseline period from the first cycle to the mth cycle as a curve B;

s35, taking the 1 st point to the m th point as a baseline data set to perform linear fitting, and calculating a baseline analytical formula y as ax + b

S36, subtracting the ordinate of the base line after calibration from the ordinate of each point on the curve B to obtain deltaRn.

And S4, obtaining the Ct value of each sample according to the calibrated baseline.

S41, taking the fluorescence value deltaRn of each line calibration and the corresponding cycle number as a new coordinate pair of the curve, and calculating 10-time standard deviation of all points in the baseline period as a threshold;

s42, taking the minimum value of the threshold values of all samples as an automatic threshold value;

and S43, calculating the Ct value according to the automatic threshold value and the base line after each sample is calibrated.

	Sample No	Sample No. 2	Sample No. three	Sample No. 4	Sample five	Sample six
							Ct value	17.54	21.20	25.36	28.73	32.26	36.94

The technical solution of the present invention is not limited to the limitations of the above specific embodiments, and all technical modifications made according to the technical solution of the present invention fall within the protection scope of the present invention.