CN116334189B - Internal standard system for monitoring qPCR reaction interference and application thereof - Google Patents
Internal standard system for monitoring qPCR reaction interference and application thereof Download PDFInfo
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention relates to the technical field of qPCR (quantitative polymerase chain reaction), and particularly discloses an internal standard system for monitoring qPCR reaction interference and application thereof. Comprising the following steps: the designed internal standard system has more sensitive reaction to the interfering substances of qPCR reaction, such as ethanol, EDTA and the like, so that quantitative or qualitative detection of qPCR can be realized, a reference basis for sample interference judgment is provided, and the reliability of a sample report is confirmed. Besides, an internal standard system in the reaction mother solution is built in, and a reference of another dimension is provided for pipetting or pipetting operation errors in the operation process, so that the reliability of a detection report is greatly improved.
Description
Technical Field
The invention relates to the technical field of qPCR, in particular to an internal standard system for monitoring qPCR reaction interference and application thereof.
Background
Conventional PCR is usually performed by an end-point method. After the nucleic acid amplification reaction is finished, the fragment size of the amplified product and the dyeing brightness of the strip are judged through dyeing treatment and electrophoresis separation, analysis is finished in a semi-quantitative or qualitative mode, and electrophoresis and dyeing are finished through additional pipetting operation because an amplification reaction tube is required to be opened, so that environmental pollution is easy to cause, false positive is caused, and the application of the method is limited, in particular to clinical examination. The fluorescent quantitative PCR technology not only realizes quantitative estimation of specific nucleic acid fragments of samples, but also has the characteristics of high sensitivity, good specificity and reliability, high degree of automation and low pollution rate, so that the fluorescent quantitative PCR gradually replaces the conventional PCR and is used for detection or in-vitro diagnosis.
The Ct value detected by fluorescence quantitative PCR (qPCR for short) is the core of quantification and is the cycle number experienced when a fluorescence signal reaches a set threshold or an inflection point of curve fitting, and the cycle number is positively correlated with the copy number of a target gene in an initial reaction, so that the Ct value can be used for calculating the content of the target gene in a sample. There are many factors affecting the Ct value, such as PCR reaction interfering substances introduced by reaction consumables or sample pretreatment, operator's operation errors, and the like. The presence of operational errors or interfering substances may cause a large gap between the Ct value of the sample detection and the actual level, or lag, or advance, affecting the accuracy of the reported value, particularly in clinical diagnosis, animal and plant quarantine inspection, or drug quality release. Therefore, various reference controls are commonly arranged in the qPCR detection reaction and are used for assisting in judging the reliability of a sample report, such as positive reference, and the detection reaction is proved to be undisturbed; negative references for confirming that the reaction system is free of contamination, etc. For nucleic acid amplification detection, it is often necessary to isolate DNA or RNA nucleic acids from various types of samples in advance and then perform the detection. Since various interfering substances such as bilirubin in stool samples and ethylenediamine tetraacetic acid (EDTA) which prevents degradation of nucleic acids by nucleases during nucleic acid extraction or ethanol washes for purification of nucleic acids interfere with PCR reactions, affecting accuracy and reliability of quantitative or qualitative reporting of qPCR, it is necessary to set up an internal reference control to monitor reaction interference introduced by the sample or nucleic acid extraction process.
Disclosure of Invention
The invention aims to provide an internal standard system for monitoring qPCR reaction interference and application thereof, so as to solve the problems in the background technology.
In order to solve the technical problems, the invention provides the following technical scheme:
an internal standard system for monitoring qPCR reaction interference, comprising: an internal standard system primer, an internal standard system probe and an internal standard system template;
the nucleotide sequence of an upstream primer of the internal standard system primer is shown as SEQ ID NO:1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO:2 is shown in the figure;
the nucleotide sequence of the internal standard system probe is shown as SEQ ID NO:3 is shown in the figure;
the nucleotide sequence of the internal standard system template is shown as SEQ ID NO: 4.
Preferably, the final concentration of the internal standard system primer is 0.1 mu M, the final concentration of the internal standard system probe is 0.1 mu M, and the final concentration of the internal standard system template in the reaction system is 1000-100000 copies/system.
Preferably, the 5 'end of the internal standard system probe is connected with a fluorescence report group, the 3' end of the internal standard system probe is connected with a fluorescence quenching group, the fluorescence report group is selected from any one of FAM, HEX, ROX or CY5, and the fluorescence quenching group is selected from any one of MGB, BHQ or TAMRA.
Preferably, the fluorescent reporter group is HEX, and the fluorescent quenching group is TAMRA.
An application of an internal standard system for monitoring the interference of qPCR reaction is that the internal standard system is used for detecting the interference substances of a sample to be detected.
Preferably, the interfering substance is any one of ethylenediamine tetraacetic acid or ethanol.
Compared with the prior art, the invention has the following beneficial effects: the internal standard system designed by the invention can be used for monitoring whether the whole qPCR process is performed correctly. In order to avoid the homology between an internal standard system and a substance to be detected and the initiation of a competition reaction and the interference of a reaction sample on a nucleic acid amplification reaction, the internal standard system primer, the internal standard system probe and the internal standard system template in the internal standard system designed by the invention have low homology with the gene sequences of mammals, plants and marine organisms, and a proper amount of the internal standard system primer, the internal standard system probe and the internal standard system template are selected and added in the nucleic acid amplification reaction to form a multiplex qPCR amplification reaction with the target gene to be detected. The designed internal standard system has more sensitive reaction to the interfering substances of qPCR reaction, such as ethanol, EDTA and the like, so that the quantitative or qualitative detection of qPCR can be provided, a reference basis for sample interference judgment is provided, and the reliability of a sample report is confirmed. Besides, an internal standard system in the reaction mother solution is built in, and a reference of another dimension is provided for pipetting or pipetting operation errors in the operation process, so that the reliability of a detection report is greatly improved.
The action mechanism of the invention is as follows: the internal reference is also a pair of PCR reactionsReference is made, but there are various arrangements that act as reference controls for different reaction processes. Such as: detecting copy number of specific gene in certain cell, adding primers and detection probes for cell reference gene in the system for confirming the process of cell sample lysis and genomic DNA extraction, or detecting relative content level of target to be detected by taking specific housekeeping gene as reference (e.g. 2) –ΔΔCT Relative quantification). Wherein, the sample to be detected is built in the detection reaction to form a multiple reaction system, so that the interference condition of the sample to be detected on the qPCR reaction can be directly reflected. However, it is a practical difficulty that multiple PCR reactions can compete with each other, such as: the advantageous internal standard system amplification reaction inhibits amplification of the gene of interest, or vice versa; secondly, the designed internal standard system needs to reflect the interference condition of the sample more sensitively than the gene to be detected.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is a graph showing the amplification of CHO cell DNA samples in example 1 of the present invention;
FIG. 2 is an amplification plot of the CHO cell internal standard system of example 1 of the present invention;
FIG. 3 is a graph showing the amplification of E.coli DNA samples in example 2 of the present invention;
FIG. 4 is an amplification graph of an E.coli internal standard system in example 2 of the present invention;
FIG. 5 is a sample amplification plot of CHO cell FAM channel in example 3 of the present invention;
FIG. 6 is a graph showing the amplification of CHO cell HEX channel samples in example 3 of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the following examples, the experimental material sources were:
CHO cell source: ATCC (american type culture collection), lot number: 63830396, cargo number: CCL-61, raw material name: CHO-K1;
CHO cell DNA extraction kit (manufacturer: tiangen organism, cat# DP-304);
bacterial DNA extraction kit (manufacturer: tiangen organism, cat# DP-302);
absolute ethanol (Shanghai Biotechnology, cat No. a500737, grade molecular biology grade);
ethylenediamine tetraacetic acid (EDTA) (Shanghai creatures, cat No. a600107, grade reagent grade);
qPCR special premix (AceQ U+Probe Master Mix) (Nanjinouzan, cat. No. Q113).
The nucleotide sequence of the internal standard system primer is as follows:
the upstream primer is shown as SEQ ID NO:1 is shown as follows: 5'-ctaccgtgatgccataccag-3';
the downstream primer is shown as SEQ ID NO:2 is shown as follows: 5'-ctgctagtatgcggctggt-3';
the nucleotide sequence of the internal standard system probe is shown as SEQ ID NO:3, shown in the following: HEX-agtgaagcacgacacagacctaccg-TAMRA;
the nucleotide sequence of the internal standard system template is shown as SEQ ID NO:4, specifically: 5'-ctaccgtgatgccataccagtgaagcacgacacagacctaccgatccagcttccacgcaccactcgaccagccgcatactagcag-3'.
The internal standard system primer adopts database (Genbank database under NCBI website) comparison, and the comparison result shows that no matching item exists for the information of the database of mammals, plants and marine organisms.
Example 1: in this example, genomic DNA was extracted from a conventional CHO cell, and amplified using a qPCR detection reaction solution to which an internal standard system was added.
1. Test material:
extracting genome DNA from cultured CHO cells according to a CHO cell DNA extraction kit specification, testing the extracted DNA by agarose gel electrophoresis and a spectrophotometer to identify DNA quality and determine DNA concentration, diluting the extracted DNA to 30 ng/mu L by using TE buffer according to the concentration determined by the spectrophotometer, preparing a standard curve by using a Qubit instrument and a CHO DNA national quantitative reference, assigning a value to the extracted CHO DNA sample to obtain the actual concentration of the finally extracted CHO DNA sample, wherein the DNA sample is used as an application sample in the embodiment, and the preservation condition is-20 ℃.
Spectrophotometry detects that the extracted CHO DNA meets the OD260/OD280 of 1.8-2.0 and the OD260/OD230 is more than 2.0, which shows that the extracted CHO DNA is complete and has no degradation and qualified purity.
2. Selecting an internal standard system and designing a primer probe:
because of the future application of the internal standard system, it may involve detecting nucleic acids of cells and microorganisms of various sources, and thus the design of the internal standard substance is required to avoid the nucleic acid sequence information of mammals, plants and marine organisms in the database as much as possible. Primer and probe were designed using primer5.0, primer probe design obeys the following principle: the product has the length of 80bp-160bp, avoids secondary structure, avoids base mismatch, has the GC content of 45% -65%, has the primer length of 18bp-25bp, has the probe length of 20bp-30bp, has the primer Tm value of 58-62 ℃ and has the probe Tm value of 68-72 ℃. The internal standard system template, the primer and the probe are all biosynthesized by Shanghai workers.
3. Real-time fluorescent quantitative PCR:
real-time fluorescent quantitative PCR was performed on ABI 7500, and the test loading samples were 6 gradient samples of 300pg/μL, 30pg/μL, 3pg/μL, 0.3pg/μL, 0.03pg/μL, 0.003pg/μL, respectively, of the assigned extracted DNA, each sample set at 3 replicates.
Each replicate system was 30. Mu.L, including 10. Mu.L of CHO DNA sample templates at different concentrations, 15. Mu.L of qPCR dedicated premix (containing UNG decontamination enzyme), 4. Mu.L of primer probe buffer and 1. Mu.L of internal standard system template.
Wherein, 4 mu L primer probe buffer solution comprises a primer, a probe and an internal standard system primer and an internal standard system probe for CHO target detection; the final concentration of these primer probes in the 30 μl system is: primer 0.2 mu M for CHO target detection and fluorescent marked probe 0.16 mu M; 0.1 mu M of internal standard system primer and 0.1 mu M of internal standard system probe;
the nucleotide sequence of the upstream primer of the primer for CHO target detection is shown as SEQ ID NO:5, specifically:
5'-gacagggtttctctgtgtag-3'；
the nucleotide sequence of a downstream primer of the primer for CHO target detection is shown as SEQ ID NO:6, specifically:
5'-cagcactcgggaggcaga-3'；
the nucleotide sequence of the probe for CHO target detection is shown as SEQ ID NO:7, specifically:
FAM-ctttggagcctatcctg-BHQ1；
1. Mu.L of internal standard system template with a final concentration of 100000 copies per system in 30. Mu.L system.
The reaction procedure is as in table 1:
TABLE 1
In fluorescent quantitative PCR, the sample detection Ct value is inversely proportional to the sample concentration, and the higher the sample concentration, the smaller the detection Ct value. FIG. 1 shows an amplification curve representing a CHO sample detection channel, and FIG. 2 shows an internal standard system detection channel. The results in FIG. 1 show that 6 gradient samples are amplified normally, and the Ct value deviation between each other is between 3.1 and 3.6, which accords with the Ct value deviation of 10 times gradient dilution. The results of FIG. 2 show that the internal standard system has higher detection precision, does not interfere with the detection of CHO genomic DNA in the system, can coexist with mammalian cell genomic DNA targets in the system, and does not affect the normal amplification thereof.
Example 2: in this example, genomic DNA was extracted from E.coli and amplified using a qPCR detection reaction solution to which an internal standard system was added.
1. Test material:
extracting genome DNA from the cultured escherichia coli bacterial liquid according to a specification of a bacterial DNA extraction kit, testing the extracted DNA by agarose gel electrophoresis and a spectrophotometer to identify DNA quality and determine DNA concentration, diluting the extracted DNA to 30 ng/mu L by using a TE buffer according to the concentration determined by the spectrophotometer, preparing a standard curve by using a Qubit instrument and a national escherichia coli DNA quantitative reference, assigning a value to the extracted escherichia coli DNA sample to obtain the actual concentration of the finally extracted escherichia coli DNA sample, wherein the DNA sample is taken as an application sample of the embodiment, and the preservation condition is-20 ℃.
The spectrophotometry detection shows that the extracted escherichia coli DNA meets the OD260/OD280 of 1.8-2.0, and the OD260/OD230 is more than 2.0, which indicates that the extracted escherichia coli DNA is complete and has no degradation and qualified purity.
2. Real-time fluorescent quantitative PCR
Real-time fluorescent quantitative PCR was performed on ABI 7500, and the test loading samples were 5 gradient samples of 300pg/μL, 30pg/μL, 3pg/μL, 0.3pg/μL, 0.03pg/μL, respectively, of the assigned E.coli DNA after extraction, each sample set at 3 replicates.
Each replicate system was 30. Mu.L, including 10. Mu.L of E.coli DNA sample templates at different concentrations, 15. Mu.L of qPCR-specific premix (containing UNG decontamination enzyme), 4. Mu.L of primer probe buffer and 1. Mu.L of internal standard system template.
Wherein 4 mu L of primer probe buffer solution comprises a primer, a probe and an internal standard system primer and an internal standard system probe for detecting the escherichia coli target; the final concentration of these primer probes in the 30 μl system is: e.coli target detection primer 0.167. Mu.M, E.coli target detection probe 0.167. Mu.M; 0.1 mu M of internal standard system primer and 0.1 mu M of internal standard system probe;
the nucleotide sequence of the upstream primer of the primer for detecting the escherichia coli target is shown as SEQ ID NO:8, specifically:
5'-ggaatcgctagtaatcgtggat-3'；
the nucleotide sequence of a downstream primer of the primer for detecting the escherichia coli target is shown as SEQ ID NO:9, specifically:
5'-agtcatgaatcacaaagtggtaagc-3'；
the nucleotide sequence of the probe for detecting the escherichia coli target is shown as SEQ ID NO:10, specifically:
FAM-aatgccacggtgaatacgttcccg-BHQ1；
1. Mu.L of internal standard system template with a final concentration of 1000 copies/system in 30. Mu.L system.
The reaction procedure is as follows in table 2:
TABLE 2
In fluorescent quantitative PCR, the sample detection Ct value is inversely proportional to the sample concentration, and the higher the sample concentration, the smaller the detection Ct value. FIG. 3 shows the amplification curve of the E.coli DNA sample detection channel, and FIG. 4 shows the internal standard system detection channel. The results in FIG. 3 show that 5 gradient samples amplify normally, with Ct value deviations between 3.1 and 3.6 from each other, consistent with Ct value deviations from 10-fold gradient dilutions. The results of FIG. 4 show that the internal standard system has higher detection precision, does not interfere with the detection of the genomic DNA of the Escherichia coli in the system, can coexist with the genomic DNA target of the bacterial cells in the system, and does not affect the normal amplification of the genomic DNA target.
Example 3: in the embodiment, a PCR reaction inhibitor with a certain concentration is added into a sample to be detected, and the effect of an internal standard system in detection is tested and observed.
1. Test material:
extracting genome DNA from cultured CHO cells according to a CHO cell DNA extraction kit specification, testing the extracted DNA by agarose gel electrophoresis and a spectrophotometer to identify DNA quality and determine DNA concentration, diluting the extracted DNA to 30 ng/mu L by using TE buffer according to the concentration determined by the spectrophotometer, preparing a standard curve by using a Qubit instrument and a CHO DNA national quantitative reference, assigning a value to the extracted CHO DNA sample to obtain the actual concentration of the finally extracted CHO DNA sample, wherein the DNA sample is used as an application sample in the embodiment, and the preservation condition is-20 ℃.
Spectrophotometry detects that the extracted CHO DNA meets the OD260/OD280 of 1.8-2.0 and the OD260/OD230 is more than 2.0, which shows that the extracted CHO DNA is complete and has no degradation and qualified purity.
A clean 15mL centrifuge tube was taken, 2.5mL of sterilized ultrapure water was added thereto, 7.5mL of absolute ethanol was then added thereto, and after 30s of vortexing with an oscillator, the tube was allowed to stand for use.
8.767g of ethylenediamine tetraacetic acid (EDTA) solid is weighed, dissolved in water, and fixed to 1L to prepare 30mM EDTA solution with final concentration for later use.
2. Real-time fluorescent quantitative PCR:
real-time fluorescent quantitative PCR was performed on ABI 7500 with 3 pg/. Mu.L CHO DNA samples, 3 pg/. Mu.L CHO DNA samples with 5% ethanol, and 3 pg/. Mu.L CHO DNA samples with 2mM EDTA, 2 replicates were set per sample, and 30. Mu.L per replicate system.
(1) When the test loading sample is 3pg/μl of CHO DNA sample, the system comprises 10 μl of CHO DNA sample template, 15 μl of qPCR special premix (containing UNG decontamination enzyme), 4 μl of primer probe buffer and 1 μl of internal standard system template;
(2) when the sample was a 3pg/μl CHO DNA sample containing 5% ethanol, the system comprised 10 μl CHO DNA sample template, 2 μl75% ethanol, 15 μl qPCR special premix (containing UNG decontamination enzyme), 2 μl primer probe buffer and 1 μl internal standard system template;
(3) when the sample was a 3pg/μl CHO DNA sample containing 2mM EDTA, the system included 10 μl CHO DNA sample template, 2 μl30mM EDTA,15 μl qPCR special premix (containing UNG decontamination enzyme), 2 μl primer probe buffer and 1 μl internal standard system template.
The primer probe buffer solution comprises a primer for detecting the CHO target, a probe, an internal standard system primer and an internal standard system probe; the final concentration of these primer probes in the 30 μl system is: primer 0.2 mu M for CHO target detection and fluorescent marked probe 0.16 mu M; 0.1 mu M of internal standard system primer and 0.1 mu M of internal standard system probe;
the nucleotide sequence of the upstream primer of the primer for CHO target detection is shown as SEQ ID NO:5, specifically:
5'-gacagggtttctctgtgtag-3'；
the nucleotide sequence of a downstream primer of the primer for CHO target detection is shown as SEQ ID NO:6, specifically:
5'-cagcactcgggaggcaga-3'；
the nucleotide sequence of the probe for CHO target detection is shown as SEQ ID NO:7, specifically:
FAM-ctttggagcctatcctg-BHQ1；
1. Mu.L of internal standard system template with a final concentration of 100000 copies per system in 30. Mu.L system.
The reaction procedure is as follows in table 3:
TABLE 3 Table 3
The amplification result data are shown in the following table 4, and as shown in fig. 5 and 6, different amounts of inhibitors (ethanol and EDTA) are added to the same sample, so that amplification abnormality can be obviously shown by an internal standard system (HEX channel) during amplification, a sample containing 5% ethanol and 2mM EDTA is included, and the HEX channel amplification curve is obviously behind that of a control sample; the sample containing 5% ethanol and 2mM EDTA has small difference between the FAM channel amplification curve and the FAM channel amplification curve of the control sample, and the Ct value is close, so that the internal standard system is sensitive to the existence of the inhibitor in the PCR system, and can be used for monitoring whether the PCR reaction process is normally carried out.
TABLE 4 Table 4
It can be seen from the above three sets of examples that the addition of the internal standard system does not affect the amplification of nucleic acids of mammalian cells such as CHO cells and microorganisms such as escherichia coli, and by introducing the internal standard system into the amplification system, more sensitive monitoring of PCR reaction inhibitors such as ethanol, EDTA and the like can be achieved to provide anomalies in the amplification reaction, helping to achieve more accurate analysis of data.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. An internal standard system for monitoring interference in a qPCR reaction, comprising: an internal standard system primer, an internal standard system probe and an internal standard system template;
the nucleotide sequence of an upstream primer of the internal standard system primer is shown as SEQ ID NO:1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO:2 is shown in the figure;
the nucleotide sequence of the internal standard system probe is shown as SEQ ID NO:3 is shown in the figure;
the nucleotide sequence of the internal standard system template is shown as SEQ ID NO: 4.
2. The internal standard system for monitoring the interference of the qPCR reaction according to claim 1, wherein the final concentration of the primer of the internal standard system is 0.1 mu M, the final concentration of the probe of the internal standard system is 0.1 mu M, and the final concentration of the template of the internal standard system in a reaction system is 1000-100000 copies/system.
3. The internal standard system for monitoring the interference of the qPCR reaction according to claim 1, wherein the 5 'end of the probe of the internal standard system is connected with a fluorescence reporting group, the 3' end of the probe of the internal standard system is connected with a fluorescence quenching group, the fluorescence reporting group is selected from any one of FAM, HEX, ROX and CY5, and the fluorescence quenching group is selected from any one of MGB, BHQ and TAMRA.
4. An internal standard system for monitoring interference of qPCR reaction according to claim 3, wherein the fluorescent reporter group is HEX and the fluorescent quenching group is TAMRA.
5. An application of an internal standard system for monitoring qPCR reaction interference, which is characterized in that the internal standard system as claimed in any one of claims 1 to 4 is used for detecting interference substances in a sample to be detected.
6. The use of an internal standard system for monitoring the interference of a qPCR reaction according to claim 5, wherein the interfering substance is any one of ethylenediamine tetraacetic acid or ethanol.
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