CN112852935A - Method for multiple detection of target nucleotide sequence based on double-labeled oligonucleotide probe melting curve and kit thereof - Google Patents

Method for multiple detection of target nucleotide sequence based on double-labeled oligonucleotide probe melting curve and kit thereof Download PDF

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CN112852935A
CN112852935A CN202110130298.8A CN202110130298A CN112852935A CN 112852935 A CN112852935 A CN 112852935A CN 202110130298 A CN202110130298 A CN 202110130298A CN 112852935 A CN112852935 A CN 112852935A
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double
primer
labeled oligonucleotide
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CN112852935B (en
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许向华
张玲华
周中人
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Shanghai Kuailing Biology Engineering Co ltd
Shanghai Miaoling Bioengineering Co ltd
Shanghai Quicking Biotech Co ltd
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Shanghai Miaoling Bioengineering Co ltd
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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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Abstract

The invention relates to a method for multiple detection of target nucleotide sequence based on double-labeled oligonucleotide probe melting curve and a kit applied by the method, the invention adopts the double-labeled oligonucleotide probe to perform melting curve analysis on multiple PCR amplification products, and particularly detects the melting curve of a hybrid double strand of at least one double-labeled oligonucleotide probe (usually two or more) with the same fluorescent group and a single-stranded nucleic acid product matched with the double-labeled oligonucleotide probe in a single fluorescence detection channel; the invention can realize the effect that one fluorescence channel carries out multiple detection and each detected target site is detected by using a single probe, is particularly suitable for detecting a plurality of targets by using a single fluorescence channel, and effectively combines double interpretation of an amplification curve and a dissolution curve, thereby ensuring the specificity of detection.

Description

Method for multiple detection of target nucleotide sequence based on double-labeled oligonucleotide probe melting curve and kit thereof
Technical Field
The application relates to the technical field of biochemistry, in particular to a method for multiple detection of a target nucleotide sequence based on a double-labeled oligonucleotide probe melting curve and a kit thereof.
Background
The multiplex nucleic acid detection is mainly used for simultaneously detecting various pathogenic microorganisms or identifying the typing identification of certain pathogenic microorganisms, certain genetic diseases and cancer genes, the most mature technology is PCR nucleic acid amplification technology at present, and specific primers of various pathogenic microorganisms are simultaneously added in the same PCR reaction tube for amplification. Can be used to detect multiple pathogens simultaneously or to identify that type of pathogen infection. The earliest technique was to perform multiplex amplification detection in electrophoretic PCR, and identify multiple amplification products by multiple bands after electrophoresis. After the advent of real-time fluorescent PCR technology, multiplex amplification was achieved in instruments with multiple fluorescent channels by placing a nucleic acid probe in each fluorescent channel at a wavelength corresponding to the fluorescent wavelength, but this technology was limited by the number of fluorescent channels in the instrument.
Melting curve analysis for amplification products was initially fluorescent dye-based real-time PCR. After the amplification reaction is completed, the reaction product is gradually heated (from below the melting temperature of the primer to over 90 ℃ or higher) under fluorescence monitoring, and the negative first derivative of the change of the dye fluorescence signal is used for plotting the temperature, so that the double-stranded DNA product has a corresponding characteristic peak at the melting temperature Tm. Theoretically, a dye melting curve in one fluorescence channel can have a plurality of characteristic peaks, but targets with close Tm cannot be distinguished by using different fluorescence dyes, namely different fluorescence dyes cannot be used for aiming at double chains, each fluorescence channel displays the characteristic peaks corresponding to all double chains Tm, and more characteristic peak results can not be obtained in a plurality of fluorescence channels than in one fluorescence channel.
At present, nucleic acid probes are used to generate melting curves to distinguish (asymmetric) amplification (single-stranded) products, and especially in different fluorescence channels, a plurality of nucleic acid probes are arranged at the same time to obtain different melting curve characteristic peaks to represent different target sequences. Fluorescent probes were used in both amplification and melting curves. Patent CN201010166995.0 discloses a fluorescence probe method for detecting the characteristic peak of melting curve of multiplex PCR, which requires that the DNA polymerase used has no exonuclease activity, and the fluorescence signal of the amplification curve is from the hybridization of probe and target sequence as the melting curve. The method requires that the fluorescent probe is not degraded in the amplification process, and correspondingly, compared with a fluorescent signal generated by the separation of a fluorescent group and a quenching group due to the degradation of the fluorescent probe, the fluorescent signal which is not degraded by the fluorescent probe is weaker. Patent CN201410206857.9 uses DNA polymerase having nucleolytic enzyme activity, and uses two probes, a fluorescent probe at the amplification stage with high Tm and a probe for melting curve analysis with low Tm, the former being degraded at the amplification stage. The two fluorescent probes achieve the technical effect of better signals of an amplification curve and a melting curve. Although the cost of this approach is high, the amplification curve has at least two effects: (1) the amplification curve is completed first, so that information can be obtained earlier; (2) the amplification curve is formed by the participation of the primers and the probes, and has specificity, and only when a plurality of groups of primer probes exist, each group can generate an amplification signal, so that the information of the melting curve is needed; in particular, amplification curve results are negative, and melting curve positives are abnormal and therefore considered suspicious and retested.
However, the prior art does not have a multiplex fluorescence nucleic acid amplification detection method which can combine the information of an amplification curve and a dissolution curve and can be applied to the practical application with low cost and high specificity, wherein the detection number is more than the number of fluorescence channels, especially the application of a single fluorescence detection channel.
Disclosure of Invention
The invention provides a method for multiple detection of target nucleotide sequence based on double-labeled oligonucleotide probe melting curve and a kit applied by the method. The single-stranded nucleic acid product is single-stranded DNA generated by asymmetric PCR, the single-stranded DNA of various target genes is generated in an amplification system, a double-labeled oligonucleotide probe which is used for labeling the single-stranded product with the same fluorescent group and the corresponding quenching group is utilized to form hybrid molecules with the single-stranded product, and the characteristic peak information of the corresponding melting temperature Tm of the single-stranded product is obtained in a melting curve. The effect that one fluorescence channel is used for multiplex detection and one probe is used for detecting each target point to be detected is achieved. The methods and kits provided herein are particularly useful for detecting multiple targets using a single fluorescence channel. In addition, targets for detection according to the present invention include DNA and RNA.
In order to detect a target gene through a melting curve characteristic peak and realize multiple nucleic acid detection, the invention adopts the following technical scheme:
a method for multiple detection of target nucleotide sequence based on double-labeled oligonucleotide probe melting curve, the reaction system comprises:
the reaction system comprises a thermostable DNA polymerase with 5' nuclease activity;
a plurality of pairs of amplification primers that match a target sequence and a plurality of double-labeled oligonucleotide probes that correspond to the same target sequence; the primer pair at least comprises a first primer pair with concentration difference, and the first primer pair comprises a high-concentration primer and a low-concentration primer;
at least one double-labeled oligonucleotide probe is arranged in the reaction system, the melting temperature Tm value of the probe is greater than the melting temperature Tm values of the two matched primers, and the real-time amplification fluorescent signals of the double-labeled oligonucleotide probes corresponding to the same target sequence can be normally detected; in the melting curve analysis of the hybridization of the labeled oligonucleotide probe to the amplified target nucleic acid single strand, a characteristic peak is generated in relation to the Tm value of the labeled oligonucleotide probe.
If the target is RNA, the reaction system of the present invention may further comprise a reverse transcriptase capable of reverse transcribing RNA to DNA in a buffer solution of the above-mentioned thermostable DNA polymerase having 5' nuclease activity.
The oligonucleotide probe in the methods of the invention comprises a reporter label and a quencher label, wherein the quencher label quenches the fluorescence emitted by the reporter label when the labeled oligonucleotide probe is in a single-stranded conformation that is not hybridized to the target sequence; when the labeled oligonucleotide probe is hybridized with the target sequence, a double-stranded structure is formed, the fluorescence of the report label cannot be quenched, and the fluorescence intensity of the report label is higher than that when the oligonucleotide probe is not hybridized with the target sequence and is in a single-stranded state; when a DNA polymerase having 5' nuclease activity encounters a dual-labeled oligonucleotide probe during extension, the reporter label is cleaved and the fluorescence intensity is higher than when the oligonucleotide probe hybridizes to the target sequence.
Generally, in order to ensure the normal display of the melting curve formed by hybridization of the amplified target nucleic acid single strands, the concentration of the double-labeled oligonucleotide probe corresponding to the same target sequence in the first primer pair should at least exceed the amount of the primer added at a low concentration in the first primer pair. The amplification curve signal of the invention mainly comes from fluorescence generated by cutting off the report label when the DNA polymerase with 5' nuclease activity encounters the double-labeled oligonucleotide probe in the extension process, so that the melting temperature Tm of at least one double-labeled oligonucleotide probe is generally higher than the melting temperature Tm of two matched primers, so that the real-time amplification fluorescence signal of the double-labeled oligonucleotide probe corresponding to the same target sequence can be normally detected.
Preferably, the concentration of the double-labeled oligonucleotide probe corresponding to the same target sequence of the first primer pair is at least 10% more than the amount of the low concentration primer added; preferably, the concentration of the double-labeled oligonucleotide probe corresponding to the same target sequence of the first primer pair is at least 30% more than the amount of the low concentration primer added; more preferably, the concentration of the double-labeled oligonucleotide probe corresponding to the same target sequence of the first primer pair is at least 100% more than the amount of the low concentration primer added.
In some embodiments (at least 2 or more than 2 dissolution peaks in the dissolution curve), in order to distinguish a plurality of different dissolution peaks, the melting temperature Tm values of a plurality of the dual-labeled fluorescent probes labeled with the same fluorescent group are different from each other by not less than 2 ℃; more preferably, the melting temperature Tm values of a plurality of the double-labeled fluorescent probes labeled with the same fluorescent group are different from each other by not less than 4 ℃.
In some embodiments of the invention, applicants have explored the effect of concentration differences between asymmetric PCR primer pairs on the detection results of the methods of the invention. Generally, the concentration ratio of the low concentration primer to the high concentration primer should be in the range of 1:3 to 1: 30; preferably, the concentration ratio of the low concentration primer to the high concentration primer is 1:4 to 1: 30; more preferably, the ratio of the concentration of the low concentration primer to the concentration of the high concentration primer is 1:4 to 1: 10.
In the present embodiment, the requirement for symmetric PCR primers requires that the difference in primer pair concentration not exceed 50%. The concentration of the symmetric PCR primers is slightly different, and the product is mainly double-stranded. The detection is focused on the amplification curve, and the corresponding fluorescence channel is set to have only one target sequence. Compared with the common PCR, the invention simultaneously has multiple amplifications and carries out melting curve analysis on the single-chain product of asymmetric amplification; comparing melting curve analysis, obtaining result from amplification curve, time consumption is much shorter.
The double-labeled oligonucleotide probe in the reaction system in the scheme of the invention can adopt a TaqMan type or molecular beacon type probe.
When detecting RNA sample, reverse transcriptase capable of doing RT-PCR is added in the reaction system.
In another aspect, the present invention provides a kit for multiplex detection of target nucleotides based on a melting curve of a ditag oligonucleotide probe, the kit comprising at least:
a thermostable DNA polymerase having 5' nuclease activity;
a plurality of pairs of amplification primers matched with a target sequence and a plurality of double-labeled oligonucleotide probes corresponding to the same target sequence, wherein the melting temperature Tm value of at least one double-labeled oligonucleotide probe is greater than the melting temperature Tm of two matched primers;
and instructions for how to perform the multiplex detection of the target nucleotide and/or a reverse transcriptase for RT-PCR.
The above description of how to perform multiplex detection of target nucleotides describes the technical solution or concept of the present invention, and guides the method for multiplex detection of target nucleotide sequences using the melting curve based on the double-labeled oligonucleotide probe of the present invention.
As described in the above technical solution of the method of the present invention, the kit of the present invention employs the detection method of the present invention, such that the species of the dual-labeled oligonucleotide probe in the kit is larger than the number of fluorescence channels used for labeling the dual-labeled oligonucleotide probe correspondingly. In a popular way, the kit detects N genes, N probes are used, M fluorescent groups for marking the N probes are used, the kit can achieve N > M, more detection possibilities are provided, and the detection specificity is ensured.
One of the more common schemes for the kit of the present invention is:
the kit comprises a plurality of pairs of amplification primers matched with a target sequence and a plurality of double-labeled oligonucleotide probes corresponding to the same target sequence, wherein the primer pairs of the kit at least comprise a first primer pair with concentration difference, and the first primer pair comprises a high-concentration primer and a low-concentration primer;
the kit further comprises a thermostable DNA polymerase having 5' nuclease activity;
the melting temperature Tm value of at least one double-labeled oligonucleotide probe in the kit is greater than the melting temperature Tm of two matched primers (the real-time amplification fluorescent signal of the double-labeled oligonucleotide probe corresponding to the same target sequence can be normally detected).
Further, the concentration of the double-labeled oligonucleotide probe corresponding to the same target sequence in the first primer pair at least exceeds the addition amount of the primer with a low concentration in the first primer pair; preferably, the concentration of the double-labeled oligonucleotide probe corresponding to the same target sequence of the first primer pair is at least 10% more than the amount of the low concentration primer added; preferably, the concentration of the double-labeled oligonucleotide probe corresponding to the same target sequence of the first primer pair is at least 30% more than the amount of the low concentration primer added; more preferably, the concentration of the double-labeled oligonucleotide probe corresponding to the same target sequence of the first primer pair is at least 100% more than the amount of the low concentration primer added.
Further, in the kit, the melting temperature Tm values of a plurality of the double-labeled fluorescent probes labeled with the same fluorescent group are different from each other by not less than 2 ℃; more preferably, the melting temperature Tm values of a plurality of the double-labeled fluorescent probes labeled with the same fluorescent group are different from each other by not less than 4 ℃.
Furthermore, in the kit, the concentration ratio of the low-concentration primer to the high-concentration primer in the primer pair with the concentration difference is 1:3-1: 30; preferably, the concentration ratio of the low concentration primer to the high concentration primer is 1:4 to 1: 30; more preferably, the ratio of the concentration of the low concentration primer to the concentration of the high concentration primer is 1:4 to 1: 10.
Further, the kit comprises at least one second primer pair with a concentration difference of not more than 50%, namely a primer pair for symmetric PCR.
The double-labeled oligonucleotide probe in the kit comprises a TaqMan type or molecular beacon type probe.
The method for multiple detection of target nucleotide sequence based on double-labeled oligonucleotide probe melting curve and the kit thereof have the advantages that: the invention can realize the effect that one fluorescence channel carries out multiple detection and each detected target site is detected by using a single probe; meanwhile, the method and the kit provided by the invention are particularly suitable for detecting a plurality of targets by utilizing a single fluorescence channel to achieve a multiple detection effect; furthermore, the invention combines the double interpretation of the amplification curve and the dissolution curve, and can better ensure the specificity of detection; furthermore, the detection scheme of the present invention allows for a combination of symmetric and asymmetric amplification within multiple target sequences, increasing the degree of freedom and flexibility of detection.
Drawings
FIG. 1 is a triple fluorescent PCR detection amplification curve (two channels) of the febrile pig in example 1;
FIG. 2 is a triple fluorescent PCR product melting curve (two channels) of the febrile pig in example 1;
FIG. 3 is the fluorescent PCR detection amplification curve (dual channel) for swine disease 2+3 in example 2;
FIG. 4 is the melting curve (double channel) of the fluorescent PCR products of swine disease 2+3 in example 2;
FIG. 5 is the amplification curve (single channel) for the triple fluorescent PCR detection of canine diseases in example 3;
FIG. 6 is a melting curve (single channel) of the canine triple fluorescent PCR product of example 3;
FIG. 7 is a 1+3 fluorescent PCR detection amplification curve (two channels) in example 4;
FIG. 8 is the melting curve (double channel) of the 1+3 fluorescent PCR product in example 4;
FIG. 9 is an amplification curve of FIPV0.4+ Bar1:20+ FELV1:5+ FIV1:10 in example 5, FIG. 9-1 is channel 1 (amplification curve for asymmetric amplification), and FIG. 9-2 is channel 2 (amplification curve for symmetric amplification);
FIG. 10 is the dissolution curve of FIPV0.4+ Bar1:20+ FELV1:5+ FIV1:10 in example 5 with the primer ratio;
FIG. 11 is an amplification curve of FIPV0.4+ Bar1:20+ FELV1:10+ FIV1:10 in example 5, FIG. 11-1 is channel 1 (amplification curve for asymmetric amplification), and FIG. 11-2 is channel 2 (amplification curve for symmetric amplification);
FIG. 12 is the dissolution curve of FIPV0.4+ Bar1:20+ FELV1:10+ FIV1:10 in example 5 with the primer ratio;
FIG. 13 shows the primer ratios FIPV0.4+ Bar1:20+ FELV1:9+ FIV1 in example 5: 9, fig. 13-1 is channel 1 (amplification curve for asymmetric amplification), fig. 13-2 is channel 2 (amplification curve for symmetric amplification);
FIG. 14 shows the primer ratios FIPV0.4+ Bar1:20+ FELV1:9+ FIV1 in example 5: 9 dissolution profile;
FIG. 15 is an amplification curve of FIPV0.4+ Bar1:20+ FELV1:8+ FIV1:8 in example 5, FIG. 15-1 is channel 1 (amplification curve for asymmetric amplification), and FIG. 15-2 is channel 2 (amplification curve for symmetric amplification);
FIG. 16 is the dissolution curve of FIPV0.4+ Bar1:20+ FELV1:8+ FIV1:8 in example 5 with the primer ratio;
FIG. 17 is an amplification curve of FIPV0.4+ Bar1:20+ FELV1:7+ FIV1:7 in the primer ratio of example 5, FIG. 17-1 is channel 1 (amplification curve of asymmetric amplification), and FIG. 17-2 is channel 2 (amplification curve of symmetric amplification);
FIG. 18 is the dissolution curve for FIPV0.4+ Bar1:20+ FELV1:7+ FIV1:7 in example 5 with the primer ratio.
FIG. 19-1 is the sensitivity of the 6.1.1 assay, channel 1 amplification curve of example 6;
FIG. 19-2 is the sensitivity of the 6.1.1 assay, channel 1 melting curve of example 6;
FIGS. 19-3 are the sensitivity of the 6.1.1 assay, channel 2 amplification curves of example 6
FIGS. 19-4 are the sensitivity of the 6.1.1 assay, channel 2 melting curves of example 6
FIGS. 19-5 are the sensitivity of the 6.1.1 assay, channel 3 amplification curves of example 6
FIG. 20-1 is the channel 1 amplification curve for the 6.1.2 test Sausanduo vaccine sample of example 6;
fig. 20-2 is a channel 1 melting curve for the 6.1.2 test samaosan vaccine sample in example 6;
FIG. 20-3 is the amplification curve for channel 2 for the 6.1.2 test Saisanduo vaccine sample of example 6;
fig. 20-4 are channel 2 melting curves for the 6.1.2 test samaosan vaccine sample in example 6;
FIGS. 20-5 are the channel 3 amplification curves for the 6.1.2 test Sausanduo vaccine sample of example 6;
in example 6, the probes for the three channel detection of 6.1.1 and 6.1.2 are channel 1: FHV (feline herpesvirus type I) probe, TM60 ℃; bb (Bordetella bronchiseptica) probe, TM66 ℃; cf (C.felis) probe, TM72 ℃; and (3) a channel 2: mf (Mycoplasma felis) probe, TM62 ℃; pack () probe, TM71 ℃; and (3) passage: FCV (feline calicivirus), RNA virus, using ordinary RT-PCR, did not produce single-stranded DNA, did not produce a melting curve.
FIG. 21 is a melting curve of 6.2 reverse transcription from RNA template in example 6 by asymmetric PCR.
Detailed Description
The following description, with reference to the accompanying drawings, is provided to facilitate a comprehensive understanding of various embodiments of the application as defined by the claims and their equivalents. These embodiments include various specific details for ease of understanding, but these are to be considered exemplary only. Accordingly, those skilled in the art will appreciate that various changes and modifications may be made to the various embodiments described herein without departing from the scope and spirit of the present application. In addition, descriptions of well-known functions and constructions will be omitted herein for brevity and clarity.
The terms and phrases used in the following specification and claims are not to be limited to the literal meaning, but are merely for the clear and consistent understanding of the application. Accordingly, it will be appreciated by those skilled in the art that the description of the various embodiments of the present application is provided for illustration only and not for the purpose of limiting the application as defined by the appended claims and their equivalents.
The technical solutions in the embodiments of the present application will be described clearly and completely with reference to the accompanying drawings in some embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. 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 application.
It is to be understood that the terminology used in the embodiments of the present application is for the purpose of describing particular embodiments only, and is not intended to be limiting of the application. The technical solution of the present invention will be described in detail below with reference to the accompanying drawings.
Example 1 triple fluorescent PCR multiplex assay for febrile pig
In this embodiment, three pathogens that cause Fever in pigs, namely, African Swine Fever Virus (ASFV), Swine Fever Virus (CSFV), And Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus (HP-PRRSV), are performed by using the method And the kit of the present invention, And the specific implementation process is as follows:
the components of the PCR system
Name of each component Final concentration
PCR Buffer
1×
MgCl2 1.5mM
Betaine 0.8M
dNTPs 0.25mM
Forward primer (Quanduo) 0.4μM
Reverse primer (quantitative) According to the proportion
Probe needle 0.4μM
Taq enzyme 0.5U
DNA template 2μL
Sterile ultrapure water Is made up to 20
Setting of reaction program
Setting a fluorescence detection channel for collecting FAM fluorescence signals and HEX fluorescence signals, putting a PCR reaction tube into a fluorescence quantitative PCR instrument to start amplification, and carrying out the following reaction procedures (taking a macrostone SLAN96 as an example):
PCR program setting
Figure BDA0002925194030000101
And detecting three gene sequences by using two fluorescence channels by using a multiplex fluorescence PCR and melting curve characteristic peak method. The specificity of detection comes from the sequence specificity of two primers and one probe. In the reaction system of this example, three sequences were amplified simultaneously, one of which was conventional PCR, and the ratio of the amounts of the two primers was 1:1, cutting off the fluorescent group of the probe in the amplification process to generate a fluorescent signal, and forming an amplification curve. The double-stranded DNA product does not change its binding state to the probe during the subsequent melting curve (temperature rise) detection and thus has no detectable signal. Two are asymmetric PCR, the ratio of the amounts of the two primers is 1:5, the probe is complementary to the strand on which the large amount of primer is located. During the amplification process, when DNA polymerase with exonuclease activity synthesizes a small amount of primer strands, the probe is degraded and a fluorescent signal is generated; when the equivalent of the primer is exhausted, the primer with a large amount still generates a new single strand, and the relative position of the fluorescent group and the quenching group is changed and a fluorescent signal is generated by the combination of the probe and the single strand. The conventional PCR product is subjected to exponential amplification, the amplification curve is S-shaped, when the primers with small asymmetric PCR equivalent are exhausted, the single-stranded DNA is generated by linear amplification, and the amplification curve is linear. In the subsequent detection of a melting curve (temperature rising process), the probe and the single-stranded hybrid chain are rapidly dissociated near TM, the change of the fluorescence signal reaches a peak value, namely, the derivative of the fluorescence intensity is plotted against the temperature change to obtain a characteristic peak.
In this embodiment, the triple fluorescent PCR detection kit for febrile pig amplifies specific DNA fragments of African Swine Fever Virus (ASFV), Swine Fever Virus (CSFV) And high Pathogenic Porcine Reproductive And Respiratory Syndrome Virus (HP-PRRSV) under the action of DNA polymerase. The genes or DNA sequences tested were: the capsid protein 72 gene (ASFV-VP72) of African swine fever virus, the non-coding region (CSFV-5' -UTR) of swine fever virus, and the non-structural protein 2 gene (HP-PRRSV-NSP2) of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). Wherein the concentration ratio of a primer pair for detecting the African swine fever is 1:1, and a fluorescent group of the probe is HEX; the concentration ratio of the primer pair for detecting the classical swine fever virus and the porcine reproductive and respiratory syndrome virus is 1:3-1:10, and the fluorescent group of the probe is FAM. The ratio of the amounts of high concentration primer and probe was 2: 1. the Tm values of the primers are all around 60 ℃, and the Tm values of the probes are 76 ℃ for African Swine Fever Virus (ASFV), 75 ℃ for swine fever virus (CSFV) and 69 ℃ for Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).
Determination of results
In this embodiment, the experimental conditions are that the Ct values of the positive controls of the two fluorescence channels should be less than 30 and an S-type amplification curve appears, and the negative controls should have no Ct value or Ct value > 34 or no S-type amplification curve. When the test obtains an effective result, the result judgment standard is that the Ct value of the detected sample of the fluorescence channel of the African swine fever virus probe is less than 30 and an S-shaped amplification curve appears, and the African swine fever virus is judged to be positive; if no S-shaped amplification curve appears, or the tested sample has no Ct value or the Ct value is more than 34, the African swine fever virus is judged to be negative; ct of the detected sample is more than or equal to 30 and less than or equal to 34, and an S-shaped amplification curve appears, so that the detected sample is judged to be suspicious; it is recommended to re-sample and extract nucleic acids and to perform the result judgment after amplification.
The Ct value of the amplification curve of the detected sample of the asymmetric amplification probe fluorescence channel is less than 30, and the characteristic peak of the TM value at 69 +/-1 ℃ is judged to be positive for the highly pathogenic porcine reproductive and respiratory syndrome virus; the tested sample has no Ct value or no obvious amplification curve and no characteristic peak of TM value at 69 +/-1 ℃, and is judged to be negative to highly pathogenic porcine reproductive and respiratory syndrome virus; ct of the sample to be detected is more than or equal to 30 and less than or equal to 34, an obvious amplification curve appears, and a characteristic peak with a TM value at 69 +/-1 ℃ is judged to be suspicious; it is recommended to re-sample and extract nucleic acids and to perform the result judgment after amplification.
The Ct value of the amplification curve of the detected sample of the asymmetric amplification probe fluorescence channel is less than 30, and the characteristic peak of the TM value at 75 +/-1 ℃ is judged to be the hog cholera virus positive; the tested sample has no Ct value or no obvious amplification curve and no characteristic peak of TM value at 75 +/-1 ℃, and is judged to be hog cholera virus negative; ct of the sample to be detected is more than or equal to 30 and less than or equal to 34, an obvious amplification curve appears, and a characteristic peak with a TM value at 75 +/-1 ℃ is judged to be suspicious; it is recommended to re-sample and extract nucleic acids and to perform the result judgment after amplification.
And interpreting the rules according to the results. The amplification curve of six-tube PCR is shown in FIG. 1, and the melting curve is shown in FIG. 2. The PCR amplification product of African swine fever is double-stranded and has no melting curve. There is an S-type amplification curve and Ct value is less than 30. The amplification curve recorded by the FAM channel is the sum of amplification signals generated by the viruses of swine fever and porcine blue ear, and has an obvious amplification curve and a Ct value less than 30. Rn of the general PCR of African swine fever is about twice that of the asymmetric PCR amplification of swine fever and porcine blue ear. The melting curve characteristic peaks of the swine fever and the blue ear of pig are 75 +/-1 ℃ and 69 +/-1 ℃ respectively. As shown in FIG. 2, three of the 6 tubes had porcine reproductive and respiratory syndrome characteristic peaks, and two tubes had swine fever characteristic peaks. The six tubes of samples all detect African swine fever virus, two tubes detect swine fever virus and porcine reproductive and respiratory syndrome virus simultaneously, one tube detects the porcine reproductive and respiratory syndrome virus, and the detection result of the embodiment is completely consistent with the prepared samples.
The kit of this example detects three swine fever diseases. Considering the detection time, whether non-pestilence infection exists or not and whether swine fever or porcine reproductive and respiratory syndrome infection exists can be judged when twenty cycles of amplification are carried out, and the pathogenic concentration of the non-pestilence infection can be estimated according to Ct. The non-pestilence PCR product was mainly double stranded DNA and the melting curve showed no characteristic peak. Melting curve data aids in the determination if swine fever or porcine reproductive and respiratory syndrome is present. The swine fever and the porcine blue ear are not usually infected at the same time, and the concentration of the pathogen can be estimated from the Ct when only one is infected. The specificity of amplification curve positivity and melting curve characteristic peak positivity is due to the sequence specificity of the primers and probes. The evidence of amplification curve positivity + melting curve positivity is more reliable than that of amplification curve positivity but takes more time to detect. The amplification curve can provide quantitative information more readily than the melting curve. The invention integrates the advantages of the two.
In the background art, the multiplex PCR kit based on melting curve characteristic peak accepted in the market adopts a technical scheme that one (single-stranded product of) asymmetric amplification reaction with higher cost is provided with two probes with high Tm and low Tm, the high Tm probe is degraded in the amplification stage, the low Tm probe is not degraded in the amplification stage, corresponding characteristic peak is generated in the subsequent melting curve detection, and one target position and two probes achieve the same technical effect as one target position and one probe in the present invention: in a protocol where one fluorescent channel is used to detect multiple genes, the amplification curve is preserved and can be used to estimate pathogen concentration in practice in general. The price of the current probe, 10OD, is about twice as high as 1 OD. Generally, 20nt (base) molecular weight 20 × 330,10 μ M20 × 330 ug/L2 × 33ug/mL 33ug/500uL (single strand Oligo DNA at 1OD approximately 33 μ g) was diluted with 500uL deionized water to obtain 10uM of mother solution, so the probe cost is lower than the probe cost in the above-mentioned technical scheme of using one more probe. In the optimization of reaction conditions, the quality control of raw materials, semi-finished products and finished products, the scheme of two probes has higher cost than the scheme of one probe.
Example 2 pig disease 2+3 fluorescent PCR multiplex assay
The method and the kit are applied to multiple detection of porcine epidemic diarrhea virus variant (PEDV) -VAR, classical strain (PEDV) -CLA, rotavirus (PRoV), transmissible gastroenteritis virus (TGEV) and delta coronavirus (PDCoV) pathogens related to porcine diarrhea. The PCR components and temperature program of PCR and melting curves were as in example 1 or fine-tuned.
The preliminary results of the multiplex swine disease PCR of this example are shown in FIGS. 3 and 4. Wherein the first channel detects the protein gene of Porcine Epidemic Diarrhea Virus (PEDV) -VAR variant-S, (PEDV) -CLA classical strain-S protein gene and rotavirus (PRoV) -VP6 gene, and the probes TM are 69 ℃, 73 ℃ and 63 ℃ respectively; the second channel detects the protein gene of transmissible gastroenteritis virus (TGEV) -M and the gene of swine T-type coronavirus (PDCoV) -N, and the probes TM are respectively at 58 ℃ and 67 ℃. The Tm values of the primers are both around 60 ℃, and the Tm value of the probe of at least one detection target position in the two channels is higher than that of the primer of the detection target position.
Results of testing plasmid-mimetic samples: the PCR amplification efficiency is not very good, and the Rn of the two channels is not high. Ct < 20. The position of the characteristic peak of the melting curve is correct, and the characteristic peaks of different genes in the same fluorescence channel are clearly distinguished. The technical effect of detecting 2+3 target positions by two fluorescence channels can be achieved.
Example 3 Triplex fluorescent PCR multiplex assay for pets
The method and the kit are applied to the multiple detection of canine parainfluenza virus, toxoplasma gondii and adenovirus II pathogens. The PCR components and temperature program of PCR and melting curves were as in example 1 or fine-tuned.
The dog parainfluenza N protein gene (CPIV-nucleococcus protein (N) gene), Toxoplasma gondii-TgShIr 28 repeat region, and the canine adenovirus 2(CAV2) E3 gene are detected in a triple way, and the TM values corresponding to the probes are respectively as follows: 58. 65 and 70 ℃. The Tm values of the primers are both around 60 ℃, and the Tm values of the probes of the two detection targets are higher than that of the primers of the detection targets. The results of parallel detection of several tubes are shown in FIGS. 5 and 6. Results of testing plasmid-mimetic samples: the PCR has better amplification efficiency, Rn is about 0.7, and Ct is less than 20. The position of the characteristic peak of the melting curve is correct, and the characteristic peaks of different genes in the same fluorescence channel are clearly distinguished. The technical effect of detecting 3 target sites by one fluorescence channel can be achieved.
Example 4 multiplex fluorescence PCR and melting curve characteristic peak method for detecting 1+3 gene sequences by two fluorescence channels
In this example, 1+3 gene sequences were detected using two fluorescence channels using multiplex fluorescence PCR and melting curve signature peak method. The PCR components and temperature program of PCR and melting curves were as in example 1 or fine-tuned.
Porcine Epidemic Diarrhea Virus (PEDV) -VAR variant-S protein gene, -CLA classical strain-S protein gene and rotavirus (PRoV) -VP6 gene, wherein the probes TM are 69 ℃, 73 ℃ and 63 ℃; the second channel detects the porcine delta coronavirus (PDCoV) -N gene and the probe TM 67 ℃. The Tm values of the primers are both around 60 ℃, and the Tm value of the probe of at least one detection target position in the two channels is higher than that of the primer of the detection target position.
The result of detecting the plasmid simulation sample is shown in fig. 7, the first channel is about ct23, the second channel is about ct25, and the PDCoV is judged to be positive by the fact that ct35 is clearly positive but does not reach the plateau region of the S-shaped curve.
As shown in FIG. 8, rotavirus (PRoV), Porcine Epidemic Diarrhea Virus (PEDV) -VAR variant and-CLA classical strain were determined to be positive by three characteristic peaks at 63 deg.C, 69 deg.C and 73 deg.C.
The technical effect of detecting 1+3 target positions by two fluorescence channels can be achieved.
Example 5 four-fold detection of feline hematological disorders
In this example, four PCR assays were performed on feline hematological diseases (feline infectious peritonitis virus FIPV, feline babesia Bar, feline leukemia virus FELV, feline immunodeficiency virus FIV), PCR components and temperature program of PCR and melting curves were as in example 1 or fine-tuned, while the concentration ratio of the matched asymmetric primers was explored and optimized.
Feline infectious peritonitis virus, feline babesia, feline leukemia virus, feline immunodeficiency virus are four blood-related viruses common to cats. Studies have shown that feline infectious peritonitis is a disease caused by infection with feline coronavirus, with a very high infection rate, generally considered as an oral-nasal infection, and the course of the disease may be paroxysmal (occurring more frequently in kittens) or slow and lasting for weeks. Malignant tumor infectious diseases caused by feline leukemia virus (FeLV) infection are mainly characterized by malignant lymphoma, myelogenous leukemia, non-regenerative anemia and the like, and have no obvious seasonality, long incubation period and short course of disease. Feline immunodeficiency virus disease was discovered in 1987 to belong to the genus lentivirus of the family retroviridae, and infected cats can develop acquired immunodeficiency syndrome, commonly known as feline aids, in cats. Babesiosis is a piriasis disease caused by protozoa of babesiaceae, which mainly attacks red blood cells of cats, and sick cats mainly show high fever, anemia, jaundice, emaciation, weakness, and the like.
In this example, primers and probes were synthesized by professional companies, wherein the primers were purified by PAGE and the probes were purified by HPLC. Except for labeling the 5 '-VIC fluorescent reporter group of the FIPV probe, each probe is labeled with FAM fluorescent reporter group at the 5' end. The 3' ends of all probes were labeled with a BHQ1 quenching group. After design and optimization, the Tm value of the primer is determined to be about 60 ℃, the Tm of the cat infectious peritonitis virus probe is 70 ℃, the Tm of the cat babesia is 75 ℃, the Tm of the cat leukemia virus is 69 ℃, the Tm of the cat immunodeficiency virus is 63 ℃, and the Tm of the probe of at least one detection target position in the two channels is higher than the Tm of the primer of the detection target position.
Determination of results
And after the operation of the fluorescence quantitative PCR instrument is finished, the melting curve analysis is carried out on the test result by using the matched software. And taking the positive control reaction tube as a reference, and taking a sample with a melting curve completely identical to the peak pattern of the positive control tube as a corresponding pathogen to detect the positivity. The results are shown in the following table and corresponding FIGS. 9-18.
FIV Probe-Cat blood quadruple plasmid sensitivity optimization FELV FIV primer ratio analysis: the optimal primer ratio is FIPV0.4+ Bar1:20+ FELV1:8+ FIV1:8, the detection limit is 2-channel FIPV-10 copies, 1-channel FIV-Bar-100 copies and FELV-1000 copies. The results of the amplification curve and the melting curve for different primer ratios are as follows.
Figure BDA0002925194030000161
Figure BDA0002925194030000171
Figure BDA0002925194030000172
Figure BDA0002925194030000173
Figure BDA0002925194030000181
Figure BDA0002925194030000182
Figure BDA0002925194030000183
The amplification curve data described above and the melting curve characteristic peak data of FIGS. 9-18 in this example are collated in the following table:
FIV probe-cat blood quadruple plasmid sensitivity optimization FELV FIV primer proportion
Figure BDA0002925194030000191
The analysis kit detects the plasmid simulation sample (the plasmid concentration gradient 5-1 is 10 respectively)5-101Template amount), the above five ratios at higher template concentrations (10)5-104) Has better effect.
Considering the sensitivity of the amplification curve of the feline infectious peritonitis virus FIPV (channel 2) and the sensitivity of the amplification curve of the feline babesia Bar, the feline leukemia virus FELV and the feline immunodeficiency virus FIV (channel 1) as well as the sensitivity of the characteristic peaks of the melting curves of Bar, FELV and FIV (channel 1), Bar1:20+ FELV1:8+ FIV1:8 are the optimal proportion.
Considering development costs, it is impossible to try various combinations of primer concentration ratios indefinitely. Generally, the concentration ratio of most primer pairs is 1:3-1:30, or 1:4-1: 10. The inventors' trial process is briefly described below:
step 1, determining the quality of each pair of primers, selecting a primer pair which can obtain good effect in a large variation range of a primer comparison example, namely, good amplification effect, and selecting the proportion which enables the primer pair to amplify weakly as a starting point; the conventional starting point is as follows: high concentration primer concentration 0.4, high: 2: 1, low: high-1: 20-1: 5, namely the concentration of the low-concentration primer is 0.02-0.08;
step 2, determining the proportion of other primer pairs: trying a plurality of pairs of primer amplification and melting curve effects at an equal ratio of 1:4-1: 10;
2.1 trying a plurality of pairs of primer amplification and melting curve effects at an equal ratio of 1:3 to 1: 30;
2.2 on the basis of 2, the proportion range of individual primer pairs is 1:3-1: 30;
and 3, fine tuning optimization.
Effect, law of parameter change: different concentration ratios have different effects. And (3) probe: low primer concentration, when the ratio is from small to large: according to the amplification curve, after the Ct value is leaned, the Rn value is also reduced; the melting curve has the characteristic peak position unchanged, and the peak height has a process from low to high and then to low. This allows for better results with relatively few experiments.
In the experiment of asymmetric amplification-melting curve, the commonly used primer comparison ratio is 1:10, occasionally 1:20, and is not common, 1:3-1:10, in example 5, the concentration (ratio) of the primer pair for symmetric amplification and one group of asymmetric amplification is fixed, the concentration (ratio) of the primer pair for the other two groups of asymmetric amplification is tried in equal ratio of 1:4-1:10, and better results are obtained with less attempts. According to the embodiments, the ratio of asymmetric primers in the multiplex amplification system of the present invention should be 1:3 to 1: 30. The applicant also carries out optimization verification on the swine fever primer ratio of the swine disease triple of the example 1, when the swine fever primer ratio is 1:8 and 1:3, characteristic peaks exist, but when the swine fever primer ratio is 1:3, another characteristic peak is not very obvious, so that the swine fever primer ratio is generally 1:8 in a kit.
The fluorescence signal of the amplification curve is mainly from the probe which is cut off in the amplification process, the characteristic peak of the melting curve is from the melting of the single strand produced by amplification and the hybrid molecule of the probe in a specific temperature range, so that the temperature derivative of the change of the fluorescence signal reaches the peak value at the temperature, namely, the amount of the single strand/probe hybrid molecule determines the height of the characteristic peak, and the two processes need enough probe. In most experiments, the amount of probe is consistent with or half of the amount of the abundant primer, far exceeding the amount of probe required to generate amplification and melting curves.
Example 6 the presence of RNA in the target in multiplex amplification
6.1 Cat respiratory tract 5 detection system
FHV (feline herpesvirus type I), Bb (Bordetella bronchiseptica), Cf (Chlamydia felis), Mf (Mycoplasma felis), FCV (feline calicivirus), probes containing 2-P2-VIC as internal standards, the signals marked by the probes correspond to three fluorescent signal channels, respectively:
channel 1: FHV, Bb, Cf;
and (3) a channel 2: mf, internal standard;
and (3) passage: FCV.
The feline calicivirus is an RNA virus, the ratio of primers is 1:1, the other four items are used for detecting DNA virus, the amount of the probe is half of the amount of the primer with larger amount, and the amount of the primer with smaller amount is 1/10 or 1/5 of the amount of the primer with the largest amount. The reaction system is shown in the following table:
25uL system Single part of uL Tm
Assist in saint V enzyme (RT + DNA Taq) 1
2*Mix 12.5
FHV-F11(10uM) 0.04
FHV-R11(10uM) 0.4
FHV-P11SD (10uM) upstream 0.2 60℃
Bb-F2(10uM) 0.4
Bb-R2(10uM) 0.04
Bb-P2(10uM) upstream 0.2 66℃
Cf-OMPA-F1(10uM) 0.04
Cf-OMPA-R1(10uM) 0.4
Cf-OMPA-P1(10uM) upstream 0.2 72℃
Mf-(16+23s)-F2(10uM) 0.4
Mf-(16+23s)-R2(10uM) 0.08
Mf- (16+23s) -PJC2rc-VIC (10uM) downstream 0.2 62℃
bag-F2 (10um) 0.02
bags-R2 (10um) 0.4
Bags 2-P2-VIC (10um) (upstream) 0.2 71℃
FCV-ORF1-F1(10uM) 0.3
FCV-ORF1-R1(10uM) 0.3
FCV-ORF1-P1-Texas(10uM) 0.2
Target + internal standard 3+1
Water (W) 3.48uL
Reverse transcription of RNA to DNA (FCV) was completed at 50 ℃ for 10min before PCR cycling. Temperature program of the whole detection process:
Figure BDA0002925194030000221
Figure BDA0002925194030000231
6.1.1 detection of plasmids
Ct, Rn of the amplification procedure are shown in the table
Figure BDA0002925194030000232
As shown in FIGS. 19-1 and 19-5, the detection sensitivity of the amplification curves of channel 1 and channel 3 reached 1 copy. Channel 2 was provided with an internal standard for measuring the efficiency of the entire reaction system, and the concentration selected corresponded to weak positive between Ct 30-35. Thus, setting the ratio of Mf primer pairs to 1:5 corresponds to increasing the amount of primers by a small amount. The copy quantity of the Mf template is changed, Rn of the amplification curve of the channel 2 is changed, Ct is basically not influenced by Mf amplification, the difference between Mf of corresponding critical concentration and blank control is not obvious, and a characteristic peak at the position of 62 ℃ of a melting curve needs to be consulted. The amplification curve of channel 2, when the pattern is good, as shown in FIGS. 19-3, also reaches 1 copy of detection sensitivity.
The melting curve shows that the characteristic peaks of the channel 1 at the positions of 60 ℃, 66 ℃ and 72 ℃ respectively show positive FHV, Bb and Cf, and the sensitivity reaches 1 copy as shown in FIG. 19-2. The characteristic peak at 71 ℃ position of channel 2 is internal standard, the internal standard concentration of each tube (including the control without added sample) is consistent, and as shown in FIGS. 19-4, six lines all have the characteristic peak at 71 ℃. The characteristic peak at 62 ℃ for channel 2 indicates Mf positive with a sensitivity of 1 copy. Channel 3 has no characteristic peaks (not shown), consistent with expectations. The PCR product of the equivalent primer pair is double-stranded DNA and cannot be combined with the probe to generate a melting curve characteristic peak.
6.1.2 Miaosan cat rhinotracheitis vaccine sample detection
The detection template was a nasal tracheitis vaccine sample from Miaosan cat to prevent feline distemper (FPV, not in five respiratory tracts of cats), feline rhinobronchitis (FHV, channel 1, TM60 ℃) and feline calicivirus (FCV, channel 3). Ct, Rn of the amplification process are shown in the following table.
Unique identification Channel Ct Rn Channel Ct Rn Channel Ct Rn
Miaosan
1 1 24.72 0.313 2 34.37 0.584 3 20.37 2.307
Miaosan 2 1 28.02 0.378 2 33.95 0.605 3 24.2 2.227
Miaosan duo 3 1 31.36 0.371 2 33.78 0.587 3 27.91 1.995
Miaosan duo 4 1 35.93 0.265 2 34.08 0.525 3 34.14 1.309
- 1 NoCt -0.007 2 32.9 0.51 3 NoCt -0.014
Channel 1, FHV positive, with the fourth sample being weak positive (Ct close to 36), as in fig. 20-1; channel 3, FCV positive (RT-PCR assay), where the fourth sample was weakly positive (Ct close to 34) as in fig. 20-5; lane 2, internal standard weakly positive (Ct33-35) as shown in FIG. 20-3.
The melting curve, channel 1, four samples all had clear characteristic peak of TM60 deg.C, as shown in FIG. 20-2; channel 2, four samples and control all had characteristic peaks at TM71 ℃, as shown in fig. 20-4; channel 3 has no characteristic peaks (not shown). After reverse transcription of the RNA template, conventional PCR is carried out, and the product is double-stranded DNA which is not combined with the probe, so that a characteristic peak of a melting curve cannot be generated near the probe TM.
The detection system for 5 cat respiratory tracts is provided with an asymmetric PCR-melting curve internal standard and comprises 5 groups of primers and probes, wherein the ratio of the primers for detecting RNA is 1:1, and the sensitivity of a melting curve and an amplification curve of each target reaches 1 copy by plasmid detection. The kit has good effect in detecting FHV and FCV nucleic acid in the nasal tracheitis vaccine sample of the Miaosan cat.
6.2 Cat respiratory disease detection System
Mf (cat mycoplasma), FCV (cat calicivirus), asymmetric PCR amplification, one channel, detecting one DNA virus and one RNA virus. The reaction system is as follows:
Figure BDA0002925194030000241
Figure BDA0002925194030000251
reverse transcription of RNA to DNA was completed at 50 ℃ for 10min before PCR cycling. Temperature program of the whole detection process:
Figure BDA0002925194030000252
the primer comparative example is Mf1:10+ FCV1:5 fixed; template quantity is respectively 104Copy, 103Copy, 102Copy (duplicate triple holes), 101Copy (duplicate triple holes), 100Copy (replicate three wells), blank (replicate two wells).
The melting curve is shown in FIG. 21. The characteristic peak of TM62 ℃ corresponds to DNA virus Mf, 104Copy, 103Copy, 102Copies (triplicate wells) had characteristic peaks, 101Copy (duplicate triple holes), 100Copy (triplicate wells), blank (duplicate wells) no characteristic peak; the characteristic peak at the temperature of TM69 ℃ corresponds to RNA virus FCV,104Copy, 103Copy, 102Copy (duplicate triple holes), 101Copies (triplicate wells) had characteristic peaks, 100One line of copies appeared to have characteristic peaks, the remaining 100Copies and blanks had no characteristic peaks.
In a multiplex PCR system, RNA is used as a template for reverse transcription and then asymmetric PCR is carried out, the product is single-stranded DNA and is combined with a probe, and a characteristic peak of a melting curve is generated near the probe TM. The target can be detected by experiments, but the experimental data is not ideal (Rn is lower). In some experiments, the position of the characteristic peak deviates from the TM, which indicates that the detected target of the RNA virus may have variation.
In addition to being limited by the efficiency of reverse transcriptase, the variation speed of RNA virus is higher than that of DNA virus, which is probably one of the reasons that the amplification curve of the scheme of multiple asymmetric RT-PCR + melting curve is not ideal enough, and the amplification efficiency can be optimized by using degenerate primers and common probes with single sequence; the melting curve of the multiple asymmetric RT-PCR + melting curve scheme can give information on whether the target to be detected exists in the samples of the multiple targets and whether the target is mutated or not.
While the invention has been described with reference to a number of illustrative embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

Claims (24)

1. A method for multiple detection of target nucleotide sequence based on double-labeled oligonucleotide probe melting curve is characterized in that the reaction system comprises:
a plurality of pairs of amplification primers that match a target sequence and a plurality of double-labeled oligonucleotide probes that correspond to the same target sequence; the primer pair at least comprises a first primer pair with concentration difference, and the first primer pair comprises a high-concentration primer and a low-concentration primer;
the reaction system also comprises a thermostable DNA polymerase with 5' nuclease activity;
the melting temperature Tm value of at least one double-labeled oligonucleotide probe in the reaction system is greater than the melting temperature Tm of two matched primers, and real-time amplification fluorescent signals of the double-labeled oligonucleotide probes corresponding to the same target sequence can be normally detected;
in the melting curve analysis of the hybridization of the labeled oligonucleotide probe to the amplified target nucleic acid single strand, a characteristic peak is generated in relation to the Tm value of the labeled oligonucleotide probe.
2. The method for multiplex detection of a target nucleotide sequence according to claim 1, wherein the reaction system further comprises a reverse transcriptase.
3. The method for multiplex detection of a target nucleotide sequence according to claim 1 or 2, characterized in that: the concentration of the double-labeled oligonucleotide probe corresponding to the same target sequence in the first primer pair at least exceeds the addition amount of the primer with the low concentration in the first primer pair.
4. The method for multiplex detection of a target nucleotide sequence according to claim 1 or 2, characterized in that: the melting temperature Tm values of a plurality of the double-labeled fluorescent probes labeled with the same fluorescent group are different from each other by not less than 2 ℃.
5. The method for multiplex detection of a target nucleotide sequence according to claim 1 or 2, characterized in that: the concentration ratio of the low-concentration primer to the high-concentration primer is 1:3-1: 30.
6. The method for multiplex detection of a target nucleotide sequence according to claim 1 or 2, characterized in that: the concentration ratio of the low-concentration primer to the high-concentration primer is 1:4-1: 10.
7. The method for multiplex detection of a target nucleotide sequence according to claim 2 or 3, wherein: the concentration of the double-labeled oligonucleotide probe corresponding to the same target sequence of the first primer pair is at least 10% more than the amount of the low concentration primer added.
8. The method for multiplex detection of a target nucleotide sequence according to claim 2 or 3, wherein: the concentration of the double-labeled oligonucleotide probe corresponding to the same target sequence of the first primer pair is at least 30% more than the amount of the low concentration primer added.
9. The method for multiplex detection of a target nucleotide sequence according to claim 1 or 2, characterized in that: the concentration of the double-labeled oligonucleotide probe corresponding to the same target sequence of the first primer pair is at least 100% more than the amount of the low concentration primer added.
10. The method for multiplex detection of a target nucleotide sequence according to claim 1 or 2, characterized in that: the reaction system at least comprises a second primer pair with the concentration difference not more than 50%.
11. The method for multiplex detection of a target nucleotide sequence according to claim 1 or 2, characterized in that: the double-labeled oligonucleotide probe in the reaction system comprises a TaqMan type or molecular beacon type probe.
12. The method for multiplex detection of a target nucleotide sequence according to claim 1 or 2, characterized in that: and (3) analyzing a sample to be detected by an amplification and melting curve, wherein the positive standard of a certain target in the sample to be detected is an amplification peak and a characteristic peak of which the melting temperature corresponds to the melting temperature of the certain target.
13. A kit for multiplex detection of target nucleotides based on a melting curve of a ditag oligonucleotide probe, comprising:
a thermostable DNA polymerase having 5' nuclease activity;
a plurality of pairs of amplification primers matched with a target sequence and a plurality of double-labeled oligonucleotide probes corresponding to the same target sequence, wherein the melting temperature Tm value of at least one double-labeled oligonucleotide probe is greater than the melting temperature Tm of two matched primers;
and instructions for how to perform the multiplex detection of the target nucleotide and/or a reverse transcriptase for RT-PCR.
14. The kit for multiplex detection of a target nucleotide according to claim 13, characterized in that: the number of double-labeled oligonucleotide probes is greater than the number of fluorescence channels used to label the double-labeled oligonucleotide probes accordingly.
15. A kit for multiplex detection of target nucleotides based on a melting curve of a ditag oligonucleotide probe, comprising:
a plurality of pairs of amplification primers matched with a target sequence and a plurality of double-labeled oligonucleotide probes corresponding to the same target sequence, wherein the primer pairs of the kit at least comprise a first primer pair with concentration difference, and the first primer pair comprises a high-concentration primer and a low-concentration primer;
the kit further comprises a thermostable DNA polymerase having 5' nuclease activity;
the kit is provided with at least one double-labeled oligonucleotide probe, and the melting temperature Tm value of the double-labeled oligonucleotide probe is larger than the melting temperature Tm values of two matched primers.
16. The kit for multiplex detection of a target nucleotide according to claim 13 or 15, characterized in that: the concentration of the double-labeled oligonucleotide probe corresponding to the same target sequence in the first primer pair at least exceeds the addition amount of the primer with the low concentration in the first primer pair.
17. The kit for multiplex detection of a target nucleotide according to claim 13 or 15, characterized in that: the melting temperature Tm values of a plurality of the double-labeled fluorescent probes labeled with the same fluorescent group are different from each other by not less than 2 ℃.
18. The kit for multiplex detection of a target nucleotide according to claim 13 or 15, characterized in that: the concentration ratio of the low-concentration primer to the high-concentration primer is 1:3-1: 30.
19. The kit for multiplex detection of a target nucleotide according to claim 13 or 15, characterized in that: the concentration ratio of the low-concentration primer to the high-concentration primer is 1:4-1: 10.
20. The kit for multiplex detection of a target nucleotide according to claim 13 or 15, characterized in that: the concentration of the double-labeled oligonucleotide probe corresponding to the same target sequence of the first primer pair is at least 10% more than the amount of the low concentration primer added.
21. The kit for multiplex detection of a target nucleotide according to claim 13 or 15, characterized in that: the concentration of the double-labeled oligonucleotide probe corresponding to the same target sequence of the first primer pair is at least 30% more than the amount of the low concentration primer added.
22. The kit for multiplex detection of a target nucleotide according to claim 13 or 15, characterized in that: the concentration of the double-labeled oligonucleotide probe corresponding to the same target sequence of the first primer pair is at least 100% more than the amount of the low concentration primer added.
23. The kit for multiplex detection of a target nucleotide according to claim 13 or 15, characterized in that: the kit at least comprises a second primer pair with the concentration difference not more than 50%.
24. The kit for multiplex detection of a target nucleotide according to claim 13 or 15, characterized in that: the double-labeled oligonucleotide probe in the kit comprises a TaqMan type or molecular beacon type probe.
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