CN110438124B - High-throughput detection probe, melting curve detection method and application thereof - Google Patents
High-throughput detection probe, melting curve detection method and application thereof Download PDFInfo
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Abstract
The invention discloses a high-throughput detection probe and a melting curve detection method and application thereof, the probe of the invention forms a molecular beacon probe with a similar hairpin structure in an amplification stage, the probe quenches by using the fluorescence of a plurality of G base pair probes, the probe is melted with target nucleic acid in a subsequent melting program segment, a Tm value is adjusted according to the base composition of a 5' tail sequence of the probe or the extension length of a downstream region of an amplicon, the probe can be marked by multicolor fluorescent groups, and the aim of higher-throughput detection is achieved by using multiple channels, wherein a single channel at least detects 8 target nucleic acids; when the detection probe is subjected to first round amplification, the target sequence specific binding sequence I is specifically bound with the target nucleic acid, the target sequence specific binding sequence II is subjected to enzyme digestion to form a second round extended primer, and through twice specific binding, the detection specificity is enhanced, a narrower melting peak is formed, the channel utilization efficiency is improved, and the detection sensitivity is also improved.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a high-throughput detection probe, a melting curve detection method and application thereof.
Background
Currently, fluorescent PCR techniques have been widely used for the detection of genetic variations, tumor mutations, and microorganisms. The probe melting curve technology is also called post-PCR technology, and utilizes a detection probe capable of being specifically combined with a target nucleic acid sequence, a melting procedure is added after an amplification procedure, double chains formed by a fluorescence labeling probe and the target nucleic acid are melted by gradually increasing the temperature, a melting peak with a specific melting point (Tm value) is formed, and a plurality of target nucleic acids can be distinguished according to the difference of the melting points. The molecular beacon probe is one of the probes commonly used in melting curves, and other probes include linear double-labeled probes, single-labeled probes, nonstandard probes, and the like. The molecular beacon probe consists of a stem region and a ring region, wherein the ring region is a specific binding region with a target nucleic acid, the stem region is an artificially added high GC sequence, and when the target nucleic acid does not exist, a fluorescent group and a quenching group are contacted with each other due to the fact that the stem region has a hairpin structure, the fluorescence background is very low, and the specificity is good. Although the molecular beacon probe has outstanding advantages, the detection flux is low, the sensitivity is not high, and when the melting points of two target sequences are less than 4, a fusion peak is easily formed, and the specific target nucleic acid cannot be judged; the market needs a melting curve detection method with independent existence of each melting peak, no fusion and good distinguishing effect, and the invention solves the problems.
Disclosure of Invention
The melting curve detection method of the detection probe can achieve high-throughput detection, 8 or more target nucleic acids can be detected simultaneously by a single channel to form melting peaks with specific Tm values, the difference between the melting peaks of the target nucleic acids is 2-6 ℃, the melting peaks can independently exist without fusion, and the distinguishing effect is good.
In order to achieve the above object, the present invention adopts the following technical solutions:
a high-flux detection probe, the 5 'end of the detection probe is marked with a fluorescent marker, and the 3' end is sealed; the 3 'end is provided with a target specific binding sequence I, and the 5' end is provided with a tail sequence; the tail sequence includes: the first 3-5 are GC sequences, the target specific binding sequence II is behind the GC sequences, the length of the target specific binding sequence II is 8-25nt, and the Tm value is 45-55 ℃; the target specific binding sequence II is consistent with a section of sequence at the downstream of the amplicon, and the downstream sequence of the amplicon is positioned between the detection probe and the downstream primer;
the target specific binding sequence I binds to the target nucleic acid during the first amplification cycle and the target specific binding sequence II is cleaved by enzyme during the first amplification cycle to form a second round of extended primers.
A melting curve detection method of a high-throughput detection probe comprises the following steps:
the design of a high-throughput detection probe,
marking a fluorescent marker at the 5 'end, and sealing the 3' end; the 3 'end is provided with a target specific binding sequence I, and the 5' end is provided with a tail sequence; the tail sequence includes: the first 3-5 are GC sequences, the target specific binding sequence II is behind the GC sequences, the length of the target specific binding sequence II is 8-25nt, and the Tm value is 45-55 ℃; the target specific binding sequence II is consistent with a section of sequence at the downstream of the amplicon, and the downstream sequence of the amplicon is positioned between the detection probe and the downstream primer;
the first round of amplification is carried out,
the target specific binding sequence II of the tail sequence at the 5 ' end is subjected to enzyme digestion in the first round of amplification, the hydroxyl is exposed at the 3 ' end and can be used as a primer for second round of extension, and a GC sequence and 4 to 6C basic groups are introduced into the 5 ' end of a downstream primer;
carrying out a second round of amplification, wherein,
after the second round of extension is finished, the probe forms a molecular beacon probe similar to a hairpin structure, the stem region of the molecular beacon consists of a GC sequence in a probe tail sequence and a complementary sequence of a GC sequence of a downstream primer 5 ', 4-6G bases are added at the 3' end of the probe, and the probe has a quenching effect on a fluorescent marker of the probe;
matching and melting the probe and the downstream sequence of the target nucleic acid to form a melting peak with a specific Tm value, and designing the difference between the melting peaks of the target nucleic acids to be 2-6 ℃ according to the base composition of the 5' tail sequence of the probe or the extension length of the downstream region of the amplicon;
synthesizing a plasmid template of a detection object, and performing PCR and on-machine detection.
The method for detecting a melting curve of the high-throughput detection probe comprises the following specific steps of: the GC content of the 5' tail sequence of the probe is adjusted, and particularly, a proper binding region of the target specific binding sequence II on the first round amplicon is selected, so that the higher the GC content of the binding region is, the higher the Tm value is.
The method for detecting a melting curve of a high-throughput detection probe as described above, wherein the Tm value of the melting peak is adjusted according to the extension length of the downstream region of the amplicon, comprises: the longer the length of the extension product of the target specific binding sequence II of the probe and the region downstream of the first round amplicon, the higher the Tm value.
The melting curve detection method of the high-throughput detection probe adopts multicolor fluorescence labeling and utilizes multi-channel detection, and each channel detects at least 8 target nucleic acids.
In the method for detecting a melting curve of a high-throughput detection probe, the fluorescent label labeled at the 5' end of the detection probe comprises: FAM, HEX, ROX, Cy 5.
In the method for detecting a melting curve of a high-throughput detection probe, the quencher labeled at the 3' end of the detection probe comprises: BHQ1, BHQ2, TAMRA, MGB.
The application of a high-throughput detection probe for detecting a plurality of target sequence mutations.
The method for detecting the human papillomavirus target nucleic acid comprises the following steps:
the 8 high-risk target sequences of the human papilloma virus comprise: HPV16, HPV33, HPV39, HPV45, HPV51, HPV52, HPV56, HPV 66;
amplifying by using universal primers HPV-F and HPV-R, wherein the sequences are shown as SEQ NO.01 and SEQ NO. 02;
designing a detection probe in a conserved region of 8 high-risk HPV types, wherein the sequence is shown as SEQ NO.03, SEQ NO.04, SEQ NO.05, SEQ NO.06, SEQ NO.07, SEQ NO.08, SEQ NO.09, SEQ NO.10, a fluorophore is marked at the 5 'end, the tail sequence at the 5' end of the probe comprises a GC sequence and a sequence consistent with the downstream of an amplicon, the GC sequence is used as a stem of a molecular beacon, cutting off a sequence consistent with the downstream of the amplicon by Taq DNA polymerase during the first round of amplification, extending a primer used as a second round of extension, introducing a GC sequence and 4-6C bases into the 5 'end of a downstream primer, obtaining a complementary GC sequence after the second round of extension by the GC sequence, adding 4-6G bases into the 3' end of the downstream primer during the upstream primer extension as another stem of the molecular beacon, and forming a molecular beacon probe similar to a hairpin structure by a detection probe after the second round of extension;
quenching the molecular beacon probe by utilizing the fluorescence of a plurality of G base pair probes, melting the molecular beacon probe and the amplicon in a subsequent melting procedure to form a melting peak with a specific Tm value, and designing the difference between the melting peaks of all target nucleic acids according to the base composition of a 5' tail sequence of the probe or the extension length of a downstream region of the amplicon to be 2-6 ℃;
artificially synthesizing the plasmid templates of the 8 HPV types, and performing PCR and computer detection.
The invention has the advantages that:
the detection probe of the invention introduces one stem of the molecular beacon probe through the tail sequence at the 5 'end of the probe, and simultaneously introduces another stem of the molecular beacon and 4-6 free G bases through the GC sequence at the 5' end of the downstream primer, and the 4-6 free G bases can play a good quenching role;
the Tm value is adjusted according to the base composition of the 5' tail sequence of the probe and the extension length of the downstream sequence of the amplicon, meanwhile, the probe can be marked by multicolor fluorophores, and the aim of higher-flux detection is achieved by utilizing multiple channels, wherein 8 or more target nucleic acids can be detected by a single channel;
the detection probe used in the invention has the advantages that during the first round of amplification, the target sequence specific binding sequence I is specifically bound with the target nucleic acid, the target sequence specific binding sequence II is cut by enzyme at the moment to form a second round of extended primer, the detection specificity is enhanced through twice specific binding, a narrower melting peak is formed, the channel utilization efficiency is improved, and the detection sensitivity is also improved.
Drawings
FIG. 1 is a detection flow chart of the probe melting curve detection method of the present invention;
FIG. 2 is a diagram of the structure of a molecular beacon generated by the present invention;
FIG. 3 is a graph showing the result of verifying the detection flux of the method for detecting a melting curve of a probe according to the present invention;
FIG. 4 is a graph of the detection flux verification result of the molecular beacon system;
FIG. 5 is a graph showing the results of detection sensitivity in the method for detecting a melting curve of a probe according to the present invention.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
A high-flux detection probe, the 5 'end of the detection probe is marked with a fluorescent marker, and the 3' end is sealed; the 3 'end is provided with a target specific binding sequence I, and the 5' end is provided with a tail sequence; the tail sequence includes: the first 3-5 are GC sequences, the target specific binding sequence II is behind the GC sequences, the length of the target specific binding sequence II is 8-25nt, and the Tm value is 45-55 ℃; the target specific binding sequence II is consistent with a section of sequence at the downstream of the amplicon, and the downstream sequence of the amplicon is positioned between the detection probe and the downstream primer; the target specific binding sequence I binds to the target nucleic acid during the first amplification cycle and the target specific binding sequence II is cleaved by enzyme during the first amplification cycle to form a second round of extended primers. As an example, the fluorescent label labeled at the 5' end of the detection probe includes: FAM, HEX, ROX, Cy 5; as an example, the quencher group labeled at the 3' end of the detection probe includes: BHQ1, BHQ2, TAMRA, MGB.
The melting curve detection principle of the invention is as follows:
in the invention, the 5 'end of the detection probe is marked with a fluorescent group, the 3' end is closed, the probe sequence comprises a tail sequence at the 5 'end and a target specific binding sequence I at the 3' end, the first 3-5 of the tail sequence are GC sequences, the subsequent target specific binding sequence II is 8-25nt in length, the Tm value is 45-55 ℃, the sequence of the sequence is consistent with a section of sequence at the downstream of an amplicon, and the downstream sequence is positioned between the probe and a downstream primer. The melting probe of the present invention is generated during the procedure, and in the first round of amplification, the 5 'tail of the probe is cleaved by Taq DNA polymerase during the extension phase, and the 3' end is exposed to a hydroxyl group, which can be used as a primer for the second round of extension. And a GC sequence and 4 to 6C bases are introduced into the 5 ' end of the downstream primer, so that after the second round of extension is finished, the probe can form a molecular beacon probe similar to a hairpin structure, as shown in FIG. 2, the stem region of the molecular beacon consists of a GC sequence in the tail sequence of the probe and a complementary sequence of the GC sequence of the downstream primer 5 ', and the 3 ' end of the probe is added with 4 to 6G bases through second extension to quench the fluorescent group of the probe. In the subsequent melting procedure, the probe is matched with the downstream sequence of the target nucleic acid and is melted to form melting peaks with specific Tm values, the difference between the melting peaks of different target nucleic acids is designed to be 2-6 ℃, the detection principle diagram of the invention is shown in figure 1, wherein n represents 4-6.
A melting curve detection method of a high-throughput detection probe comprises the following steps:
the design of a high-throughput detection probe,
marking a fluorescent marker at the 5 'end, and sealing the 3' end; the 3 'end is provided with a target specific binding sequence I, and the 5' end is provided with a tail sequence; the tail sequence includes: the first 3-5 are GC sequences, the target specific binding sequence II is behind the GC sequences, the length of the target specific binding sequence II is 8-25nt, and the Tm value is 45-55 ℃; the target specific binding sequence II is consistent with a section of sequence at the downstream of the amplicon, and the downstream sequence of the amplicon is positioned between the detection probe and the downstream primer; preferably, the probes are fluorescently labeled in multiple colors, using multiple channels for detection of at least 8 target nucleic acids per channel.
The first round of amplification is carried out,
the target specific binding sequence II of the tail sequence at the 5 ' end is subjected to enzyme digestion in the first round of amplification, the hydroxyl is exposed at the 3 ' end and can be used as a primer for second round of extension, and a GC sequence and 4 to 6C basic groups are introduced into the 5 ' end of a downstream primer;
carrying out a second round of amplification, wherein,
after the second round of extension is finished, the probe forms a molecular beacon probe similar to a hairpin structure, as shown in fig. 2, the stem region of the molecular beacon consists of a GC sequence in a probe tail sequence and a complementary sequence of a GC sequence of a downstream primer 5 ', 4-6G bases are added to the 3' end of the probe, and the probe has a quenching effect on a fluorescent marker of the probe;
matching and melting the probe and the downstream sequence of the target nucleic acid to form a melting peak with a specific Tm value, and designing the difference between the melting peaks of the target nucleic acids to be 2-6 ℃ according to the base composition of the 5' tail sequence of the probe or the extension length of the downstream region of the amplicon;
the specific method for adjusting the Tm value of the melting peak according to the base composition of the 5' tail sequence of the probe comprises the following steps: the GC content of the 5' tail sequence of the probe is adjusted, and particularly, a proper binding region of the target specific binding sequence II on the first round amplicon is selected, so that the higher the GC content of the binding region is, the higher the Tm value is.
The specific method for adjusting the Tm value of the melting peak according to the extension length of the downstream region of the amplicon is as follows: the longer the length of the extension product of the target specific binding sequence II of the probe and the region downstream of the first round amplicon, the higher the Tm value.
Synthesizing a plasmid template of a detection object, and performing PCR and on-machine detection.
The following experiments for verifying the detection performance of the high-throughput probe melting curve method, using 8 high-risk types of Human Papilloma Virus (HPV) as examples:
preparation of the experiment:
(1) the method system design of the invention: 8 high-risk types of Human Papilloma Virus (HPV) are respectively HPV16, HPV33, HPV39, HPV45, HPV51, HPV52, HPV56 and HPV66, universal primers SEQ NO.01 and SEQ NO.02 are used, and detection probes are designed in conserved regions of the 8 high-risk HPV types, and the sequences are shown as SEQ NO.03, SEQ NO.04, SEQ NO.05, SEQ NO.06, SEQ NO.07, SEQ NO.08, SEQ NO.09 and SEQ NO. 10. The 5' tail sequence of the probe comprises a GCCG sequence and a target nucleic acid specific binding sequence II, underlined, wherein, the GCCG sequence is used as one stem of the molecular beacon, the target nucleic acid specific binding sequence II is cut off by Taq enzyme in the first round of amplification, the target nucleic acid specific binding sequence II is used as a primer for the second round of extension for extension, the GCCG sequence and 4C bases are also introduced into the 5 'end of the downstream primer, the GCCG sequence is used as the other stem of the molecular beacon, 4 free G bases can be added at the 3' end of the downstream primer in the second round of extension, thus, after the second round is finished, the molecular beacon probe similar to a hairpin structure is formed, the probe replaces a quenching group with a plurality of G bases, and in a subsequent melting procedure, the probe and an amplicon are melted to form a melting peak with a specific Tm value, and the difference between the melting peaks of different target nucleic acids is designed to be 2-6 ℃.
(2) Designing a molecular beacon detection system: universal primers HPV-F and HPV-R are used, and the sequences are shown as SEQ NO.01 and SEQ NO. 02. Probes are designed according to conserved sequences of HPV16, HPV33, HPV39, HPV45, HPV51, HPV52, HPV56 and HPV66, the sequences are shown as SEQ NO.11, SEQ NO.12, SEQ NO.13, SEQ NO.14, SEQ NO.15, SEQ NO.16, SEQ NO.17 and SEQ NO.18, the probes are labeled with fluorescent FAM, the labeled quenching BHQ1, and bold bases are stem sequences of the molecular beacon probes. The primer probe sequences used in the present invention are shown in Table 2.
TABLE 2 primer and Probe sequences for use in the invention
Note: "P" represents a probe of the invention; "P1" represents a molecular beacon probe; lower case, italic, bold letters indicate the base introduced; underlined bases indicate target specific binding sequence II.
(3) Preparation of plasmid template: 8 kinds of HPV type plasmids are synthesized and diluted to 1 copies/. mu.l-1000 copies/. mu.l for standby.
(4) Preparation of PCR reaction system
TABLE 3 preparation of PCR reaction systems of the present invention
Deionized water was added to 25. mu.l.
TABLE 4 molecular Beacon Probe reaction System formulation
Deionized water was added to 25. mu.l.
(5) And (4) adding samples and loading on a machine.
Comparative example: molecular beacon probe system:
the establishment process of the molecular beacon probe system comprises the following steps: universal primers HPV-F and HPV-R are used, and the sequences are shown as SEQ NO.01 and SEQ NO. 02. Probes are designed according to conserved sequences of HPV16, HPV33, HPV39, HPV45, HPV51, HPV52, HPV56 and HPV66, the sequences are shown as SEQ NO.11, SEQ NO.12, SEQ NO.13, SEQ NO.14, SEQ NO.15, SEQ NO.16, SEQ NO.17 and SEQ NO.18, the probes are labeled with fluorescent FAM, the labeled quenching BHQ1, and bold bases are stem sequences of the molecular beacon probes. PCR reaction systems were prepared according to the amounts shown in Table 4. 8 HPV type plasmids are synthesized and diluted to 1 copies/. mu.l-1000 copies/. mu.l. Setting a reaction program, adding samples and loading on a machine.
Experiment one: high-throughput melting curve detection method high-throughput verification experiment,
8 HPV construction plasmids are used as templates, the concentration is 1000 copies/mu l, 5 mu l is taken as a template, each plasmid is subjected to 4 parallel reactions, and a template-free control (NTC) is arranged at the same time.
Experiment two: sensitivity experiment of the high-flux melting curve detection method,
using 8 HPV construction plasmids as templates, at concentrations of 100 copies/. mu.l, 10 copies/. mu.l, 1 copies/. mu.l, 5. mu.l were used as templates, and each concentration was subjected to 4 parallel reactions, while a no-template control (NTC) was set.
The above experiment, the procedure for the system of the invention, the first round of amplification, was as follows: 3 minutes at 95 ℃ for one cycle; 2min at 40 ℃ and 2min at 58 ℃ (day light at this stage), 50 cycles. And (3) second round extension: 20min at 45 ℃. The procedure for the molecular beacon system was as follows, 95 ℃ for 3 minutes, one cycle; 2min at 40 ℃ and 2min at 58 ℃ (day light at this stage), 50 cycles.
The melting curve analysis program was: denaturation at 95 ℃ for 2min, heat preservation at 40 ℃ for 3min, and melting curve analysis from 45 ℃ to 85 ℃. The experiments were performed on a SLAN96P real-time fluorescent PCR instrument.
(6) Analysis of Experimental results
The result of the first experiment: the probe provided by the invention has narrower peak shape of the melting curve and higher channel utilization rate, 8 HPV types can be effectively detected by a single channel, and the result is shown in figure 3, and the different melting peaks can not be fused and have good discrimination. The results of the molecular beacon probe system of the comparative example are shown in FIG. 4, the discrimination effect between the types is poor, the peak shape is wide, and particularly, HPV39 and HPV45 generate serious fusion peaks. The above results show that the probe melting curve method of the present invention has higher discrimination and more sufficient detection channel utilization.
The second experiment results: the sensitivity test results of the system of the present invention are shown in FIG. 5, the solid line represents 50 copies/reaction, the dotted line represents 5 copies/reaction, and 5 copies/reaction can be detected in plasmid samples of 8 HPV types. The melting curve method has high detection sensitivity and higher sensitivity to low-content microorganisms.
The invention provides a high-throughput detection probe and a melting curve detection method and application thereof, the melting curve detection method of the detection probe can achieve high-throughput detection, a single channel can simultaneously detect 8 or more target nucleic acids to form melting peaks with specific Tm values, the difference between the melting peaks of the target nucleic acids is 2-6 ℃, the melting peaks can independently exist without fusion, and the distinguishing effect is good.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.
Sequence listing
<110> Hefei Ou Chuang Gene Biotech Co., Ltd
<120> high-throughput detection probe, melting curve detection method thereof and application thereof
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Claims (6)
1. A melting curve detection method of a high-throughput detection probe is characterized by comprising the following steps:
the design of a high-throughput detection probe,
labeling a fluorescent marker at the 5 'end of the detection probe, and labeling a quenching group at the 3' end of the detection probe; the 3 'end is provided with a target specific binding sequence I, and the 5' end is provided with a tail sequence; the tail sequence includes: the first 3-5 are GC sequences, the target specific binding sequence II is behind the GC sequences, the length of the target specific binding sequence II is 8-25nt, and the Tm value is 45-55 ℃; the target specific binding sequence II is consistent with the downstream sequence of the amplicon, the downstream sequence of the amplicon is positioned between a detection probe and a downstream primer, the detection site of the probe is positioned between an upstream primer and a downstream primer of the amplified target nucleic acid, and the upstream primer and the downstream primer are amplified to form the amplicon;
carrying out the first round of PCR amplification,
the target specific binding sequence II of the tail sequence at the 5 ' end is subjected to enzyme digestion during the first round of amplification, the hydroxyl group is exposed at the 3 ' end and can be used as a primer for the second round of extension, and 4 to 6C basic groups and GC sequences are sequentially introduced from 5 ' to 3 ' of the 5 ' end of the downstream primer;
carrying out a second round of PCR amplification,
after the second round of extension is finished, the probe forms a molecular beacon probe with a hairpin structure, the stem region of the molecular beacon consists of a GC sequence in a probe tail sequence and a complementary sequence of a GC sequence of a downstream primer 5 ', 4-6G bases are added at the 3' end of the probe, and the probe has a quenching effect on a fluorescent marker of the probe;
matching and melting the probe and the downstream sequence of the target nucleic acid to form a melting peak with a specific Tm value, and designing the difference between the melting peaks of the target nucleic acids to be 2-6 ℃ according to the base composition of the 5' tail sequence of the probe or the extension length of the downstream region of the amplicon;
and (6) performing detection on the machine.
2. The method for melting curve detection of high throughput detection probe according to claim 1, wherein the specific method for adjusting the Tm value of the melting peak according to the base composition of the 5' tail sequence of the probe comprises: the GC content of the 5' tail sequence of the probe is adjusted, and particularly, a proper binding region of the target specific binding sequence II on the first round amplicon is selected, so that the higher the GC content of the binding region is, the higher the Tm value is.
3. The method for melting curve detection of a high-throughput detection probe according to claim 1, wherein the method for adjusting the Tm value of the melting peak according to the elongation of the region downstream of the amplicon comprises: the longer the length of the extension product of the target specific binding sequence II of the probe and the region downstream of the first round amplicon, the higher the Tm value.
4. The melting curve detection method of high-throughput detection probe according to claim 1, wherein the multi-color fluorescent label is used, and at least 8 target nucleic acids are detected in each channel by multi-channel detection.
5. The melting curve detection method of high-throughput detection probe according to claim 1, wherein the fluorescent label labeled at the 5' end of the detection probe comprises: FAM, HEX, ROX, Cy 5.
6. The melting curve detection method of a high-throughput detection probe according to claim 1, wherein the quencher labeled at the 3' end of the detection probe comprises: BHQ1, BHQ2, TAMRA, MGB.
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