CN116904564A - Fluorescent real-time detection system, method, multiplex primer and kit for simultaneously detecting n target genes - Google Patents
Fluorescent real-time detection system, method, multiplex primer and kit for simultaneously detecting n target genes Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
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- C12Q2600/00—Oligonucleotides characterized by their use
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The application provides a fluorescence real-time detection system for simultaneously detecting n target genes, wherein a weight probe is designed for complementation with the target genes of a sample to be detected and release of a reporter gene; determining whether the n target genes exist in the sample to be detected by detecting and analyzing the fluorescence intensity and the weight number sent by the reporter genes; wherein, X quenching groups and Y quenching genes are marked on the weight probe; and n/3 is more than or equal to X and less than or equal to n is more than or equal to Y. The detection system and the detection method provide a weighted probe, which can realize the simultaneous detection of a plurality of target nucleic acid sequences under the condition of a few kinds and quantity of fluorescent groups; according to the application, 1-2 fluorescent groups can be used for simultaneously detecting 4-6 target nucleic acid sequences, the limit of the number of fluorescent channels in the existing equipment is overcome, and multiple target nucleic acid sequences, even more than ten and tens of target nucleic acid sequences can be simultaneously detected on one existing equipment.
Description
Technical Field
The application relates to the technical field of molecular biology, in particular to a fluorescence real-time detection system, a method, a multiplex primer and a kit for simultaneously detecting n target genes.
Background
Multiplex PCR (polymerase chain reaction) refers to a PCR reaction in which two or more pairs of primers are added to the same PCR reaction system to simultaneously amplify a plurality of kinds of nucleic acid fragments. In the real-time PCR method, the target nucleic acid sequence can be detected by using a fluorescent-labeled oligonucleotide probe.
For multiplex real-time fluorescent PCR reactions, each probe can be labeled with a different fluorescent group; thus, the target nucleic acid sequence specifically recognized by each probe can be detected based on the unique fluorescent signal carried by each probe. However, due to the limitation of the instrument, the number of the maximum fluorescence detection channels which can be detected in the monograph detection is not more than 6; in the detection mode of melting curve analysis after amplification, a step of analyzing the melting point after PCR amplification is needed, but the number of fluorescent channels is also reduced.
WadleS et al (Biotechniques 2016, 61 (3): 123-8) studied a five-fold PCR system that used 5 mediator probes and 5 fluorescent probes in total. In contrast, the conventional five-fold real-time PCR method requires only 5 fluorescent probes. Under the condition of the existing equipment, the condition is complex and the cost is high.
US 2015/0074887 A1 discloses a real-time PCR assay that can be used for melting curve analysis, with detection of a target nucleic acid sequence being achieved by 3 probes. When the method disclosed in this patent is used for multiplex real-time PCR, it is necessary to design a plurality of probes for each target nucleic acid sequence, which leads to complexity of the reaction system and instability of the result.
CN109837333a discloses a fluorescent real-time detection reagent and method for simultaneously detecting multiple target genes, wherein detection of target nucleic acid sequences is achieved by grouping the target genes. When the method disclosed by the patent is adopted for multiplex real-time PCR, tm values of PCR amplification products need to be controlled, so that the difference between Tm values of PCR amplification products of the same group and labeled with the same fluorescent label is more than 1 ℃, the control requirement on the actual operation process is very high, the same design and grouping of primers are carried out aiming at different targets to be detected by carefully analyzing oligonucleotide sequences, the screening requirement on the target genes to be detected is met, and the method has no popularity.
Also, the requirement for multiple wells and sites is critical for real-time fluorescence detection, both for the number of simultaneous assays and for the primers.
Aiming at the above situation, a new real-time PCR detection method needs to be developed, so that more target genes can be conveniently and rapidly detected under the conditions of controllable fluorescent channels and loci, and meanwhile, the method has broad spectrum and can be used in a large range.
Disclosure of Invention
In the present application, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the nucleic acid chemistry laboratory procedures used herein are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present application, definitions and explanations of related terms are provided below.
As used herein, the terms "target gene", "target nucleic acid" refer to a target nucleic acid sequence to be detected, and the terms are used interchangeably.
As used herein, the term "weighted probe" refers to a single stranded nucleic acid molecule comprising an oligonucleotide sequence (also known as a mediator sequence) and a specific sequence (i.e., a target nucleic acid sequence) in the 5 'to 3' direction. In the present application the oligonucleotide sequence does not contain a sequence complementary to the target nucleic acid sequence, and the specific sequence contains a sequence complementary to the target nucleic acid sequence. Thus, the oligonucleotide sequence hybridizes or anneals to the target gene to be detected via a specific sequence under conditions that allow hybridization, annealing or amplification of the nucleic acid; while the oligonucleotide sequence does not hybridize to the target nucleic acid sequence under the same conditions.
The term "specific sequence" refers to a sequence that is capable of specifically hybridizing or annealing to a nucleic acid sequence to be detected, as it comprises a sequence complementary to the sequence to be detected, under conditions that allow hybridization, annealing or amplification of the nucleic acid. Likewise, a specific sequence or specific sequence hybridizes or anneals only to a particular complementary target nucleic acid sequence and not to other nucleic acid sequences under conditions that allow hybridization, annealing or amplification of the nucleic acids.
In the present application, the term "oligonucleotide sequence" refers to a sequence in the weighted probe that is not complementary to the target nucleic acid sequence, which is located at the 5' end of the weighted probe.
In the application, a weight probe is designed for each target gene to be detected, and the weight probe is designed with a unique specific sequence and an oligonucleotide sequence according to the complementary target nucleic acid sequence; and also fluorescent probes matched with the weighting probes.
In the application, multiple repeated oligonucleotide sequences are designed on the weight probes, and the actual multiple repeated sequences (complementary to the weight probes) on the fluorescent probes are analyzed according to the sequence combination of the repeated numbers when the fluorescence intensity is detected, so that the situation of the target nucleic acid sequence complementary to the corresponding weight probes is analyzed.
In the present application, the term "complementary" means that two nucleic acid sequences are capable of forming hydrogen bonds and forming a duplex according to the base pairing rules. That is, under conditions that allow hybridization, annealing or amplification of nucleic acids, two nucleic acid sequences that are "complementary" will specifically hybridize or anneal and form a duplex.
In the present application, the term "fluorescent probe" refers to an oligonucleotide sequence that indicates a fluorescent group and is capable of generating a fluorescent signal upon release.
Detection system
The application provides a fluorescence real-time detection system for simultaneously detecting n target genes, wherein a weight probe is designed for complementation with the target genes of a sample to be detected and releasing a reporter gene; determining whether the n target genes exist in the sample to be detected by detecting and analyzing the fluorescence intensity and the weight number sent by the reporter genes; wherein, X quenching groups and Y quenching genes are marked on the weight probe; and n/3 is more than or equal to X and less than or equal to n is more than or equal to Y.
Also, to detect fluorescent signals, the system further includes a fluorescent probe having a reporter gene labeled thereon, and the fluorescent probe includes a sequence complementary to the oligonucleotide.
The weight probe is marked with a quenching group, the fluorescent probe is marked with a reporting group, the reporting group can emit a signal, and the quenching group can absorb or quench the signal emitted by the reporting group.
In the conventional multiplex fluorescence real-time detection method, a fluorescent probe is designed for each target gene, and the color of fluorescence emitted by each fluorescent probe is different. And detecting the finally emitted fluorescent color, so as to judge whether the corresponding target gene exists in the sample.
The weight probe provided by the application is designed to contain a specific sequence and an oligonucleotide sequence, wherein the specific sequence is complementary with a target gene, the oligonucleotide sequence comprises a plurality of groups of short oligonucleotide sequences, and each group of short oligonucleotide sequences is marked with a quenching group; and the short oligonucleotide sequences of each set may be identical or different.
The term "short oligonucleotide sequence" in the present application means that the length is shorter than the oligonucleotide sequence and the oligonucleotide sequence mainly consists of the short oligonucleotide sequence. In the present application, a short oligonucleotide sequence is referred to as a set of short oligonucleotide sequences. According to the application, a plurality of groups of short oligonucleotide sequences can be designed according to actual detection requirements, each oligonucleotide sequence can comprise a plurality of groups of short oligonucleotide sequences, and each group of short oligonucleotide sequences in the same oligonucleotide sequence can be the same sequence or different sequences.
In the present application, each oligonucleotide sequence comprises n sets of short oligonucleotide sequences, n being an integer of 1 or more, for example 1,2,3,4,5,6,7,8 or more.
In the present application, the oligonucleotide sequences on the weighted probes are labeled with a quenching group, preferably the quenching group is labeled on a short oligonucleotide sequence.
In the present application, the short oligonucleotide sequences may be of any length, for example, each set of short oligonucleotide sequences may be 10-20nt, such as 10-15nt,15-20nt,13-18nt.
In order to improve the specificity, the 3' -end of the weighting probe is further provided with a tail sequence, the tail sequence does not hybridize with the target nucleic acid sequence under any condition, and the tail sequence can be of any length, for example, the tail sequence can be 8-15nt, such as 8nt,9nt,10nt,11nt,12nt,13nt,14nt,15nt, and optimally 10nt.
In the application, the fluorescent probe is a segment of oligonucleotide sequence which indicates a fluorescent group and can generate a fluorescent signal after being released; and the oligonucleotide sequences on the fluorescent probes may hybridize or anneal to the short oligonucleotide sequences on the weighted probes under conditions that allow hybridization, annealing or amplification of the nucleic acids.
The 3 'end of the fluorescent probe is also provided with a tail sequence, the tail sequence of the 3' end of the fluorescent probe does not hybridize with the oligonucleotide sequence on the weight probe under any condition, and the tail sequence of the 3 'end of the fluorescent probe can be of any length, for example, the tail sequence of the 3' end of the fluorescent probe can be 10-20nt, for example, 10-15nt,15-20nt,13-18nt,8-15nt.
In the application, the tail sequence of the 3 '-end of the fluorescent probe and the tail sequence of the 3' -end of the weighting probe can be the same sequence or different sequences.
In the application, the detection system designs a weight probe and a fluorescent probe according to a target gene, and then under the condition of allowing nucleic acid hybridization, annealing or amplification, the specific sequences on the target gene and the weight probe in a sample to be detected are complementary; the oligonucleotide sequences on the fluorescent probe and the weighted probe are complementary. Subsequent extension occurs under extension conditions, and the fluorescent groups on the fluorescent probe are released and fluorescent signals are emitted. And counting the fluorescent signal intensity, analyzing the data, and carrying out weight analysis according to the number of fluorescent groups on the hybridized fluorescent probes on the weight probes with complementary different target nucleic acid sequences, so as to judge the condition of the target genes in the sample.
In the application, the fluorescent probe is complementary with the oligonucleotide sequence on the weight probe, so that the number of fluorescent genes on the fluorescent probe and the number of quenching groups marked on the weight probe are the same, and the types are fluorescence-quenching types; i.e., the reporter is capable of signaling and the quencher on the complementary fluorescent probe is capable of absorbing or quenching the signaling from the reporter.
The fluorescent probe provided by the application is a section of oligonucleotide sequence, and the section of oligonucleotide sequence is marked with a fluorescent group. Accordingly, the oligonucleotide sequences of the fluorescent probes of the present application are complementary to the short oligonucleotide sequences on the weighted probes under conditions that allow hybridization, annealing or amplification of the nucleic acids.
In the present application, the sample to be tested may be obtained from any source, including but not limited to prokaryotes, eukaryotes or viruses or viroids. The sample to be tested may also be any form of nucleic acid sequence, such as genomic sequences, artificially isolated or fragmented sequences, synthetic sequences, etc.
Detection method
The application provides a single tube non-diagnosis method for detecting n target genes, which adopts the detection system principle and comprises the following specific steps:
step 1: providing n target genes to be detected with the same order of magnitude;
step 2: providing a weight probe and a fluorescent probe for each target gene to be detected, and annealing the weight probe and the fluorescent probe;
the weight probe is marked with a quenching gene, and the fluorescent probe is marked with a reporter gene;
step 3: amplifying a target gene to be detected, a weight probe and a fluorescent probe; thus, the fluorescent groups are separated and release fluorescent signals, and the fluorescent signals are detected and weight analysis is carried out, so that the condition of the target gene is detected.
Wherein, n target genes to be detected in the step 1 are amplified by adopting an asymmetric PCR method until the target genes are amplified in the same order of magnitude, and then subsequent reactions are carried out.
The primer design of the asymmetric PCR in the step 1 can be designed according to the specific condition of the target genes, the application is not particularly limited, and only the final n target genes to be detected are required to be of equal orders of magnitude.
In the detection method and the detection system, the weight probe and the fluorescent probe can be annealed first and then subjected to isothermal amplification with the target gene. In the application, isothermal amplification is carried out at normal temperature by adopting isothermal amplification enzyme.
In the application, the isothermal amplification enzyme is Bst polymerase.
Each of the weighted probes comprises a specific sequence and an oligonucleotide sequence; the specific sequence is complementary with a target gene of a sample to be detected; and the oligonucleotide is marked with a quenching gene; the fluorescent probe is marked with a reporter gene, and the fluorescent probe comprises a sequence complementary to the oligonucleotide.
The weight probe is marked with X quenching groups and Y quenching groups; the fluorescent probe is marked with X reporting groups and Y reporting groups; and n/3 is more than or equal to X and less than or equal to n is more than or equal to Y.
Multiplex detection
The present application provides a non-diagnostic method for rapid detection of influenza a, influenza b and novel coronaviruses using the fluorescent real-time detection system.
The non-diagnostic method for rapidly detecting influenza A, influenza B and novel coronaviruses comprises the following specific steps:
step 1: providing target genes of equal order: the M gene of the influenza A virus, the HA gene of the influenza B virus, the ORFlab gene of the novel coronavirus, the N gene of the novel coronavirus and the humanized internal reference gene are amplified to equal orders of magnitude through asymmetric PCR;
step 2: providing a weight probe and a fluorescent probe for each target gene to be detected, and annealing the weight probe and the fluorescent probe;
the weight probe is marked with a quenching gene, and the fluorescent probe is marked with a reporter gene;
step 3: isothermal amplification is carried out on the target gene to be detected, the weight probe and the fluorescent probe; thus, the fluorescent groups are separated and release fluorescent signals, and the fluorescent signals are detected and weight analysis is carried out, so that the condition of the target gene is detected.
Wherein, the multiplex primer used in the method comprises:
the influenza A virus M gene specific primer pair is as follows:
forward primer: 5'-CTTCTAACCGAGGTCGAAACGTA-3'
Reverse primer: 5'-GGTGACAGGATTGGTCTTGTCTTTA-3'
The influenza A virus M gene fluorescence weighting probe is as follows:
5'-AACTGGTCCTTCGTCGCACTCGCCCTGGTGACAGGATTGGTCTTGTCTTTATCCACTTTTTTTTTT-3'
the specific primer pair of the influenza B virus HA genes is as follows:
forward primer: 5'-AAATACGGTGGATTAAACAAAAGCAA-3'
Reverse primer: 5'-CCAGCAATAGCTCCGAAGAAA-3'
The influenza B virus HA gene fluorescence weighting probe is as follows:
5'-TACATCTGCGTCTAGCAACTGGTCCTTCGTCGAACATACCTACCAGCAATAGCTCCGAAGAAACCCCTTTCCTTTTTTTTTT-3'
the novel coronavirus ORFlab gene specific primer pair is:
forward primer: 5'-CCCTGTGGGTTTTACACTTAA-3'
Reverse primer: 5'-ACGATTGTGCATCAGCTGA-3'
The novel coronavirus ORFlab gene fluorescent weighting probe comprises the following components:
5'-TACATCTGCGTCTAGCAACTGGTCCTTCGTCGTACATCTGCGTCTAGCAACTGGTCCTTCGTCGAACTGTACTGACGATTGTGCATCAGCTGACTGAAGCATGGTTTTTTTTTT-3'
the novel coronavirus N gene specific primer pair is as follows:
forward primer: 5'-GGGGAACTTCTCCTGCTAGAAT-3'
Reverse primer: 5'-CAGACATTTTGCTCTCAAGCTG-3'
The novel coronavirus N gene fluorescence weighting probe comprises the following components:
5'-TACATCTGCGTCTAGCAACTGGTCCTTCGTCGGACATCGATACAGACATTTTGCTCTCAAGCTGGTTCAATCTTTTTTTTTT-3'
the specific primer pair of the humanized reference gene is as follows:
forward primer: 5'-TCAAGAAGGTGGTGAAGCAGG-3'
Reverse primer: 5'-CAGCGTCAAAGGTGGAGGAGT-3'
The fluorescence weighting probe of the humanized reference gene is as follows:
5'-AACTGGTCCTTCGTCGATTGTTAGGTCAGCGTCAAAGGTGGAGGAGTGGGTGTCGCTTTTTTTTTT-3'
fluorescent probe 1:5'-CGACGAAGGACCAGTTTTTTTTTTT-3'
Fluorescent probe 2:5'-GCTAGACGCAGATGTATTTTTTTTT-3'
Fluorescent probe 3:5'-CGACGAAGGACCAGTTTTTTTTTTT-3'
Fluorescent probe 4:5'-GCTAGACGCAGATGTATTTTTTTTT-3'.
Wherein ". Cndot.tttttt" of the sequence 3' in the above primer is the tail sequence, and ". Cndot.cndot.cndot.is the number of" T "without limitation.
Wherein, the fluorescent weighting probe of the influenza A virus M gene marks a BHQ1 quenching group at the 5' end;
the 20 th base (T) label of the influenza B virus HA gene fluorescence weighting probe is marked with a BHQ1 quenching group at the 5' end;
the novel coronavirus ORFlab gene fluorescent weighting probe marks a BHQ1 quenching group at the 20 th base (T) from the 5 'end, marks a BHQ1 quenching group at the 52 th base (T) from the 5' end;
the novel coronavirus N gene fluorescence weighting probe marks a BHQ1 quenching group at the 20 th base (T) from the 5' end;
the fluorescent weighting probe of the humanized reference gene marks a BHQ1 quenching group at the 5' end;
the fluorescent probe 1 marks a FAM fluorescent group at 15 th base (T) from the 5' end;
the 3 rd base (T) of the fluorescent probe 2 is marked with a FAM fluorescent group at the 5' end;
the fluorescent probe 3 marks HEX fluorescent groups at 15 th base (T) from the 5' end;
the fluorescent probe 4 labels the HEX fluorescent group at the 3 rd base (T) from the 5' end.
Kit for detecting a substance in a sample
A kit for rapid combined detection of novel coronaviruses, influenza A viruses and influenza B viruses, which comprises a reagent I and a reagent II; the reagent I comprises SuperMMLV Reverse Transcriptase, taq enzyme and MgCl 2 dNTPs,10 Xbuffer and the multiplex primer according to any one of claims 10 to 12; the reagent II is 10 Xbuffer and the multiplex probe; the reagent III is isothermal amplification enzyme, mgCl 2 ，10×buffer，dNTP。
In the reagent I, the concentration of Taq polymerase is 0.03-0.05U/. Mu.L, mgCl 2 The concentration of dNTPs is 4-5 mM, the concentration of dNTPs is 0.15-0.25 mM, and the concentration of the M gene specific primer pair of the influenza A virus, the HA gene specific primer pair of the influenza B virus, the N gene specific primer pair of the novel coronavirus and the humanized internal reference gene specific primer pair are all 0.3-0.5 mu M.
The concentrations of the influenza A virus M gene specific probe, the influenza B virus HA gene specific probe, the novel coronavirus N gene specific probe, the humanized internal reference gene specific probe and the fluorescent probes 1 to 4 in the reagent II are all 0.1-0.3 mu M.
The concentration of the isothermal amplification enzyme in the reagent III is 0.03-0.05U/. Mu.L, 10 Xbuffer (Mg 2+ The final concentration is 60 nmol/L), and the concentration of dNTP is 0.15-0.25 mM.
In the application, the kit also comprises a negative quality control product and a positive quality control product, wherein the negative quality control product is normal saline, and the positive quality control product is pseudovirus comprising a target fragment of an influenza A virus M gene, pseudovirus comprising a target fragment of an influenza B virus HA gene, novel coronavirus ORFlFlgene, pseudovirus comprising a target fragment of an N gene and a target fragment of a humanized internal reference gene.
Wherein the pseudovirus concentration is 1.0X10 2 ～1.0×10 4 copies/μL。
The reagent IPCR amplification conditions of the kit are as follows: reverse transcription at 42 ℃ for 30min, 5min at 95 ℃, denaturation at 95 ℃ for 20s, annealing at 60 ℃ for 1min, extension at 72 ℃ for 30s, thermal convection PCR reaction for 50 cycles, 10min at 72 ℃ and preservation at 4 ℃;
the reagent II probe annealing conditions were: 90℃2min,85℃1min,80℃1min,75℃1min,70℃1min,65℃1min,60℃1min,55℃1min,50℃1min,45℃1min,40℃1min,37℃5min,25℃5min,4℃;
the fluorescent PCR detection conditions of the reagent III are as follows: 25 ℃,5s,500 cycles.
The working conditions of the various enzymes of the application can be determined by a person skilled in the art by conventional methods and can generally involve the following factors: temperature, pH of buffer, composition, concentration, ionic strength, etc. Alternatively, conditions recommended by the manufacturer of the enzyme may be used.
Various technical features of the technical solutions of the present application may be modified, replaced, or combined based on the principles described in detail in the present application by those skilled in the art without departing from the spirit and scope of the present application. All such modifications and variations are intended to be included within the scope of the claims or the equivalents thereof.
Advantageous effects of the application
Compared with the prior art, the technical scheme of the application has the following beneficial effects:
(1) The detection system and the detection method provide a weight probe, which can realize the simultaneous detection of multiple target genes under the condition of few types and quantity of fluorescent groups;
(2) According to the application, 1-2 fluorescent groups can be used for simultaneously detecting 4-6 target nucleic acid sequences, so that the limit of the number of fluorescent channels in the existing equipment is overcome, and multiple target genes, even more than ten and tens of target genes can be simultaneously detected on one existing equipment;
(3) Only two kinds of fluorescence can be used for marking and detecting 5 genes, so that crosstalk between fluorescence spectrums of different colors is avoided, and the detection cost can be reduced;
(4) And (3) adding the sample into a hole for detection, namely, all primer pairs and probe frameworks are arranged in one detection hole site, and adding the sample can simultaneously detect and distinguish the A flow (M gene), the B flow (HA gene), the novel crown (ORFlab gene, N gene) and the internal reference (GADPH gene), thereby improving the detection efficiency.
The application provides a high-efficiency, low-cost and simple multiplex detection method. The maximum number of target nucleic acid sequences that can be detected by the method of the application is not limited by the number of types of fluorophores, and multi-site detection is achieved in a small number of wells.
Drawings
In order to more clearly illustrate the embodiments of the application or the technical solutions in the prior art, the drawings which are required in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are some embodiments of the application and that other drawings may be obtained from these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of an overall inspection system according to the present application
FIG. 2 is a diagram; design structure diagram aiming at HumanGAPDH reference gene weight probe
FIG. 3 is a diagram; design structure diagram for SARS-CoV-2N gene weight probe
FIG. 4 is a diagram; design structure diagram for SARS-CoV-2ORF gene weight probe
Fig. 5 is: design structure diagram aiming at Influenzaam gene weight probe
FIG. 6 is a diagram; design structure diagram aiming at Influenza BHA gene weight probe
Fig. 7 is: new coronavirus detection experimental graph.
1-weights means that the reference gene was detected, 2-weights means that the SARS-CoV-2N gene was detected, 3-weights means that the reference gene and the SARS-CoV-2N gene were detected, 4-weights means that the SARS-CoV-2ORF gene was detected, 5-weights means that the reference gene and the SARS-CoV-2ORF gene were detected, 6-weights means that the SARS-CoV-2N gene and the SARS-CoV-2ORF gene were detected, 7-weights
ts represents the detection of the reference gene, SARS-CoV-2N gene and SARS-CoV-2ORF gene.
Fig. 8 is: influenza A virus and influenza B virus detection experimental diagram
1-weights represent the detection of influenza A virus M gene, 2-weights represent the detection of influenza B virus HA gene, and 3-weights represent the detection of influenza A virus M gene and influenza B virus HA gene
Detailed Description
The application will now be described with reference to the following examples, which are intended to illustrate the application, but not to limit it. It should be understood that these embodiments are merely illustrative of the principles and technical effects of the present application and are not intended to represent all the possibilities of the present application. The application is not limited to the materials, reaction conditions or parameters mentioned in these examples. Other technical solutions may be implemented by those skilled in the art using other similar materials or reaction conditions in accordance with the principles of the present application. Such solutions do not depart from the basic principles and concepts described herein and are intended to be within the scope of the present application.
Example 1: real-time PCR assay using weighted probes and fluorescent probes
In this example, a real-time PCR assay system using a weight probe and a fluorescent probe and a corresponding test method were established with an influenza A virus M gene, an influenza B virus HA gene, a novel coronavirus ORFlab gene, a novel coronavirus N gene, and a humanized reference gene.
The influenza A virus M gene specific primer pair is as follows:
forward primer: 5'-CTTCTAACCGAGGTCGAAACGTA-3'
Reverse primer: 5'-GGTGACAGGATTGGTCTTGTCTTTA-3'
The influenza A virus M gene fluorescence weighting probe is as follows:
5'-AACTGGTCCTTCGTCGCACTCGCCCTGGTGACAGGATTGGTCTTGTCTTTATCCACTTTTTTTTTT-3'
the specific primer pair of the influenza B virus HA genes is as follows:
forward primer: 5'-AAATACGGTGGATTAAACAAAAGCAA-3'
Reverse primer: 5'-CCAGCAATAGCTCCGAAGAAA-3'
The influenza B virus HA gene fluorescence weighting probe is as follows:
5'-TACATCTGCGTCTAGCAACTGGTCCTTCGTCGAACATACCTACCAGCAATAGCTCCGAAGAAACCCCTTTCCTTTTTTTTTT-3'
the novel coronavirus ORFlab gene specific primer pair is:
forward primer: 5'-CCCTGTGGGTTTTACACTTAA-3'
Reverse primer: 5'-ACGATTGTGCATCAGCTGA-3'
The novel coronavirus ORFlab gene fluorescent weighting probe comprises the following components:
5'-TACATCTGCGTCTAGCAACTGGTCCTTCGTCGTACATCTGCGTCTAGCAACTGGTCCTTCGTCGAACTGTACTGACGATTGTGCATCAGCTGACTGAAGCATGGTTTTTTTTTT-3'
the novel coronavirus N gene specific primer pair is as follows:
forward primer: 5'-GGGGAACTTCTCCTGCTAGAAT-3'
Reverse primer: 5'-CAGACATTTTGCTCTCAAGCTG-3'
The novel coronavirus N gene fluorescence weighting probe comprises the following components:
5'-TACATCTGCGTCTAGCAACTGGTCCTTCGTCGGACATCGATACAGACATTTTGCTCTCAAGCTGGTTCAATCTTTTTTTTTT-3'
the specific primer pair of the humanized reference gene is as follows:
forward primer: 5'-TCAAGAAGGTGGTGAAGCAGG-3'
Reverse primer: 5'-CAGCGTCAAAGGTGGAGGAGT-3'
The fluorescence weighting probe of the humanized reference gene is as follows:
5'-AACTGGTCCTTCGTCGATTGTTAGGTCAGCGTCAAAGGTGGAGGAGTGGGTGTCGCTTTTTTTTTT-3'
fluorescent probe 1:5'-CGACGAAGGACCAGTTTTTTTTTTT-3'
Fluorescent probe 2:5'-GCTAGACGCAGATGTATTTTTTTTT-3'
Fluorescent probe 3:5'-CGACGAAGGACCAGTTTTTTTTTTT-3'
Fluorescent probe 4:5'-GCTAGACGCAGATGTATTTTTTTTT-3'.
The primers and probes used in this experiment were synthesized by Shanghai Mulberry root Biotechnology services Co. BstDNA polymerase and its corresponding buffers were purchased from New England Biolabs (NEB). dNTP mixtures were purchased from Beijing Tiangen Biotechnology Co. Oligonucleotides complementary to the probes were synthesized by Shanghai Sang Gong Biotechnology services Inc. Single step RT-PCR kits were purchased from Shanghai Bioengineering services Inc. RNA extraction kits were purchased from the company of the technology of the tenaton. MS2-VLPs encapsulating SARS-CoV-2, influenza a and influenza b sequences were from the national clinical laboratory center (beijing, china). Clinical samples were from the middle and day friendly hospitals (Beijing, china).
The non-diagnostic method for rapidly detecting influenza A, influenza B and novel coronaviruses comprises the following specific steps:
step 1: providing target genes of equal order: the M gene of the influenza A virus, the HA gene of the influenza B virus, the ORFlab gene of the novel coronavirus, the N gene of the novel coronavirus and the humanized internal reference gene are amplified to equal orders of magnitude through asymmetric PCR;
step 2: providing a weight probe and a fluorescent probe for each target gene to be detected, and annealing the weight probe and the fluorescent probe;
the weight probe is marked with a quenching gene, and the fluorescent probe is marked with a reporter gene;
step 3: after annealing the weighted probe and the fluorescent probe, adding a detected virus chain complementary to the weighted probe, polymerizing the base pairs under the action of DNA polymerase, and replacing the fluorescent probe and emitting fluorescence.
Nucleotide extraction and Single Strand formation
Viral RNA was extracted and purified from different MS2-VLPs and clinical samples using an RNA extraction kit (TianlongScience and Technology Co. Ltd, xi 'an, china) based on the GeneRotex96 nucleic acid extraction and purification system (Tianlong Science and Technology Co. Ltd, xi' an, china). Next, five pairs of primers were added to a 200. Mu.l PCR tube of a1 XAbstrartOne-step RT-PCR mixture (1 XAbstrartOne-step RT-PCR Mix: abstart Taq with dNTP, 1 XOne-stepRT-PCR buffer, 3.3mM Mg2+, 1 Xsolution I). The concentration ratio of the upstream primer to the downstream primer is 8:1, single strand formation by asymmetric PCR, can be further detected. The conditions of the detailed asymmetric PCR reaction system are shown in Table S1 and Table S2.
TABLE S1 preparation Table of asymmetric PCR reaction System
| Final concentration | Volume (mu L) | |
| Abstart Taq | 1* | 2 |
| One-step RT-PCR buffer（5×） | 1* | 10 |
| Solution I（10×） | 1* | 5 |
| 1-ORFlab-F(50uM) | 0.2uM | 0.2 |
| 1-ORFlab-R(50uM) | 1.6uM | 1.6 |
| 2-N-F(12.5uM) | 0.01uM | 0.04 |
| 2-N-R(12.5uM) | 0.08uM | 0.32 |
| NCF23(50uM) | 0.1uM | 0.1 |
| NCR24(50uM) | 0.8uM | 0.8 |
| 3-A-F(50uM) | 0.3uM | 0.3 |
| 3-A-R(50uM) | 2.4uM | 2.4 |
| 4-B-F(50uM) | 0.6uM | 0.6 |
| 4-B-R(50uM) | 0.06uM | 0.06 |
| RNA | —— | 15 |
| H2O | Make up to 50 mu L | 11.58 |
TABLE S2 asymmetric PCR reaction conditions
Wherein:
annealing 3 weight probes, fluorescent probe 1 and fluorescent probe 2 respectively, and adding 3 DNA-input, dNTP, bstpolymerase to complement 10 Xbuffer and ddH 2 And O is subjected to fluorescence detection.
Table 3: reaction system for forming annealed probe 1 by annealing weight probe 1 and fluorescent probe 1
| Final concentration (nmol/. Mu.L) | Volume (mu L) | |
| Fluorescent probe 1 | 166.7 | 6 |
| Fluorescent probe 2 | — | 0 |
| Weight probe 1 | 166.7 | 5 |
| 10×buffer | 1× | 5 |
| ddH2O | — | Complement to 50 |
Table 4: reaction system for forming annealing probe 2 by annealing weight probe 2, fluorescent probe 1 and fluorescent probe 2
| Final concentration (nmol/. Mu.L) | Volume (mu L) | |
| Fluorescent probe 1 | 166.7 | 6 |
| Fluorescent probe 2 | 166.7 | 6 |
| Weight probe 2 | 166.7 | 5 |
| 10×buffer | 1× | 5 |
| ddH2O | Complement to 50 |
Table 5: reaction system for forming annealed probe 3 by annealing weight probe 3, fluorescent probe 1 and fluorescent probe 2
| Final concentration (nmol/. Mu.L) | Volume (mu L) | |
| Fluorescent probe 1 | 166.7 | 12 |
| Fluorescent probe 2 | 166.7 | 12 |
| Weight probe 3 | 166.7 | 5 |
| 10×buffer | 1× | 5 |
| ddH2O | Complement to 50 |
Table 6: reaction system for forming annealing probe 4 by annealing weight probe 4, fluorescent probe 3 and fluorescent probe 4
| Final concentration (nmol/. Mu.L) | Volume (mu L) | |
| Fluorescent probe 3 | 166.7 | 12 |
| Fluorescent probe 4 | 166.7 | 12 |
| Weight probe 4 | 166.7 | 5 |
| 10×buffer | 1× | 5 |
| ddH2O | Complement to 50 |
Table 7: reaction system for forming annealing probe 5 by annealing weight probe 5, fluorescent probe 3 and fluorescent probe 4
| Final concentration (nmol/. Mu.L) | Volume (mu L) | |
| Fluorescent probe 3 | 166.7 | 6 |
| Fluorescent probe 4 | 166.7 | 6 |
| Weight probe 5 | 166.7 | 5 |
| 10×buffer | 1× | 5 |
| ddH2O | Complement to 50 |
Table 8: annealing reaction condition of weight probe and fluorescent probe
| Temperature (temperature) | Time | Cycle number |
| 90℃ | 2min | 1 |
| 85℃ | 1min | 1 |
| 80℃ | 1min | 1 |
| 75℃ | 1min | 1 |
| 70℃ | 1min | 1 |
| 65℃ | 1min | 1 |
| 60℃ | 1min | 1 |
| 55℃ | 1min | 1 |
| 50℃ | 1min | 1 |
| 45℃ | 1min | 1 |
| 40℃ | 1min | 1 |
| 37℃ | 5min | 1 |
| 25℃ | 5min | 1 |
| 4℃ | Forever | 1 |
Table 9: isothermal amplification reaction system
| Final concentration | Volume (mu L) | |
| Hybrid annealing probe | 20nmol/μL | 30 |
| dNTP | 3 | |
| Bst polymerase（8000units/mL） | 20units | 2.5 |
| 10×buffer | 1× | 2 |
| Viral RNA | 10 | |
| ddH2O | — | Complement to 50 |
Table 10: isothermal amplification reaction conditions
| Temperature (temperature) | Time | Cycle number |
| 25℃ | 5s | 500 |
Under the above conditions, as shown in fig. 7 and 8, the detection method provided by the application can be used for judging whether the target gene exists or not by using the fluorescence color and the fluorescence intensity.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.
Sequence listing
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Claims (10)
1. The fluorescent real-time detection system for simultaneously detecting n target genes is characterized in that a weight probe is designed for complementation with the target genes of a sample to be detected and releasing a reporter gene; determining whether the n target genes exist in the sample to be detected by detecting and analyzing the fluorescence intensity and the weight number sent by the reporter genes; wherein, the liquid crystal display device comprises a liquid crystal display device,
the weight probe is marked with X quenching groups and Y quenching genes; and n/3 is more than or equal to X and less than or equal to n is more than or equal to Y.
2. The fluorescent real-time detection system for simultaneously detecting n target genes according to claim 1, wherein each of the weight probes comprises a specific sequence and an oligonucleotide sequence;
the specific sequence is complementary with a target gene of a sample to be detected; and the oligonucleotide is marked with a quenching gene;
preferably, the detection system further comprises a fluorescent probe, wherein the fluorescent probe is marked with a reporter gene, and the fluorescent probe comprises a sequence complementary to the oligonucleotide.
3. A method for single tube detection of non-diagnostic n target genes using the fluorescent real-time detection system according to any one of claims 1-2.
4. A single tube method for detecting non-diagnostic n target genes as claimed in claim 3, wherein the method steps are as follows:
step 1: providing n target genes to be detected with the same order of magnitude;
step 2: providing a weight probe and a fluorescent probe for each target gene to be detected, and annealing the weight probe and the fluorescent probe;
the weight probe is marked with a quenching gene, and the fluorescent probe is marked with a reporter gene;
step 3: amplifying a target gene to be detected, a weight probe and a fluorescent probe; and detecting the fluorescent signal;
preferably, each of the weight probes comprises a specific sequence and an oligonucleotide sequence;
the specific sequence is complementary with a target gene of a sample to be detected; and the oligonucleotide is marked with a quenching gene;
the fluorescent probe is marked with a reporter gene, and the fluorescent probe comprises a sequence complementary to the oligonucleotide.
5. The method for single tube detection of non-diagnosis of n target genes according to claim 4, wherein the weight probe is marked with X quenching groups and Y quenching genes; the fluorescent probe is marked with X reporter groups and Y reporter genes;
and n/3 is more than or equal to X and less than or equal to n is more than or equal to Y.
6. The single tube non-diagnostic method for detecting n target genes according to claim 4 or 5, wherein the n target genes to be detected of equal order of magnitude are obtained by performing asymmetric PCR on a sample.
7. A method for rapid detection of influenza a, influenza b and novel coronaviruses non-diagnostic using the fluorescent real-time detection system of any one of claims 1-2.
8. A multiplex primer for use in the non-diagnostic method of claim 7.
9. The multiplex primer of claim 8, wherein the multiplex primer comprises: specific primer pairs and fluorescent weighting probes for influenza A virus M gene, influenza B virus HA gene, novel coronavirus ORFlab gene, novel coronavirus N gene and humanized internal reference gene;
the influenza A virus M gene specific primer pair is as follows:
forward primer: 5'-CTTCTAACCGAGGTCGAAACGTA-3'
Reverse primer: 5'-GGTGACAGGATTGGTCTTGTCTTTA-3'
The influenza A virus M gene fluorescence weighting probe is as follows:
5'-AACTGGTCCTTCGTCGCACTCGCCCTGGTGACAGGATTGGTCTTGTCTTTATCCACTTTTTTTTTT-3'
the specific primer pair of the influenza B virus HA genes is as follows:
forward primer: 5'-AAATACGGTGGATTAAACAAAAGCAA-3'
Reverse primer: 5'-CCAGCAATAGCTCCGAAGAAA-3'
The influenza B virus HA gene fluorescence weighting probe is as follows:
5'-TACATCTGCGTCTAGCAACTGGTCCTTCGTCGAACATACCTACCAGCAATAGCTCCGAAGAAACCCCTTTCCTTTTTTTTTT-3'
the novel coronavirus ORFlab gene specific primer pair is:
forward primer: 5'-CCCTGTGGGTTTTACACTTAA-3'
Reverse primer: 5'-ACGATTGTGCATCAGCTGA-3'
The novel coronavirus ORFlab gene fluorescent weighting probe comprises the following components:
5'-TACATCTGCGTCTAGCAACTGGTCCTTCGTCGTACATCTGCGTCTAGCAACTGGTCCTTCGTCGAACTGTACTGACGATTGTGCATCAGCTGACTGAAGCATGGTTTTTTTTTT-3'
the novel coronavirus N gene specific primer pair is as follows:
forward primer: 5'-GGGGAACTTCTCCTGCTAGAAT-3'
Reverse primer: 5'-CAGACATTTTGCTCTCAAGCTG-3'
The novel coronavirus N gene fluorescence weighting probe comprises the following components:
5'-TACATCTGCGTCTAGCAACTGGTCCTTCGTCGGACATCGATACAGACATTTTGCTCTCAAGCTGGTTCAATCTTTTTTTTTT-3'
the specific primer pair of the humanized reference gene is as follows:
forward primer: 5'-TCAAGAAGGTGGTGAAGCAGG-3'
Reverse primer: 5'-CAGCGTCAAAGGTGGAGGAGT-3'
The fluorescence weighting probe of the humanized reference gene is as follows:
5'-AACTGGTCCTTCGTCGATTGTTAGGTCAGCGTCAAAGGTGGAGGAGTGGGTGTCGCTTTTTTTTTT-3'
fluorescent probe 1:5'-CGACGAAGGACCAGTTTTTTTTTTT-3'
Fluorescent probe 2:5'-GCTAGACGCAGATGTATTTTTTTTT-3'
Fluorescent probe 3:5'-CGACGAAGGACCAGTTTTTTTTTTT-3'
Fluorescent probe 4:5'-GCTAGACGCAGATGTATTTTTTTTT-3';
preferably, the influenza a virus M gene fluorescent weighting probe is labeled with a BHQ1 quencher group at the 5' end;
the 20 th base (T) label of the influenza B virus HA gene fluorescence weighting probe is marked with a BHQ1 quenching group at the 5' end;
the novel coronavirus ORFlab gene fluorescent weighting probe marks a BHQ1 quenching group at the 20 th base (T) from the 5 'end, marks a BHQ1 quenching group at the 52 th base (T) from the 5' end;
the novel coronavirus N gene fluorescence weighting probe marks a BHQ1 quenching group at the 20 th base (T) from the 5' end;
the fluorescent weighting probe of the humanized reference gene marks a BHQ1 quenching group at the 5' end;
the fluorescent probe 1 marks a FAM fluorescent group at 15 th base (T) from the 5' end;
the 3 rd base (T) of the fluorescent probe 2 is marked with a FAM fluorescent group at the 5' end;
the fluorescent probe 3 marks HEX fluorescent groups at 15 th base (T) from the 5' end;
the fluorescent probe 4 labels the HEX fluorescent group at the 3 rd base (T) from the 5' end.
10. A kit for rapid combined detection of novel coronaviruses, influenza A viruses and influenza B viruses is characterized by comprising a reagent I and a reagent II; the reagent I comprises SuperMMLV Reverse Transcriptase, taq enzyme and MgCl 2 dNTPs,10 Xbuffer and the multiplex primer according to any one of claims 10 to 12; the reagent II is 10 Xbuffer and the multiplex fluorescent probe according to claim 9 or 10; the reagent III is isothermal amplification enzyme, mgCl 2 ，10×buffer，dNTP；
Preferably, the kit's reagent IPCR amplification conditions are: reverse transcription at 42 ℃ for 30min, 5min at 95 ℃, denaturation at 95 ℃ for 20s, annealing at 60 ℃ for 1min, extension at 72 ℃ for 30s, thermal convection PCR reaction for 50 cycles, 10min at 72 ℃ and preservation at 4 ℃;
the reagent II probe annealing conditions were: 90℃2min,85℃1min,80℃1min,75℃1min,70℃1min,65℃1min,60℃1min,55℃1min,50℃1min,45℃1min,40℃1min,37℃5min,25℃5min,4℃; the fluorescent PCR detection conditions of the reagent III are as follows: 25 ℃,5s,500 cycles;
preferably, in the reagent I, the concentration of Taq polymerase is 0.03-0.05U/. Mu.L, mgCl 2 The concentration of dNTPs is 4-5 mM, the concentration of dNTPs is 0.15-0.25 mM, and the concentration of the M gene specific primer pair of the influenza A virus, the HA gene specific primer pair of the influenza B virus, the N gene specific primer pair of the novel coronavirus and the humanized internal reference gene specific primer pair are all 0.3-0.5 mu M;
preferably, the concentrations of the influenza A virus M gene specific probe, the influenza B virus HA gene specific probe, the novel coronavirus N gene specific probe, the humanized internal reference gene specific probe and the fluorescent probes 1 to 4 in the reagent II are all 0.1-0.3 mu M;
preferably, the concentration of isothermal amplification enzyme in the reagent III is 0.03-0.05U/. Mu.L, and the concentration of dNTP is 0.15-0.25 mM;
preferably, the kit further comprises a negative quality control and a positive quality control, wherein the negative quality control is normal saline, and the positive quality control is pseudoviruses comprising a target fragment of an influenza A virus M gene, a target fragment of an influenza B virus HA gene, a novel coronavirus ORFlab gene, a target fragment of an N gene and a target fragment of a humanized reference gene;
preferably, the pseudovirus concentration is 1.0X10 2 ～1.0×10 4 copies/μL。
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