WO2019153675A1 - 一种双重致病菌的比色传感新方法 - Google Patents

一种双重致病菌的比色传感新方法 Download PDF

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WO2019153675A1
WO2019153675A1 PCT/CN2018/099324 CN2018099324W WO2019153675A1 WO 2019153675 A1 WO2019153675 A1 WO 2019153675A1 CN 2018099324 W CN2018099324 W CN 2018099324W WO 2019153675 A1 WO2019153675 A1 WO 2019153675A1
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sequence
nucleotide sequence
complementary
seq
nucleic acid
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PCT/CN2018/099324
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English (en)
French (fr)
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罗云波
许文涛
黄昆仑
田晶晶
杜再慧
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中国农业大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention belongs to the field of biological detection technology, and particularly relates to a novel colorimetric sensing method for dual pathogenic bacteria.
  • the traditional bacterial detection method is mainly based on the physiological and biochemical characteristics of bacteria. After the steps of pre-enrichment, selective plate separation and biochemical identification, it takes 5-7 days from sampling to determination. The detection period is long, the operation is cumbersome and the workload is large. . It has been more than half a century to identify bacteria using the specificity of the antigen-antibody reaction, but the screening of microbial antibodies is cumbersome and the final detection specificity is not high.
  • the continuous improvement and development of molecular biology detection technology has overcome the problems of tedious and time-consuming experimentation of traditional detection methods, and has also led to the rapid development of rapid detection methods for microorganisms, but the disadvantage of molecular biology methods is that the results are not visualized. It is not easy to analyze the results.
  • the new colorimetric sensing method established by the invention overcomes the deficiencies of the existing detection technologies and realizes accurate, rapid, simple and efficient detection and analysis of microorganisms.
  • the acid sequence is located at the 5' or 3' end of the upstream primer; the complementary sequence A and the nucleotide sequence of the complementary sequence B are complementary and/or inversely complementary; the linker comprises a polymerase binding inhibitor Structure and/or structure that inhibits new strand extension during nucleic acid amplification in vitro; the downstream primer includes a nucleotide sequence that specifically amplifies the target to be tested;
  • the A and B are only used to distinguish different complementary sequences and are not used for sorting.
  • the complementation includes complementary or reverse complementarity as defined by the prior art or common general knowledge, and/or complementarity or reverse complementation according to complementary principles as defined by the prior art or common general knowledge.
  • the polymerase includes a polymerase that can be used in in vitro nucleic acid amplification techniques.
  • the nucleotide sequence capable of specifically amplifying the target to be tested specifically includes a primer sequence designed according to a characteristic sequence of the target to be tested; the feature sequence includes a feature sequence defined by prior art or common knowledge; the design The design method described in the prior art or common knowledge is included.
  • the method further includes at least one of the following 1)-2):
  • the in vitro nucleic acid amplification technique comprises an ultra-fast PCR reaction comprising: 90-98 ° C, 2-6 s; 50-60 ° C, 2-8 s; a total of 20-40 cycles ;
  • the reaction process of the ultra-fast PCR reaction comprises: 95 ° C, 4 s; 58 ° C, 6 s; a total of 30 cycles;
  • the concentration of the upstream primer and the downstream primer in the reaction system of the ultra-fast PCR reaction is 10 times or more of a common PCR concentration; specifically, 20 times; the reaction system of the ultra-fast PCR reaction further includes DNA.
  • a polymerase the concentration of the DNA polymerase is more than 10 times the concentration of a common PCR, and specifically, 60 times;
  • the linker comprises a compound having a long chain structure.
  • the connecting arm is oxyethyleneglycol
  • the chemical structure of oxyethyleneglycol is:
  • the method further includes at least one of the following 1)-8):
  • the upstream primer comprises: a primer obtained by ligating the nucleotide sequences shown by SEQ ID NO: 1 and SEQ ID NO: 2 in the Sequence Listing through a tether;
  • the downstream primer comprises the nucleotide sequence shown in SEQ ID NO: 3 in the Sequence Listing;
  • the upstream primer comprises: substituting and/or deleting and/or adding a nucleotide sequence represented by SEQ ID NO: 1 and/or SEQ ID NO: 2 in the sequence listing by one or several nucleotides and a primer obtained by ligating a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 1 and/or SEQ ID NO: 2 in the Sequence Listing by a tether;
  • the downstream primer comprises a substitution and/or deletion and/or addition of one or several nucleotides of the nucleotide sequence shown by SEQ ID NO: 3 in the Sequence Listing and SEQ ID NO: 3 in the Sequence Listing a nucleotide sequence having the same function as the nucleotide sequence shown;
  • the upstream primer comprises: a primer obtained by ligating the nucleotide sequences shown by SEQ ID NO: 4 and SEQ ID NO: 5 in the Sequence Listing through a tether;
  • the downstream primer comprises the nucleotide sequence shown in SEQ ID NO: 6 in the Sequence Listing;
  • the upstream primer comprises: substituting and/or deleting and/or adding a nucleotide sequence represented by SEQ ID NO: 4 and/or SEQ ID NO: 5 in the Sequence Listing by one or several nucleotides and a primer obtained by ligating a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 4 and/or SEQ ID NO: 5 in the Sequence Listing by a tether;
  • the downstream primer comprises a substitution and/or deletion and/or addition of one or several nucleotides of the nucleotide sequence shown by SEQ ID NO: 6 in the Sequence Listing and SEQ ID NO: 6 in the Sequence Listing.
  • the nucleotide sequences shown have the same functional nucleotide sequence.
  • the function includes achieving specific amplification or detecting a target to be tested.
  • the upstream primer is:
  • the upstream primers are commercially available directly, and the preparation method thereof belongs to the prior art.
  • Another object of the present invention is to provide a detection method comprising a nucleic acid self-assembly color reaction, the reaction system of the nucleic acid self-assembly color reaction comprising a hairpin sequence, characterized in that the hairpin sequence comprises: All or part of the nucleotide sequence, complementary sequence C and complementary sequence D of the G-quadruplex functional nucleic acid sequence; all or part of the nucleotide sequence constituting the G-quadruplex functional nucleic acid sequence is located in the hairpin sequence a 5' end and/or a 3' end; the complementary sequence C is complementary and/or inversely complementary to the complementary sequence C of another hairpin sequence; the complementary sequence D is linked to the target to be tested or to the target to be tested Acid sequence complementary and/or reverse complementary;
  • the C and D are only used to distinguish different complementary sequences and are not used for sorting.
  • the G-quadruplex functional nucleic acid sequence comprises a G-quadruplex functional nucleic acid sequence as defined by the prior art or common general knowledge; in particular, the G-quadruplex functional nucleic acid sequence comprises the self-assembly of the sequence Forming a G-quadruplex functional nucleic acid having horseradish peroxidase activity, the G-quadruplex functional nucleus
  • the acid can catalyze the formation of ABTS-, which is a blue-green color of ABTS 2- and H 2 O 2 .
  • the hairpin sequence comprises cleaving a G-quadruplex functional nucleic acid sequence at a ratio of 25% and/or 75%, and the split sequence is added to the 5' end and/or 3 of the card issue sequence, respectively.
  • the split sequence is added to the 5' end and/or 3 of the card issue sequence, respectively.
  • the T base is added after the splitting; the T base is added at the 5' end and/or the 3' end of the cleaved sequence;
  • the G-quadruplex functional nucleic acid sequence includes the nucleotide sequence shown by SEQ ID NO: 7 and/or SEQ ID NO: 8 in the Sequence Listing;
  • the complementary sequence D may be complementary or inversely complementary to the complementary sequence A and/or the complementary sequence B of the present invention
  • the card issue sequence includes at least one of the following 1)-4):
  • nucleotide sequence shown by SEQ ID NO: 9 in the sequence listing and/or the nucleotide sequence shown by SEQ ID NO: 9 is substituted and/or deleted and/or added by one or several nucleotides and a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 9 in the Sequence Listing;
  • nucleotide sequence shown by SEQ ID NO: 10 in the sequence listing and/or the nucleotide sequence shown by SEQ ID NO: 10 is substituted and/or deleted and/or added by one or several nucleotides and a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 10 in the Sequence Listing;
  • nucleotide sequence shown by SEQ ID NO: 11 in the sequence listing and/or the nucleotide sequence shown by SEQ ID NO: 11 is substituted and/or deleted and/or added by one or several nucleotides and a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 11 in the Sequence Listing;
  • nucleotide sequence shown by SEQ ID NO: 12 in the sequence listing and/or the nucleotide sequence shown in SEQ ID NO: 12 is substituted and/or deleted and/or added by one or several nucleotides and A nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 12 in the Sequence Listing.
  • nucleic acid self-assembly color reaction further comprises at least one of the following 1) to 2):
  • the reaction conditions of the nucleic acid self-assembly color reaction include incubation at 37 ° C for 20 min;
  • the final concentration of the hairpin sequence is 2 ⁇ M.
  • a method of amplifying the object to be tested, and then detecting the object to be tested by any of the methods of the present invention is a method of amplifying the object to be tested, and then detecting the object to be tested by any of the methods of the present invention.
  • the complementary sequence D of the present invention is complementary or inversely complementary to the complementary sequence A and/or the complementary sequence B of the present invention.
  • the method further includes at least one of the following 1)-4):
  • the object to be tested contains the object to be tested; and specifically, when the color of the reaction system is blue-green, it is determined that the object to be tested contains the object to be tested;
  • the signal is amplified by 2-4 times;
  • the microarray method may be used to determine whether the object to be tested contains the object to be tested or contains several objects to be tested; the microarray method includes: The hairpin sequence is separately placed in different micropores for reaction, and then according to the reaction result, it is judged whether the object to be tested is contained or contains several objects to be tested.
  • the color of the reaction liquid in the micropore changes or turns blue-green, The total number of micropores containing the target to be tested; the color change or the blue-green color is the total number of types of the target to be tested contained in the test object.
  • the type of the hairpin sequence includes: the complementary sequence D in the hairpin sequence is the same type of hairpin as the hairpin sequence complementary or inversely complementary to the complementary sequence A and/or B in the upstream primer.
  • the sequences, otherwise, are different types of hairpin sequences; the upstream primer nucleotide sequences are identical to the same upstream primer.
  • the type of the upstream primer or the downstream primer includes: the primer pair that can amplify the same target to be the same type of upstream primer or the downstream primer; otherwise, the primer pair that can amplify different target to be tested is a different kind of upstream primer or Downstream primers.
  • kit and/or biosensor comprising at least one of the following 1) to 2):
  • the upstream primer comprising: a complementary sequence A, a tether, a complementary sequence B, and a nucleotide sequence capable of specifically amplifying a target to be detected;
  • the tether is located in the complementary sequence A and Between the complementary sequences B, the nucleotide sequence capable of specifically amplifying the target to be tested is located at the 5' end or the 3' end of the upstream primer;
  • the complementary sequence A and the nucleotide of the complementary sequence B Sequence complementary and/or reverse complementary;
  • the linker comprises a structure that inhibits polymerase binding and/or a structure that inhibits new strand elongation during nucleic acid amplification in vitro;
  • the downstream primer includes specific amplification to be tested The nucleotide sequence of the target;
  • a hairpin sequence comprising: all or part of a nucleotide sequence, a complementary sequence C and a complementary sequence D constituting a G-quadruplex functional nucleic acid sequence; said all of the G-quadruplex functional nucleic acid sequences Or a partial nucleotide sequence is located at the 5' end and/or the 3' end of the hairpin sequence; the complementary sequence C is complementary and/or inversely complementary to the complementary sequence C of another hairpin sequence;
  • the target to be tested or the nucleotide sequence linked to the target to be tested is complementary and/or reverse complementary.
  • the complementation includes complementary or reverse complementarity as defined by the prior art or common general knowledge, and/or complementarity or reverse complementation according to complementary principles as defined by the prior art or common general knowledge;
  • the polymerase includes a polymerase that can be used in in vitro nucleic acid amplification techniques
  • the nucleotide sequence capable of specifically amplifying the target to be tested specifically includes a primer sequence designed according to a characteristic sequence of the target to be tested; the feature sequence includes a feature sequence defined by prior art or common knowledge; the design Including the design methods described in the prior art or common knowledge;
  • the connecting arm comprises a compound having a long chain structure
  • the connecting arm is oxyethyleneglycol
  • the chemical structure of oxyethyleneglycol is:
  • the upstream primer and the downstream primer include at least one of the following 1)-8):
  • the upstream primer comprises: a primer obtained by ligating the nucleotide sequences shown by SEQ ID NO: 1 and SEQ ID NO: 2 in the Sequence Listing through a tether;
  • the downstream primer comprises the nucleotide sequence shown in SEQ ID NO: 3 in the Sequence Listing;
  • the upstream primer comprises: substituting and/or deleting and/or adding a nucleotide sequence represented by SEQ ID NO: 1 and/or SEQ ID NO: 2 in the sequence listing by one or several nucleotides and a primer obtained by ligating a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 1 and/or SEQ ID NO: 2 in the Sequence Listing by a tether;
  • the downstream primer comprises a substitution and/or deletion and/or addition of one or several nucleotides of the nucleotide sequence shown by SEQ ID NO: 3 in the Sequence Listing and SEQ ID NO: 3 in the Sequence Listing a nucleotide sequence having the same function as the nucleotide sequence shown;
  • the upstream primer comprises: a primer obtained by ligating the nucleotide sequences shown by SEQ ID NO: 4 and SEQ ID NO: 5 in the Sequence Listing through a tether;
  • the downstream primer comprises the nucleotide sequence shown in SEQ ID NO: 6 in the Sequence Listing;
  • the upstream primer comprises: substituting and/or deleting and/or adding a nucleotide sequence represented by SEQ ID NO: 4 and/or SEQ ID NO: 5 in the Sequence Listing by one or several nucleotides and a primer obtained by ligating a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 4 and/or SEQ ID NO: 5 in the Sequence Listing by a tether;
  • the downstream primer comprises a substitution and/or deletion and/or addition of one or several nucleotides of the nucleotide sequence shown by SEQ ID NO: 6 in the Sequence Listing and SEQ ID NO: 6 in the Sequence Listing a nucleotide sequence having the same function as the nucleotide sequence shown;
  • the function includes achieving specific amplification or detecting a target to be tested.
  • the upstream primer is:
  • the C and D are only used to distinguish different complementary sequences, and are not used for sorting
  • the G-quadruplex functional nucleic acid sequence comprises a G-quadruplex functional nucleic acid sequence as defined by the prior art or common general knowledge; in particular, the G-quadruplex functional nucleic acid sequence comprises the self-assembly of the sequence Forming a G-quadruplex functional nucleic acid having horseradish peroxidase activity, which is capable of catalyzing ABTS 2- and H 2 O under the induction of hemin 2 generates the reaction solution was blue-green substance ABTS -.
  • the hairpin sequence comprises cleaving a G-quadruplex functional nucleic acid sequence at a ratio of 25% and/or 75%, and the split sequence is added to the 5' end and/or 3 of the card issue sequence, respectively.
  • the split sequence is added to the 5' end and/or 3 of the card issue sequence, respectively.
  • the T base is added after the splitting; the T base is added at the 5' end and/or the 3' end of the cleaved sequence;
  • the G-quadruplex functional nucleic acid sequence includes the nucleotide sequence shown by SEQ ID NO: 7 and/or SEQ ID NO: 8 in the Sequence Listing;
  • the complementary sequence D may be complementary or inversely complementary to the complementary sequence A and/or B of claim 1;
  • the card issue sequence includes at least one of the following 1)-4):
  • nucleotide sequence shown by SEQ ID NO: 9 in the sequence listing and/or the nucleotide sequence shown by SEQ ID NO: 9 is substituted and/or deleted and/or added by one or several nucleotides and a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 9 in the Sequence Listing;
  • nucleotide sequence shown by SEQ ID NO: 10 in the sequence listing and/or the nucleotide sequence shown by SEQ ID NO: 10 is substituted and/or deleted and/or added by one or several nucleotides and a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 10 in the Sequence Listing;
  • nucleotide sequence shown by SEQ ID NO: 11 in the sequence listing and/or the nucleotide sequence shown by SEQ ID NO: 11 is substituted and/or deleted and/or added by one or several nucleotides and a nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 11 in the Sequence Listing;
  • nucleotide sequence shown by SEQ ID NO: 12 in the sequence listing and/or the nucleotide sequence shown in SEQ ID NO: 12 is substituted and/or deleted and/or added by one or several nucleotides and A nucleotide sequence having the same function as the nucleotide sequence shown by SEQ ID NO: 12 in the Sequence Listing.
  • kit and/or biosensor includes the following 1)-3):
  • a final object of the invention is to provide the use of any of the methods of the invention, a kit of any of the invention and/or a biosensor.
  • the application includes at least one of the following 1)-4):
  • the microorganism includes Salmonella and/or Staphylococcus aureus.
  • the application does not include the diagnosis and treatment of the disease described in Article 25 of the Chinese Patent Law.
  • a new method of dual colorimetric sensing based on ultra-fast PCR established by the invention:
  • the method establishes an ultra-fast PCR reaction system, which reduces the conventional PCR process, which takes about 3 hours, to 5 minutes, and significantly reduces the time spent on the PCR reaction;
  • the ultra-fast PCR reaction system is equipped with a nucleic acid self-assembly color-developing module, which not only amplifies the reaction signal again, but also facilitates the ultra-sensitive detection of pathogenic bacteria; and solves the problem that the traditional PCR results are difficult to visually detect;
  • an ultra-rapid polymerase chain reaction (PCR) amplification primer is designed according to the virulence gene of Salmonella and Staphylococcus aureus, and a nucleic acid self-assembly color-developing module is combined to establish a A new dual colorimetric sensing method based on ultra-fast PCR for ultrasensitive detection of Salmonella and Staphylococcus aureus.
  • PCR polymerase chain reaction
  • the detection method and the biosensor established by the invention are faster and more sensitive than the traditional method, and have the advantages of high specificity, high sensitivity, reliable detection result, etc., which can simplify the analysis and detection steps, shorten the analysis time, and more importantly, make Online real-time detection is possible, easy to carry and field work, and has a very good application prospect in the field of microbial detection including food safety and rapid detection.
  • the detection method and the biosensor established by the invention can simultaneously realize the dual specific detection of Salmonella and Staphylococcus aureus, the detection has good specificity, high sensitivity, reliable detection result, can be discerned by the naked eye, and the detection process is quick and convenient. It is of great significance in daily monitoring or market screening.
  • the detection methods and biosensors of the present invention have detection sensitivities of 10 cfu/mL and 10 cfu/mL for detection of Salmonella and Staphylococcus aureus respectively; in addition, specific test results indicate that the detection method established by the present invention and The biosensor has no cross-reaction to Shigella and Escherichia coli, and can simultaneously achieve dual specific detection of Salmonella and Staphylococcus aureus.
  • the nucleic acid self-assembly color-developing module of the invention is non-enzymatic reaction, the reaction system component is simpler, the reaction process is simpler, the step of terminating the enzymatic reaction is reduced, and the constant temperature reaction simplifies the requirement for the enzymatic reaction temperature. Significantly reduce economic costs, shorten reaction time, and help to meet the requirements of fast and simple detection.
  • Figure 1 is a structural diagram of an ultra-fast PCR device
  • Figure 2 is a graph showing the results of verification of the amplification effect of the dual ultra-fast PCR reaction; wherein Lane 1 is a negative control without product purification (double ultra-fast PCR reaction system without adding Salmonella and Staphylococcus aureus genome); Lane 2 is Positive samples without product purification (double ultra-fast PCR reaction system with Salmonella and Staphylococcus aureus genomes added); Lane 3 is a negative control with product purification (double ultra-fast PCR reaction without Salmonella or Staphylococcus aureus genome) Lane 4; Lane 4 is a positive sample purified by the product (a dual ultra-fast PCR reaction system with the addition of Salmonella and Staphylococcus aureus genomes);
  • Figure 3 is a standard curve of Salmonella
  • Figure 4 is a standard curve of Staphylococcus aureus
  • Figure 5 is a graph showing the results of specific experiments, wherein 1 is micropores 1, 2 is micropores 2, 3 is micropores 3, and 4 is micropores 4; a is a result of detecting Salmonella and Shigella samples; b is a graph showing the results of detection of Escherichia coli and Staphylococcus aureus samples; c is a graph showing the results of detection of Salmonella and Staphylococcus aureus samples.
  • Example 1 Establishment of a new method based on ultra-fast PCR for dual colorimetric sensing for detecting Salmonella and Staphylococcus aureus
  • the strain information used in this example is shown in Table 1.
  • the nucleotide sequences of the designed primers are shown in Table 2 and the Sequence Listing.
  • nucleotide sequence on the left side of the oxyethyleneglycol bridge of the upstream primer Primer 1 is the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing, and the nucleotide sequence on the right side of the tether is in the order of In the nucleotide sequence shown by SEQ ID NO: 2 in the list, the chemical structure of the tether is:
  • nucleotide sequence of the downstream primer Primer 2 is the nucleotide sequence shown by SEQ ID NO: 3 in the Sequence Listing.
  • nucleotide sequence on the left side of the oxyethyleneglycol bridge of the upstream primer Primer 3 is the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing, and the nucleotide sequence on the right side of the tether is in the order of In the nucleotide sequence shown by SEQ ID NO: 5 in the list, the chemical structure of the tether is the same as that of Primer 1.
  • nucleotide sequence of the downstream primer Primer 4 is the nucleotide sequence shown by SEQ ID NO: 6 in the Sequence Listing.
  • the hairpin sequence 1-4 (Hairpin 1, Hairpin 2, Hairpin 3, Hairpin 4) is the nucleotide sequence of the two G-quadruplex functional nucleic acid sequences: SEQ ID NO: 7 in the sequence listing.
  • the nucleotide sequence TGGG TGGG TAGGG CGGG shown by AGGG CGGG TGGG TGGG and SEQ ID NO: 8 was cleaved at a ratio of about 25% to 75%, and added to both ends of a hairpin probe (HairpinProbe). And a T-base protected cleaved G-quadruplex sequence was added.
  • the split G-quadruplex is close to each other in distance, under the induction of hemin, the formation of horseradish peroxidase
  • the active G-quadruplex functional nucleic acid functions to catalyze the color development of ABTS 2- and H 2 O 2 .
  • the nucleotide sequence of the hairpin sequence Hairpin 1 in Table 2 is the nucleotide sequence shown in SEQ ID NO: 9 in the Sequence Listing; the nucleotide sequence of Hairpin 2 is shown in SEQ ID NO: 10 in the Sequence Listing.
  • Ex Taq DNA polymerase 10 x Ex Taq Buffer (20 mM Mg2+ Plus) and dNTP Mixture (2.5 mM) were purchased from TAKaRa. Hemin and 2,2-diaza-bis(3-ethyl-benzothiazole-6-sulfonic acid) diamine salt (ABTS2-) were purchased from Aladdin Chemical Co. (Sigma-Aldrich Chemical Co.). ). The experimental water was taken from the Milli-Q pure water system. Other reagents were purchased from Sinopharm Group.
  • the main structure of the ultra-fast PCR device is shown in Figure 1.
  • the specific structure, connection method, working principle and working process include:
  • the ultra-fast PCR device uses a Light Cycler model capillary (20uL, 04 929 292 001, Roche) as the PCR sample chamber. By rapid centrifugation, the samples are collected at one end of each capillary. After centrifugation, the capillary with the sample is fixed. On a plastic stand.
  • the plastic bracket is connected to a stepping motor (42JSF630AS-1000, Just Motioin Control), and the capillary sample chamber fixed on the plastic bracket is driven by the stepping motor between a high temperature water bath at 95 ° C and a medium temperature water bath at 58 ° C. Cyclic conversion to achieve reaction temperature changes and control during ultra-fast PCR reactions.
  • the stepping motor is powered by a switching power supply (S-100-24, Elecall), and the frequency or time of the above-mentioned cyclic conversion of the stepping motor is realized by a DC servo motor driver (YZ-ACSD60, Moving) and Labview (version 2014). control. Temperature measurement is achieved using a thermocouple encapsulated in a capillary. The amplification and linearization process of the thermocouple signal is transmitted by a temperature transmitter (SBWR-2260, K, Yuancheng) and processed by the chicken UNO v1.0 chip. The chicken UNO chip converts the received analog signal into a digital signal, which is then executed by the PC IDE (version 1.8.1) module.
  • the Salmonella and Staphylococcus aureus were cultured overnight in LB medium, and the genomic DNA in Salmonella and Staphylococcus aureus was extracted by New Industry's bacterial genomic DNA extraction kit.
  • One microliter of genomic DNA was mixed and used as a template in Table 3.
  • Primers 1-4 (Primer 1, Primer 2, Primer 3, Primer 4) in Table 3 are specifically primers 1-4 (Primer 1, Primer 2, Primer 3, Primer 4) listed in Table 2 above.
  • Photographing system Molecular Imager Gel Doc XR (Bio-Rad). And use PCR product purification test
  • the kit removes primer dimers, unreacted primers and reaction impurities.
  • Fig. 2 The results of the amplification effect of the dual ultra-fast PCR reaction are shown in Fig. 2.
  • the results of Fig. 2 show that the double ultra-fast PCR reaction system can effectively amplify the dual pathogenic bacteria; and the purification of the PCR product purification kit effectively removes Primer dimer, unreacted primer and reaction impurities.
  • Hairpin Probe The four hairpin probes listed in Table 2 above (Hairpin Probe): Hairpin 1, Hairpin 2, Hairpin 3, Hairpin 4 were respectively dissolved in ultrapure water to 100 ⁇ M, heated at 95 ° C for 5 min, and then slowly lowered to room temperature;
  • Salmonella and Staphylococcus aureus were cultured overnight in LB medium, diluted in a gradient, and counted on a plate at concentrations of 10 1 cfu/ml, 10 2 cfu/ml, 10 3 cfu/mL, and 10 4 cfu/mL.
  • 10 5 cfu/mL of Salmonella or Staphylococcus aureus was extracted from the genome by New Industry's bacterial genomic DNA extraction kit. The genomics extracted from the same concentration of Salmonella broth and Staphylococcus aureus broth are mixed (according to a volume ratio of 1:1, that is, 1 uL each), and then used as a template according to the above step (3).
  • the ultra-fast PCR reaction performs a dual ultra-fast PCR reaction.
  • the reaction system (10 ⁇ l) after completion of the reaction was evenly divided into 2 portions, one of which was added with the ultrapure aqueous solution of Hairpin 1 and Hairpin 2 prepared above, and the other was added with the ultrapure aqueous solution of Hairpin 3 and Hairpin 4 prepared above;
  • Self-assembly buffer (8 mM Na 2 HPO 4 , 2.5 mM NaH 2 PO 4 , 0.15 M NaCl, 2 mM MgCl 2 , pH 7.4) was added to each of the two systems, and each hairpin probe was added to each.
  • the final concentration of (HairpinProbe) was 2 ⁇ M, and both systems were 10 ⁇ l; both were incubated at 37 ° C for 20 min to obtain nucleic acid self-assembly products;
  • nucleic acid self-assembly chromogenic system including: take 10 ⁇ L of nucleic acid self-assembly reaction product, add 1 ⁇ L hemin (hemo) stock solution (10 ⁇ M), 32 ⁇ L G-quadruplex induction buffer (100mM2-(4-morpholine) Ethylenesulfonic acid (MES), 40 mM KCl, with a volume percentage of 0.05% Triton X-100, pH 5.5), 23 ⁇ L of ultrapure water; incubate at 37 ° C for 20 min; add 8 ⁇ L of ABTS 2 stock solution (20 mM) and 8 ⁇ L of hydrogen peroxide ( The H 2 O 2 ) stock solution (20 mM) was incubated for 5 min at room temperature in the dark.
  • hemin (hemo) stock solution 10 ⁇ M
  • G-quadruplex induction buffer 100mM2-(4-morpholine) Ethylenesulfonic acid (MES), 40 mM KCl
  • the detection limits of Salmonella and Staphylococcus aureus were determined to be 10 cfu/mL and 10 cfu/mL, respectively, indicating that the new detection method established by the present invention has high sensitivity.
  • the Salmonella broth at a concentration of 10 cfu/mL and the S. aureus broth at 10 cfu/mL were respectively detected by a conventional plate test method and a new method established by the present invention, and the test results are shown in Table 5, and the present invention was established.
  • the new detection method (the detection process is consistent with the above sensitivity experiment process, the different places only extract the genomic DNA of Salmonella bacillus liquid and Staphylococcus aureus liquid with the concentration of 10 cfu/mL, after extraction, each 1 ⁇ L is mixed
  • the average number of colonies detected as a template is close to the average number of colonies detected by the conventional flat panel detection method, indicating that the new detection method established by the present invention has high accuracy.
  • Salmonella, Staphylococcus aureus, Shigella and Escherichia coli were cultured overnight in LB medium to prepare 10 cfu/mL of Salmonella bacterium, 10 cfu/mL of Staphylococcus aureus, 100 cfu/mL.
  • the genomic DNA in different bacterial liquids was extracted from the Herculillus bacterial solution and the 100 cfu/mL E. coli bacterial solution using New Industry's bacterial genomic DNA extraction kit.
  • Hairpin Probe The four hairpin probes listed in Table 2 above: Hairpin 1, Hairpin 2, Hairpin 3, and Hairpin 4 were each dissolved in ultrapure water to 100 ⁇ M, heated at 95 ° C for 5 min, and then slowly cooled to room temperature for use.
  • the three groups of reaction systems (10 ⁇ l) after completion of the reaction were equally divided into 4 portions, and the first portion of each reaction system was added to 3 microwells 1 each having a number of 1 (Hairpin 1 and Hairpin 2 super The pure aqueous solution is dissolved in the micropores 1 in advance), and the second portion of each reaction system is separately added to the three micropores 2 of number 2 (the ultrapure aqueous solution of Hairpin 3 and Hairpin 4 is dissolved in the micropores 2 in advance).
  • each reaction system was added to 3 micropores 3 and 3 micropores 4 respectively (micropores 3 and micropores 4 did not place any Hairpin as a negative control), and then in each microwell Self-assembly buffer (8 mM Na 2 HPO 4 , 2.5 mM NaH 2 PO 4 , 0.15 M NaCl, 2 mM MgCl 2 , pH 7.4) was added, and the final concentration of each hairpin probe (HairpinProbe) was 2 ⁇ M. Each microwell was 10 ⁇ l, and each was incubated at 37 ° C for 20 min to obtain a nucleic acid self-assembly product;
  • the experimental results are shown in Fig. 5.
  • the detection method established by the invention has no cross-reaction to Shigella and Escherichia coli, and can simultaneously achieve dual specific detection of Salmonella and Staphylococcus aureus.

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Abstract

本申请提供了一种致病菌比色传感新方法，包括体外核酸扩增和检测方法，其中体外核酸扩增中使用依次由互补序列、连接臂、互补序列和特异性扩增待测目标的核酸序列组成的引物进行超快速PCR反应，检测中使用互补序列和G-四链体组成的发夹序列实现自组装显色。

Description

一种双重致病菌的比色传感新方法
本申请要求于2018年02月08日提交中国专利局、申请号为201810128946.4、发明名称为“一种双重致病菌的比色传感新方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物检测技术领域，具体涉及一种双重致病菌的比色传感新方法。
背景技术
传统的细菌检测方法主要根据细菌的生理生化特征，经过前增菌、选择性平板分离、生物化学鉴定等步骤，从取样到确定结果需要5-7天，检测周期长，操作繁琐，工作量大。利用抗原抗体反应的特异性，对细菌进行鉴别，已有了半个多世纪的历史，但是微生物抗体的筛选十分繁琐，并且最终的检测特异性不高。分子生物学检测技术的不断完善和发展，克服了传统检测方法实验操作繁琐、耗时长等问题，也使得针对微生物开展的快速检测方法得到迅速发展，但是分子生物学方法的缺点在于结果不可视化，不容易分析结果。
发明内容
本发明建立的比色传感新方法，克服了现有检测技术的不足，实现了对微生物进行准确、快速、简单高效的检测和分析。
本发明的一个目的是提供一种检测方法，所述方法包括体外核酸扩增技术,所述体外核酸扩增技术的反应体系包括上游引物和下游引物，所述上游引物包括：互补序列A、连接臂、互补序列B和可特异性扩增待测目标的核苷酸序列；所述连接臂位于所述互补序列A和互补序列B之间，所述可特异性扩增待测目标的核苷酸序列位于所述上游引物的5’端或3’端；所述互补序列A和所述互补序列B的核苷酸序列互补和/或反向互补；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；所述下游引物包括可特异性扩增待测目标的核苷酸序列；
所述A、B只用于区别不同的互补序列，不用于排序。
所述互补包括现有技术或公知常识所定义的互补或反向互补，和/或根据现有技术或公知常识所定义的互补原则进行互补或反向互补。
所述聚合酶包括可用于体外核酸扩增技术的聚合酶。
所述可特异性扩增待测目标的核苷酸序列具体包括根据待测目标的特征序列所设计的引物序列；所述特征序列包括现有技术或公知常识所定义的特征序列；所述设计包括现有技术或公知常识所记载的设计方法。
具体的，所述方法还包括下述1)-2)中的至少一种：
1)所述体外核酸扩增技术包括超快速PCR反应，所述超快速PCR反应的反 应过程包括：90-98℃，2-6s；50-60℃，2-8s；共20-40个循环；
具体的，所述超快速PCR反应的反应过程包括：95℃，4s；58℃，6s；共30个循环；
具体的，所述超快速PCR反应的反应体系中上游引物和下游引物的浓度为普通PCR浓度的10倍以上；还具体的，为20倍；所述超快速PCR反应的反应体系中还包括DNA聚合酶，所述DNA聚合酶的浓度为普通PCR浓度的10倍以上，还具体的，为60倍；
2)所述连接臂包括具长链结构的化合物。
再具体的，所述连接臂为oxyethyleneglycol，oxyethyleneglycol的化学结构为：
Figure PCTCN2018099324-appb-000001
具体的，所述方法还包括下述1)-8)中的至少一种：
1)所述上游引物包括：将序列表中SEQ ID NO：1和SEQ ID NO：2所示的核苷酸序列通过连接臂连接后得到的引物；
2)所述下游引物包括序列表中SEQ ID NO：3所示的核苷酸序列；
3)所述上游引物包括：将序列表中SEQ ID NO：1和/或SEQ ID NO：2所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：1和/或SEQ ID NO：2所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；
4)所述下游引物包括将序列表中SEQ ID NO：3所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：3所示核苷酸序列具有相同功能的核苷酸序列；
5)所述上游引物包括：将序列表中SEQ ID NO：4和SEQ ID NO：5所示的核苷酸序列通过连接臂连接后得到的引物；
6)所述下游引物包括序列表中SEQ ID NO：6所示的核苷酸序列；
7)所述上游引物包括：将序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；
8)所述下游引物包括将序列表中SEQ ID NO：6所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：6所示核苷酸序列具有相同功能的核苷酸序列。
所述功能包括可实现特异性扩增或检测待测目标。
还具体的，所述上游引物为：
AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol-CTCTCTCTTTCCCT CTCTCTCTCTTTTTTGTGAAATTATCGCCACGTTCGGGCAA和/或
Figure PCTCN2018099324-appb-000002
所述上游引物公众可直接购买得到，其制备方法属于现有技术。
本发明的另一个目的是提供一种检测方法，所述方法包括核酸自组装显色反应,所述核酸自组装显色反应的反应体系包括发卡序列，其特征在于，所述发卡序列包括：构成G-四链体功能核酸序列的全部或部分核苷酸序列、互补序列C和互补序列D；所述构成G-四链体功能核酸序列的全部或部分核苷酸序列位于所述发卡序列的5’端和/或3’端；所述互补序列C与另一个发卡序列的互补序列C互补和/或反向互补；所述互补序列D与待测目标或与待测目标连接的核苷酸序列互补和/或反向互补；
所述C、D只用于区别不同的互补序列，不用于排序。
所述G-四链体功能核酸序列包括现有技术或公知常识所定义的G-四链体功能核酸序列；具体的，所述G-四链体功能核酸序列包括所述序列经自组装后能形成具有类辣根过氧化物酶活性的G-四链体功能核酸，所述G-四链体功能核
酸在氯高铁血红素(hemin)的诱导下，能催化ABTS 2-与H 2O 2生成使反应液呈蓝绿色的物质ABTS-。
具体的，所述发卡序列包括将G-四链体功能核酸序列按照25％和/或75％的比例劈裂，劈裂后的序列分别添加在所述发卡序列的5’端和/或3’端；再具体的，所述劈裂后还需添加T碱基；所述T碱基添加在劈裂后的序列的5’端和/或3’端；
具体的，所述G-四链体功能核酸序列包括序列表中SEQ ID NO：7和/或SEQ ID NO：8所示的核苷酸序列；
具体的，所述互补序列D可与本发明所述的互补序列A和/或互补序列B互补或反向互补；
再具体的，所述发卡序列包括下述1)-4)中的至少一种：
1)序列表中SEQ ID NO：9所示的核苷酸序列和/或SEQ ID NO：9所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：9所示核苷酸序列具有相同功能的核苷酸序列；
2)序列表中SEQ ID NO：10所示的核苷酸序列和/或SEQ ID NO：10所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：10所示核苷酸序列具有相同功能的核苷酸序列；
3)序列表中SEQ ID NO：11所示的核苷酸序列和/或SEQ ID NO：11所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：11所示核苷酸序列具有相同功能的核苷酸序列；
4)序列表中SEQ ID NO：12所示的核苷酸序列和/或SEQ ID NO：12所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：12所示核苷酸序列具有相同功能的核苷酸序列。
还具体的，所述核酸自组装显色反应还包括下述1)-2)中所述的至少一种：
1)所述核酸自组装显色反应的反应条件包括于37℃孵育20min；
2)所述核酸自组装显色反应的反应体系中，所述发夹序列的终浓度为2μM。
本发明的再一个目的是提供一种检测方法，所述方法包括先通过本发明任
一所述方法扩增待测目标，然后再通过本发明任一所述方法检测待测目标。
具体的，所述方法中，本发明所述互补序列D与本发明所述的互补序列A和/或互补序列B互补或反向互补。
具体的，所述方法还包括下述1)-4)中至少一种：
1)通过最终反应体系的颜色变化判断待测物中是否含有待测目标；
具体的，当反应体系的颜色发生变化时，判断待测物中含有待测目标；再具体的，当反应体系的颜色为蓝绿色时，判断待测物中含有待测目标；
2)通过最终反应体系的颜色来制作标准曲线的方法来计算待测物中的待测目标的浓度；
3)通过增加反应体系中的发夹序列的种类来放大待测目标的检测信号；
具体的，每增加一种，信号放大2-4倍；
4)通过增加反应体系中的上游引物或下游引物的种类，和同时增加发夹序列的种类来实现双重或多重检测；
具体的，当所述检测为双重或多重检测时，可通过微阵列法判断待测物中是否含有待测目标或含有几种待测目标；所述微阵列法包括，将所述不同种类的发夹序列分别单独放置于不同的微孔中进行反应，然后根据反应结果判断是否含有待测目标或含有几种待测目标，当微孔中的反应液颜色发生变化或变为蓝绿色时，判断含有待测目标；发生颜色变化或变为蓝绿色的微孔总数为待测物中含有的待测目标的种类总数。
所述发夹序列的种类包括：所述发夹序列中的互补序列D与同一个所述上游引物中的互补序列A和/或B互补或反向互补的发夹序列为同一种类的发夹序列，否则，为不同种类的发夹序列；所述上游引物核苷酸序列相同的为同一个上游引物。
所述上游引物或下游引物的种类包括：可扩增同一待测目标的引物对为同一种类的上游引物或下游引物；反之，可扩增不同待测目标的引物对为不同种类的上游引物或下游引物。
本发明的还一个目的是提供一种试剂盒和/或生物传感器，所述试剂盒和/或生物传感器包括下述1)-2)所述中的至少一种：
1)上游引物和下游引物，所述上游引物包括：互补序列A、连接臂、互补序列B和可特异性扩增待测目标的核苷酸序列；所述连接臂位于所述互补序列A和互补序列B之间，所述可特异性扩增待测目标的核苷酸序列位于所述上游引物的5’端或3’端；所述互补序列A和所述互补序列B的核苷酸序列互补和/或反向互补；所述 连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；所述下游引物包括可特异性扩增待测目标的核苷酸序列；
2)发卡序列，所述发卡序列包括：构成G-四链体功能核酸序列的全部或部分核苷酸序列、互补序列C和互补序列D；所述构成G-四链体功能核酸序列的全部或部分核苷酸序列位于所述发卡序列的5’端和/或3’端；所述互补序列C与另一个发卡序列的互补序列C互补和/或反向互补；所述互补序列D与待测目标或与待测目标连接的核苷酸序列互补和/或反向互补。
所述互补包括现有技术或公知常识所定义的互补或反向互补，和/或根据现有技术或公知常识所定义的互补原则进行互补或反向互补；
所述聚合酶包括可用于体外核酸扩增技术的聚合酶；
所述可特异性扩增待测目标的核苷酸序列具体包括根据待测目标的特征序列所设计的引物序列；所述特征序列包括现有技术或公知常识所定义的特征序列；所述设计包括现有技术或公知常识所记载的设计方法；
具体的，所述连接臂包括具长链结构的化合物；
再具体的，所述连接臂为oxyethyleneglycol，oxyethyleneglycol的化学结构为：
Figure PCTCN2018099324-appb-000003
具体的，所述上游引物和下游引物包括下述1)-8)中的至少一种：
1)所述上游引物包括：将序列表中SEQ ID NO：1和SEQ ID NO：2所示的核苷酸序列通过连接臂连接后得到的引物；
2)所述下游引物包括序列表中SEQ ID NO：3所示的核苷酸序列；
3)所述上游引物包括：将序列表中SEQ ID NO：1和/或SEQ ID NO：2所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：1和/或SEQ ID NO：2所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；
4)所述下游引物包括将序列表中SEQ ID NO：3所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：3所示核苷酸序列具有相同功能的核苷酸序列；
5)所述上游引物包括：将序列表中SEQ ID NO：4和SEQ ID NO：5所示的核苷酸序列通过连接臂连接后得到的引物；
6)所述下游引物包括序列表中SEQ ID NO：6所示的核苷酸序列；
7)所述上游引物包括：将序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID  NO：4和/或SEQ ID NO：5所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；
8)所述下游引物包括将序列表中SEQ ID NO：6所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：6所示核苷酸序列具有相同功能的核苷酸序列；
所述功能包括可实现特异性扩增或检测待测目标。
再具体的，所述上游引物为：
Figure PCTCN2018099324-appb-000004
和/或TGAGGTAGTAGGTTGTATAGTT-oxyethyleneglycol-AACTATACAACC TACTACCTCAAAAAAAAAAAAGCACATAACAAGCG
所述C、D只用于区别不同的互补序列，不用于排序；
所述G-四链体功能核酸序列包括现有技术或公知常识所定义的G-四链体功能核酸序列；具体的，所述G-四链体功能核酸序列包括所述序列经自组装后能形成具有类辣根过氧化物酶活性的G-四链体功能核酸，所述G-四链体功能核酸在氯高铁血红素(hemin)的诱导下，能催化ABTS 2-与H 2O 2生成使反应液呈蓝绿色的物质ABTS -。
具体的，所述发卡序列包括将G-四链体功能核酸序列按照25％和/或75％的比例劈裂，劈裂后的序列分别添加在所述发卡序列的5’端和/或3’端；再具体的，所述劈裂后还需添加T碱基；所述T碱基添加在劈裂后的序列的5’端和/或3’端；
具体的，所述G-四链体功能核酸序列包括序列表中SEQ ID NO：7和/或SEQID NO：8所示的核苷酸序列；
具体的，所述互补序列D可与权利要求1所述的互补序列A和/或B互补或反向互补；
再具体的，所述发卡序列包括下述1)-4)中的至少一种：
1)序列表中SEQ ID NO：9所示的核苷酸序列和/或SEQ ID NO：9所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：9所示核苷酸序列具有相同功能的核苷酸序列；
2)序列表中SEQ ID NO：10所示的核苷酸序列和/或SEQ ID NO：10所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：10所示核苷酸序列具有相同功能的核苷酸序列；
3)序列表中SEQ ID NO：11所示的核苷酸序列和/或SEQ ID NO：11所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：11所示核苷酸序列具有相同功能的核苷酸序列；
4)序列表中SEQ ID NO：12所示的核苷酸序列和/或SEQ ID NO：12所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID  NO：12所示核苷酸序列具有相同功能的核苷酸序列。
具体的，所述试剂盒和/或生物传感器包括下述1)-3)：
1)AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge–CTCTCT CTTTCCCTCTCTCTCTCTTTTTTGTGAAATTATCGCCACGTTCGGGCAA和/或
Figure PCTCN2018099324-appb-000005
2)TCATCGCACCGTCAAAGGAACC和/或GATAAAGAAGAAACCAGCAG；
3)序列表中SEQ ID NO：9、SEQ ID NO：10SEQ ID NO：11、SEQ ID NO：12所示的核苷酸序列中的至少一种。其中，oxyethyleneglycol的化学结构为：
Figure PCTCN2018099324-appb-000006
本发明的最后一个目的是提供本发明任一所述方法、本发明任一所述试剂盒和/或生物传感器的应用。
具体的，所述应用包括下述1)-4)中的至少一种应用：
1)检测微生物；
2)制备检测微生物的产品和/或相关产品中的应用；
3)双重或多重微生物的检测；
4)制备用于双重或多重微生物检测的产品和/或相关产品中的应用。
具体的，所述微生物包括沙门氏菌和/或金黄色葡萄球菌。
可选的，所述任一应用不包括中国专利法第二十五条所述的疾病的诊断和治疗方法。
本发明建立的一种基于超快速PCR的双重比色传感新方法：
(1)该方法建立了超快速PCR反应体系，将耗时3小时左右的传统PCR过程缩减到5分钟，显著减少了PCR反应的用时；
(2)将超快速PCR反应体系搭载核酸自组装显色模块，不仅再次放大了反应信号、有利于实现致病菌的超灵敏检测；而且解决了传统PCR结果难于可视化检测的难题；
(3)将显色体系置于微孔中，经过微孔阵列排序，解决了致病菌的双重检测问题。
本发明的一个具体实施例根据沙门氏菌、金黄色葡萄球菌的毒力基因，设计超快速聚合酶链式反应(Polymerase Chain Reaction,PCR)的扩增引物，结合核酸自组装显色模块，整合建立一种基于超快速PCR的双重比色传感新方法，用于沙门氏菌、金黄色葡萄球菌的超灵敏检测。
本发明具有下述有益技术效果：
1)本发明所建立的检测方法和生物传感器比传统方法更快捷、更灵敏，具备特异性强、灵敏度高、检测结果可靠等优点，可以简化分析检测步骤，缩短分析时间，更重要的是使在线实时检测成为可能，便于携带和野外作业，在包括食品安全和快速检测领域的微生物检测领域，具有非常好的应用前景。
2)本发明所建立的检测方法和生物传感器可同时实现对沙门氏菌和金黄色葡萄球菌的双重特异性检测，检测的特异性好、灵敏度高，检测结果可靠、肉眼即可辨别，检测过程快捷方便，在日常监控或市场筛查等方面具有重要意义。具体的，本发明所建立的检测方法和生物传感器对沙门氏菌、金黄色葡萄球菌检测的检测灵敏度分别为10cfu/mL，10cfu/mL；此外，特异性试验结果表明，本发明所建立的检测方法和生物传感器对志贺氏杆菌、大肠杆菌无交叉反应，可同时实现沙门氏菌、金黄色葡萄球菌的双重特异性检测。
3)本发明核酸自组装显色模块为非酶反应，反应体系成分更简单、反应过程也更简单，减少了终止了酶促反应的步骤，且恒温反应简化了对酶促反应温度的要求，显著降低了经济成本、缩短了反应时间，有助于达到快速简捷检测的要求。
附图说明
图1为超快速PCR装置的结构图；
图2为双重超快速PCR反应的扩增效果验证结果图；其中，泳道1为未经过产物纯化的阴性对照(不添加沙门氏菌、金黄色葡萄球菌基因组的双重超快速PCR反应体系)；泳道2为未经过产物纯化的阳性样品(添加沙门氏菌、金黄色葡萄球菌基因组的双重超快速PCR反应体系)；泳道3为经过产物纯化的阴性对照(不添加沙门氏菌、金黄色葡萄球菌基因组的双重超快速PCR反应体系)；泳道4为经过产物纯化的阳性样品(添加沙门氏菌、金黄色葡萄球菌基因组的双重超快速PCR反应体系)；
图3为沙门氏菌的标准曲线图；
图4为金黄色葡萄球菌的标准曲线图；
图5为特异性实验结果图，其中1为微孔1,2为微孔2,3为微孔3,4为微孔4；a为对沙门氏菌与志贺氏杆菌样品进行检测的结果图；b为对大肠杆菌与金黄色葡萄球菌样品进行检测的结果图；c为对沙门氏菌与金黄色葡萄球菌样品进行检测的结果图。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中未作具体说明的分子生物学实验方法，均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行，或者按照试剂盒和产品说明书进行。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、一种用于检测沙门氏菌、金黄色葡萄球菌的基于超快速PCR的双重比色传感新方法的建立
(一)实验材料
本实施例所采用的菌株信息见表1，所设计的引物的核苷酸序列见表2和序列表。
表1
Figure PCTCN2018099324-appb-000007
表2
Figure PCTCN2018099324-appb-000008
表2中，上游引物Primer 1的连接臂(oxyethyleneglycol bridge)左侧的核苷酸序列为序列表中SEQ ID NO：1所示的核苷酸序列，连接臂右侧的核苷酸序列为序列表中SEQ ID NO：2所示的核苷酸序列，连接臂的化学结构为：
Figure PCTCN2018099324-appb-000009
表2中，下游引物Primer 2的核苷酸序列为序列表中SEQ ID NO：3所示的核苷酸序列。
表2中，上游引物Primer 3的连接臂(oxyethyleneglycol bridge)左侧的核苷酸序列为序列表中SEQ ID NO：4所示的核苷酸序列，连接臂右侧的核苷酸序列为序列表中SEQ ID NO：5所示的核苷酸序列，连接臂的化学结构与Primer 1的连接臂的化学结构相同。
表2中，下游引物Primer 4的核苷酸序列为序列表中SEQ ID NO：6所示的核苷酸序列。
表2中，发卡序列1-4(Hairpin 1、Hairpin 2、Hairpin 3、Hairpin 4)为将2种G-四链体功能核酸序列：序列表中SEQ ID NO：7所示的核苷酸序列AGGG CGGG TGGG TGGG和SEQ ID NO：8所示的核苷酸序列TGGG TGGG TAGGG CGGG，按照约25％，75％的比例劈裂，分别添加在发卡探针(HairpinProbe)的两端。且添加T碱基保护劈裂的G-四链体序列。经过“引发剂”(Intiator)促发核酸自组装，使劈裂的G-四链体在距离上相互靠近，在氯高铁血红素(hemin)的诱导下，形成具有类辣根过氧化物酶活性的G-四链体功能核酸，发挥催化ABTS 2-与H 2O 2显色的功能。具体的，表2中发卡序列Hairpin 1的核苷酸序列为序列表中SEQ ID NO：9所示的核苷酸序列；Hairpin 2的核苷酸序列为序列表中SEQ ID NO：10所示的核苷酸序列；Hairpin 3的核苷酸序列为序列表中SEQ ID NO：11所示的核苷酸序列；Hairpin4的核苷酸序列为序列表中SEQ ID NO：12所示的核苷酸序列。
表2中所列的序列均为人工合成。
Ex Taq DNA聚合酶,10×Ex Taq Buffer(20mM Mg2+Plus)与dNTP Mixture(2.5mM)均购自宝生物公司(TAKaRa)。氯高铁血红素(Hemin)与2，2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二胺盐(ABTS2-)购自阿拉丁试剂(Sigma-Aldrich Chemical Co.)。实验用水均来自Milli-Q纯水系统。其他试剂均购自国药集团。
(二)超快速PCR装置的搭建
超快速PCR装置的主要结构如图1所示，其具体的结构、连接方式和工作原理、工作过程包括：
超快速PCR装置采用Light Cycler型号的毛细管(20uL，04 929 292 001，Roche)作为PCR样品室，通过快速离心的方式，样品会分别聚集到各个毛细管一端，离心完成后带有样品的毛细管被固定在塑料支架上。塑料支架连接到步进电机(42JSF630AS-1000，Just Motioin Control)上，由该步进电机带动固定在塑料支架上的毛细管样品室在95℃的高温水浴锅和58℃的中温水浴锅之间循环转换，实现超快速PCR反应过程中的反应温度变化及控制。所述步进电机由开关电源(S-100-24，Elecall)进行供电，采用直流伺服电机驱动器(YZ-ACSD60，Moving)以及Labview(version 2014)实现步进电机的上述循环转换的频率或时间的控制。温度测量利用封装在毛细管中的热电偶来实现。热电偶信号的放大及线性化处理过程则利用温度变送器(SBWR-2260，K，Yuancheng)进行传递并采用Arduino UNO v1.0芯片进行处理。 Arduino UNO芯片将接收到的温度的模拟信号转换成数字信号，然后由Arduino IDE(version 1.8.1)模块执行运算。
(三)双重超快速PCR反应
1)配制双重超快速PCR反应体系，具体见表3：
表3
Figure PCTCN2018099324-appb-000010
将沙门氏菌与金黄色葡萄球菌在LB培养基中过夜培养活化，采用New Industry公司的细菌基因组DNA提取试剂盒分别提取沙门氏菌菌液和金黄色葡萄球菌菌液中的基因组DNA，将提取到的两种基因组DNA各取1μL混合，作为表3中的模板。表3中的引物1-4(Primer 1、Primer 2、Primer 3、Primer 4)具体为上述表2中所列的引物1-4(Primer 1、Primer 2、Primer 3、Primer 4)。
2)双重超快速PCR反应过程：
按照表3，在冰上配制10微升反应体系，迅速置于步骤(二)搭建的超快速PCR反应装置中进行温度控制，温度控制及循环数见表4：
表4
Figure PCTCN2018099324-appb-000011
3)双重超快速PCR反应的扩增效果验证：
完成上述双重超快速PCR反应过程后，使用2％溴化乙锭预染色的琼脂糖凝胶电泳验证双重超快速PCR反应体系的扩增效果，电泳条件：130V for 25min，
拍照系统：Molecular Imager Gel Doc XR(Bio-Rad)。并使用PCR产物纯化试
剂盒(上海生工)去除引物二聚体、未反应的引物与反应杂质。
双重超快速PCR反应的扩增效果验证结果见图2，图2结果表明：双重超快速PCR反应体系实现了双重致病菌的有效扩增；且经过PCR产物纯化试剂盒的纯化，有效去除了引物二聚体、未反应的引物与反应杂质。
(四)核酸自组装显色模块的建立与双重致病菌的可视化检测
1)灵敏度实验
沙门氏菌、金黄色葡萄球菌各自的标准曲线的绘制：
将上述表2所列的4条发夹探针(HairpinProbe)：Hairpin 1，Hairpin 2，Hairpin3，Hairpin 4分别用超纯水溶解至100μM，于95℃加热5min，后缓慢降至室温；
将沙门氏菌与金黄色葡萄球菌在LB培养基中过夜培养活化，将梯度稀释、经过平板计数，浓度分别为10 1cfu/ml,10 2cfu/ml,10 3cfu/mL,10 4cfu/mL,10 5cfu/mL的沙门氏菌菌液或金黄色葡萄球菌菌液采用New Industry公司的细菌基因组DNA提取试剂盒分别提取基因组。将从相同浓度的沙门氏菌菌液和金黄色葡萄球菌菌液中提取到的基因组混合(按照体积比1:1进行混合，即各取1uL)后作为模板，按照上述步骤(三)所述的双重超快速PCR反应进行双重超快速PCR反应。将完成反应后的反应体系(10微升)平均分成2份，其中一份加入上述制备的Hairpin 1、Hairpin2的超纯水溶液，另一份中加入上述制备的Hairpin3、Hairpin4的超纯水溶液；然后再分别在两份体系中加入自组装缓冲液(8mM Na 2HPO 4,2.5mM NaH 2PO 4,0.15M NaCl,2mM MgCl 2,pH 7.4)，并使每份中每一种发夹探针(HairpinProbe)的终浓度为2μM，且两份体系均为10微升；均于37℃孵育20min，得核酸自组装产物；
建立核酸自组装显色体系，体系包括：取10μL核酸自组装反应产物，加入1μL氯高铁血红素(hemin)储液(10μM)，32μL G-四链体诱导缓冲液(100mM2-(4-morpholine)ethanesulfonic acid(MES),40mMKCl,与体积百分数为0.05％Triton X-100,pH 5.5)，23μL超纯水；于37℃孵育20min；加入8μL ABTS2-储液(20mM)与8μL过氧化氢(H 2O 2)储液(20mM)于室温避光孵育5min。反应完成后利用分光光度计检测反应液在415nm的OD值，绘制沙门氏菌、金黄色葡萄球菌各自的标准曲线图，绘制结果如图3和图4所示。
根据绘制的标准曲线与3σ原则，确定沙门氏菌与金黄色葡萄球菌的检测限分别为：10cfu/mL，10cfu/mL，说明本发明所建立的检测新方法灵敏度高。
标准曲线的绘制和检测限的确定方法按照文献Macdougall,D.,Crummett,W.B.,1980.Anal.Chem.52(14),2242-2249.所记载的方法进行。
2)准确度实验
加标回收检测实验：
将浓度为10cfu/mL的沙门氏菌菌液与10cfu/mL的金黄色葡萄球菌菌液分别用传统的平板检测法和本发明建立的新方法进行检测，检测结果如表5所示，本发明所建立的检测新方法(检测过程同上述灵敏度实验过程一致，不同的地方只是提取的是浓度均为10cfu/mL的沙门氏菌菌液和金黄色葡萄球菌菌液的基因组DNA，提取后，各取1μL混合后作为模板)所检测出的平均菌落数接近于传统平板检测法检测出的平均菌落数，说明本发明所建立的检测新方法准确度高。
表5
Figure PCTCN2018099324-appb-000012
Figure PCTCN2018099324-appb-000013
3)特异性实验
将沙门氏菌、金黄色葡萄球菌、志贺氏杆菌与大肠杆菌在LB培养基中过夜培养活化，制备成10cfu/mL的沙门氏菌菌液、10cfu/mL的金黄色葡萄球菌菌液、100cfu/mL的志贺氏杆菌菌液、100cfu/mL的大肠杆菌菌液，采用New Industry公司的细菌基因组DNA提取试剂盒分别提取不同菌液中的基因组DNA。将从沙门氏菌菌液与志贺氏杆菌菌液中提取的基因组各取1μL混合作为模板,计为反应体系1；将从大肠杆菌菌液与金黄色葡萄球菌菌液中提取的基因组各取1μL混合作为模板,计为反应体系2；将从沙门氏菌菌液与金黄色葡萄球菌菌液中提取的基因组各取1μL混合作为模板,计为反应体系3，按照上述步骤(三)所述的双重超快速PCR反应(除模板做相应的替换外，其它均一致)进行3组反应体系的双重超快速PCR反应。
将上述表2所列的4条发夹探针(HairpinProbe)：Hairpin 1，Hairpin 2，Hairpin3，Hairpin 4分别用超纯水溶解至100μM，于95℃加热5min，后缓慢降至室温备用。
分别将完成反应后的3组反应体系(10微升)平均分成4份，每组反应体系的第一份分别加入到3个编号均为1的微孔1中(Hairpin 1与Hairpin 2的超纯水溶液提前溶解在微孔1中)，每组反应体系的第二份分别加入到3个编号均为2的微孔2中(Hairpin 3与Hairpin 4的超纯水溶液提前溶解在微孔2中)，每组反应体系的其余两份分别添加到3个微孔3与3个微孔4中(微孔3与微孔4则不放置任何Hairpin作为阴性对照)，再分别在每个微孔中加入自组装缓冲液(8mM Na 2HPO 4,2.5mM NaH 2PO 4,0.15M NaCl,2mM MgCl 2,pH 7.4)，并使每一种发夹探针(HairpinProbe)的终浓度为2μM，每个微孔均为10微升，均于37℃孵育20min，得核酸自组装产物；
在每个微孔中加入1μLhemin储液(10μM)，32μL G-四链体诱导缓冲液(100mM 2-(4-morpholine)ethanesulfonic acid(MES),40mMKCl,与体积百分数为0.05％Triton X-100,pH 5.5)，23μL超纯水；于37℃孵育20min；加入8μL ABTS 2-储液(20mM)与8μL过氧化氢(H 2O 2)储液(20mM)于室温避光孵育5min。
实验结果如图5所示，本发明所建立的检测方法对志贺氏杆菌、大肠杆菌无交叉反应，可同时实现沙门氏菌、金黄色葡萄球菌的双重特异性检测。
以上所述实施例仅表达了本发明的实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制，但凡采用等同替换或等效变换的形式所获得的技术方案，均应落在本发明的保护范围之内。
Figure PCTCN2018099324-appb-000014
Figure PCTCN2018099324-appb-000015
Figure PCTCN2018099324-appb-000016

Claims (10)

  1. 一种检测方法，所述方法包括体外核酸扩增技术,所述体外核酸扩增技术的反应体系包括上游引物和下游引物，其特征在于，所述上游引物包括：互补序列A、连接臂、互补序列B和可特异性扩增待测目标的核苷酸序列；所述连接臂位于所述互补序列A和互补序列B之间，所述可特异性扩增待测目标的核苷酸序列位于所述上游引物的5’端或3’端；所述互补序列A和所述互补序列B的核苷酸序列互补和/或反向互补；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增反应过程中新链延伸的结构；所述下游引物包括可特异性扩增待测目标的核苷酸序列。
  2. 根据权利要求1所述的方法，其特征在于，所述方法还包括下述1)-2)中的至少一种：
    1)所述体外核酸扩增技术包括超快速PCR反应，所述超快速PCR反应的反应过程包括：90-98℃，2-6s；50-60℃，2-8s；共20-40个循环；
    2)所述连接臂包括具长链结构的化合物。
  3. 根据权利要求1和/或2所述的方法，其特征在于，所述方法还包括下述1)-8)中的至少一种：
    1)所述上游引物包括：将序列表中SEQ ID NO：1和SEQ ID NO：2所示的核苷酸序列通过连接臂连接后得到的引物；
    2)所述下游引物包括序列表中SEQ ID NO：3所示的核苷酸序列；
    3)所述上游引物包括：将序列表中SEQ ID NO：1和/或SEQ ID NO：2所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：1和/或SEQ ID NO：2所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；
    4)所述下游引物包括将序列表中SEQ ID NO：3所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：3所示核苷酸序列具有相同功能的核苷酸序列；
    5)所述上游引物包括：将序列表中SEQ ID NO：4和SEQ ID NO：5所示的核苷酸序列通过连接臂连接后得到的引物；
    6)所述下游引物包括序列表中SEQ ID NO：6所示的核苷酸序列；
    7)所述上游引物包括：将序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：4和/或SEQ ID NO：5所示核苷酸序列具有相同功能的核苷酸序列通过连接臂连接后得到的引物；
    8)所述下游引物包括将序列表中SEQ ID NO：6所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：6所示核苷酸序列具有相同功能的核苷酸序列。
  4. 一种检测方法，所述方法包括核酸自组装显色反应,所述核酸自组装显色反应 的反应体系包括发卡序列，其特征在于，所述发卡序列包括：构成G-四链体功能核酸序列的全部或部分核苷酸序列、互补序列C和互补序列D；所述构成G-四链体功能核酸序列的全部或部分核苷酸序列位于所述发卡序列的5’端和/或3’端；所述互补序列C与另一个发卡序列的互补序列C互补和/或反向互补；所述互补序列D与待测目标或与待测目标连接的核苷酸序列互补和/或反向互补。
  5. 根据权利要求4所述的方法，其特征在于，所述发卡序列包括下述1)-4)中的至少一种：
    1)序列表中SEQ ID NO：9所示的核苷酸序列和/或SEQ ID NO：9所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：9所示核苷酸序列具有相同功能的核苷酸序列；
    2)序列表中SEQ ID NO：10所示的核苷酸序列和/或SEQ ID NO：10所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：10所示核苷酸序列具有相同功能的核苷酸序列；
    3)序列表中SEQ ID NO：11所示的核苷酸序列和/或SEQ ID NO：11所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：11所示核苷酸序列具有相同功能的核苷酸序列；
    4)序列表中SEQ ID NO：12所示的核苷酸序列和/或SEQ ID NO：12所示核苷酸序列经过一个或几个核苷酸的取代和/或缺失和/或添加且与序列表中SEQ ID NO：12所示核苷酸序列具有相同功能的核苷酸序列。
  6. 根据权利要求4和/或5所述的方法，其特征在于，所述核酸自组装显色反应还包括下述1)-2)中所述的至少一种：
    1)所述核酸自组装显色反应的反应条件包括于37℃孵育20min；
    2)所述核酸自组装显色反应的反应体系中，所述发夹序列的终浓度为2μM。
  7. 一种检测方法，其特征在于，所述方法包括先通过权利要求1、2和/或3所述方法扩增待测目标，然后再通过权利要求4、5和/或6所述方法检测待测目标；具体的，其中，权利要求4所述互补序列D与权利要求1所述的互补序列A和/或互补序列B互补或反向互补。
  8. 根据权利要求7所述的方法，其特征在于，所述方法还包括下述1)-4)中至少一种：
    1)通过最终反应体系的颜色变化判断待测物中是否含有待测目标；
    2)通过最终反应体系的颜色来制作标准曲线的方法来计算待测物中的待测目标的浓度；
    3)通过增加反应体系中的发夹序列的种类来放大待测目标的检测信号；
    4)通过增加反应体系中的上游引物或下游引物的种类，和同时增加发夹序列的种类来实现双重或多重检测。
  9. 一种试剂盒和/或生物传感器，其特征在于，所述试剂盒和/或生物传感器包括下述1)-2)所述中的至少一种：
    1)上游引物和下游引物，所述上游引物包括：互补序列A、连接臂、互补序列B和可特异性扩增待测目标的核苷酸序列；所述连接臂位于所述互补序列A和互补序列B之间，所述可特异性扩增待测目标的核苷酸序列位于所述上游引物的5’端或3’端；所述互补序列A和所述互补序列B的核苷酸序列互补和/或反向互补；所述连接臂包括可抑制聚合酶结合的结构和/或可抑制体外核酸扩增过程中新链延伸的结构；所述下游引物包括可特异性扩增待测目标的核苷酸序列；
    2)发卡序列，所述发卡序列包括：构成G-四链体功能核酸序列的全部或部分核苷酸序列、互补序列C和互补序列D；所述构成G-四链体功能核酸序列的全部或部分核苷酸序列位于所述发卡序列的5’端和/或3’端；所述互补序列C与另一个发卡序列的互补序列C互补和/或反向互补；所述互补序列D与待测目标或与待测目标连接的核苷酸序列互补和/或反向互补。
  10. 权利要求1、2和/或3所述方法、权利要求4和/或5所述方法、权利要求6和/或7所述方法、权利要求8和/或9所述试剂盒和/或生物传感器的应用。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114214392A (zh) * 2021-11-17 2022-03-22 广东省科学院生态环境与土壤研究所 一种基于核酸四面体的冠状病毒定量检测方法及其应用

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108251514A (zh) * 2018-02-08 2018-07-06 中国农业大学 一种双重致病菌的比色传感新方法
CN109082459A (zh) * 2018-07-18 2018-12-25 桂林理工大学 Taq DNA连接酶和量子点信号放大的DNA电化学传感器检测单核苷酸多态性的方法
CN109207615B (zh) * 2018-10-12 2021-06-29 广东省生态环境技术研究所 一种免标记荧光检测金黄色葡萄球菌mecA基因的方法及检测试剂盒
CN111154843B (zh) * 2020-04-07 2020-07-28 中国农业大学 一种基于超速pcr与功能核酸显色的定量检测方法
CN113493823A (zh) * 2020-04-07 2021-10-12 中国农业大学 一种基于超速pcr与功能核酸的可视化检测方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1274847A (zh) * 2000-06-02 2000-11-29 上海长征医院 标记信号放大探针的制备方法
CN102827836A (zh) * 2012-06-11 2012-12-19 中国科学院成都生物研究所 一种寡核苷酸探针以及用其对靶分子进行检测的方法
CN103993100A (zh) * 2014-06-17 2014-08-20 东南大学 一种提高基因连接测序准确性的方法
CN105018474A (zh) * 2014-08-22 2015-11-04 江苏省原子医学研究所 一种基于g-四链体-氯血红素dna酶的探针及其应用
CN108251514A (zh) * 2018-02-08 2018-07-06 中国农业大学 一种双重致病菌的比色传感新方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1274847A (zh) * 2000-06-02 2000-11-29 上海长征医院 标记信号放大探针的制备方法
CN102827836A (zh) * 2012-06-11 2012-12-19 中国科学院成都生物研究所 一种寡核苷酸探针以及用其对靶分子进行检测的方法
CN103993100A (zh) * 2014-06-17 2014-08-20 东南大学 一种提高基因连接测序准确性的方法
CN105018474A (zh) * 2014-08-22 2015-11-04 江苏省原子医学研究所 一种基于g-四链体-氯血红素dna酶的探针及其应用
CN108251514A (zh) * 2018-02-08 2018-07-06 中国农业大学 一种双重致病菌的比色传感新方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114214392A (zh) * 2021-11-17 2022-03-22 广东省科学院生态环境与土壤研究所 一种基于核酸四面体的冠状病毒定量检测方法及其应用
CN114214392B (zh) * 2021-11-17 2024-06-11 广东省科学院生态环境与土壤研究所 一种基于核酸四面体的冠状病毒定量检测方法及其应用

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