CN116426619B - Multiple target nucleotide detection kit, method and application - Google Patents
Multiple target nucleotide detection kit, method and application Download PDFInfo
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- 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
- C12Q1/68—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
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/44—Staphylococcus
- C12R2001/445—Staphylococcus aureus
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/63—Vibrio
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a multi-target nucleotide detection kit, a method and application thereof, belonging to the field of biotechnology, and the whole reaction system comprises: at least one pair of specific primer pairs, wherein 1 RNA base is introduced into the middle part of the specific primer, and the 3' -end of the primer is blocked; at least one blocking probe P blocked at the 3 'end, near the 3' end portion sequence specific for target binding, the 5 'end portion sequence being an artificially introduced tag sequence, the 5' end modifying fluorescent group or quenching group; artificially introduced substrate PS: a 3' end modified quenching group or a fluorescent group. The method can detect more than 3 targets in a single fluorescent channel, detect more than ten targets and even more than twenty targets in a single tube, has the advantages of low design difficulty, good specificity, high sensitivity, low cost and the like, and has extremely high clinical application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a multiple target nucleotide detection kit, a method and application.
Background
The multiple fluorescence quantitative PCR (Multiple fluorescence quantitative PCR) technology is based on the fluorescence quantitative PCR technology, and utilizes the combination of fluorescent groups of different channels to realize the real-time quantitative detection of a plurality of targets by combining the detection capability of an instrument to the fluorescence of different channels.
The chemiluminescent principle of Real-time qPCR can be divided into 2 general categories:
one type is a multiplex PCR technology based on fluorescent dye, after the non-specific fluorescent dye is embedded into DNA double-strand minor groove, fluorescent light with a certain wavelength is generated after the excitation of specific excitation light, a melting curve analysis method is developed based on the fluorescent dye, the characteristics of different DNA sequences with different Tm values are utilized, after the PCR reaction is finished, the double strand is gradually melted into a single strand through temperature programming, when the temperature corresponding to the specific melting of the double strand is reached, the fluorescence intensity is greatly reduced, and double-strand products with different lengths or double strands with different GC contents with the same length in PCR can be analyzed by utilizing the principle.
Another class is specific probes that label fluorophores, including Taqman hydrolysis probes and molecular beacons. The TaqMan hydrolysis probe (Hydrolysis probes) is a commonly used probe in a multiplex fluorescence PCR system, one end of the probe is marked with a fluorescent group, the other end is marked with a quenching group, different TaqMan hydrolysis probes can be formed by marking different fluorescent groups and corresponding quenching groups at the tail ends of different sequences, and the probes and corresponding amplification primers are added into the same reaction system, so that the common detection of a plurality of targets can be realized. Molecular beacons (Molecular beacons) are another commonly used probe in multiplex PCR systems, and based on the principle of fluorescence resonance energy transfer (Fluorescence resonance energy transfer, FRET), when no specific target is present in the system, molecular beacons spontaneously form stem-loop structures, quenching groups and fluorophores are in proximity to each other so that FRET occurs and fluorescence does not occur. If a specific target is present in the system, under certain conditions, the stem-loop structure will open and renature with the target, thereby generating a fluorescent signal. Then the simultaneous detection of multiple targets can be realized by adding several target-specific molecular beacons in the same reaction system.
Because the fluorescent groups emit fluorescence of a single wavelength, and the degree of distinction of fluorescent signals emitted by different fluorescent groups by a PCR instrument is limited, the existing PCR instrument can only detect 4-5 different fluorescent signals. In contrast, in the fluorescent dye-based multiplex PCR technology, only one fluorescent dye is usually used in the same reaction system because the dye does not have specific recognition capability. Therefore, the conventional RT-PCR detection kit based on the probe method only carries out amplification detection on at most 4 targets in a one-tube reaction system. Although there are also some technical methods for multiplex target amplification, it is generally necessary to introduce a plurality of primers and probes into a tube system, and the presence of a large number of primer fragments in the same system leads to explosive growth of primer dimers, while presenting a very high risk of non-specific and false positive detection, and presenting a great challenge in terms of primer design and long-term stability of the premixed system.
Disclosure of Invention
In order to solve the problems, the invention provides a multi-target nucleotide detection technology and application, solves the defects of low flux, multiple amplification primer dimer, non-specific amplification and the like of the existing fluorescent quantitative PCR platform, provides a multi-PCR detection method with high detection flux, strong multi-detection capability, high specificity and low cost for clinic, and can be widely applied to detection of various infectious diseases.
According to the method for detecting the multiple target nucleotide, the system has no open 3' -end, and primer extension can be started only when a target sequence exists, so that the formation of primer dimer is eliminated, non-specific amplification is reduced, the design difficulty of the primer is reduced, and the specificity of the whole detection flow is improved.
In the present invention, "Tm" refers to the temperature at which dsDNA melts to half.
In the present invention, all references to range values include the endpoints.
In one aspect, the invention provides a multiplex target nucleotide detection kit.
The kit is a real-time fluorescence PCR kit.
The kit comprises:
(1) At least one pair of specific primer pairs: 1 RNA base is introduced into the middle part of the specific primer, and the 3' -end of the primer is blocked;
(2) At least one blocking probe P: the 3 'end is blocked, the sequence specificity and target binding are close to the 3' end, the 5 'end is a tag sequence, and the 5' end modifies a fluorescent group or a quenching group;
(3) Artificially introduced substrate PS: a 3' end modified quenching group or a fluorescent group.
The blocking is the addition of blocking groups.
Preferably, the kit further comprises:
(4) A thermostable Taq DNA polymerase having 5 '. Fwdarw.3' DNA polymerase activity and 5 '. Fwdarw.3' exonuclease activity;
(5) Endonucleases that specifically cleave RNA bases of RNA and DNA hybrid strands.
Preferably, the Taq DNA polymerase is a DNA polymerase isolated from the thermophilic bacterium Thermus aquaticus (thermus aquaticus).
Preferably, the endonuclease is thermostable RNase HII, and is derived from artificially modified thermophilic bacteriaThermus thermophilus、Pyrococcus abyssi). Depending on the reaction temperature, different RNase HII may be used. In some embodiments, the reaction temperature is from 50 ℃ to 70 ℃, preferably from 55 ℃ to 65 ℃.
Preferably, the working concentration of the endonuclease is 0.5mU/uL to 20mU/uL.
The blocking probe P is combined with the target sequence at a position between the specific primer pair, and the Tm value of the blocking probe P combined with the target sequence is higher than that of the specific primer.
The tag sequence is cut by polymerase in the presence of the target sequence, is combined with a substrate PS and extends to generate a characteristic melting peak related to the Tm value of the substrate PS in melting curve analysis.
The length of the specific primer sequence is between 16bp and 30bp, the Tm value is between 45 ℃ and 65 ℃ and the specific primer sequence comprises the endpoint value. Preferably, the length of the specific primer sequence is 17bp-30bp, 18bp-30bp, 19bp-30bp, 20bp-30bp, 23bp-30bp, 25bp-30bp, 28bp-30bp, 16bp-18bp, 16bp-20bp, 16bp-25bp; the Tm value is 45-55 ℃, 55-65 ℃, 50-60 ℃, 48-62 ℃, 52-63 ℃, 52-60 ℃, 55-63 ℃ or 45-65 ℃.
The sequence length for blocking the combination of the probe P and the target is between 20bp and 35bp, the Tm value is between 55 ℃ and 75 ℃ and the sequence length comprises the endpoint value. Preferably, the sequence length for blocking the combination of the probe P and the target is 20bp-35bp, 22bp-35bp, 23bp-35bp, 25bp-35bp, 28bp-35bp, 32bp-35bp, 20bp-28bp or 22bp-26bp; the Tm value is 60-75 ℃, 65-75 ℃, 70-75 ℃, 68-75 ℃, 55-65 ℃, 55-70 ℃, 58-66 ℃ or 62-73 ℃.
The length of the tag sequence carried by the blocking probe P is 5bp-25bp, the Tm value is 20-45 ℃ and the tag sequence comprises the endpoint value. Preferably, the blocking probe P has a tag sequence with the length of 8bp-25bp, 10bp-25bp, 12bp-25bp, 15bp-25bp, 19bp-25bp, 22bp-25bp, 24bp-25bp, 5bp-15bp, 8bp-29bp or 17bp-19bp; the Tm value is 25-45 ℃, 28-45 ℃, 32-45 ℃, 35-45 ℃, 38-45 ℃, 20-38 ℃, 26-40 ℃, 28-38 ℃, 28-32 ℃ or 30-32 ℃.
The sequence length of the substrate PS is between 10bp and 70bp and comprises an endpoint value, and the Tm value is between 50 ℃ and 90 ℃ and comprises an endpoint value. Preferably, the sequence length of the substrate PS is 20bp-70bp, 30bp-70bp, 40bp-70bp, 50bp-70bp, 60bp-70bp, 10bp-20bp, 10bp-50bp, 20bp-50bp or 30bp-40bp; the Tm value is 60-90 ℃, 70-90 ℃, 80-90 ℃, 50-60 ℃, 50-70 ℃, 50-80 ℃, 55-85 ℃, 68-82 ℃, 75-80 ℃ or 78-83 ℃.
Preferably, the labeled fluorescent groups at the 5 'end of the blocking probe P and the 3' end of the substrate PS are selected from FAM, VIC, TET, CAL Gold 540, JOE, HEX, TAMRA, ROX, CY3, CY5, and the labeled quenching groups are selected from DABCYL, BHQ-1, BHQ-2, ECLIPE.
Preferably, the specific primer and the blocking group labeled at the 3' end of the blocking probe P are selected from the group consisting of: phosphate, BHQ1, BHQ2, BHQ3, C3 Spacer, spacer 9,3'C6 Spacer,dSpacer,PC Spacer or Spacer 18.
The tag sequence of the 5 '-end marked fluorescent group or the quenching group of the blocking probe is cut off in the common amplification cycle, and the cut tag sequence can be specifically combined with the corresponding substrate of the 3' -end marked quenching group or the fluorescent group and is extended under the action of the thermostable Taq DNA polymerase to generate a double-chain substrate of fluorescence quenching.
In the single channel, different targets are distinguished by corresponding characteristic peaks of double-stranded products quenched by fluorescence with different Tm values at different positions in a melting curve. The melting curve procedure is as follows: maintaining at 95 ℃ for 2min and at 50 ℃ for 2min, and then collecting fluorescent signals at 50-85 ℃ at a heating rate of 0.02 ℃/s-0.2 ℃/s.
Preferably, the kit is a kit for the combined detection of food-borne pathogenic bacteria, the type of food-borne pathogenic bacteria comprising one or more of salmonella, staphylococcus aureus, escherichia coli O157, listeria monocytogenes, vibrio and yersinia small intestine.
Further preferably, the primer sequence in the kit is selected from SEQ ID NO.1-12.
Further preferably, the sequence of probe P in the kit is selected from SEQ ID NO.13-18.
Further preferably, the sequence of the substrate PS in the kit is selected from SEQ ID NO.19-24.
Preferably, the kit is a kit for the combined detection of lower respiratory tract pathogens, wherein the lower respiratory tract pathogens comprise one or more of pseudomonas aeruginosa, acinetobacter baumannii, klebsiella pneumoniae, haemophilus influenzae, legionella pneumophila and streptococcus pneumoniae.
Further preferably, the primer sequence in the kit is selected from SEQ ID NO.25-36.
Further preferably, the sequence of probe P in the kit is selected from SEQ ID NO.37-42.
Further preferably, the sequence of the substrate PS in the kit is selected from SEQ ID NO.43-48.
In another aspect, the invention provides a method of detecting multiple target nucleotides.
The detection method is completed by the kit.
The PCR reaction program in the detection method is set as follows:
in yet another aspect, the invention provides the use of the aforementioned kit for detecting food-borne pathogenic bacteria.
The food-borne pathogenic bacteria type comprises one or more of salmonella, staphylococcus aureus, escherichia coli O157, listeria monocytogenes, vibrio and yersinia small intestine.
Preferably, the sequence of the primer required for multiplex PCR detection in the detection method is selected from SEQ ID NO.1-12.
Preferably, the sequence of blocking probe P required for multiplex PCR detection in the detection method is selected from SEQ ID NO.13-18.
Preferably, the sequence of the substrate PS required for multiplex PCR detection in the detection method is selected from SEQ ID NO.19-24.
Preferably, the salmonella, staphylococcus aureus and escherichia coli O157 are detected in the same channel, sharing FAM fluorescent group label, listeria monocytogenes, vibrio and yersinia small intestine are detected in the same channel, sharing VIC fluorescent group label.
In yet another aspect, the invention provides the use of the aforementioned kit for detecting a lower respiratory pathogen.
The lower respiratory tract pathogenic bacteria comprise one or more of pseudomonas aeruginosa, acinetobacter baumannii, klebsiella pneumoniae, haemophilus influenzae, legionella pneumophila and streptococcus pneumoniae.
Preferably, the sequence of the primer required for multiplex PCR detection in the detection method is selected from SEQ ID NO.25-36.
Preferably, the sequence of blocking probe P required for multiplex PCR detection in the detection method is selected from SEQ ID NO.37-42.
Preferably, the sequence of the substrate PS required for multiplex PCR detection in the detection method is selected from SEQ ID NO.43-48.
Preferably, the pseudomonas aeruginosa, the acinetobacter baumannii and the klebsiella pneumoniae are detected in the same channel, the 5 'end of the probe is marked by a BHQ3 quenching group, and the 3' end of the substrate is marked by a CY5 fluorescent group; the haemophilus influenzae, the Legionella pneumophila and the streptococcus pneumoniae are detected in the same channel, the 5 'end of the probe is marked by a BHQ2 quenching group, and the 3' end of the substrate is marked by a ROX fluorescent group.
The technical principle of the invention is as follows: the endonuclease specifically cuts RNA base when the specific primer is combined with the target sequence to generate an extendable specific primer, the thermostable Taq DNA polymerase cuts off the tag sequence of the 5 '-end marked fluorescent group of the blocking probe in the extension process, the 3' -end of the cut tag sequence is free hydroxyl, can be specifically combined with the 3 '-end part sequence of the substrate of the corresponding 3' -end marked quenching group, and extends under the action of the thermostable Taq DNA polymerase to generate a double-chain substrate of fluorescence quenching.
Then in the temperature rising stage of the melting curve, along with the rising of the temperature, double chains of the double-chain substrate subjected to fluorescence quenching are dissociated and separated near the Tm value, so that a fluorescent group is far away from the quenching group to generate a fluorescent signal, a melting curve negative characteristic peak is generated, and different target genes marked by the same fluorescent group are obviously distinguished by single tube detection by utilizing the different positions of the melting curve negative characteristic peaks with different Tm values.
The invention has the beneficial effects that:
(1) The multiplex PCR detection method of the invention cuts the label sequence of the fluorescent group or the quenching group marked on the 5' end of the probe in the similar common Taqman probe method, the 3' end is provided with free extendable hydroxyl, the free extendable hydroxyl is combined with the substrate with the quenching group or the fluorescent group modified on the 3' end to generate a double-chain quenching product, and the melting peaks among different targets are effectively distinguished by the special design of the probe and the substrate, thereby solving the flux limitation problem of the current fluorescent quantitative platform, simplifying experimental operation, reducing detection cost and realizing the detection of more times in a single tube.
(2) The key of distinguishing different targets in the same channel is substrates with different Tm values, so that the scheme is low in design difficulty and easy to realize.
(3) The detection method disclosed by the disclosure is stronger in specificity, and besides the specificity of the primer probe, the specificity of the cut label sequence and the corresponding substrate is also clear, and the background signal is clear and the interpretation is clear and simple.
(4) The detection method disclosed by the invention has the advantages of high sensitivity, strong anti-interference capability, high detection speed and short detection time, and the whole detection process can be completed within 60-80 min.
(5) The method aims at breaking through the bottleneck of the detection flux of the fluorescent quantitative platform, detecting more than 3 weight targets can be realized through a single channel, and target detection of more than ten weight targets or even more than twenty weight targets can be realized through a single tube.
Drawings
FIG. 1 is a graph showing the results of a test of clinical sample 1 using a conventional Taqman hydrolysis probe method.
Fig. 2 is a graph of test results of clinical sample 1 using the methods of the present disclosure.
FIG. 3 shows the results of single tube detection of the food-borne pathogens Salmonella, staphylococcus aureus, escherichia coli O157, listeria monocytogenes, vibrio, yersinia small intestine by the method of the present invention.
FIG. 4 shows the NTC results of single tube detection of the food-borne pathogens Salmonella, staphylococcus aureus, escherichia coli O157, listeria monocytogenes, vibrio, yersinia small intestine by the method of the present invention.
FIG. 5 shows the results of six simulated samples of Pseudomonas aeruginosa, acinetobacter baumannii, klebsiella pneumoniae, haemophilus influenzae, legionella pneumophila and Streptococcus pneumoniae detected by the method of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
Example 1 multiple target nucleotide detection method
This example detects food borne pathogenic bacteria by multiplex PCR.
The primer used in this example is a blocking primer with RNA base modification, the 3' end of the probe P is blocked, the 5' end of the probe P is modified with a fluorescent group, and the 3' end of the substrate PS is modified with a quenching group.
(1) Specific detection primers were designed using oligo 7.0 software based on the corresponding gene sequences of Salmonella, staphylococcus aureus, escherichia coli O157, listeria monocytogenes, vibrio, yersinia small intestine, and related specific sequences were as follows:
note that: the underlined lower case letters of the sequence indicate that the base is an RNA base where G indicates that the base is a ribonucleotide of G, the other part of the sequence is a deoxyribonucleotide, and C3 is a blocking group C3 Spacer.
(2) Jointly labeling the 5' -end of a fluorescence blocking probe of a salmonella, staphylococcus aureus and escherichia coli O157 target as FAM; jointly labeling the 5' -end of a fluorescence blocking probe of a target of Listeria monocytogenes, vibrio and yersinia small intestine as VIC; the 3' end of the 6 sites collectively marks C3 Spacer. The blocking probe is divided into two parts, a part sequence close to the 5' end of the blocking probe can be reversely complementary with a part sequence close to the 3' end of the substrate, a part sequence close to the 3' end of the blocking probe can be specifically combined with a target sequence, the position of the blocking probe is between corresponding specific primers of the target, and the sequence of the blocking probe is as follows:
| target pathogen name | Probe name | Sequence information | SEQ ID NO. |
| Salmonella bacteria | SM-P | FAM-GAAATGTAACTAGTGATCCATCAAATTAGCGGAGGCTTCCGG-C3 | 13 |
| Staphylococcus aureus | SA-P | FAM-AGGAATAGCAGCAAATGCATCACAAACAGATAAYGGCGT-C3 | 14 |
| Escherichia coli O157 | O157-P | FAM-TATGGTTCTTGCCTTGGCCTTTAAAATGTAAACAACGGTC-C3 | 15 |
| Listeria monocytogenes | DZ-P | VIC-CTGCCTTCACGACTCACCAGCATCTCCGCCTGCAAGTCC-C3 | 16 |
| Vibrio genus | HJ-P | VIC-CAGGATGATCACCGATGTAGTGAATCGCTTCTGCT-C3 | 17 |
| Yersinia small intestine | YeF-P | VIC-CGCGTCGTGACCAACCTGCCGGCACATAATAAGTCGCC-C3 | 18 |
(3) BHQ1 was co-labeled with 3' -ends of substrates of Salmonella, staphylococcus aureus, escherichia coli O157, listeria monocytogenes, vibrio and Yersinia small intestine targets. The sequence of the quenching group part near the 3 'end of the substrate can be reversely complementary with the sequence of the fluorescent label part at the 5' end, and the sequence of the substrate is as follows:
| target pathogen name | Substrate name | Sequence information | SEQ ID NO. |
| Salmonella bacteria | SM-PS | AAGCTATCTGATCACTAGTTACATTTC-BHQ1 | 19 |
| Staphylococcus aureus | SA-PS | AATGAGAAACATACGCTGCTGCTATTCCT-BHQ1 | 20 |
| Escherichia coli O157 | 157-PS | CTCAAAGCGTGCTATATACTGCTACGCTTGAGGCAAGAACCATA-BHQ1 | 21 |
| Listeria monocytogenes | DZ-PS | ATTTTCGTGAGTCGTGAAGGCAG-BHQ1 | 22 |
| Vibrio genus | HJ-PS | ACGGATGACTGGAATCCTGTGACAGGATGA-BHQ1 | 23 |
| Yersinia small intestine | YeF-PS | CCCAAGTTGAAGACTCTCCGGTTGGTCACGACGCG-BHQ1 | 24 |
It should be noted that in this embodiment, the fluorophore used includes VIC, FAM, the blocking group used includes C3 Spacer, the quenching group used includes BHQ1, and in other embodiments, the fluorophore used may also be ROX, CY5, etc., the blocking group used may also be Spacer 9, C6 Spacer, etc., and the quenching group used may also be BHQ2, BHQ3, MGB, etc.
(4) Collecting clinical food sample, extracting genomic DNA with commercial nucleic acid extraction kit, and extracting DNA with ultravioletSpectrophotometry to determine concentration and purity, its DNA OD 260 /OD 280 The value of (2) should be 1.8-2.0, the concentration should be 5-50 ng/. Mu.L, the sample DNA quality inspection is failed to be used for detection, and after the DNA quality inspection is qualified, the extracted sample DNA is preserved at-20 ℃ for standby.
(5) Calculating the configuration reaction number according to the number of samples to be detected, if the number of samples is n, carrying out n+2 reactions (including a positive control reaction and a template-free control reaction), preparing a reaction system mixed solution by additionally configuring 1-2 reactions, subpackaging into PCR tubes, wherein each tube is 15 mu L, and 5mu L of template is additionally added, and the specific PCR amplification reaction system is shown in the following table:
wherein the 10 Xprimer probe Mix comprises the following components:
| sequence name | Nucleic acid working concentration/nM | 1 reaction addition/. Mu.L |
| SM-F | 200 | 0.04 |
| SM-R | 200 | 0.04 |
| SM-P | 100 | 0.02 |
| SM-PS | 100 | 0.02 |
| SA-F | 200 | 0.04 |
| SA-R | 200 | 0.04 |
| SA-P | 100 | 0.02 |
| SA-PS | 100 | 0.02 |
| O157-F | 200 | 0.04 |
| O157-R | 200 | 0.04 |
| O157-P | 100 | 0.02 |
| O157-PS | 100 | 0.02 |
| DZ-F | 200 | 0.04 |
| DZ-R | 200 | 0.04 |
| DZ-P | 100 | 0.02 |
| DZ-PS | 100 | 0.02 |
| HJ-F | 200 | 0.04 |
| HJ-R | 200 | 0.04 |
| HJ-P | 100 | 0.02 |
| HJ-PS | 100 | 0.02 |
| YeF-F | 200 | 0.04 |
| YeF-R | 200 | 0.04 |
| YeF-P | 100 | 0.02 |
| YeF-PS | 100 | 0.02 |
| Supplement TE | - | 1.28 |
| Co-production | - | 2 |
(6) The PCR reaction tube was placed on a real-time fluorescence PCR instrument for testing, and the reaction program was set as shown in the following table:
(7) Based on the determination result of the positions of the melting peaks of the target genes, the positions of the melting peaks of the targets are as follows:
(8) A clinical sample 1 is selected, and the detection of food-borne pathogenic bacteria of the clinical sample 1 is carried out by using a traditional Taqman hydrolysis probe method and the method disclosed by the disclosure, and the results are shown in fig. 1 and 2. The results show that: two food-borne pathogens, namely Listeria monocytogenes and Vibrio, exist in the clinical sample, and the results of the two detection methods are consistent. However, the conventional Taqman hydrolysis probe method requires two-tube detection, and the detection method can realize 6-weight detection in one tube.
Example 2 multiplex detection of lower respiratory pathogens.
The kit was designed as described in example 1 for the lower respiratory tract pathogens Pseudomonas aeruginosa, acinetobacter baumannii, klebsiella pneumoniae, haemophilus influenzae, legionella pneumophila and Streptococcus pneumoniae.
In addition to the application direction, the 5 '-end of the probe P was modified with a quenching group and the 3' -end of the substrate PS was modified with a fluorescent group, which were different from those of example 1.
(1) According to the corresponding gene sequences of pseudomonas aeruginosa, acinetobacter baumannii, klebsiella pneumoniae, haemophilus influenzae, legionella pneumophila and streptococcus pneumoniae, specific detection primers are designed by utilizing oligo 7.0 software, and the related specific sequences are as follows:
note that: the underlined lower case letters of the sequence indicate that the base is an RNA base where G indicates that the base is a ribonucleotide of G, the other part of the sequence is a deoxyribonucleotide, and dSpacer is a blocking group.
(2) The 5' -end of a fluorescence blocking probe of a pathogen pseudomonas aeruginosa, acinetobacter baumannii and klebsiella pneumoniae target is jointly marked as BHQ3; jointly labeling 5' -ends of fluorescent blocking probes of targets of haemophilus influenzae, legionella pneumophila and streptococcus pneumoniae as BHQ2; the 3' end of the 6 sites collectively marks dSpacer. The blocking probe is divided into two parts, a part sequence close to the 5' end of the blocking probe can be reversely complementary with a part sequence close to the 3' end of the substrate, a part sequence close to the 3' end of the blocking probe can be specifically combined with a target sequence, the position of the blocking probe is between corresponding specific primers of the target, and the sequence of the blocking probe is as follows:
(3) The 3' end of a target substrate of pathogenic bacteria pseudomonas aeruginosa, acinetobacter baumannii and klebsiella pneumoniae is jointly marked as CY5; the 3' -end of the target substrate of haemophilus influenzae, legionella pneumophila and streptococcus pneumoniae is jointly marked as ROX. The substrate sequence is as follows:
(4) Six simulated plasmid samples of Pseudomonas aeruginosa, acinetobacter baumannii, klebsiella pneumoniae, haemophilus influenzae, legionella pneumophila and Streptococcus pneumoniae were synthesized from the biological engineering (Shanghai) Co., ltd, and the single-dose detection results are shown in FIG. 5. The result shows that the technical method can realize multiple detection on the detection of the pathogenic bacteria of the lower respiratory tract, and the same fluorescent channel targets can be clearly distinguished. Based on the determination result of the positions of the melting peaks of the target genes, the positions of the melting peaks of the targets are as follows:
the foregoing disclosure is only illustrative of some of the preferred embodiments of the present application and is not intended to limit the scope of the claims hereof, as persons of ordinary skill in the art will understand that all or part of the processes for accomplishing the foregoing embodiments may be practiced with equivalent changes which may be made by the claims herein and which fall within the scope of the invention.
Claims (11)
1. A multiplex target nucleotide detection kit, comprising:
(1) At least one pair of specific primer pairs: introducing 1 RNA base in the middle of the specific primer, and blocking the 3' -end of the primer;
(2) At least one blocking probe P: the 3 'end is blocked, the sequence specificity and target binding are close to the 3' end, the 5 'end part sequence is a tag sequence, and the 5' end modifies a fluorescent group or a quenching group;
(3) Substrate PS:3' end modified quenching group or fluorescent group;
(4) A thermostable Taq DNA polymerase having 5 '. Fwdarw.3' DNA polymerase activity and 5 '. Fwdarw.3' exonuclease activity and a thermostable endonuclease that specifically cleaves RNA bases of RNA and DNA hybrid strands;
the sequence of the blocking probe near the 5 'end label part is reversely complementary with the sequence of the substrate near the 3' end part;
the position where the blocking probe P is combined with the target sequence is between the specific primer pair, and the Tm value of the blocking probe P combined with the target sequence is higher than that of the specific primer;
the length of the specific primer sequence is between 16bp and 30bp and comprises an endpoint value, and the Tm value is between 45 ℃ and 65 ℃ and comprises an endpoint value;
the length of the tag sequence carried by the blocking probe P is 5bp-25bp, the Tm value is 20-45 ℃ and the tag sequence comprises an endpoint value;
the sequence length of the substrate PS is between 10bp and 70bp and comprises an endpoint value, and the Tm value is between 50 ℃ and 90 ℃ and comprises an endpoint value.
2. The kit of claim 1, wherein the sequence length that blocks the binding of probe P to the target is between 20bp and 35bp inclusive and the Tm value is between 55 ℃ and 75 ℃ inclusive.
3. The kit according to claim 1, wherein the fluorescent groups marked on the 5 '-end of the blocking probe P and the 3' -end of the substrate PS are one or more of FAM, VIC, TET, CAL Gold 540, JOE, HEX, TAMRA, ROX, CY3, CY5, and the marked quenching groups are one or more of DABCYL, BHQ-1, BHQ-2, BHQ-3, ECLIPE.
4. The kit according to claim 1, wherein the blocking group labeled on the 3' end of the specific primer and blocking probe P is selected from the group consisting of: phosphate, BHQ1, BHQ2, BHQ3, C3 Spacer, spacer 9,3' C6 Spacer, dsspacer, PC Spacer or Spacer 18.
5. The kit of claim 1, wherein the endonuclease specifically cleaves RNA bases when the specific primer binds to the target sequence to produce an extendable specific primer, wherein during extension the thermostable Taq DNA polymerase cleaves a tag sequence that blocks the 5 'end-labeled fluorescent group of the probe, and wherein the cleaved tag sequence specifically binds to the substrate of the corresponding 3' end-labeled quenching group and is extended by the thermostable Taq DNA polymerase to produce a fluorescence quenched double-stranded substrate.
6. The kit of claim 5, wherein the differentiation of different targets is performed in a single channel by the different melting peak positions in the melting curve of the corresponding fluorescence quenched double-stranded products of different Tm values.
7. The kit of claim 6, wherein the melting curve procedure is: maintaining at 95 ℃ for 2min and at 50 ℃ for 2min, and then collecting fluorescent signals at 50-85 ℃ at a heating rate of 0.02 ℃/s-0.2 ℃/s.
8. The kit according to claim 1, wherein the sequences of the specific primer, blocking probe P and substrate PS are selected from the group consisting of: SEQ ID NO.1-2, SEQ ID NO.13 and SEQ ID NO.19; or SEQ ID NO.3-4, SEQ ID NO.14 and SEQ ID NO.20; or SEQ ID No.5-6, SEQ ID No.15 and SEQ ID No.21; or SEQ ID No.7-8, SEQ ID No.16 and SEQ ID No.22; or at least two of SEQ ID NO.11-12, SEQ ID NO.18 and SEQ ID NO. 24; the kit is a kit for jointly detecting food-borne pathogenic bacteria, wherein the types of the food-borne pathogenic bacteria comprise at least two of salmonella, staphylococcus aureus, escherichia coli O157, listeria monocytogenes and yersinia small intestine.
9. The kit according to claim 1, wherein the sequences of the specific primer, blocking probe P and substrate PS are selected from the group consisting of: SEQ ID No.25-26, SEQ ID No.37 and SEQ ID No.43; or SEQ ID No.27-28, SEQ ID No.38 and SEQ ID No.44; or SEQ ID NO.29-30, SEQ ID NO.39 and SEQ ID NO.45; or SEQ ID NO.31-32, SEQ ID NO.40 and SEQ ID NO.46; or SEQ ID No.33-34, SEQ ID No.41 and SEQ ID No.47; or at least two of SEQ ID NO.35-36, SEQ ID NO.42 and SEQ ID NO. 48; the kit is a kit for jointly detecting lower respiratory tract pathogenic bacteria, wherein the lower respiratory tract pathogenic bacteria comprise at least two of pseudomonas aeruginosa, acinetobacter baumannii, klebsiella pneumoniae, haemophilus influenzae, legionella pneumophila and streptococcus pneumoniae.
10. Use of a kit according to any one of claims 1-8 for detecting food-borne pathogenic bacteria in a food product.
11. A method for detecting food-borne pathogenic bacteria in a food, comprising detecting the food using the kit of any one of claims 1 to 8.
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| CN103703013A (en) * | 2011-02-14 | 2014-04-02 | 斯威夫特生物科学公司 | Polynucleotide primers and probes |
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