CN117402989A - PCR amplification primer pair and probe for bacterial drug resistance gene detection and application thereof - Google Patents
PCR amplification primer pair and probe for bacterial drug resistance gene detection and application thereof Download PDFInfo
- Publication number
- CN117402989A CN117402989A CN202311595357.4A CN202311595357A CN117402989A CN 117402989 A CN117402989 A CN 117402989A CN 202311595357 A CN202311595357 A CN 202311595357A CN 117402989 A CN117402989 A CN 117402989A
- Authority
- CN
- China
- Prior art keywords
- probe
- pcr amplification
- seq
- primer pair
- drug resistance
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000000523 sample Substances 0.000 title claims abstract description 253
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 112
- 238000012408 PCR amplification Methods 0.000 title claims abstract description 111
- 238000001514 detection method Methods 0.000 title claims abstract description 73
- 206010059866 Drug resistance Diseases 0.000 title claims abstract description 71
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 56
- YZBQHRLRFGPBSL-RXMQYKEDSA-N carbapenem Chemical compound C1C=CN2C(=O)C[C@H]21 YZBQHRLRFGPBSL-RXMQYKEDSA-N 0.000 claims abstract description 13
- 108010059993 Vancomycin Proteins 0.000 claims abstract description 9
- 230000003321 amplification Effects 0.000 claims abstract description 9
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 9
- 229960003165 vancomycin Drugs 0.000 claims abstract description 9
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 claims abstract description 9
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 claims abstract description 9
- 108090000204 Dipeptidase 1 Proteins 0.000 claims abstract description 6
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 claims abstract description 6
- 102000006635 beta-lactamase Human genes 0.000 claims abstract description 6
- 229960003085 meticillin Drugs 0.000 claims abstract description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 22
- 125000006853 reporter group Chemical group 0.000 claims description 9
- 238000010791 quenching Methods 0.000 claims description 8
- 230000000171 quenching effect Effects 0.000 claims description 8
- 206010034133 Pathogen resistance Diseases 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 claims description 2
- 238000003752 polymerase chain reaction Methods 0.000 abstract description 9
- 244000005700 microbiome Species 0.000 abstract description 4
- 239000000203 mixture Substances 0.000 description 34
- 238000006243 chemical reaction Methods 0.000 description 26
- 239000013612 plasmid Substances 0.000 description 21
- 238000011144 upstream manufacturing Methods 0.000 description 18
- 238000012216 screening Methods 0.000 description 13
- 238000012163 sequencing technique Methods 0.000 description 12
- 239000000243 solution Substances 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 238000013461 design Methods 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 238000012795 verification Methods 0.000 description 6
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 244000052769 pathogen Species 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 108010085238 Actins Proteins 0.000 description 3
- 102100035071 Vimentin Human genes 0.000 description 3
- 208000022362 bacterial infectious disease Diseases 0.000 description 3
- PHIQHXFUZVPYII-UHFFFAOYSA-N carnitine Chemical compound C[N+](C)(C)CC(O)CC([O-])=O PHIQHXFUZVPYII-UHFFFAOYSA-N 0.000 description 3
- 230000037029 cross reaction Effects 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 238000007480 sanger sequencing Methods 0.000 description 3
- 239000004575 stone Substances 0.000 description 3
- 101150114434 vanA gene Proteins 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 230000036457 multidrug resistance Effects 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 101150037181 vanB gene Proteins 0.000 description 2
- 102100022900 Actin, cytoplasmic 1 Human genes 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 241000588921 Enterobacteriaceae Species 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 206010048723 Multiple-drug resistance Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108010010677 Phosphodiesterase I Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 101150018417 VIM gene Proteins 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000003436 cytoskeletal effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 210000003632 microfilament Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 108010068698 spleen exonuclease Proteins 0.000 description 1
- 210000003699 striated muscle Anatomy 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
Classifications
-
- 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
- C12Q1/686—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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
-
- 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
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of microorganism detection, and particularly relates to a PCR (polymerase chain reaction) amplification primer pair and a probe for detecting bacterial drug resistance genes and application thereof. Comprises one or more of the following primer pairs and probes: (1) PCR amplification primer pairs and probes for detecting ultra-broad spectrum beta-lactamase drug resistance genes; (2) A PCR amplification primer pair and a probe for detecting carbapenem drug resistance genes; (3) PCR amplification primer pairs and probes for detecting the methicillin-resistant gene; (4) PCR amplification primer pair and probe for detecting vancomycin drug resistance gene. The PCR amplification primer pair and the probe for detecting the bacterial drug resistance genes can accurately detect the drug resistance genes.
Description
Technical Field
The invention belongs to the technical field of microorganism detection, and particularly relates to a PCR (polymerase chain reaction) amplification primer pair and a probe for detecting bacterial drug resistance genes and application thereof.
Background
The disease caused by bacterial infection is a common disease and frequently-occurring disease which seriously endanger human health, and is also one of the main complications and lethal reasons of the late stage of various organ diseases. The antibacterial medicine plays an important role in treating bacterial infectious diseases, and the death rate related to infection is obviously reduced. However, with the abuse of clinical antibiotics, the emergence and widespread bacterial resistance present serious challenges for clinical anti-infective therapy, severely affecting human and animal health. As well as environmental and economic problems, bacterial resistance has become a hotspot of global common concern.
At present, bacterial resistance is very severe, especially in medical, animal sources and environments. The clinical bacterial drug Resistance is very severe, and the detection rate of various common Multi-drug Resistance (MDR) bacteria and broadly-resistant (XDR) bacteria is relatively high. According to the data of Chinese bacterial drug resistance monitoring net (CHINET), most of clinical isolates in China show a trend of increasing the drug resistance rate of antibiotics year by year in 2022 in 2005, and the situation of multiple drug resistance is serious. National bacterial resistance monitoring networks (calss) reports also show that the detection rate of a variety of important bacterial resistance remains high. The most common are methicillin-resistant staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), ultra-broad-spectrum beta-lactamase Enterobacteria (ESBLs), carbapenem-resistant enterobacteriaceae (CRE), and the like.
Therefore, the detection of bacterial drug resistance has very important significance in the aspects of timely and accurate treatment of bacterial infection, epidemiological research of pathogenic bacteria, reduction of occurrence and transmission of bacterial drug resistance and the like.
Disclosure of Invention
In view of the above problems, it is an object of the present invention to provide a primer set and a probe for PCR amplification for detecting a bacterial drug resistance gene, wherein the primer set and the probe are designed by different bacterial drug resistance genes, and can accurately detect a specific bacterial drug resistance gene.
In order to achieve the above purpose, the present invention may adopt the following technical scheme:
in one aspect, the invention provides a PCR amplification primer pair and a probe for detecting bacterial drug resistance genes, which comprise one or more of the following primer pairs and probes: (1) The PCR amplification primer pair and the probe are used for detecting the ultra-broad spectrum beta-lactamase drug resistance gene, the PCR amplification primer pair is shown as SEQ ID NO.1 and SEQ ID NO.2, and the probe is shown as SEQ ID NO. 3; (2) A PCR amplification primer pair and a probe for detecting carbapenem resistance gene, wherein the PCR amplification primer pair and the probe comprise one or more of the following sequences: (a) The PCR amplification primer pair is shown as SEQ ID NO.4 and SEQ ID NO.5, and the probe is shown as SEQ ID NO. 6; (b) The PCR amplification primer pair is shown as SEQ ID NO.7 and SEQ ID NO.8, and the probe is shown as SEQ ID NO. 9; (c) The PCR amplification primer pair is shown as SEQ ID NO.10 and SEQ ID NO.11, and the probe is shown as SEQ ID NO. 12; (d) The PCR amplification primer pair is shown as SEQ ID NO.13 and SEQ ID NO.14, and the probe is shown as SEQ ID NO. 15; (e) The PCR amplification primer pair is shown as SEQ ID NO.16 and SEQ ID NO.17, and the probe is shown as SEQ ID NO. 18; (f) The PCR amplification primer pair is shown as SEQ ID NO.19 and SEQ ID NO.20, and the probe is shown as SEQ ID NO. 21; (3) The PCR amplification primer pair and the probe are used for detecting the methicillin-resistant gene, the PCR amplification primer pair is shown as SEQ ID NO.22 and SEQ ID NO.23, and the probe is shown as SEQ ID NO. 24; (4) A PCR amplification primer pair and a probe for detecting vancomycin drug resistance genes, wherein the PCR amplification primer pair and the probe comprise one or more of the following sequences: (a) The PCR amplification primer pair is shown as SEQ ID NO.25 and SEQ ID NO.26, and the probe is shown as SEQ ID NO. 27; (b) The PCR amplification primer pair is shown as SEQ ID NO.28 and SEQ ID NO.29, and the probe is shown as SEQ ID NO. 30.
The invention also provides a PCR amplification detection reagent for detecting the bacterial drug resistance genes, which comprises the PCR amplification primer pair and the probe for detecting the bacterial drug resistance genes.
In still another aspect, the present invention provides a kit for detecting bacterial drug resistance genes, which comprises the above-mentioned primer pair for detecting bacterial drug resistance genes, a probe, or the above-mentioned reagent for detecting bacterial drug resistance genes.
In still another aspect, the invention provides an application of the PCR amplification primer pair and the probe for detecting the bacterial drug resistance genes in preparing detection products for detecting the bacterial drug resistance genes.
In still another aspect, the invention provides an application of the PCR amplification primer pair, the probe or the PCR amplification detection reagent or the PCR amplification detection kit for detecting the bacterial drug resistance genes in detecting the bacterial drug resistance genes.
In yet another aspect, the present invention provides a method for detecting a bacterial drug resistance gene comprising: using the PCR amplification primer pair, the probe or the PCR amplification detection reagent or the PCR amplification detection kit for detecting the bacterial drug resistance genes to carry out PCR amplification detection on a sample to be detected; the amplification conditions for PCR include: the first stage: 95 ℃,2 minutes, 1 cycle; and a second stage: 95 ℃,10 seconds, 60 ℃,30 seconds, 40 cycles; fluorescence signals were collected at 60℃in the second stage.
The beneficial effects of the invention at least comprise: the PCR amplification primer pair and the probe for detecting the bacterial drug resistance gene can accurately detect the bacterial drug resistance gene.
Drawings
FIG. 1 shows a first generation of sequencing results of sample 1 (OXA-23);
FIG. 2 shows a sample 2 (mecA) generation sequencing result;
FIG. 3 shows a sample 2 (NDM) generation sequencing result;
FIG. 4 shows a sample 3 (NDM) generation sequencing result;
FIG. 5 shows a sample 4 (OXA-48) generation of sequencing results;
FIG. 6 shows a sample 4 (CTX-M) generation sequencing result;
FIG. 7 shows a sample 5 (CTX-M) generation sequencing result;
FIG. 8 shows a sample 6 (CTX-M) generation sequencing result;
FIG. 9 shows a sample 1 (mecA) generation sequencing result;
FIG. 10 shows the detection of sample 1 by the detection method of the present invention;
FIG. 11 shows a sample 2 detected by the detection method of the present invention;
FIG. 12 shows the detection of sample 3 by the detection method of the present invention;
FIG. 13 shows the detection of sample 4 by the detection method of the present invention;
FIG. 14 shows the detection of sample 5 by the detection method of the present invention;
FIG. 15 shows the detection of sample 6 by the detection method of the present invention;
FIG. 16 shows the detection of sample 7 by the detection method of the present invention;
FIG. 17 is an electrophoretogram of 5 negative samples.
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context clearly differs, singular forms of expression include plural forms of expression. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to indicate the presence of a feature, number, operation, component, part, element, material, or combination. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, elements, materials or combinations thereof may be present or added. As used herein, "/" may be interpreted as "and" or "as appropriate.
An embodiment of the invention provides a PCR amplification primer pair and a probe for detecting bacterial drug resistance genes, which comprise one or more of the following primer pairs and probes: (1) The PCR amplification primer pair and the probe are used for detecting the ultra-broad spectrum beta-lactamase drug resistance gene, the PCR amplification primer pair is shown as SEQ ID NO.1 and SEQ ID NO.2, and the probe is shown as SEQ ID NO. 3; (2) A PCR amplification primer pair and a probe for detecting carbapenem resistance gene, wherein the PCR amplification primer pair and the probe comprise one or more of the following sequences: (a) The PCR amplification primer pair is shown as SEQ ID NO.4 and SEQ ID NO.5, and the probe is shown as SEQ ID NO. 6; (b) The PCR amplification primer pair is shown as SEQ ID NO.7 and SEQ ID NO.8, and the probe is shown as SEQ ID NO. 9; (c) The PCR amplification primer pair is shown as SEQ ID NO.10 and SEQ ID NO.11, and the probe is shown as SEQ ID NO. 12; (d) The PCR amplification primer pair is shown as SEQ ID NO.13 and SEQ ID NO.14, and the probe is shown as SEQ ID NO. 15; (e) The PCR amplification primer pair is shown as SEQ ID NO.16 and SEQ ID NO.17, and the probe is shown as SEQ ID NO. 18; (f) The PCR amplification primer pair is shown as SEQ ID NO.19 and SEQ ID NO.20, and the probe is shown as SEQ ID NO. 21; (3) The PCR amplification primer pair and the probe are used for detecting the methicillin-resistant gene, the PCR amplification primer pair is shown as SEQ ID NO.22 and SEQ ID NO.23, and the probe is shown as SEQ ID NO. 24; (4) A PCR amplification primer pair and a probe for detecting vancomycin drug resistance genes, wherein the PCR amplification primer pair and the probe comprise one or more of the following sequences: (a) The PCR amplification primer pair is shown as SEQ ID NO.25 and SEQ ID NO.26, and the probe is shown as SEQ ID NO. 27; (b) The PCR amplification primer pair is shown as SEQ ID NO.28 and SEQ ID NO.29, and the probe is shown as SEQ ID NO. 30.
The PCR amplification primer pair and the probe in the invention are designed according to the bacterial drug resistance gene. Specifically, in the PCR amplification primer pair and probe for detecting the carbapenem-resistant gene, (a) the KPC gene of the carbapenem-resistant gene is detected by the primer pair and probe of group, (b) the IMP gene of the carbapenem-resistant gene is detected by the primer pair and probe of group, (c) the VIM gene of the carbapenem-resistant gene is detected by the primer pair and probe of group, (d) the NDM gene of the carbapenem-resistant gene is detected by the primer pair and probe of group, (e) the OXA-23 gene of the carbapenem-resistant gene is detected by the primer pair and probe of group, (f) the OXA-48 gene of the carbapenem-resistant gene is detected by the primer pair and probe of group; among the PCR amplification primer pair and probe for detecting vancomycin resistance gene, (a) the vancomycin resistance gene VanA gene is detected by the primer pair and probe of group, and the vancomycin resistance gene VanB gene is detected by the primer pair and probe of group.
The invention further provides a PCR amplification detection reagent for detecting bacterial drug resistance genes, which comprises the PCR amplification primer pair and the probe for detecting bacterial drug resistance genes.
The PCR amplification detection reagent for detecting a bacterial drug resistance gene described above may be used in combination with an auxiliary detection reagent, which is a reagent known in the art, such as a buffer, a ligase, etc., to detect a bacterial drug resistance gene.
In some specific embodiments, the PCR amplification detection reagent may further comprise a PCR amplification primer pair of the human reference gene, and a probe, wherein the PCR amplification primer pair of the human reference gene is shown as SEQ ID NO.31 and SEQ ID NO.32, and the probe is shown as SEQ ID NO. 33.
In some embodiments, the PCR amplification detection reagents described above may further include a fluorescent reporter group and a fluorescent quencher group. The 5 'end of the probe is marked with a report group, the 3' end is marked with a fluorescence quenching group, the probe is only specifically combined with the template, and the combining site is between the two primers. When the probe is complete, the fluorescent energy of the reporter group is absorbed by the quenching group, so that the instrument cannot collect signals, as the reaction progresses, taq enzyme encounters the probe, and the activity of the 3 '. Fwdarw.5' exonuclease is utilized to cut off the probe, so that the fluorescent energy of the reporter group cannot be absorbed by the quenching group, and fluorescent signals are generated.
In some embodiments, the fluorescent reporter group may be ROX, FAM, CY5 or VIC; the fluorescence quenching group is BHQ1 or BHQ2.
Still another embodiment of the present invention provides a PCR amplification detection kit for detecting a bacterial drug resistance gene, which includes the above-described PCR amplification primer pair, probe, or the above-described PCR amplification detection reagent for detecting a bacterial drug resistance gene.
It should be noted that the above-mentioned PCR amplification primer pair, probe or PCR amplification detection reagent for detecting bacterial drug resistance gene may be prepared in the form of a kit, which is known in the art, for example, a kit form including a reagent bottle, a specification, etc.
The invention also provides an application of the PCR amplification primer pair and the probe for detecting the bacterial drug resistance genes in preparing detection products for detecting the bacterial drug resistance genes.
The detection product may be a detection reagent or a detection kit.
The invention also provides an application of the PCR amplification primer pair, the probe or the PCR amplification detection reagent or the PCR amplification detection kit for detecting the bacterial drug resistance genes in detecting the bacterial drug resistance genes.
In another embodiment of the present invention, a method for detecting a bacterial drug resistance gene is provided, comprising: using the PCR amplification primer pair, the probe or the PCR amplification detection reagent or the PCR amplification detection kit for detecting the bacterial drug resistance genes to carry out PCR amplification detection on a sample to be detected; the amplification conditions for PCR include: the first stage: 95 ℃,2 minutes, 1 cycle; and a second stage: 95 ℃,10 seconds, 60 ℃,30 seconds, 40 cycles; fluorescence signals were collected at 60℃in the second stage.
For a better understanding of the present invention, the content of the present invention is further elucidated below in connection with the specific examples, but the content of the present invention is not limited to the examples below.
1. Primer probe design
(one) detection Gene validation
The genes detected were determined by reference as follows: ultra-broad spectrum beta-lactamase drug resistance gene: CTX-M; carbapenem drug resistance gene: KPC, IMP, VIM, NDM, OXA-23 and OXA-48; methicillin-resistant gene: mecA; vancomycin resistance gene: vanA, vanB; and the human internal reference gene beta-actin is arranged, and the beta-actin is a main protein component in striated muscle fibers, is also a main component of muscle filaments and cytoskeletal microfilaments, is almost expressed in all eukaryotic cells, is widely existing in tissues or cells of mammals, and is the best choice of RT-PCR standardized internal reference.
(II) Probe primer sequence design
The design of the primer probe is assisted by means of software such as Beacon Designer, oligo, primer Express 3.0 and the like, the detected gene sequence is imported into the software, the design experience of the research and development team probe is combined according to the primer probe design principle, and the design is carried out by setting the software. In order to increase the success rate of designing the probe primers and shorten the experiment time, 3 groups of probe primers are designed for each gene, and finally, a group of probe primers with the best effect is selected from the 3 groups of probe primers, and 1 group of probe primers are designed for the reference gene.
In addition, the experimental method of the invention is a multiplex fluorescence real-time quantitative PCR method; the TaqMan probe method is a quantitative PCR technology with high specificity, and the working principle of the TaqMan probe method is that a pair of PCR primers and a probe exist in a PCR reaction system, the 5 'end of the probe is marked with a reporter group, the 3' end of the probe is marked with a fluorescence quenching group, the probe is specifically combined with a template, and the combining site of the probe is arranged between the two primers. When the probe is complete, the fluorescent energy of the reporter group is absorbed by the quenching group, so that the instrument cannot collect signals, as the reaction progresses, taq enzyme encounters the probe, and the probe is cut off by utilizing the activity of 5 '. Fwdarw.3' exonuclease, so that the fluorescent energy of the reporter group cannot be absorbed by the quenching group, and fluorescent signals are generated. When designing the primer probes, the 5' modification of different gene probes is respectively 4 different report groups, so that fluorescence with different wavelengths can be generated, and at most 4 probes with different fluorescence can be placed in one reaction hole. After the design is completed, the designed primer probe sequence is subjected to specificity comparison on NCBI, the specificity is strong when the specificity is 100%, other sequences consistent with the specificity are not generated, and the screening design result is shown in the following table 1.
TABLE 1 primer pair and Probe case
(III) Synthesis of Probe primer
The probe primers were synthesized by general biosystems.
(IV) Gene plasmid Synthesis
The general biosystems synthesized plasmids of the genes (mock samples).
2. Primer probe screening
In the embodiment of the invention, a group of primer probes with the best result is selected from 3 groups of primer probes (shown in table 1) designed by each gene, and the method specifically comprises the following steps:
preparation of probe primer
Taking CTX-M as an example to prepare a primer probe mix, preparing a first group of primer probes: 2ul of upstream primer (F1) +2ul of downstream primer (R1) +2ul of probe (P1) +14ul of DEPC water, and after the addition, mixing and centrifuging; preparing a second group of primer probes: 2ul of upstream primer (F2) +2ul of downstream primer (R2) +2ul of probe (P2) +14ul of DEPC water, and after the addition, mixing and centrifuging; preparing a third group of primer probes: 2ul of upstream primer (F3) +2ul of downstream primer (R3) +2ul of probe (P3) +14ul of DEPC water, and after the addition, mixing and centrifuging; the preparation method of other gene primer probes is the same as that of the above.
(II) preparing a reaction solution
10ul Taq Pro HS Universal U+Probe Master MIX (from Novain) +3ul ddH2O, mixing, and centrifuging, wherein the above method is 1 reaction well configuration method, and multiplying corresponding reaction number according to experimental scheme.
(III) dilution of plasmid
The commonly synthesized plasmids were diluted to 2.5 x 10-7 ng/ul, 10-8 ng/ul, and 10-9 ng/ul, respectively, with 0.1TE for use.
(IV) sample addition
The reaction solution, probe primer mix, and plasmids were sequentially added into eight rows, and the sample addition method was as follows: each reaction well was dosed: 6 wells of each group of probe primers of 13ul reaction solution Mix, 2ul probe primer Mix and 5ul diluted plasmid are prepared, 2 plasmids with the concentration of 2.5 x 10-7 ng/ul, 2 plasmids with the concentration of 2 10-8 ng/ul and 2 plasmids with the concentration of 10-9 ng/ul; three sets of primer probes for each gene; and (5) centrifuging after the addition is finished.
(V) boarding machine
The instrument used is macro stone 96P; amplification conditions for fluorescent quantitative PCR were as follows: the first stage: 95 ℃,2 minutes, 1 cycle; and a second stage: fluorescence signals were collected at 60 ℃ in the second stage at 95 ℃,10 seconds, 60 ℃,30 seconds, 40 cycles.
(sixth) experiment results
The manual threshold was adjusted to 100 and the results are shown in tables 2 to 12.
TABLE 2CTX Probe screening cases
TABLE 3KPC Probe screening cases
TABLE 4 screening cases of IPM probes
TABLE 5VIM Probe screening cases
TABLE 6NDM Probe screening cases
TABLE 7OXA-23 Probe screening cases
TABLE 8OXA-48 Probe screening cases
TABLE 9MECA Probe screening cases
TABLE 10VanA Probe screening cases
TABLE 11VanB Probe screening cases
Table 12 internal reference probe test conditions
In summary, the preliminary selected probes are shown in Table 13 below.
TABLE 13 preliminary screening of Probe conditions
3. Adjusting the proportion of primer probes
In the embodiment of the invention, better effect is achieved by adjusting the proportion of the primer probes, and the specific steps are as follows:
preparation of probe primer
Taking CTX-M as an example, the second set of probe primers was selected to be optimal by the above experiments, which used the second set of probe primers to prepare primer probe mix in different ratios, respectively, as follows:
①F:R:P:H 2 o=1:1:1:7, i.e., 2ul of upstream primer (F2) +2ul of downstream primer (R2) +2ul of probe (P2) +14ul of DEPC water, and after addition, mixing well and centrifuging;
②F:R:P:H 2 o=1:1:0.5:7.5, i.e., 2ul upstream primer (F2) +2ul downstream primer (R2) +1ul probe (P2) +15ul DEPC water, and after addition, mixing well and centrifuging;
③F:R:P:H 2 O＝0.5:0.5:0.58.5, i.e., 1ul of upstream primer (F2) +1ul of downstream primer (R2) +1ul of probe (P2) +17ul of DEPC water, and after addition, mixing well and centrifuging;
④F:R:P:H 2 o=0.5:0.5:0.25:8.75, i.e., 1ul of upstream primer (F2) +1ul of downstream primer (R2) +0.5ul of probe (P2) +17.5ul of DEPC water, and after addition, mixing well and centrifuging.
(II) preparing a reaction solution Mix
10ul Taq Pro HS Universal U+Probe Master MIX (from Novain) +3ul ddH2O, mixing, and centrifuging, wherein the above method is 1 reaction well configuration method, and multiplying corresponding reaction number according to experimental scheme.
(III) dilution of plasmid
The commonly synthesized plasmids were diluted to 2.5 x 10-7 ng/ul and 2.5 x 10-8 ng/ul, respectively, with 0.1TE for use.
(IV) sample addition
The reaction solution, the probe primer Mix and the plasmids are sequentially added into eight rows, and the sample adding method is as follows: each reaction well was dosed: 13ul of reaction solution Mix, 2ul of probe primer Mix, 5ul of diluted plasmid; 4 wells of probe primers of each group are prepared, 2 plasmids with the concentration of 2.5X10-7 ng/ul and 2 plasmids with the concentration of 2.5X10-8 ng/ul; 4 different primer probe ratios per gene. And (5) centrifuging after the addition is finished.
(V) boarding machine
The instrument used is macro stone 96P; amplification conditions for fluorescent quantitative PCR were as follows: the first stage: 95 ℃,2 minutes, 1 cycle; and a second stage: fluorescence signals were collected at 60 ℃ in the second stage at 95 ℃,10 seconds, 60 ℃,30 seconds, 40 cycles.
(sixth) experiment results
The manual threshold was adjusted to 100 and the results are shown in tables 14 to 24.
TABLE 14CTX-M Probe ratio case
TABLE 15KPC Probe proportion case
TABLE 16IMP Probe ratio case
TABLE 17VIM Probe ratio case
TABLE 18NDM Probe ratio case
TABLE 19OXA-23 probe ratio
TABLE 20OXA-48 probe ratio
TABLE 21MECA Probe proportion case
TABLE 22VANA Probe ratio case
TABLE 23VANB Probe ratio conditions
Table 24 internal reference probe ratio
In summary, the ratio of the primer probe was determined as shown in Table 25 below.
Table 25 primer probe ratio case
4. Probe grouping
In the embodiment of the invention, the gene probes with different fluorescence are divided into one group, and the gene specific primer probes of one group are added into the same PCR reaction tube at the same time, so that the probe primers in one group can not interfere with each other, and the genes of one group can be detected at the same time and are divided into 3 groups.
Group A: mecA (FAM), OXA-48 (VIC), CTX-M (ROX), VIM (CY 5);
group B: KPC (FAM), vanA (VIC), IMP (ROX), NDM (CY 5);
group C: reference genes beta-actin (VIC), vanB (ROX), OXA-23 (CY 5).
Probe primer preparation:
the preparation is carried out according to the proportion of the primer probe (table 25), and the specific steps are as follows:
group A: firstly, respectively preparing probe primer mixtures of all genes, wherein the probe primer mixtures are MecA-mix:1ul of upstream primer (F1) +1ul of downstream primer (R1) +0.5ul of probe (P1); OXA-48-mix:1ul of upstream primer (F3) +1ul of downstream primer (R3) +0.5ul of probe (P3); CTX-M-mix:1ul of upstream primer (F2) +1ul of downstream primer (R2) +1ul of probe (P2); VIM-mix:2ul of upstream primer (F3) +2ul of downstream primer (R3) +2ul of probe (P3). Mixing the above 4 mix, adding 6ul water, mixing, and centrifuging.
Group B: firstly, respectively preparing probe primer mixtures of all genes, and mixing KPC-mix:1ul of upstream primer (F3) +1ul of downstream primer (R3) +1ul of probe (P3); vanA-mix:2ul of upstream primer (F3) +2ul of downstream primer (R3) +2ul of probe (P3); IMP-mix:2ul of upstream primer (F3) +2ul of downstream primer (R3) +1ul of probe (P3); NDM-mix:1ul of upstream primer (F2) +1ul of downstream primer (R2) +1ul of probe (P2). Mixing the above 4 mix, adding 3ul water, mixing, and centrifuging.
Group C: firstly, respectively preparing probe primer mixtures of all genes, and performing internal reference-mix: 1ul of upstream primer (F) +1ul of downstream primer (R) +1ul of probe (P); vanB-mix:1ul of upstream primer (F1) +1ul of downstream primer (R1) +0.5ul of probe (P1); OXA-23-mix:1ul of upstream primer (F2) +1ul of downstream primer (R2) +0.5ul of probe (P2). Mixing the above 3 mix, adding 12ul water, mixing, and centrifuging.
(II) preparing a reaction solution Mix
10ul Taq Pro HS Universal U+Probe Master MIX (from Novain) +3ul ddH2O, mixing, and centrifuging, wherein the above method is 1 reaction well configuration method, and multiplying corresponding reaction number according to experimental scheme.
(III) dilution of plasmid
The commonly synthesized plasmids were diluted to 2.5 x 10-7 ng/ul and 2.5 x 10-8 ng/ul, respectively, with 0.1TE for use.
(IV) sample addition
The reaction solution, the probe primer Mix and the plasmids are sequentially added into eight rows, and the sample adding method is as follows: each reaction well was dosed: 13ul of reaction solution Mix, 2ul of probe primer Mix, 5ul of diluted plasmid; 4 wells were made of each set of probe primers, 2 plasmids at a concentration of 2.5X10-7 ng/ul and 2 plasmids at a concentration of 2.5X10-8 ng/ul.
(V) boarding machine
The instrument used is macro stone 96P; amplification conditions for fluorescent quantitative PCR were as follows: the first stage: 95 ℃,2 minutes, 1 cycle; and a second stage: fluorescence signals were collected at 60 ℃ in the second stage at 95 ℃,10 seconds, 60 ℃,30 seconds, 40 cycles.
(sixth) experiment results
The results of the different sets of experiments are shown in tables 26 to 28 below.
Table 26A group reaction conditions
Table 27 group B reaction conditions
Table 28 group C reaction conditions
The experimental results were combined to determine the groupings as shown in Table 29 below.
Table 29 primer probe grouping case
5. Performance verification
Accuracy (one)
The detection selects a third party detection unit (Sanger sequencing method) as an comparison method, positive sequencing results are shown in figures 1 to 9, negative sequencing results are shown in an electrophoresis result which is shown in figure 17,5, the electrophoresis result is carried out after the amplification of the first-generation primers of samples, no band exists in the results, and no target result is detected after the sequencing. By using the detection method of the present invention, drug resistance genes of 12 clinical samples detected by Sanger sequencing were detected, and the detection conditions are shown in the following FIGS. 10 to 16 and Table 30; evaluation criteria: interpreting the experimental result according to the interpretation standard provided by the kit; result judgment criteria: the consistency of the detection result with the detection result of the third party laboratory=100%; evaluation results: the consistency rate of the results is 100%, and the accuracy meets the requirements (see table 31);
table 30 accuracy verification case
| Sequence number | The method detects the result | Sanger sequencing assay results | Consistency of |
| Sample 1 | OXA-23 | OXA-23 | Consistency of |
| Sample 2 | mecA、NDM | mecA、NDM | Consistency of |
| Sample 3 | NDM | NDM | Consistency of |
| Sample 4 | OXA-48、CTX-M | OXA-48、CTX-M | Consistency of |
| Sample 5 | CTX-M | CTX-M | Consistency of |
| Sample 6 | CTX-M | CTX-M | Consistency of |
| Sample 7 | mecA | mecA | Consistency of |
| Sample 8 | Negative of | Negative of | Consistency of |
| Sample 9 | Negative of | Negative of | Consistency of |
| Sample 10 | Negative of | Negative of | Consistency of |
| Sample 11 | Negative of | Negative of | Consistency of |
| Sample 12 | Negative of | Negative of | Consistency of |
Table 31 method compliance verification
/>
(II) lower detection limit
The verification method comprises the following steps: performing gradient dilution to the detection limit concentration of 1100copies/ml by using a constant value sample, and performing repeated measurement on the concentration sample for 20 times in the same batch; judgment standard: at least 18 times of target nucleic acid must be detected, and the detection rate is more than or equal to 90 percent (standard source, molecular diagnosis test procedure performance verification guideline); evaluation results: the detection rate of the result is more than 90%, and the lower detection limit meets the requirements, and is shown in the following table 32.
Table 32 detects the lower limit condition
(III) Cross
The number of samples is more than or equal to 20, and the repetition number is more than or equal to 3; sample: pathogens having homology to the nucleic acid sequence of the test subject and being susceptible to the same or similar clinical symptoms typically include microorganisms having similar genetic structures, normal flora in the specimen, co-infectious microorganisms causing similar disease states or clinically relevant; the verification process comprises the following steps: detecting pathogen nucleic acids that may potentially produce cross-reactions; judgment standard: the established reagent is negative for detection of all pathogens that may potentially produce cross-reactions; evaluation results: the detection results are all negative, and the established reagent can not cross react with the following 20 pathogens, and the false detection can not occur in the report, and the cross reaction meets the requirements (see tables 48 to 50).
TABLE 48OXA-23, VANB crossover case
Table 49KPC, NDM, vanA, IMP crossover case
Table 50MECA, OXA-48, VIM, CTX-M crossover cases
/>
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (9)
1. The PCR amplification primer pair and the probe for detecting the bacterial drug resistance genes are characterized by comprising one or more of the following primer pairs and probes:
(1) The PCR amplification primer pair and the probe are used for detecting the ultra-broad spectrum beta-lactamase drug resistance gene, the PCR amplification primer pair is shown as SEQ ID NO.1 and SEQ ID NO.2, and the probe is shown as SEQ ID NO. 3;
(2) A PCR amplification primer pair and a probe for detecting carbapenem resistance gene, wherein the PCR amplification primer pair and the probe comprise one or more of the following sequences: (a) The PCR amplification primer pair is shown as SEQ ID NO.4 and SEQ ID NO.5, and the probe is shown as SEQ ID NO. 6; (b) The PCR amplification primer pair is shown as SEQ ID NO.7 and SEQ ID NO.8, and the probe is shown as SEQ ID NO. 9; (c) The PCR amplification primer pair is shown as SEQ ID NO.10 and SEQ ID NO.11, and the probe is shown as SEQ ID NO. 12; (d) The PCR amplification primer pair is shown as SEQ ID NO.13 and SEQ ID NO.14, and the probe is shown as SEQ ID NO. 15; (e) The PCR amplification primer pair is shown as SEQ ID NO.16 and SEQ ID NO.17, and the probe is shown as SEQ ID NO. 18; (f) The PCR amplification primer pair is shown as SEQ ID NO.19 and SEQ ID NO.20, and the probe is shown as SEQ ID NO. 21;
(3) The PCR amplification primer pair and the probe are used for detecting the methicillin-resistant gene, the PCR amplification primer pair is shown as SEQ ID NO.22 and SEQ ID NO.23, and the probe is shown as SEQ ID NO. 24;
(4) A PCR amplification primer pair and a probe for detecting vancomycin drug resistance genes, wherein the PCR amplification primer pair and the probe comprise one or more of the following sequences: (a) The PCR amplification primer pair is shown as SEQ ID NO.25 and SEQ ID NO.26, and the probe is shown as SEQ ID NO. 27; (b) The PCR amplification primer pair is shown as SEQ ID NO.28 and SEQ ID NO.29, and the probe is shown as SEQ ID NO. 30.
2. A PCR amplification detection reagent for bacterial drug resistance genes, which is characterized by comprising the PCR amplification primer pair and the probe for drug resistance gene detection according to claim 1.
3. The PCR amplification detection reagent for detecting bacterial drug resistance genes according to claim 2, comprising a PCR amplification primer pair of a reference gene and a probe, wherein the PCR amplification primer pair of the reference gene is shown as SEQ ID NO.31 and SEQ ID NO.32, and the probe is shown as SEQ ID NO. 33.
4. The reagent for PCR amplification detection of bacterial resistance gene according to claim 2 or 3, further comprising a fluorescent reporter group and a fluorescent quencher group.
5. The reagent for PCR amplification detection of bacterial drug resistance gene according to claim 4, wherein the fluorescent reporter group is ROX, FAM, CY5 or VIC; the fluorescence quenching group is BHQ1 or BHQ2.
6. The PCR amplification detection kit for detecting the bacterial drug resistance gene is characterized by comprising the PCR amplification primer pair for detecting the bacterial drug resistance gene, the probe or the PCR amplification detection reagent for detecting the bacterial drug resistance gene according to claims 2 to 5.
7. The use of the PCR amplification primer pair and the probe for detecting the bacterial drug resistance genes as defined in claim 1 in the preparation of detection products for detecting the bacterial drug resistance genes.
8. Use of the PCR amplification primer pair, the probe for detecting bacterial drug resistance gene according to claim 1 or the PCR amplification detection reagent for detecting bacterial drug resistance gene according to claims 2 to 5 or the PCR amplification detection kit according to claim 6 for detecting bacterial drug resistance gene.
9. A method for detecting a bacterial drug resistance gene, characterized in that a sample to be detected is subjected to PCR amplification detection using the PCR amplification primer pair for bacterial drug resistance gene detection of claim 1, a probe, or the PCR amplification detection reagent for detecting a bacterial drug resistance gene of claims 2 to 5 or the PCR amplification detection kit of claim 6; the amplification conditions for PCR include: the first stage: 95 ℃,2 minutes, 1 cycle; and a second stage: 95 ℃,10 seconds, 60 ℃,30 seconds, 40 cycles; fluorescence signals were collected at 60℃in the second stage.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311595357.4A CN117402989A (en) | 2023-11-27 | 2023-11-27 | PCR amplification primer pair and probe for bacterial drug resistance gene detection and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311595357.4A CN117402989A (en) | 2023-11-27 | 2023-11-27 | PCR amplification primer pair and probe for bacterial drug resistance gene detection and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117402989A true CN117402989A (en) | 2024-01-16 |
Family
ID=89500048
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311595357.4A Pending CN117402989A (en) | 2023-11-27 | 2023-11-27 | PCR amplification primer pair and probe for bacterial drug resistance gene detection and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117402989A (en) |
-
2023
- 2023-11-27 CN CN202311595357.4A patent/CN117402989A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2001504330A (en) | Species-specific, genus-specific and universal probes and primers for rapid detection and identification of common bacterial and fungal pathogens and related antibiotic resistance genes from clinical specimens for diagnosis in microbiological laboratories | |
| WO2023109032A1 (en) | Multiple nucleic acid detection system, and preparation method therefor and use thereof | |
| CN111004862B (en) | Primer and probe for rapidly detecting and identifying cryptococcus and application thereof | |
| CN107460242B (en) | Detection kit capable of simultaneously detecting multiple high-drug-resistance genes and application thereof | |
| CN113005226A (en) | Oligonucleotide and kit for detecting SARS-CoV-2 | |
| CN117363767B (en) | Probe combination, primer set and kit for real-time fluorescence PCR detection of target genes and application of probe combination and primer set and kit | |
| CN111690760A (en) | Method for accurately detecting cryptococcus neoformans | |
| CN112359125A (en) | Method for rapidly detecting cryptococcus gatherensis | |
| US20110287965A1 (en) | Methods and compositions to detect clostridium difficile | |
| CN117512204B (en) | Primer and probe combination for multiplex detection of aspergillus, cryptococcus and yersinia, kit and application | |
| CN113584191A (en) | Primer, probe and kit for multiplex PCR detection of 7 drug-resistant genes | |
| CN117551812A (en) | Composition for detecting PaBV type 2, paBV type 4 and PaBV type 5, kit and application thereof | |
| CN117402989A (en) | PCR amplification primer pair and probe for bacterial drug resistance gene detection and application thereof | |
| CN109457020A (en) | Resistance detection composition, Resistance detection kit and Resistance detection method | |
| CN115725754A (en) | Primer probe combination and kit for detecting three pneumonia pathogens | |
| CN113564269A (en) | Probe composition for preventing reverse transcription of bacterial conserved region and application thereof | |
| CN104561341B (en) | Primers and kit for detecting macrolide drug-resistance genes of bacteria | |
| CN113215329A (en) | Primer, probe and kit for multiplex PCR detection of 7 respiratory subtype influenza viruses | |
| KR102520374B1 (en) | Method and Kit for Identifying the serotype of Actinobacillus pleuropneumoniae causing pneumonia in Pigs using PNA probe | |
| KR102502129B1 (en) | Method and Kit for Identifying the serotype of Actinobacillus pleuropneumoniae causing pneumonia in Pigs using PNA probe | |
| US20230071189A1 (en) | Bacterial quantification method | |
| CN107523618A (en) | The PCR detection kit of drug-fast bacteria comprising production carbapenem and its application | |
| CN109207623B (en) | Fluorescent PCR (polymerase chain reaction) primer, probe and detection kit for identifying three sporothrix bacteria | |
| KR20230137097A (en) | Primer set for detecting Mycoplasma gallicepticum and detection method using the same | |
| CN117965771A (en) | Primer probe set for detecting cow mastitis pathogen by multiplex fluorescence quantitative PCR and detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |