CN116287325A - Primer probe composition, kit and application for identifying carbapenem drug-resistant acinetobacter baumannii - Google Patents
Primer probe composition, kit and application for identifying carbapenem drug-resistant acinetobacter baumannii Download PDFInfo
- Publication number
- CN116287325A CN116287325A CN202211320569.7A CN202211320569A CN116287325A CN 116287325 A CN116287325 A CN 116287325A CN 202211320569 A CN202211320569 A CN 202211320569A CN 116287325 A CN116287325 A CN 116287325A
- Authority
- CN
- China
- Prior art keywords
- acinetobacter baumannii
- resistant
- gene
- carbapenem
- probe
- 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
- 241000588626 Acinetobacter baumannii Species 0.000 title claims abstract description 87
- 239000000523 sample Substances 0.000 title claims abstract description 80
- YZBQHRLRFGPBSL-RXMQYKEDSA-N carbapenem Chemical compound C1C=CN2C(=O)C[C@H]21 YZBQHRLRFGPBSL-RXMQYKEDSA-N 0.000 title claims abstract description 40
- 239000000203 mixture Substances 0.000 title claims abstract description 31
- 229940079593 drug Drugs 0.000 title claims abstract description 21
- 239000003814 drug Substances 0.000 title claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 34
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 34
- 101150106096 gltA gene Proteins 0.000 claims abstract description 22
- 238000006243 chemical reaction Methods 0.000 claims abstract description 20
- 238000007847 digital PCR Methods 0.000 claims abstract description 17
- 239000002773 nucleotide Substances 0.000 claims description 15
- 125000003729 nucleotide group Chemical group 0.000 claims description 15
- 206010059866 Drug resistance Diseases 0.000 claims description 14
- 101100281953 Homo sapiens GAPDH gene Proteins 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- 230000003321 amplification Effects 0.000 claims description 8
- 208000015181 infectious disease Diseases 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 8
- 108020004707 nucleic acids Proteins 0.000 claims description 8
- 150000007523 nucleic acids Chemical class 0.000 claims description 8
- 102000039446 nucleic acids Human genes 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 108020004414 DNA Proteins 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 238000012216 screening Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 4
- 238000000605 extraction Methods 0.000 claims description 4
- 238000010791 quenching Methods 0.000 claims description 4
- 230000000171 quenching effect Effects 0.000 claims description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 239000008280 blood Substances 0.000 abstract description 6
- 210000004369 blood Anatomy 0.000 abstract description 6
- 229940124350 antibacterial drug Drugs 0.000 abstract description 3
- 238000012986 modification Methods 0.000 abstract description 2
- 230000004048 modification Effects 0.000 abstract description 2
- 238000004904 shortening Methods 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 10
- 238000003752 polymerase chain reaction Methods 0.000 description 6
- 238000003908 quality control method Methods 0.000 description 6
- 238000012408 PCR amplification Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 101150042350 gltA2 gene Proteins 0.000 description 4
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 230000017531 blood circulation Effects 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 108010068385 carbapenemase Proteins 0.000 description 2
- 229940041011 carbapenems Drugs 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 241000589291 Acinetobacter Species 0.000 description 1
- 241000304886 Bacilli Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000588697 Enterobacter cloacae Species 0.000 description 1
- 241000194032 Enterococcus faecalis Species 0.000 description 1
- 241000194031 Enterococcus faecium Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 101001066129 Homo sapiens Glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 241001147736 Staphylococcus capitis Species 0.000 description 1
- 241000191963 Staphylococcus epidermidis Species 0.000 description 1
- 241000192087 Staphylococcus hominis Species 0.000 description 1
- 241000122973 Stenotrophomonas maltophilia Species 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009640 blood culture Methods 0.000 description 1
- 208000037815 bloodstream infection Diseases 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 229940032049 enterococcus faecalis Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000011218 segmentation Effects 0.000 description 1
- 238000010206 sensitivity analysis Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 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
- C12Q2600/158—Expression markers
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- 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 discloses a primer probe composition for identifying carbapenem-resistant Acinetobacter baumannii, a kit and application thereof, wherein the primer probe composition comprises a primer pair and a probe for detecting the gltA gene of the Acinetobacter baumannii and the OXA-23 drug-resistant gene of the Acinetobacter baumannii, and the primer probe composition has higher sensitivity and specificity to the carbapenem-resistant Acinetobacter baumannii. The kit comprising the primer probe composition adopts a detection method of microdroplet digital PCR, and can realize the purpose of directly qualitatively determining the presence of carbapenem Acinetobacter baumannii in a whole blood sample by microdroplet treatment of a PCR reaction system to be analyzed in a sample with a lower level copy number, thereby shortening the time required for detection and prompting the clinical modification of a treatment scheme and the reasonable selection of antibacterial drugs.
Description
Technical Field
The invention belongs to the technical field of identification of non-fermented gram-negative bacilli, and particularly relates to a primer probe composition, a kit and application for identifying carbapenem-resistant acinetobacter baumannii.
Background
In non-fermented gram-negative bacillus, the blood flow infection rate caused by Acinetobacter baumannii is high, and Carbapenem drug-resistant Acinetobacter baumannii (Carbapenem-resistant Acinetobacter Baumanni, CRAB) is mainly used, so that diagnosis is not timely, and the clinical treatment difficulty and the patient death rate are high, which becomes a great challenge in the current medical and health industry.
The mechanism of drug resistance of CRAB is quite complex, and the OXA-type carbapenemase is one of the main reasons for drug resistance of Acinetobacter baumannii to carbapenems. The detection method of Acinetobacter baumannii mainly comprises a blood culture method, a PCR (polymerase chain reaction) method or a qPCR (quantitative polymerase chain reaction) method. The main technical problems of the existing method are as follows:
1. the sensitivity is low: the result of the detection of the low-concentration template cannot be determined (such as antibiotics, low concentration of bacteria in the sample and certain bactericidal substances in blood are used before the sample is collected), and the possibility of missing the sample with low concentration exists; 2. the culture method is time-consuming and usually needs to be cultured for more than 18 hours, and the result cannot be obtained in the same day, so that the treatment can be delayed.
At present, no carbapenemase typing experiment suitable for Acinetobacter baumannii recommended by the American clinical and laboratory standards institute (Clinical and Laboratory Standards Institute, CLSI) or approved by expert consensus is available, so a rapid identification method for the carbapenems drug-resistant Acinetobacter baumannii is established, and the method has very important clinical significance and value for compensating the lack of the enzyme production typing experiment suitable for Acinetobacter baumannii and shortening the conventional reporting time, thereby helping clinic to select antibacterial drugs for patients suffering from blood stream infection CRAB as soon as possible and timely, and formulating reasonable treatment and medication schemes.
Disclosure of Invention
In order to overcome the defects of the prior art, one of the purposes of the invention is to provide a primer probe composition for identifying carbapenem drug-resistant Acinetobacter baumannii, which has high sensitivity and strong specificity, can realize the specific amplification detection of Acinetobacter baumannii and drug-resistant gene OXA-23 thereof, and is suitable for screening and diagnosis of infection CRAB; the second object of the invention is to provide an application of a primer probe composition for identifying carbapenem-resistant acinetobacter baumannii in preparing a product for screening carbapenem-resistant acinetobacter baumannii infection; the invention further aims to provide a kit for identifying carbapenem-resistant acinetobacter baumannii, which has high accuracy and good convenience, combines microdroplet and PCR amplification technology, and is suitable for early screening, auxiliary diagnosis and prognosis evaluation of infection.
One of the purposes of the invention is realized by adopting the following technical scheme:
a primer probe composition for identifying carbapenem-resistant acinetobacter baumanii, which comprises a primer pair and a probe for detecting the gltA gene of the acinetobacter baumanii and the OXA-23 drug-resistant gene of the acinetobacter baumanii;
the nucleotide sequence of the primer pair for detecting the acinetobacter baumannii gltA gene comprises sequences shown in SEQ ID NO.1 and SEQ ID NO. 2; the nucleotide sequence of the probe of the acinetobacter baumannii gltA gene comprises a sequence shown in SEQ ID NO. 3;
the nucleotide sequence of the primer pair for detecting the Acinetobacter baumannii OXA-23 drug resistance gene comprises sequences shown in SEQ ID NO.4 and SEQ ID NO. 5; the nucleotide sequence of the probe of the Acinetobacter baumannii OXA-23 drug resistance gene comprises a sequence shown in SEQ ID NO. 6.
Further, the primer probe composition further comprises an internal reference primer pair for detecting an internal reference gene and a corresponding internal reference probe; the two ends of the reference probe are respectively marked with a fluorescent group and a quenching group, and the fluorescent groups of the reference probe are different from the fluorescent groups of probes respectively used for detecting the gltA gene of the acinetobacter baumannii and the OXA-23 drug resistance gene of the acinetobacter baumannii.
Further, the reference gene is a human GAPDH gene; the nucleotide sequences of the internal reference primer pair of the human GAPDH gene comprise sequences shown as SEQ ID NO.7 and SEQ ID NO. 8; the nucleotide sequence of the internal reference probe of the human GAPDH gene comprises the sequence shown in SEQ ID NO. 9.
Further, the 5' end of the probe for detecting the acinetobacter baumannii gltA gene and the acinetobacter baumannii OXA-23 drug resistance gene is marked with any one of FAM, ROX, VIC and CY5 fluorescent groups; the 3' end of the probe is labeled with any one of MGB, BHQ and TAMRA quenching groups.
The second purpose of the invention is realized by adopting the following technical scheme:
the application of the primer probe composition for identifying the carbapenem-resistant acinetobacter baumannii in preparing products for screening carbapenem-resistant acinetobacter baumannii infection.
The third purpose of the invention is realized by adopting the following technical scheme:
a kit for identifying carbapenem-resistant acinetobacter baumannii comprises the primer probe composition for identifying carbapenem-resistant acinetobacter baumannii.
Further, at least one of a nucleic acid extraction reagent and a PCR reaction reagent is also included.
Further, the detection method of the kit comprises the following steps:
extracting genome of thallus in a sample to be detected, taking the extracted genome as a DNA template, adding the primer probe composition for identifying carbapenem drug-resistant Acinetobacter baumannii to obtain detection liquid, performing microdroplet treatment on the detection liquid, performing microdroplet digital PCR amplification, detecting fluorescent signals and judging the result.
Specifically, the microdroplet digital PCR amplification procedure is:
1) Reacting for 4-6 min at 90-96 ℃ and circulating for 1-2 times;
2) Reacting for 15-25 s at 90-95 ℃, then circulating for 0.5-2 min at 50-70 ℃ and 40-50 times;
3) And the reaction is kept at 30-40 ℃ until the end of the reaction.
Further, the detection liquid comprises the following components: the primer probe composition for identifying carbapenem-resistant Acinetobacter baumannii, 2 XddPCR Mix, DNA polymerase, DNA template and water.
Compared with the prior art, the invention has the beneficial effects that:
(1) The primer probe composition for identifying carbapenem-resistant acinetobacter baumanii selects gltA gene and OXA-23 gene to detect acinetobacter baumanii and drug resistance thereof, and human GAPDH gene is used as an internal reference gene for simulating clinical blood flow infection samples; through sequence comparison, a primer probe is designed for a corresponding gene specific section, so that the primer probe composition has higher sensitivity and specificity for carbapenem drug-resistant Acinetobacter baumannii.
(2) The kit provided by the invention comprises the primer probe composition, and can be used for directly and rapidly detecting Acinetobacter baumannii and the drug-resistant gene OXA-23 thereof from a whole blood sample of a patient infected with CRAB by blood flow, so that the problem of lack of enzyme-producing typing experiments applicable to Acinetobacter baumannii is solved, the conventional reporting time is shortened, and a method is provided for determining a reasonable treatment scheme in clinic earlier. Specifically, the kit adopts a detection method of microdroplet digital PCR, a PCR reaction system to be analyzed is subjected to microdroplet treatment, each generated microdroplet contains one or more or does not contain nucleic acid target molecules to be detected, after independent PCR amplification reaction is carried out on tens of thousands of microdroplets, fluorescent signals of each microdroplet are collected and are subjected to statistical analysis, so that absolute quantification of pathogen DNA or RNA is realized, based on the advantages of digital PCR, the purpose of directly determining the nature of carbapenem Acinetobacter in a whole blood sample can be realized in a sample with a lower level copy number, the time required for detection is shortened, and the modification of a treatment scheme and the reasonable selection of antibacterial drugs can be prompted clinically. Compared with the traditional fluorescent quantitative PCR method, the quantitative mode of the ddPCR technology is independent of a standard curve, and the sensitivity and the accuracy are higher than those of the traditional method.
(3) Specifically, the detection method of the kit is to generate microdroplets by using detection liquid to realize the segmentation of a reaction system; then the amplification is finished by a common PCR instrument; and reading fluorescent signals by a photographing mode, and calculating the initial copy number concentration of the target molecules according to the number of positive microdroplets and the Poisson distribution. The experiment does not need a chip, and the instrument and consumable cost is lower.
Detailed Description
The present invention will be further described with reference to the following specific embodiments, and it should be noted that, without conflict, any combination of the embodiments or technical features described below may be used to form new embodiments.
A primer probe composition for identifying carbapenem-resistant acinetobacter baumanii, which comprises a primer pair and a probe for detecting the gltA gene of the acinetobacter baumanii and the OXA-23 drug-resistant gene of the acinetobacter baumanii, and a primer pair and a probe for detecting an internal reference gene;
the nucleotide sequence of the primer pair for detecting the acinetobacter baumannii gltA gene comprises sequences shown in SEQ ID NO.1 and SEQ ID NO. 2; the nucleotide sequence of the probe of the acinetobacter baumanii gltA gene comprises a sequence shown in SEQ ID NO.3, specifically 5'- (HEX) -TTCGATGCGAAAGTTCGTGCTCATACTATG- (BHQ 1) -3';
the nucleotide sequence of the primer pair for detecting the Acinetobacter baumannii OXA-23 drug resistance gene comprises sequences shown in SEQ ID NO.4 and SEQ ID NO. 5; the nucleotide sequence of the probe of the Acinetobacter baumannii OXA-23 drug resistance gene comprises a sequence shown in SEQ ID NO.6, and specifically 5'- (FAM) -TGAAGCTTTCTGCAGTCCCAGTCTATCAGG- (BHQ 1) -3'.
The reference gene is a human GAPDH gene; the nucleotide sequences of the internal reference primer pair of the human GAPDH gene comprise sequences shown as SEQ ID NO.7 and SEQ ID NO. 8; the nucleotide sequence of the internal reference probe of the human GAPDH gene comprises a sequence shown in SEQ ID NO.9, specifically 5'- (CY 5) -TCCCCCCCACCCCCATAGGC- (BHQ 3) -3'.
A kit for identifying carbapenem-resistant acinetobacter baumannii comprises the primer probe composition for identifying carbapenem-resistant acinetobacter baumannii.
1. Detection method of kit
The specific operation steps of A are as follows:
1. experimental materials
1. Main instrument equipment: droplet generator, PCR amplification instrument, and droplet analysis software
2. The main reagent comprises:
(1)2×ddPCR Mix
(2) DNA polymerase
2. Detection step
1. Sample preparation
Corresponding clinical isolates were collected: escherichia coli, klebsiella pneumoniae, staphylococcus epidermidis, enterococcus faecium, staphylococcus aureus, pseudomonas aeruginosa, staphylococcus capitis, pseudomonas maltophilia, staphylococcus hominis, enterococcus faecalis, enterobacter cloacae, and OXA-23 drug-resistant Acinetobacter baumannii, and extracting the above strains and human genome nucleic acids respectively for primer specificity analysis; wherein the OXA-23 drug-resistant Acinetobacter baumannii sample is used as a positive quality control, the human genome sample is used as a negative quality control, and the non-enzyme water is used as a blank control.
2. Triple digital PCR detection system establishment
The system configuration is performed in the reagent preparation area. The configuration table of the triple digital PCR detection system of the acinetobacter baumannii gltA, OXA-23 and human GAPDH genes is shown in table 1 (25 ul reaction system):
TABLE 1 digital PCR triple reaction System
| Reagent name | Volume (mu L) |
| 2×ddPCR Mix | 12.5 |
| DNA polymerase | 0.6 |
| gltA upstream primer (50. Mu.M) | 0.25 |
| gltA downstream primer (50. Mu.M) | 0.25 |
| gltA probe (5. Mu.M) | 1.25 |
| Oxa-23 upstream primer (50. Mu.M) | 0.25 |
| Oxa-23 downstream primer (50. Mu.M) | 0.25 |
| Oxa-23 probe (5. Mu.M) | 1.25 |
| GAPDH upstream primer (50. Mu.M) | 0.25 |
| GAPDH downstream primer (50. Mu.M) | 0.25 |
| GAPDH probe (5. Mu.M) | 1.25 |
| DNA template | 0.5-5ng |
| RNase Free Water | to 25 |
3. Sample addition
a, adding samples in a sample preparation area, and respectively adding the nucleic acid extracted in the step 1 into a reaction system shown in the step 2; b, transferring the prepared sample tube into a micro-centrifuge, and centrifuging at 4000rpm for 5 seconds; the bottom of the tube was flicked to mix the reaction solution uniformly while removing bubbles, and centrifuged at 4000rpm for 5 seconds.
4. Droplet generation and PCR amplification
1) Generating droplets according to instructions of the droplet generator;
2) Transferring the generated microdroplet to a PCR instrument for amplification; the reaction procedure: 95 ℃/5min,1 cycle; 94 ℃/20s,60 ℃/1min,45 cycles; 35 deg.C/Hold.
5. Droplet reading
(1) After the PCR amplification is finished, information acquisition is carried out on the digital PCR reaction by utilizing droplet reading software;
(2) The results are analyzed and interpreted in droplet analysis software.
B result analysis and interpretation
1. Threshold setting
The threshold line of fluorescence is set according to the fluorescence value of the negative droplets in the digital PCR system. The threshold line requires a significant distinction between negative and positive amplification results.
2. Primer-specific assay results
The primer specificity detection results are shown in Table 2, so that the primer probe group has good specificity.
TABLE 2 primer specificity detection results
Note that: "-" indicates that the gene test result is negative, and "+" indicates that the gene test result is positive.
2. Sensitivity analysis detection
Plasmids (engineering) containing the gltA gene and the OXA-23 gene of Acinetobacter baumannii were used as samples for the primer sensitivity test, and plasmids were subjected to gradient dilution with TE buffer, and were added to the reaction system shown in Table 3 in accordance with 1000copies/T, 100copies/T, 50copies/T, 20copies/T, 10copies/T, 5copies/T, 2copies/T, and a blank (no enzyme water instead of plasmid), respectively, for the test.
TABLE 3 digital PCR Dual reaction System
The detection results are shown in Table 4, which shows that the digital PCR detection system of the present invention can detect 10copies/T at the lowest.
TABLE 4 sensitivity test results
Note that: "-" indicates that the gene test result is negative, and "+" indicates that the gene test result is positive.
3. Detection method for simulated clinical sample
The specific operation steps of A are as follows:
sample preparation
(1) Collecting clinically separated sensitive and OXA-23 drug-resistant Acinetobacter baumannii strains, and culturing to obtain corresponding bacterial liquid;
(2) Adding different types of Acinetobacter baumannii bacteria liquid into blood of healthy people to be used as a simulated clinical sample; a blood sample of a healthy person is used as a negative quality control product;
(3) Performing nucleic acid extraction on the simulated clinical samples according to the instructions of the commercial nucleic acid extraction kit;
b detection analysis
The nucleic acid samples obtained in the step 1 were added to the triple reaction system constructed in the example 2, respectively, and detection analysis was performed according to the example 2.
C quality control
One of the following conditions is not met, the experiment is regarded as invalid and the detection needs to be repeated:
the number of digital PCR droplets generated is not less than 90% of theoretical value;
blank control: HEX, FAM, CY5 three channels (representing the Acinetobacter baumannii gltA gene, the Acinetobacter baumannii OXA-23 drug resistance gene, and the human GAPDH gene, respectively) all had no positive microdroplet;
negative quality control: the CY5 channel (representing the human GAPDH gene) has positive droplets; HEX and FAM channels (respectively representing the gltA gene and the OXA-23 drug resistance gene of Acinetobacter baumannii) have no positive microdroplet;
d, analyzing and interpreting results
After the above quality control conditions were satisfied, the results were interpreted according to table 5.
TABLE 5 interpretation of results
E detection result
The detection results are shown in Table 6, and show that the digital PCR reaction system can rapidly and accurately detect Acinetobacter baumannii and OXA-23 drug-resistant strains thereof.
Table 6 test results for simulated clinical samples
Note that: "-" indicates that the gene test result is negative, and "+" indicates that the gene test result is positive.
In summary, the kit comprising the primer probe composition has higher sensitivity and specificity to carbapenem drug-resistant Acinetobacter baumannii, and the detection method of the microdroplet digital PCR can reduce the omission of low-concentration samples, can report at an earlier stage of infection, and further shortens the detection period; sensitivity and specificity meet clinical use requirements.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention are intended to be within the scope of the present invention as claimed.
Claims (10)
1. The primer probe composition for identifying carbapenem-resistant acinetobacter baumannii is characterized by comprising a primer pair and a probe for detecting the gltA gene of the acinetobacter baumannii and the OXA-23 drug-resistant gene of the acinetobacter baumannii;
the nucleotide sequence of the primer pair for detecting the acinetobacter baumannii gltA gene comprises sequences shown in SEQ ID NO.1 and SEQ ID NO. 2; the nucleotide sequence of the probe of the acinetobacter baumannii gltA gene comprises a sequence shown in SEQ ID NO. 3;
the nucleotide sequence of the primer pair for detecting the Acinetobacter baumannii OXA-23 drug resistance gene comprises sequences shown in SEQ ID NO.4 and SEQ ID NO. 5; the nucleotide sequence of the probe of the Acinetobacter baumannii OXA-23 drug resistance gene comprises a sequence shown in SEQ ID NO. 6.
2. The primer probe composition for identifying carbapenem-resistant acinetobacter baumannii of claim 1, further comprising an internal reference primer pair for detecting an internal reference gene and a corresponding internal reference probe; the two ends of the reference probe are respectively marked with a fluorescent group and a quenching group, and the fluorescent groups of the reference probe are different from the fluorescent groups of probes respectively used for detecting the gltA gene of the acinetobacter baumannii and the OXA-23 drug resistance gene of the acinetobacter baumannii.
3. The primer probe composition for identifying carbapenem-resistant acinetobacter baumannii of claim 2, wherein said reference gene is a human GAPDH gene; the nucleotide sequences of the internal reference primer pair of the human GAPDH gene comprise sequences shown as SEQ ID NO.7 and SEQ ID NO. 8; the nucleotide sequence of the internal reference probe of the human GAPDH gene comprises the sequence shown in SEQ ID NO. 9.
4. The primer probe composition for identifying carbapenem-resistant acinetobacter baumanii according to claim 1, wherein the 5' -end of the probe for detecting the gltA gene of acinetobacter baumanii and the OXA-23 drug-resistant gene of acinetobacter baumanii is labeled with any one of FAM, ROX, VIC and CY5 fluorescent groups; the 3' end of the probe is labeled with any one of MGB, BHQ and TAMRA quenching groups.
5. Use of the primer probe composition for identifying carbapenem-resistant acinetobacter baumannii according to any one of claims 1 to 4 in the preparation of a product for screening carbapenem-resistant acinetobacter baumannii infection.
6. A kit for identifying carbapenem-resistant acinetobacter baumanii, comprising the primer probe composition for identifying carbapenem-resistant acinetobacter baumanii of any one of claims 1 to 4.
7. The kit for identifying carbapenem-resistant acinetobacter baumannii of claim 6, further comprising at least one of a nucleic acid extraction reagent and a PCR reaction reagent.
8. Kit for identifying carbapenem-resistant acinetobacter baumannii according to claim 6 or 7, wherein the detection method comprises the following steps:
extracting genome of thallus in a sample to be detected, adding the primer probe composition for identifying carbapenem drug-resistant acinetobacter baumannii according to any one of claims 1-3 to obtain detection liquid, performing microdroplet treatment on the detection liquid, performing microdroplet digital PCR amplification, detecting fluorescent signals and judging the result.
9. The kit for identifying carbapenem-resistant acinetobacter baumannii of claim 8, wherein said procedure for microdroplet digital PCR amplification is:
1) Reacting for 4-6 min at 90-96 ℃ and circulating for 1-2 times;
2) Reacting for 15-25 s at 90-95 ℃, then circulating for 0.5-2 min at 50-70 ℃ and 40-50 times;
3) And the reaction is kept at 30-40 ℃ until the end of the reaction.
10. The kit for identifying carbapenem-resistant acinetobacter baumannii of claim 8, wherein said detection solution comprises the following components: the primer probe composition for identifying carbapenem-resistant acinetobacter baumannii of any of claims 1 to 3, 2 x ddPCR Mix, DNA polymerase, DNA template and water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211320569.7A CN116287325A (en) | 2022-10-26 | 2022-10-26 | Primer probe composition, kit and application for identifying carbapenem drug-resistant acinetobacter baumannii |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211320569.7A CN116287325A (en) | 2022-10-26 | 2022-10-26 | Primer probe composition, kit and application for identifying carbapenem drug-resistant acinetobacter baumannii |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116287325A true CN116287325A (en) | 2023-06-23 |
Family
ID=86783868
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211320569.7A Pending CN116287325A (en) | 2022-10-26 | 2022-10-26 | Primer probe composition, kit and application for identifying carbapenem drug-resistant acinetobacter baumannii |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116287325A (en) |
-
2022
- 2022-10-26 CN CN202211320569.7A patent/CN116287325A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113512602B (en) | Blood stream infection pathogen multiple gene detection system and kit and application thereof | |
| CN111647654B (en) | Primer composition, kit and method for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutation | |
| WO2023109032A1 (en) | Multiple nucleic acid detection system, and preparation method therefor and use thereof | |
| CN111073961A (en) | High-throughput detection method for gene rare mutation | |
| CN112226538B (en) | Primer probe combination, kit and method for novel coronavirus detection | |
| CN110904250B (en) | Multiplex fluorescent quantitative PCR primer, kit and detection method for detecting multiple bacteria | |
| CN112941210A (en) | Kit and method for detecting drug-resistant mutation of mycobacterium tuberculosis rifampicin and isoniazid | |
| JPWO2016167365A1 (en) | Mental illness biomarker | |
| EP4006172A1 (en) | Composition for detecting pathogens, and kit and method therefor | |
| CN110577987A (en) | Detection method of CGG (glutamic acid G) repetitive sequence of FMR1 gene and application thereof | |
| CN115948532A (en) | SMA detection kit based on digital PCR technology | |
| CN115992276A (en) | Primer and kit for detecting urinary tract infection pathogens and drug resistance genes and application of primer and kit | |
| CN108048565A (en) | A kind of primer for detecting ApoE gene pleiomorphisms and its detection method and application | |
| CN110846408A (en) | Primer combination for detecting TTN gene mutation and application thereof | |
| CN103789440A (en) | Spinal muscular atrophy-related gene mutation detection method, related detection probe composition and detection kit as well as related application | |
| CN111235268B (en) | SNP locus genotype detection reagent and application in corresponding kit and kit | |
| CN116144811B (en) | Multiplex primer set, method and kit for detecting cerebrospinal fluid pathogen | |
| CN116356054A (en) | Kit for detecting common pathogenic microorganisms and drug resistance genes and detection method | |
| CN116083555A (en) | Biomarker combination for predicting or detecting keratoconus, detection agent and application | |
| CN116287325A (en) | Primer probe composition, kit and application for identifying carbapenem drug-resistant acinetobacter baumannii | |
| CN111349718A (en) | Primer group for pathogenic nucleic acid amplification, pathogenic nucleic acid detection library construction method and pathogenic detection method | |
| CN111690736A (en) | Warfarin medication gene detection kit and use method thereof | |
| CN112029851A (en) | Method and kit for detecting gene polymorphism of clopidogrel medication and application of kit | |
| CN117467781B (en) | SNP (Single nucleotide polymorphism) marker, PCR (polymerase chain reaction) primer pair and probe pair related to brucella compound neonomine drug resistance, kit and application of SNP marker and PCR primer pair and probe pair | |
| CN116121358B (en) | Composition for psychotropic drug gene detection, kit and use 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 |