CN116162718A - Primer probe combination, kit and method for identifying gram-positive bacteria - Google Patents
Primer probe combination, kit and method for identifying gram-positive bacteria Download PDFInfo
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- 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
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- 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]
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Abstract
The invention discloses a primer probe combination, a kit and a method for identifying gram-positive bacteria, wherein the primer probe combination comprises a forward primer 1, a forward primer 2, a reverse primer 1, a reverse primer 2 and a sequence shown by a probe; the forward primer 1 has a sequence shown as SEQ ID NO.1, the forward primer 2 has a sequence shown as SEQ ID NO.2, the reverse primer 1 has a sequence shown as SEQ ID NO.3, the reverse primer 2 has a sequence shown as SEQ ID NO.4, and the probe has a sequence shown as SEQ ID NO. 5. The primer probe combination for identifying the gram-positive bacteria uses a bacterial ribosome RNA region as a target region, and the bacterial sequence in the region is downloaded and compared, so that the optimal gram-positive bacteria primer and probe are screened and designed in a specific conservation region, and the type of the gram-positive bacteria is rapidly and accurately detected.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a primer probe combination, a kit and a method for identifying gram-positive bacteria.
Background
Gram-positive bacteria are a large variety of bacteria, often broadly referred to as a large class of bacteria that appear bluish purple under microscopic examination after gram staining, and encompass most of the genera pyococcus (staphylococcus, streptococcus, enterococcus), tubercle bacillus, tetanus bacillus, anthrax bacillus, and the like. In recent years, gram positive bacterial infection has an upward trend year by year, and the etiology of the diseases caused by the gram positive bacterial infection is complex, and the gram positive bacterial infection is one of main pathogenic bacteria causing common diseases and frequently encountered diseases in hospitals, and is dominant in hospital acquired bacteremia infection, and can generate exotoxins to cause diseases and even death of people, thereby seriously threatening the health of the human beings and the global public health safety. In order to effectively control gram-positive bacteria infection, the gram-positive bacteria is clinically determined as the infectious pathogen through preliminary screening, and then preliminary medication comments are given according to the characteristics that most gram-positive bacteria are sensitive to medicines such as penicillin, vancomycin, first (or second) generation cephalosporin, clindamycin and the like, so that the pathogenic identification range can be narrowed, diagnosis can be quickly made, and the method is very important for further separating and identifying specific strains through biochemical experiments.
At present, the common gram-positive bacteria identification method is a gram-staining method, and the method is invented in 1884 and generally comprises four steps of primary staining, mordant staining, decoloring and counterstaining, wherein the identification principle depends on a unique cell wall structure of the gram-positive bacteria, and gaps can not appear when ethanol is decolored by virtue of a network layer structure containing thicker cell walls and more peptidoglycan, so that a complex of crystal violet and iodine formed after crystal violet primary staining and iodine liquid mordant staining is left in the cell wall, and the complex is blue-violet under microscopic examination. Although gram staining methods have been used for more than a hundred years, the procedures are cumbersome, the used organic solvents are easy to pollute the environment, false positives caused by incomplete decolorization or excessive staining and false negative results formed by cell wall permeability change caused by death or autolysis of cell culture for too long exist in experiments, and great trouble is caused to doctors and patients. Besides the gram staining method, the gram positive bacteria identification method also comprises a bacterial separation culture method, a biochemical reaction method and a serological method, so that the gram positive bacteria can be systematically and comprehensively identified, but the defect that the detection time is long (2-5 days) exists. Therefore, there is a need to develop a new method for identifying gram-positive bacteria, which provides a basis for diagnosing and treating clinical gram-positive bacteria infection.
With the development of molecular biology technology, polymerase Chain Reaction (PCR) technology based on nucleic acid detection, real-time fluorescence PCR, nested PCR and the like are widely applied by virtue of the advantages of rapid detection, strong specificity, high sensitivity and the like. The real-time fluorescent PCR is a method based on PCR, and the total amount of products after each cycle is monitored by fluorescent chemical substances in PCR amplification reaction.
Therefore, the primer probe combination, the kit and the method which can rapidly and accurately detect the gram-positive bacteria type are of great significance.
Disclosure of Invention
The invention mainly aims to provide a primer probe combination, a kit and a method for identifying gram-positive bacteria, so as to overcome the defects in the prior art.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention comprises the following steps:
in a first aspect of embodiments of the present invention, there is provided a primer probe combination for identifying gram positive bacteria, the primer probe combination comprising the sequences shown in SEQ ID NO.1-SEQ ID NO. 5.
Further, the primer probe combination comprises a forward primer 1, a forward primer 2, a reverse primer 1, a reverse primer 2 and a sequence shown by a probe; wherein, the liquid crystal display device comprises a liquid crystal display device,
the forward primer 1 has a sequence shown as SEQ ID NO.1, the forward primer 2 has a sequence shown as SEQ ID NO.2, the reverse primer 1 has a sequence shown as SEQ ID NO.3, the reverse primer 2 has a sequence shown as SEQ ID NO.4, and the probe has a sequence shown as SEQ ID NO. 5.
In a second aspect of the embodiment of the invention, a real-time fluorescence PCR detection kit for identifying gram-positive bacteria is provided, which comprises a real-time fluorescence PCR amplification reagent, a positive reference substance, a negative reference substance, nuclease-free water and any one of the primer probe combinations.
Further, the positive control includes a streptococcus pneumoniae inactivated sample and a human whole genome DNA sample.
Further, the negative control is NaCl physiological saline with the mass percent concentration of 0.9 percent.
Further, the human DNA internal reference primer probe combination comprises sequences shown in SEQ ID NO.6-SEQ ID NO. 8.
In a third aspect of embodiments of the present invention, there is provided a method for identifying gram-positive bacteria for the purpose of not diagnosing and treating a disease, comprising the steps of:
s1, extracting a human whole genome DNA sample for later use;
s2, mixing a positive quality control product, a negative quality control product, a real-time fluorescence PCR amplification reagent and any one of the primer probe combinations, and then adding an EDTA solution to fix the volume to obtain a mixture;
s3, performing PCR reaction on the mixture obtained in the step S2, and collecting fluorescent signals;
s4, detecting the fluorescent signal, wherein if the Ct value is less than 30, the detection result is gram-positive bacteria; if the Ct value is more than or equal to 30, the detection result is not gram positive bacteria.
Further, the method for extracting the human whole genome DNA sample comprises the following steps: taking a clinical negative sample, extracting genome DNA according to a trace sample genome DNA extraction kit DP316 specification of Tiangen biological company, taking the clinical sample if the sample is a sputum sample, adding an equal volume of 1-10% NaOH solution, liquefying at room temperature until no solid state and wiredrawing state exist, and extracting the genome DNA.
Further, the final concentration of EDTA solution in step S2 is 0.1 to 1mM.
Further, the conditions of the PCR reaction in step S3 are as follows: denaturation at 95℃for 3min, nucleic acid melting; annealing at 95 ℃,25s, extension at 60 ℃,35s, running 40 cycles and collecting fluorescent signals.
The fluorescent probe PCR detection method is adopted, a fluorescent group (donor) and a quenching group (acceptor) are respectively marked at the 5' end and the 3' end of the probe, when the probe is complete, the fluorescent group and the quenching group do not fluoresce due to the fluorescence resonance energy transfer effect, when the primer extends to the 5' end of the probe, under the Taq enzymatic hydrolysis effect, the fluorescent group and the quenching group are separated to excite fluorescence, the accumulation of the reaction product quantity is changed through a fluorescent signal, and the progress of the amplification reaction is monitored in real time, and the detection method is high in detection sensitivity and strong in specificity. Compared with the traditional bacterial separation culture, biochemical reaction and serological detection method, the time consumption is about 2-5 days, and the detection method can finish the amplification reaction only by 1-2 hours, so that the time consumption is shortened; compared with a gram staining method, the detection method does not need to use an organic solvent, and is more environment-friendly; compared with the traditional PCR, the detection method has the advantages that the fluorescent probe is adopted, the specificity is stronger, the detection result is more accurate, the subsequent complicated agarose gel electrophoresis analysis of the reaction product is not needed, the operation is simpler and more convenient, the cross contamination of the reaction product is avoided, and the nucleic acid electrophoresis apparatus and gel imaging apparatus are omitted; compared with mass spectrum detection, the detection method is simpler and more convenient to operate, and an experimenter can independently complete the operation through simple training.
The invention provides a new primer probe combination, a kit and a method for identifying gram-positive bacteria, wherein the primer probe combination for identifying gram-positive bacteria takes a bacterial ribosome RNA area as a target area, and the optimal gram-positive bacteria primer and probe are screened and designed in a specific conservation area by downloading and comparing bacterial sequences in the area, can be realized on staphylococcus aureus, staphylococcus epidermidis, staphylococcus saprophyticus, staphylococcus hemolyticus, staphylococcus capitis, staphylococcus silvery, staphylococcus xylosus, staphylococcus saccharolyticus, staphylococcus mimicus, staphylococcus chromogenes, staphylococcus singapore, staphylococcus nepalensis, staphylococcus amber, staphylococcus sebum, staphylococcus picos, staphylococcus schneider, staphylococcus wovens, staphylococcus pasteurella, staphylococcus other, staphylococcus kresoxim, staphylococcus oto, staphylococcus lugdunensis, staphylococcus albert, staphylococcus hominis, staphylococcus gallinarum, staphylococcus felis, staphylococcus suis, staphylococcus macerans, staphylococcus felis, staphylococcus macerans, staphylococcus Streptococcus pneumoniae, streptococcus agalactiae, streptococcus azides, streptococcus faecium, streptococcus avis, streptococcus intermedius, gordon, streptococcus stomatitis, streptococcus thermophilus, streptococcus pyogenes, streptococcus mutans, streptococcus febrile, streptococcus faecalis, streptococcus lactis, streptococcus hemolyticus, streptococcus Bobipus, streptococcus ragmitis, streptococcus australis, streptococcus equisimilis, streptococcus suis, streptococcus equi, streptococcus iniae, streptococcus equi, streptococcus equisimilis, streptococcus gallic acid-decomposing Streptococcus, streptococcus Himalayans, streptococcus Paris, streptococcus infant, streptococcus maculons, streptococcus macerans, streptococcus Ma Kaka, streptococcus faecalis, streptococcus miehei, streptococcus paracasei, streptococcus paratuberis, streptococcus pyogenes, enterococcus avium, enterococcus raffinum, enterococcus faecalis, enterococcus faecium, enterococcus equi, enterococcus alkalescense, enterococcus lactis, enterococcus seawater, enterococcus anii, enterococcus coli, enterococcus cecum, enterococcus casselifaciens, enterococcus canis, enterococcus davidiana, enterococcus very different, enterococcus durans, enterococcus gallinarum, streptococcus vestibuli, enterococcus aureus, enterococcus peroxide, enterococcus haii, enterococcus hulfii, enterococcus italica, enterococcus morbus, enterococcus hawaii, enterococcus helveticus enterococcus mundtii, enterococcus olivaceus, enterococcus light-colored, enterococcus nanchangii, enterococcus olivaceus, enterococcus light-colored, enterococcus mongolicus, enterococcus peruvian, enterococcus suis, enterococcus reesei, enterococcus murinus, enterococcus saccharolyticus, enterococcus northsonii, enterococcus termitis, enterococcus tha, enterococcus oligosacchari, enterococcus enteritidis, enterococcus urinae, enterococcus villosa, tubercle bacillus, diphtheria bacillus, tetanus, actinomyces tunica, propionibacterium acnes, micrococcus luteus, hueplerian body, bacillus anthracis, bacillus, and the like.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
In a first aspect of embodiments of the present invention, there is provided a primer probe combination for identifying gram positive bacteria, the primer probe combination comprising the sequences shown in SEQ ID NO.1-SEQ ID NO. 5. Preferably, the primer probe combination comprises a forward primer 1, a forward primer 2, a reverse primer 1, a reverse primer 2 and a sequence shown by a probe; wherein, the forward primer 1 has a sequence shown as SEQ ID NO.1, the forward primer 2 has a sequence shown as SEQ ID NO.2, the reverse primer 1 has a sequence shown as SEQ ID NO.3, the reverse primer 2 has a sequence shown as SEQ ID NO.4, and the probe has a sequence shown as SEQ ID NO. 5.
In a second aspect of the embodiment of the invention, a real-time fluorescence PCR detection kit for identifying gram-positive bacteria is provided, which comprises a real-time fluorescence PCR amplification reagent, a positive reference substance, a negative reference substance, nuclease-free water and any one of the primer probe combinations.
The invention is described in detail below with reference to specific examples:
1. kit composition
1. Primer probe
Designing a universal primer and a probe of gram-positive bacteria according to the sequence of the bacterial ribosomal RNA gene; by aligning the introns of the human RPP30 gene, internal primers and probes for human DNA were designed (see Table 1).
TABLE 1 primer, probe sequence information
2. Real-time fluorescent PCR amplification reagents: probe qPCR Mix, with UNG.
3. Positive control: streptococcus pneumoniae inactivated sample + human whole genomic DNA sample.
4. Negative control: 0.9% NaCl physiological saline.
5. Nuclease-free water.
2. Whole genome DNA extraction
Taking 100-200 mu L of blood/alveolar lavage liquid/cerebrospinal fluid/pharyngeal swab samples (50 uL for sputum sample extraction), adding an equal volume of 1-10% NaOH solution into the sputum samples before extraction, liquefying at room temperature for 15min until no solid state exists, liquefying when the silk is pulled, and extracting genome DNA according to the specification of a microsamples genome DNA extraction kit DP316 of Tiangen biological company.
3. Real-time fluorescence PCR reaction system
The real-time fluorescence PCR reaction system is prepared according to the following scheme:
1. the kit is provided with 1 reaction system group, 1 positive quality control group and 1 negative quality control group, 3 reaction groups are mixed into a tube, the mixture is carried out according to the primer probes in the table, and then EDTA solution with the final concentration of 0.1-1 mM is added into the mixed primer probes;
2. the final concentration of FAM fluorescent probe in the real-time fluorescent PCR reaction system is 0.05uM, the final concentrations of upper and lower primers of gram positive bacteria are 0.16uM, the internal reference primer probe is 0.05uM, and the addition amount of the mixed primer probe in the real-time fluorescent PCR reaction system is 2.1 mu L;
3. real-time fluorescent PCR amplification reagents: 12.5. Mu.L;
4. clinical sample template: 3 μL;
5. nuclease-free water made up the system to 25 μl.
4. Real-time fluorescent PCR reaction conditions
The real-time fluorescence PCR reaction is carried out on an ABI 7500 real-time fluorescence PCR instrument, FAM and ROX fluorescence signals are collected in real time in each cycle, and the invention can avoid pollution caused by the last reaction product by using an amplification reagent containing UNG enzyme. The specific real-time fluorescence PCR amplification conditions were performed as follows: melting nucleic acid at 95 ℃ for 3 min; 95℃25s,60℃35s, 40 cycles were run and fluorescence signals were collected.
5. Nucleic acid quality control preparation
1. Positive nucleic acid quality control: staphylococcus aureus, streptococcus pneumoniae, listeria monocytogenes, enterococcus faecalis, enterococcus faecium, streptococcus agalactiae, enterococcus gallinarum, and enterococcus casseliflavus are purchased from north nano biotechnology limited; mycobacterium tuberculosis complex, hui's organisms, streptococcus parahaemolyticus, streptococcus tiger, streptococcus viridae, corynebacterium silvery, staphylococcus haemolyticus, streptococcus gordonii, propionibacterium acnes, and Staphylococcus wovens are positive samples for clinical sequencing. The gram positive bacteria nucleic acid quality control and clinical inactivation positive sample are prepared by extracting nucleic acid according to the specification of a microsample genome DNA extraction kit DP316 of Tiangen biological company.
Negative nucleic acid quality control: pertussis, legionella pneumophila, klebsiella pneumoniae, pseudomonas aeruginosa, acinetobacter mansoni, neisseria meningitidis, haemophilus influenzae, moraxella catarrhalis, aspergillus niger, aspergillus fumigatus, candida albicans, candida tropicalis liquor from North Nabiotechnology Co., ltd; viral nucleic acid quality control coronavirus HKU1 (FNRV 2853) was purchased from biotechnology limited, complex hundred australia; novel coronaviruses (M591002-0001) were purchased from the company Shanghai, inc.; parainfluenza virus type 1 (BDS-IQC-281), influenza B virus (BDS-IQC-073), rhinovirus (BDS-IQC-318), enterovirus (BDS-IQC-115) were purchased from Bandwidth Biotechnology Inc. The DNA nucleic acid quality control product is prepared by extracting nucleic acid according to the specification of a microsample genome DNA extraction kit DP316 of Tiangen biology company, and extracting RNA nucleic acid quality control product according to the specification of Chejj Viral RNA Mini Kit (52906).
6. Real-time fluorescence PCR detection of nucleic acid quality control and clinical samples
Respectively carrying out real-time fluorescence PCR detection on the extracted nucleic acid by using the primer probe combination, wherein the detection results show that gram positive bacteria are positive, and the Ct value of the detection results is less than 30 (see Table 2); the detection results of gram-negative bacteria, fungi, viruses and other negative samples are negative (see table 3), and specific Ct value detection results are shown in the following table.
Table 2. Real-time fluorescence PCR detection of gram positive bacteria detection results:
table 3. Real-time fluorescent PCR control sample detection results:
7. analysis of results
1. Analysis of detection results requires: the blank control test requires that the Ct value has no detection signal before 30, otherwise, the Ct value needs to be detected again; when a sample to be detected is detected, firstly, ensuring that the Ct value of the internal reference of the ROX channel DNA is less than or equal to 36, otherwise, the detection result is invalid, and carrying out extraction or sample adding experiments again, if the Ct value of the FAM channel of the sample to be detected is less than 30, judging that the detection result is positive, and if the Ct value is more than or equal to 30, judging that the detection result is negative.
2. Different clinical samples of 10 patients are detected by using the kit, the detection result of the kit is consistent with the detection result of metagenomic sequencing, and the specific detection result is shown in the following table 4.
TABLE 4 real-time fluorescence PCR detection results of clinical metagenomic sequencing samples
While only certain embodiments of the present invention have been described, it will be apparent to those skilled in the art that other modifications and improvements can be made without departing from the inventive concept of the present invention.
Claims (10)
1. A primer probe combination for identifying gram positive bacteria, characterized in that: the primer probe combination comprises sequences shown as SEQ ID NO.1-SEQ ID NO. 5.
2. The primer probe combination of claim 1, wherein: the primer probe combination comprises a forward primer 1, a forward primer 2, a reverse primer 1, a reverse primer 2 and a sequence shown by a probe; wherein, the liquid crystal display device comprises a liquid crystal display device,
the forward primer 1 has a sequence shown as SEQ ID NO.1, the forward primer 2 has a sequence shown as SEQ ID NO.2, the reverse primer 1 has a sequence shown as SEQ ID NO.3, the reverse primer 2 has a sequence shown as SEQ ID NO.4, and the probe has a sequence shown as SEQ ID NO. 5.
3. A real-time fluorescence PCR detection kit for identifying gram-positive bacteria is characterized in that: comprising a real-time fluorescent PCR amplification reagent, a positive control, a negative control, nuclease-free water, and the primer probe combination of any one of claims 1-2.
4. The real-time fluorescent PCR detection kit as claimed in claim 3, wherein: the positive control comprises a streptococcus pneumoniae inactivated sample and a human whole genome DNA sample.
5. The real-time fluorescent PCR detection kit as claimed in claim 3, wherein: the negative control is 0.9% NaCl physiological saline.
6. The real-time fluorescent PCR detection kit as claimed in claim 3, wherein: the human DNA internal reference primer probe combination comprises sequences shown as SEQ ID NO.6-SEQ ID NO. 8.
7. A method for identifying gram-positive bacteria not for the purpose of disease diagnosis and treatment, comprising the steps of:
s1, extracting a human whole genome DNA sample for later use;
s2, mixing a positive quality control product, a negative quality control product, a real-time fluorescence PCR amplification reagent and the primer probe combination according to any one of claims 1-2, and then adding an EDTA solution to fix the volume to obtain a mixture;
s3, performing PCR reaction on the mixture obtained in the step S2, and collecting fluorescent signals;
s4, detecting the fluorescent signal, wherein if the Ct value is less than 30, the detection result is gram-positive bacteria; if the Ct value is more than or equal to 30, the detection result is not gram positive bacteria.
8. The method of claim 7, wherein the method of extracting a human whole genomic DNA sample comprises: and (3) taking a clinical negative sample, extracting genome DNA, if the sample is a sputum sample, adding NaOH solution with the concentration of 1-10% by volume and mass percent into the clinical negative sample, liquefying at room temperature until no solid state and wiredrawing state exist, and extracting the genome DNA.
9. The method according to claim 7, wherein: the final concentration of EDTA solution in step S2 is 0.1-1 mM.
10. The method according to claim 7, wherein the conditions for the PCR reaction in step S3 are: denaturation at 95℃for 3min, nucleic acid melting; annealing at 95 ℃,25s, extension at 60 ℃,35s, running 40 cycles and collecting fluorescent signals.
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