CN109439780A - Moraxelle catarrhalis fluorescence PCR detection reagent kit - Google Patents
Moraxelle catarrhalis fluorescence PCR detection reagent kit Download PDFInfo
- Publication number
- CN109439780A CN109439780A CN201811585687.4A CN201811585687A CN109439780A CN 109439780 A CN109439780 A CN 109439780A CN 201811585687 A CN201811585687 A CN 201811585687A CN 109439780 A CN109439780 A CN 109439780A
- Authority
- CN
- China
- Prior art keywords
- kit
- moraxelle catarrhalis
- pcr reaction
- nucleic acid
- 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
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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
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 present invention relates to a kind of using kit existing for moraxelle catarrhalis nucleic acid in real time fluorescent PCR method specific detection sample.Using kit of the invention, moraxelle catarrhalis nucleic acid can be whether there is in quick test sample；The kit is limited to the every reaction system of 20 copies to the detection of moraxelle catarrhalis nucleic acid, and entire reaction can be completed in 2 hours, has important application value in fields such as disease surveillance, clinical diagnosises.
Description
Technical field
The present invention relates to a kind of reagents using moraxelle catarrhalis nucleic acid in real time fluorescent PCR method specific detection sample
Box belongs to the in-vitro diagnosis field of moraxelle catarrhalis.
Background technique
Moraxelle catarrhalis (Moraxella Catarrhalis) is Gram-negative non-zymocyte.The bacterium can cause a variety of
Disease, such as meningitis, endocarditis, urethritis, baby and children's eye conjunctivitis, keratitis.In otitis media acuta, maxillary sinus
In scorching and bronchus and pulmonary infection, this bacterium is important and common pathogen.In otitis media acuta, 6% ~ 25.6% can be therefrom
The bacterium is found in the liquid of ear.From the nasal discharge of children acute maxillary sinusitis, from acute and chronic tracheitis, bronchitis, lung
Inflammation, in the case that the basis of especially original hypoimmunity is sick (such as connective tissue disease, septicaemia etc.), from phlegm and Airway secretion
Moraxelle catarrhalis is separated in object.Moraxelle catarrhalis can cause pneumonia, clinical characters mainly have high fever, expiratory dyspnea,
The symptoms such as pulse acceleration, accelerated breathing, expectoration.At the beginning of this disease is mainly in last month of spring in winter, and it is more common in old man, and has susceptible basis more
Disease, the most common are patients with COPD, most of pulmonary infections are spread by air flue approach.
In the detection, PCR method will be substituted since its fast and convenient feature gradually shows with Bacteria Culture and serology
The trend of traditional detection method based on detection.And for PCR method, the specificity of primer be its detection specificity and
The basis of sensibility.
The present invention be directed to respectively moraxelle catarrhalis gene order the special target sequence design primer of conservative region and
Taqman probe, using the method for real-time PCR, for whether there is the core of moraxelle catarrhalis in quick test sample
Acid.
Summary of the invention
The present invention solves the technical problem of whether there is moraxelle catarrhalis in rapidly and accurately test sample, provide
A kind of fluorescent PCR kit of specific detection moraxelle catarrhalis.
The technical problem to be solved by the present invention is to what is be achieved through the following technical solutions:
The kit of moraxelle catarrhalis nucleic acid in a kind of specific detection sample, component includes PCR reaction solution, enzyme mixation, yin
Property quality-control product and positive quality control product.Wherein PCR reaction solution mainly contain above-mentioned primer and probe, reaction buffer, Mg2+ and
DNTP etc., enzyme mixation mainly contain hot start Taq polymerase, and negative quality-control product is no RNase and DNase water, and positive quality control product is
The recombinant plasmid in the site of specific amplification containing moraxelle catarrhalis.
Primer in PCR reaction solution for nucleic acid amplification is that P1 and P2, P1 and P2 are for moraxelle catarrhalis gene group
The specific primer that specific and conserved sequence is designed and filtered out through preliminary experiment；For fluorescence signal monitoring in PCR reaction solution
Oligonucleotide probe is Probe1, and Probe1 is to design for moraxelle catarrhalis gene group specific and conserved sequence and through pre- reality
The specific probe filtered out is tested, wherein fluorescent reporter group X1 is ROX, and fluorescent quenching group Y1 is 1) Eclipse(is shown in Table.
The primer and probe sequence of the detection moraxelle catarrhalis of table 1
Application method it is a further object to provide this fluorescence PCR detection reagent kit in detection moraxelle catarrhalis, packet
It includes:
The PCR reaction system that kit is selected is 20 μ l, including 2 × PCR buffer, 10 μ l, 1.0 μ of 10mmol/L dNTP
L, each 0.5 μ l of 25 μm of ol/L primers, 10 μm of 0.2 μ l of ol/L probe, 0.4 μ l of hot start Taq polymerase mixed liquor, sample DNA 2
μ l adds aqua sterilisa to whole 20 μ l of system.
The PCR reaction cycle parameter of kit is 94 DEG C, 2min；Into the cycle stage: 94 DEG C of denaturation 10s, 56 DEG C are annealed
50s, 72 DEG C of extension 15s, coreaction 40 circulations.
Quality control: experiment should set up positive and negative control every time, and negative control is without Ct value (or Ct value is 0), positive control
Value≤30 Ct, otherwise experimental result is invalid.
As a result interpretation:
It is positive: " S " type amplification curve, value≤35 Ct occur；It is suspicious: " S " type amplification curve, but Ct value > 35 occur；It is negative: not have
It occurs " S " type amplification curve or although curve has been more than threshold value, but be not in " S " type；To suspect results, experiment should be repeated,
If repeating experiment or " S " type amplification curve occur, negative control is not polluted, be can determine whether as the positive.
Sample requirement: clinical sample type includes throat swab, sputum and bacterial cultures；It should be with ice after clinical sample acquisition
Transport, -20 DEG C of preservations, is unable to multigelation；DNA is extracted from clinical sample, it is proposed that is tried using commercialization stable and reliable for performance
Agent box, specific method should detect immediately referring to corresponding commodity kit specification, the DNA of extraction, otherwise, Ying Jiang DNA packing
- 80 DEG C afterwards~-20 DEG C preservations.
Kit provided by the invention has specificity well, can detect moraxelle catarrhalis, but cannot detect non-card
The nucleic acid of his Moraxella pathogen；Monitoring lower-cut to moraxelle catarrhalis nucleic acid is the 20 every reaction systems of copy；Only need 2 hours
Detection can be completed, experimental basis can be provided for the disease surveillance of moraxelle catarrhalis and clinical diagnosis.
Detailed description of the invention
Fig. 1 is the amplification curve diagram of substance real-time fluorescence PCR detection moraxelle catarrhalis positive criteria product.From left to right 5
Curve is respectively (2 × 10 after the dilution of moraxelle catarrhalis DNA ladder degree6-2×102Copies/ μ l) amplification.Abscissa is
Reaction cycle number, ordinate are the Δ Rn value of different recurring number fluorescent assay signals.
Fig. 2 is the amplification curve diagram that substance real-time fluorescence PCR detection system detects 10 kinds of bacterial genomes DNA.It is 10 kinds thin
Bacterium includes: moraxelle catarrhalis and 9 kinds of negative control microorganisms.There is S type amplification curve in moraxelle catarrhalis, and other 9 kinds of cause of diseases
Microorganism does not occur S type amplification curve.
Specific embodiment
Detailed description of the preferred embodiments of the present invention combined with specific embodiments below.It should be pointed out that listing here
Embodiment be merely exemplary the purpose of explanation, and it should not be constructed as any limitation on the scope of the present invention.Wherein make
The reagents such as kit, buffer are only the reagent being specifically chosen in the specific embodiment, it should be appreciated that those skilled in the art
Member can select as needed the corresponding reagent of other companies to achieve the object of the present invention.
1, the design and synthesis of primer and Taqman probe
The moraxelle catarrhalis genome sequence in Genbank and domestic and foreign literature is analyzed using Blast tool, is selected respectively
Its stable conservative region is selected as detection target sequence, and for detection target sequence design and synthesis primer and probe (see Table 1).
Primer and probe is synthesized by the Japanese Dalian TaKaRa treasured biotech firm, and the end of detection probe 5 ' the label ROX of moraxelle catarrhalis is glimmering
Light group, 3 ' end label Eclipse fluorescent quenching groups.
2, the preparation of strain is detected
Moraxelle catarrhalis used in the present embodiment and other negative control bacterial strains (streptococcus pneumonia, haemophilus influenzae,
Staphylococcus aureus, hemolytic streptococcus, Klebsiella Pneumoniae, Escherichia coli, pseudomonas aeruginosa, legionella pneumophilia are green
Purulence bacillus) buy in Nat'l Pharmaceutical & Biological Products Control Institute.
3, the extracting of bacterial strain DNA
It selects Qiagen company QIAamp DNA Mini Kit(article No.: 51306) extracting bacterial strain DNA.Specific steps kit ginseng
Examine operational manual.
4, the screening of primer and probe
Using the moraxelle catarrhalis of the primer and probe Detection and Extraction of design and the genomic DNA of negative control bacterial strain, through repeatedly
Experiment, filtering out sensitivity, specificity and the optimal primer combination of probe of repeatability, (see sequence table, moraxelle catarrhalis forward direction is drawn
Object P1, reverse primer P2 and probe Probe1).
5, the building and preparation of standard items
Using P1 and P2 primer amplification moraxelle catarrhalis genomic DNA, PCR product is cloned into pMD-18T carrier, converts DH α
Escherichia coli are measured respectively using ultraviolet-uisible spectrophotometer in wavelength using alkaline lysis method of extracting positive colony plasmid
At 260nm, at 280nm DNA absorptance, then calculate plasmid concentration and purity.Then 10 times of gradient dilutions are every to 200 copies
Microlitre, prepare the positive criteria product of moraxelle catarrhalis.
6, reaction condition optimization
The elements such as primer, probe, enzyme are optimized one by one, determining reaction system are as follows: 2 × PCR buffer, 10 μ l, 10mmol/L
1.0 μ l of dNTP, each 0.5 μ l of 25 μm of ol/L primers, 10 μm of 0.2 μ l of ol/L probe, 0.4 μ l of Takara polymerase mixture,
Template 2ul adds aqua sterilisa to whole 20 μ l of system.
According to amplified fragments length, the annealing temperature of primer and probe and enzyme viability, mainly to reaction annealing temperature
It is optimized with extension of time, finally determining loop parameter are as follows: 94 DEG C, 2min；Into the cycle stage: 94 DEG C of denaturation 10s,
56 DEG C of annealing 50s, 72 DEG C of extension 15s, 40 recycle, and each circulate in annealing stage acquisition fluorescence signal.
Data are analyzed by identical conditions after amplification, determine the Ct value of each sample.
7, the evaluation of detection limit
The detection limit of kit provided by the invention, positive criteria product concentration are evaluated with the positive criteria product in above-mentioned 5 are as follows: 2 ×
106copies/µl、2×105copies/µl、2×104copies/µl、2×103copies/µl、2×102Copies/ μ l, this
Inventing the kit provided is the 20 every reaction systems of copy to the Monitoring lower-cut of moraxelle catarrhalis nucleic acid.
8, the evaluation of detection specificity
The specificity of this kit is had rated using above-mentioned bacterial strains DNA as template.It is visible when being detected to moraxelle catarrhalis DNA
Amplification curve is specified, when detecting to above-mentioned 9 kinds other pharyngeal common pathogenic microorganisms DNA, does not generate positive amplification curve, explanation
Cross reaction is not present between probe and primer that we use and other bacterial strains that we select.
Although above in a preferred manner, illustrating certain embodiment party of the invention by specific embodiment
Formula, but it will be understood by a person skilled in the art that, the present invention is not limited to embodiments disclosed above, but can be according to this
The knowledge of technical field that the present invention belongs to modifies to it, makes an amendment without departing from the scope of protection of present invention.For example,
Fluorescent real time PCR used in the present invention also can according to need using the fluorescence pointed out in embodiment listed in specification
Mark substance other than group and fluorescent quenching group, such as Tet, HEX, TAMRA, ROX, Cy3, TxRd, JOE marker；Or
It is double miscellaneous using other label systems except Taqman technology, such as MGB probe, molecular beacon MB probe, scorpion shape probe, fluorescence
Hand over the fluorescence probes labelling techniques such as probe；Or the unsaturated dyestuff such as method such as SYBR Green I and LC Green are fitted into using dyestuff
Equal saturable dyes, as long as it uses specific primer sequence of the present invention, it can qualitative or quantitative testing goal gene
Presence, and then specifically detect the presence of moraxelle catarrhalis.So the change that is understood of these those skilled in the art and
The replacement of customary means is also fallen within the scope of the present invention.Protection scope of the present invention should be by appended claims institute
It limits.
Applicant: Jiangsu and wound Biotechnology Co., Ltd
Sequence table
<120>moraxelle catarrhalis fluorescence PCR detection reagent kit
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>moraxelle catarrhalis (Moraxella catarrhalis)
<400> 1
caarccaacc atgcacaatg t 21
<210> 2
<211> 21
<212> DNA
<213>moraxelle catarrhalis (Moraxella catarrhalis)
<400> 2
ataggcgaca tctgctgtcc a 21
<210> 3
<211> 25
<212> DNA
<213>moraxelle catarrhalis (Moraxella catarrhalis)
<400> 3
ctgaatatgc cgtgcgtaca ggtcg 25
Claims (3)
1. a kind of kit for moraxelle catarrhalis nucleic acid in specific detection sample is mixed including PCR reaction solution, enzyme
Liquid, negative quality-control product and positive quality control product, the primer sequence in PCR reaction solution for nucleic acid amplification reaction are as follows:
P1:5`-CAARCCAACCATGCACAATGT-3`,
P2:5`-ATAGGCGACATCTGCTGTCCA-3`,
Sequence in PCR reaction solution for the oligonucleotide probe of fluorescence signal monitoring is as follows:
Probe1:5`-X1-CTGAATATGCCGTGCGTACAGGTCG-Y1-3`,
Probe1 fluorescent reporter group X1 is ROX, and fluorescent quenching group Y1 is Eclipse.
2. kit as described in claim 1, it is further characterized in that PCR reaction system is 20 μ l, including 2 × PCR buffer
10 μ l, 1.0 μ l of 10mmol/L dNTP, each 0.5 μ l of 25 μm of ol/L primers, 10 μm of 0.2 μ l of ol/L probe, thermal starting Taq
0.4 μ l of enzyme mixation, 2 μ l of sample DNA add aqua sterilisa to whole 20 μ l of system.
3. kit as described in claim 1, it is further characterized in that PCR reaction cycle parameter are as follows:
94 DEG C, 2min；Into the cycle stage: 94 DEG C of denaturation 10s, 56 DEG C of annealing 50s, 72 DEG C of extension 15s, coreaction 40 are followed
Ring.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811585687.4A CN109439780A (en) | 2018-12-25 | 2018-12-25 | Moraxelle catarrhalis fluorescence PCR detection reagent kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811585687.4A CN109439780A (en) | 2018-12-25 | 2018-12-25 | Moraxelle catarrhalis fluorescence PCR detection reagent kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109439780A true CN109439780A (en) | 2019-03-08 |
Family
ID=65535228
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811585687.4A Pending CN109439780A (en) | 2018-12-25 | 2018-12-25 | Moraxelle catarrhalis fluorescence PCR detection reagent kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109439780A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112481401A (en) * | 2020-12-25 | 2021-03-12 | 郑州安图生物工程股份有限公司 | Kit for simultaneously detecting streptococcus pneumoniae, legionella pneumophila and moraxella catarrhalis |
| CN115029454B (en) * | 2021-11-16 | 2023-06-20 | 江汉大学 | MNP (MNP) marking site of Moraxella catarrhalis, primer composition, kit and application of MNP marking site |
-
2018
- 2018-12-25 CN CN201811585687.4A patent/CN109439780A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112481401A (en) * | 2020-12-25 | 2021-03-12 | 郑州安图生物工程股份有限公司 | Kit for simultaneously detecting streptococcus pneumoniae, legionella pneumophila and moraxella catarrhalis |
| CN112481401B (en) * | 2020-12-25 | 2024-02-20 | 郑州安图生物工程股份有限公司 | Kit for simultaneously detecting streptococcus pneumoniae, legionella pneumophila and moraxella catarrhalis |
| CN115029454B (en) * | 2021-11-16 | 2023-06-20 | 江汉大学 | MNP (MNP) marking site of Moraxella catarrhalis, primer composition, kit and application of MNP marking site |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103642936B (en) | The specific detection of 55 type adenoviruss primed probe combination and test kit | |
| Millar et al. | Molecular diagnostics of medically important bacterial infections | |
| Maaroufi et al. | Real-time PCR for determining capsular serotypes of Haemophilus influenzae | |
| CN107746879A (en) | Detect RPA primers, probe, kit and the detection method of staphylococcus aureus | |
| CN103642910B (en) | The primer of detection by quantitative Klebsiella pneumonia and probe and application thereof | |
| CN102660645B (en) | The specific detection of streptococcus pneumoniae and hemophilus influenza primed probe combination and test kit | |
| CN109680079A (en) | Detect RPA primer, probe, kit and the method for vibrio parahemolyticus | |
| EP2557156A1 (en) | Primer and probe for detecting chlamydia trachomatis, and method for detecting chlamydia trachomatis using same | |
| CN112481401B (en) | Kit for simultaneously detecting streptococcus pneumoniae, legionella pneumophila and moraxella catarrhalis | |
| CN109439780A (en) | Moraxelle catarrhalis fluorescence PCR detection reagent kit | |
| CN102634596B (en) | The specific detection of Bordetella pertussis and Bordetella parapertussis primed probe combination and test kit | |
| CN105567802A (en) | Fluorescence PCR (polymerase chain reaction) detection kit for Chlamydia pneumoniae | |
| CN109371150A (en) | Pseudomonas aeruginosa fluorescence PCR detection reagent kit | |
| CN110468223B (en) | Primer, probe, kit and method for detecting nucleic acid of mycoplasma pneumoniae and bauxiella based on dUTP/UNG method | |
| CN105624285A (en) | Mycoplasma pneumoniae fluorescent PCR detection reagent kit | |
| CN102643920B (en) | The specific detection of Neisseria meningitidis and discriminating primed probe combination and test kit | |
| CN105624284A (en) | Klebsiella pneumonia fluorescent PCR detection reagent kit | |
| CN105624286A (en) | Legionella pneumophila fluorescence PCR detection kit | |
| CN104342487A (en) | Mycoplasma nucleic acid isothermal amplification method | |
| WO2021135495A1 (en) | Primer combination and kit for detecting staphylococcus aureus | |
| CN109355413A (en) | Staphylococcus aureus fluorescence PCR detection reagent kit | |
| CN103642901B (en) | The specific detection of Neisseria meningitidis Z group primed probe combination and test kit | |
| CN105648054A (en) | Vibrio cholerae fluorescence PCR detection kit | |
| CN110512013B (en) | Method for identifying three corynebacteria by using high-resolution melting curve method | |
| CN105624283A (en) | Fluorescence PCR detection kit for stenotrophomonas maltophilia |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190308 |
|
| WD01 | Invention patent application deemed withdrawn after publication |