CN109355413A - Staphylococcus aureus fluorescence PCR detection reagent kit - Google Patents
Staphylococcus aureus fluorescence PCR detection reagent kit Download PDFInfo
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- CN109355413A CN109355413A CN201811585672.8A CN201811585672A CN109355413A CN 109355413 A CN109355413 A CN 109355413A CN 201811585672 A CN201811585672 A CN 201811585672A CN 109355413 A CN109355413 A CN 109355413A
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- staphylococcus aureus
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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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- 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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Abstract
The present invention relates to a kind of using kit existing for real time fluorescent PCR method specific detection Gold Samples staphylococcus aureus nucleic acid.Utilize kit of the invention, staphylococcus aureus 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 staphylococcus aureus nucleic acid, entire reaction can be completed in 2 hours, have important application value in fields such as disease surveillance, clinical diagnosises.
Description
Technical field
The present invention relates to a kind of using real time fluorescent PCR method specific detection Gold Samples staphylococcus aureus nucleic acid
Kit belongs to the in-vitro diagnosis field of staphylococcus aureus.
Background technique
Staphylococcus aureus (Staphylococcus Aureus) is a kind of important pathogen of the mankind, is under the jurisdiction of Portugal
Grape Coccus is the representative of gram-positive bacteria.The bacterium can draw local suppurative infection；Internal organs can also be caused to infect, such as lung
Inflammation, pericarditis, endocarditis etc. or even the general infections such as septicemia, pyemia.The pathogenicity power master of staphylococcus aureus
To depend on the toxin and aggressive enzyme (such as hemotoxin, leukocidin, plasma-coagulase, DNA that it is generated
Enzyme, enterotoxin etc.).Toxin disease is presented with: food poisoning, latent through 30min to 8h after feed pollution enterotoxin food
Phase may occur in which the acute gastroenteritiss symptom such as Nausea and vomiting, abdominal pain and diarrhea, morbidity can be self-healing for 1-2 days, prognosis
Well.Toxic shock syndrome is mainly shown as high fever, low blood pressure, erythematous rash with furfur and shock etc., trouble more than half
Person has vomiting, diarrhea, myalgia, conjunctiva and mucous membrane hyperemia, liver and kidney dysfunction etc., there is the performance of Cardiac Involvemant once in a while.
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 staphylococcus aureus gene sequence the special target sequence design primer of conservative region and
Taqman probe, using the method for real-time PCR, for whether there is staphylococcus aureus in quick test sample
Nucleic acid.
Summary of the invention
The present invention solves the technical problem of in rapidly and accurately test sample whether there is staphylococcus aureus,
A kind of fluorescent PCR kit of specific detection staphylococcus aureus is provided.
The technical problem to be solved by the present invention is to what is be achieved through the following technical solutions:
A kind of kit of specific detection Gold Samples staphylococcus aureus nucleic acid, component include PCR reaction solution, enzyme mixing
Liquid, negative 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, positive matter
Control product are the recombinant plasmid that target sequence is detected containing staphylococcus aureus.
Primer in PCR reaction solution for nucleic acid amplification is that P1 and P2, P1 and P2 are for staphylococcus aureus gene
The specific primer that group specific and conserved sequence is designed and filtered out through preliminary experiment；It is supervised in PCR reaction solution for fluorescence signal
The oligonucleotide probe of survey is Probe1, and Probe1 is to design for staphylococcus aureus gene group specific and conserved sequence
And the specific probe filtered out through preliminary experiment, wherein fluorescent reporter group X1 is ROX, and fluorescent quenching group Y1 is Eclipse
(see Table 1).
The primer and probe sequence of the detection staphylococcus aureus of table 1
It is a further object to provide this fluorescence PCR detection reagent kits in the application side of detection staphylococcus aureus
Method, comprising:
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, 2 μ of sample DNA
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 be detected immediately referring to corresponding commodity kit specification, the DNA of extraction, otherwise, should be dispensed DNA
- 80 DEG C afterwards~-20 DEG C preservations.
Kit provided by the invention has specificity well, can detect staphylococcus aureus, but cannot detect
The nucleic acid of non-staphylococcus aureus pathogen；Monitoring lower-cut to staphylococcus aureus nucleic acid is the 20 every reactants of copy
System；Only need detection to can be completed in 2 hours, can be provided for the disease surveillance of staphylococcus aureus and clinical diagnosis test according to
According to.
Detailed description of the invention
Fig. 1 is the amplification curve diagram of substance real-time fluorescence PCR detection S. aureus-positive standard items.From left to right
5 curves be respectively (2 × 10 after L-form staphylococcus aureus gradient dilution6-2×102Copies/ μ l) amplification.It is horizontal
Coordinate is reaction cycle number, and ordinate is 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: staphylococcus aureus and 9 kinds of negative control microorganisms.There is S type amplification curve in staphylococcus aureus, and other
9 kinds of pathogenic microorganisms do 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 staphylococcus aureus gene group sequence in Genbank and domestic and foreign literature is analyzed using Blast tool, point
The conservative region for not selecting its stable 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 detection probe 5 ' of staphylococcus aureus is held
Mark ROX fluorophor；3 ' end mark fluorescent Eclipse quenching groups.
2, the preparation of strain is detected
(escherichia coli, verdigris are false for staphylococcus aureus used in the present embodiment and other negative control bacterial strains
Monad, enterobacter cloacae, staphylococcus epidermis, enterococcus faecalis, Klebsiella Pneumoniae, A group streptococcus, hemolytic grape ball
Bacterium, staphylococcus saprophyticus) 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 staphylococcus aureus of the primer and probe Detection and Extraction of design and the genomic DNA of negative control bacterial strain, warp
It tests repeatedly, filters out sensitivity, specificity and the optimal primer combination of probe of repeatability (see sequence table, Staphylococcus aureus
Bacterium forward primer P1, reverse primer P2 and probe Probe1).
5, the building and preparation of standard items
Using P1 and P2 primer amplification staphylococcus aureus gene group DNA, PCR product is cloned into pMD-18T carrier, is converted
DH5 α Escherichia coli are measured respectively using ultraviolet-uisible spectrophotometer in wave using alkaline lysis method of extracting positive colony plasmid
At long 260nm, at 280nm DNA absorptance, then calculate plasmid concentration and purity.Then 10 times of gradient dilutions to 200 copy
Every microlitre, prepare the positive criteria product of staphylococcus aureus.
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 of Takara polymerase mixture
L, 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 staphylococcus aureus 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 equal when being detected to L-form staphylococcus aureus
It can be seen that clear amplification curve does not generate positive amplification curve when detecting to above-mentioned 9 kinds other pharyngeal common pathogenic microorganisms DNA,
Illustrate that there is no cross reactions between the probe that we use and primer 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 reporter group and fluorescent quenching group, such as Tet, HEX, TAMRA, ROX, Cy3, TxRd, JOE label
Object；Or using other label systems except Taqman technology, for example, it is MGB probe, molecular beacon MB probe, scorpion shape probe, glimmering
The fluorescence probes labelling techniques such as light double cross probe；Or the unsaturated dyestuff such as method such as SYBR Green I and LC are fitted into using dyestuff
The saturable dyes such as Green, as long as it uses specific primer sequence of the present invention, it can qualitative or quantitative testing goal
The presence of gene, and then specifically detect the presence of staphylococcus aureus.So these those skilled in the art are understood
Change and the replacement of customary means also fall within the scope of the present invention.Protection scope of the present invention should be by appended right
Claim is limited.
Sequence table
<110>Jiangsu and wound Biotechnology Co., Ltd
<120>staphylococcus aureus fluorescence PCR detection reagent kit
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213>staphylococcus aureus (Saccharomonospora azurea)
<400> 1
cttcatatgt gttaagtctt gcagc 25
<210> 2
<211> 28
<212> DNA
<213>staphylococcus aureus (Saccharomonospora azurea)
<400> 2
catcatacaa atacctcata ttatccat 28
<210> 3
<211> 29
<212> DNA
<213>staphylococcus aureus (Saccharomonospora azurea)
<400> 3
ccagaacaat tgaataaagc gagtgaatt 29
Claims (3)
1. a kind of kit for specific detection Gold Samples staphylococcus aureus nucleic acid, including PCR reaction solution, enzyme
Mixed liquor, 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`-CTTCATATGTGTTAAGTCTTGCAGC-3`,
P2:5`-CATCATACAAATACCTCATATTATCCAT-3`,
Sequence in PCR reaction solution for the oligonucleotide probe of fluorescence signal monitoring is as follows:
Probe1:5`-X1-CCAGAACAATTGAATAAAGCGAGTGAATT-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 is buffered
10 μ l of liquid, 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 startings
0.4 μ l of Taq enzyme mixed liquor, 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.
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| CN201811585672.8A CN109355413A (en) | 2018-12-25 | 2018-12-25 | Staphylococcus aureus fluorescence PCR detection reagent kit |
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| CN201811585672.8A CN109355413A (en) | 2018-12-25 | 2018-12-25 | Staphylococcus aureus fluorescence PCR detection reagent kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110894535A (en) * | 2019-12-26 | 2020-03-20 | 苏州德思普生物科技有限公司 | Staphylococcus aureus detection primer group, kit and method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110894535A (en) * | 2019-12-26 | 2020-03-20 | 苏州德思普生物科技有限公司 | Staphylococcus aureus detection primer group, kit and method |
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Application publication date: 20190219 |