CN105400878B

CN105400878B - One group of specific primer and probe for staphylococcus epidermis real-time fluorescence PCR detection

Info

Publication number: CN105400878B
Application number: CN201510915707.XA
Authority: CN
Inventors: 车团结; 沈颂东; 尤崇革; 谢小冬; 李琳; 李亚鹏
Original assignee: Suzhou Baiyuan Gene Technology Co Ltd
Current assignee: Suzhou Baiyuan Gene Technology Co Ltd
Priority date: 2015-12-10
Filing date: 2015-12-10
Publication date: 2018-12-28
Anticipated expiration: 2035-12-10
Also published as: CN105400878A

Abstract

The present invention provides one group of specific primer and probe for being used for staphylococcus epidermis real-time fluorescence PCR detection, one of the nucleotides sequence of the specific primer and probe is classified as 1) -3): 1), upstream primer: 5'- TCGGATCTCCATCAACT -3', downstream primer: 5'- TCAAACAACGCAACCAG -3', probe: 5'- TTTTGTCACATCATCCACTGG -3'；The Target Nucleotide Sequence of the primer and probe amplification is referring to SEQ ID No.2 in sequence table；2), the sequence complementary with the 1) sequence.Primer and probe provided by the present invention can staphylococcus epidermis content to clinical infection carry out qualitative and quantitative analysis.

Description

One group for staphylococcus epidermis real-time fluorescence PCR detection specific primer and Probe

Technical field

The invention belongs to technical field of microorganism detection method, and in particular to one group is used for staphylococcus epidermis real-time fluorescence The specific primer and probe of PCR detection.

Background technique

Staphylococcus epidermis (Staphylococcus epidermidis) SE is widely distributed in nature, it is a kind of heavy The opportunist wanted is the glucose coccus of Coagulase-negative Staphylococci.Nosocomial infection caused by SE is multiple location, is caused a disease Property with Bacterial Invasion, bacterium amount, virulence and immunity of organisms be different and different, septicemia caused by especially SE, easily primary Disease is covered, once find sick critical, poor prognosis.In addition, clinical infection caused by SE is apt to occur in hemopathic granular cell The patient of deficiency disease and solid malignant, mainly due to largely using immunosuppressor and broad-spectrum antibiotic, also and seriously Incident respiratory tract, formality wound, tracheotomy, indwelling catheter and ductus venosus etc. infect during protopathy long-term inpatients It is related.SE has drug resistance to a variety of antibiotic, causes the difficulty of clinical treatment.Recently as the extensive use of antibiotic, The progress of medical technology, the bacterium increasingly increase from the separation rate of various clinical samples, even more than staphylococcus aureus and occupy The first place of gram-positive bacteria becomes the important pathogenic bacteria of nosocomial infection.

The diagnosis of SE infection is still a problem so far.It is the contaminated bacteria of common Bacteria Culture since SE is widely present, The clinical manifestation of different parts infection is relied primarily at present and is found in blood, cerebrospinal fluid, urine, the smear of secretion and culture Pathogen as diagnosis basis.The method need to be by the processes such as Zengjing Granule, selection screening, biochemical identification, serotype, consumption Shi Yizhou or so is unfavorable for fast and accurately detecting staphylococcus epidermis.With immuno-enzymatic test, immunodiffusion, cream The result of glue agglutination test and the methods of immunofluorescence technique and enzyme-linked immunosorbent assay detection SE are still undesirable.Develop in recent years Real-time fluorescence quantitative PCR (the Fluorescence quantitative PCR) technology got up is stepped from Standard PCR qualitative detection The step of quantization realizes leap of the round pcr from qualitative to quantitative, avoids cross contamination in normal PCR Quantitative measurement Problem, it is technologically advanced relative to Standard PCR, easy to operate, realizes real-time monitoring, absolute quantitation and the mesh quickly detected , while having many advantages, such as that high sensitivity, specificity are good, operation is convenient.

Summary of the invention

It is an object of the invention to design one group of staphylococcus epidermis specific primer and probe sequence, it is wide to establish a kind of energy The method of the general qualitative or quantitative PCR detection of fluorescence quick, sensitive, that specificity is good applied to staphylococcus epidermis detection.This Invention uses gene clone technology, and staphylococcus epidermis is assembled GAP-associated protein GAP (Accumulation associated Protein) genetic fragment is inserted into carrier pMD18-T, the recombinant plasmid containing Aap genetic fragment is obtained, in this, as mark Quasi- product.A group-specific primers and probe are designed and synthesized according to the genetic fragment coding gene sequence of staphylococcus epidermis Aap, Optimize PCR reaction condition, establishes using real-time fluorescence quantitative polymerase chain reaction as the detection method of platform, and to the side established Method is assessed.

The present invention is achieved by the following technical programs:

Draw the first purpose of the invention is to provide one group of specificity for staphylococcus epidermis real-time fluorescence PCR detection The nucleotides sequence of object and probe, the specific primer and probe is classified as one of (1)-(3):

(1), upstream primer: 5'- TCGGATCTCCATCAACT -3'

Downstream primer: 5'- TCAAACAACGCAACCAG -3'

Probe: 5'- TTTTGTCACATCATCCACTGG -3'

The Target Nucleotide Sequence of the primer and probe amplification is referring to SEQ ID No.2 in sequence table；

(2), the sequence complementary with (1) sequence.

Experiments verify that by sequence described in (1) to 5 ' end and 3 ' extreme directions extend one to several bases or delete one to The primer sequence that several bases obtain can also expand above-mentioned Target Nucleotide Sequence well.

Preferably, 5 ' end fluorescent reporter groups of the probe are FAM, TET, JOE, HEX or VIC, 3 ' hold label It is fluorescent quenching group TAMRA, DABCYL or BHQ.

It is described a second object of the present invention is to provide the real-time fluorescence quantitative PCR detection kit of staphylococcus epidermis Kit is the qualitative or quantitative detection kit of staphylococcus epidermis；The kit includes primer described in claim 1 And probe；

Preferably, the immue quantitative detection reagent box further includes standard items, the standard items the preparation method comprises the following steps: by epidermis Staphylococcus conservative gene segment is inserted into carrier pMD18-T, and conversion carries out inducing expression into Escherichia coli, is contained The recombinant plasmid of staphylococcus epidermis conservative gene segment, in this, as standard items；The staphylococcus epidermis conservative gene piece Section is referring to SEQ ID No.1 in sequence table；

As further preferred, the staphylococcus epidermis conservative gene segment is with staphylococcus epidermis reference culture DNA is template, carries out PCR amplification acquisition using following primer:

Upstream primer: 5'-TTTAACACGCTCTTCACCTG-3'

Downstream primer: 5'- GTAGTCAAACAACGCAACCA -3'；

As most preferably, the condition of the PCR amplification are as follows: 94 DEG C of initial denaturation 5min；94℃ 30s,60℃ 30s,72℃ 1min, 35 circulations；72℃ 10min.

Third object of the present invention is to provide above-mentioned specific primers and probe, kit in qualitatively or quantitatively detection table Application in skin staphylococcus content.

Fourth object of the present invention is to provide the real-time fluorescence quantitative PCR detection method of staphylococcus epidermis, and step is such as Under:

(1) standard items are prepared: staphylococcus epidermis conservative gene segment is inserted into carrier pMD18-T, are converted to big Inducing expression is carried out in enterobacteria, the recombinant plasmid containing staphylococcus epidermis conservative gene segment is obtained, in this, as standard Product；The staphylococcus epidermis conservative gene segment is referring to SEQ ID No.1 in sequence table；

(2) Specification Curve of Increasing；

(3) sample to be tested and standard items are used using specific primer and probe described in claim 1 identical anti- System is answered to carry out real-time fluorescent PCR amplification；

(4) rapid quantitative detection of staphylococcus epidermis is carried out by the Ct value of standard curve and sample to be tested.

Preferably, the staphylococcus epidermis conservative gene segment is obtained by following primer amplification:

Upstream primer is 5'-TTTAACACGCTCTTCACCTG-3'；

Downstream primer is 5'- GTAGTCAAACAACGCAACCA -3'；

Preferably, the condition of staphylococcus epidermis conservative gene segment described in PCR amplification are as follows: 94 DEG C of initial denaturation 5min； 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 35 circulations；72℃ 10min；

Preferably, the dosage of primer and probe described in step (3) is 1.6 μ l；

Preferably, the reaction condition of real-time fluorescent PCR amplification described in step (3) are as follows: 94 DEG C initial denaturation 2 minutes, 94 DEG C 15 seconds, 60 DEG C of 40s and collect fluorescence signal, 40 circulations.

Fifth object of the present invention is to provide the qualitative PCR detection methods of staphylococcus epidermis, and steps are as follows:

(1) real-time fluorescent PCR amplification is carried out to sample to be tested using specific primer and probe described in claim 1；

(2) qualitative detection is carried out to staphylococcus epidermis by the Ct value of sample to be tested: when Ct value is less than 38, for sun Property；It otherwise, is feminine gender；

Preferably, the dosage of primer and probe described in step (1) is 1.6 μ l；

Preferably, the reaction condition of real-time fluorescent PCR amplification described in step (1) are as follows: 94 DEG C initial denaturation 2 minutes, 94 DEG C 15 seconds, 60 DEG C of 40s and collect fluorescence signal, 40 circulations.

Sixth object of the present invention is to provide the application methods of mentioned reagent box, and steps are as follows:

(2) Specification Curve of Increasing；

Upstream primer is 5'-TTTAACACGCTCTTCACCTG-3'；

Downstream primer is 5'- GTAGTCAAACAACGCAACCA -3'.

Preferably, the dosage of primer and probe described in step (3) is 1.6 μ l；

7th purpose of the invention is to provide the application method of kit, and steps are as follows:

(2) qualitative detection is carried out to staphylococcus epidermis by the Ct value of sample to be tested: when Ct value is less than 38, for sun Property；It otherwise, is feminine gender.

Preferably, the dosage of primer and probe described in step (1) is 1.6 μ l；

The present invention is provided to staphylococcus epidermis carry out qualitative and quantitative analysis primer and probe, by extract to The cDNA that the RNA reverse transcription of test sample obtains, can also be by the genomic DNA of extraction staphylococcus epidermis, in conjunction with real-time Fluorescence quantitative PCR detection technique and prepared standard items can reach accurate quantitative analysis detection sample mesocuticle staphylococcus content Purpose.Primer and probe provided by the present invention can staphylococcus epidermis content to clinical infection carry out qualitative, quantitative point Analysis, is of great significance to the content for judging clinical infection staphylococcus epidermis, and the present invention will play weight in clinical detection field It acts on.

Detailed description of the invention

Attached drawing is used to provide further understanding of the present invention, and constitutes part of specification, with reality of the invention It applies example to be used to explain the present invention together, not be construed as limiting the invention.In the accompanying drawings:

Fig. 1 is primer and probe system optimization experimental result；Wherein, a represents primer as 1.6 μ l, and probe is 1.6 μ l；B generation Table primer is 1.0 μ l, and probe is 1.6 μ l；C represents primer as 1.0 μ l, and probe is 0.8 μ l；D represents primer as 2.0 μ l, probe For 0.8 μ l；E represents primer as 1.6 μ l, and probe is 0.8 μ l；F represents primer as 2.0 μ l, and probe is 1.0 μ l；G represent primer as 1.0 μ l, probe are 0.8 μ l；H represents primer as 1.0 μ l, and probe is 1.0 μ l；I represents primer as 2.0 μ l, and probe is 1.6 μ l；

Fig. 2 is sensitivity experiment result；Wherein, a represents plasmid concentration as 1.00 × 10⁸Copies/ml, b represent plasmid Concentration is 1.00 × 10⁷Copies/ml, c represent plasmid concentration as 1.00 × 10⁶Copies/ml, d represent plasmid concentration as 1.00 ×10⁵Copies/ml, e represent plasmid concentration as 1.00 × 10⁴Copies/ml, f represent plasmid concentration as 1.00 × 10³Copies/ml, g represent plasmid concentration as 1.00 × 10²Copies/ml, h represent negative control；

Fig. 3 is specificity experiments result；Standard amplification curve wherein occur is staphylococcus epidermis plasmid, is not marked Quasi- amplification curve be staphylococcus aureus, staphylococcus saprophyticus, streptococcus pyogenes, escherichia coli, proteus mirabilis, Pseudomonas aeruginosa, haemophilus influenzae, Neisseria gonorrhoeae, clostridium perfringen, enterococcus faecalis, beta hemolytic streptococcus, Shigella flexneri, Neisseria meningitidis and negative control；

Fig. 4 is staphylococcus epidermis standard curve.

Specific embodiment

With reference to the accompanying drawing and the present invention is described in detail in specific embodiment.Embodiment below is convenient for preferably Understand the present invention, but does not limit the present invention.Experimental method in following embodiments is unless otherwise specified conventional method. The primer, probe and sequencing efforts used are synthesized and are completed by Sangon Biotech (Shanghai) Co., Ltd..

The preparation of 1 SE-Aap gene standard items of embodiment

Establish real time fluorescence quantifying PCR method it may first have to which external standards needed for preparation method, standard items should wrap Containing highly conserved, special sequence, it is ensured that the high specific of reaction.Aap gene has very high conservative.This research uses Staphylococcus epidermis Aap gene is as target sequence.The present embodiment mainly uses round pcr to expand staphylococcus epidermis Aap gene, It is connected in plasmid vector pMD18-T using gene recombination technology, constructs recombinant plasmid pMD18-T-SE-Aap, go forward side by side Row corresponding PCR identification and sequencing identification, are most used as the standard items to method for building up through quantitative afterwards, are the method for next step and comment Estimate and lays the foundation.

One, the preparation of template DNA

Staphylococcus epidermis (reference culture) genomic DNA is extracted, the template as the amplification of Aap gene PCR:

(1) 1ml staphylococcus epidermis bacteria suspension is taken, 8000r/min is centrifuged 5 minutes, abandons supernatant；

(2) 400 μ l suspension bacteria liquid of the TE buffer precipitating of 10% sucrose is added, the lysozyme 20 of 20mg/ml is added after mixing μ l, 37 DEG C of effect 45min；

(3) 30 μ l 10%SDS and 3 μ l 10mg/ml Proteinase Ks, 55 DEG C of water-bath 1h are added；

(4) phenol/chloroform/isoamyl alcohol (volume ratio 25:24:1) that 500 μ l are added mixes, and 12000r/min is centrifuged 10min；

(5) supernatant is taken, chloroform/isoamyl alcohol (volume ratio 24:1) that 500 μ l are added mixes, and 12000r/min is centrifuged 10min；

(6) supernatant is taken, the dehydrated alcohol of 50 μ l 3M NaAc and 800 μ l, -20 DEG C of standing 30min is added, centrifugation is gone Clearly；

(7) ethanol washing that precipitating is 70% with percent by volume, it is dry；

(8) it is dissolved in 50 μ l distilled waters, -20 DEG C of preservations.

Two, the PCR amplification of Aap genetic fragment

1, the design and synthesis of primer

The present invention is by carrying out bioinformatics comparison point to ncbi database mesocuticle staphylococcus A ap gene complete sequence Analysis is chosen and the conservative fragments sequence (sequence in sequence table SEQ ID No.1) of design primer and probe is suitble to be target, application It is fixed to devise one group of real-time fluorescence for 7 software of 3 software of Primer express, 5 software of Primer Premier and Oligo Measure the peripheral primer of PCR primer and probe and one group of correlated series.

The extension increasing sequence that this patent is chosen is as follows:

5’-TTTAACACGCTCTTCACCTGGTTTTAAATCAGGATTGAATTCACGTTTCTTGTCGAATGGAATTTC TTCCGTTGACGTAATCGGATCTCCATCAACTGGACCATATTTTGTCACATCATCCACTGGTGGTGTGACTACTTCGC CTGTATCAGGATTTTTAACTCCTGGCTTACCTGGTTGCGTTGTTTGACTAC-3’

The periphery primer sequence is as follows:

Upstream primer: SE-Aap-109F 5'-TTTAACACGCTCTTCACCTG-3'

Downstream primer: SE-Aap-302R 5'- GTAGTCAAACAACGCAACCA -3'；

Amplified fragments size is 194bp.

2, PCR reaction system and reaction condition

Using DNA as template, using above-mentioned periphery primer SE-Aap-109F/ SE-Aap-302R as amplimer, use is following System and reaction condition carry out PCR amplification.

PCR reaction system:

10×PCR buffer 5μl

DNTP(2.5 μM) 4 μ l

4 μ l of primers F (5 μM)

R(5 μM of primer) 4 μ l

2 μ l of template

1 μ l of Taq enzyme (5U)

ddH₂O 30μl

50 μ l of total volume.

Wherein primer uses SE-Aap-109F/ SE-Aap-302R, and Taq enzyme uses hundred Tyke Power Taq of Beijing Plus DNA Polymerse, PCR amplification instrument are the serial 96 grads PCR instrument of hundred Tyke iCycling of Beijing.

Amplification program/reaction condition:

94 DEG C of initial denaturation 5min；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 35 circulations；72 DEG C of 10min take 3 μ L Amplified production carries out 1% agarose electrophoresis, detects PCR product size, then using the DNA gel recycling of Axygen company production Kits recycle remaining pcr amplification product.

Three, the building and conversion of recombinant plasmid pMD18-T-SE-Aap

1, connection reaction: by pcr amplification product that above-mentioned purifying obtains and the Dalian pMD18-T(treasured biotech firm) connect It connects, is prepared using following linked system:

pMD18-T vector 1μL

Recycle 2 μ L of DNA

Solution1 5μL

ddH₂O 2μL

10 μ L of total volume.

It prepares and completes to be placed on 16 DEG C overnight, be attached reaction.

2, the conversion of pMD18-T- SE-Aap plasmid and PCR identification

(1) the DH5 α competent cell frozen is taken out from -70 DEG C of ultra low temperature freezer, being placed on ice chest solves it naturally Freeze；

(2) it takes 10 μ L of connection product to be added in the DH5 α competent cell of 50 μ L, gently shakes up postposition ice bath 30 minutes；

Heat shock 90 seconds in (3) 42 DEG C of water-baths, cooled on ice 2min is set after heat shock immediately；

(4) after LB liquid medium (without ampicillin) mixing of 400 μ l of pre-cooling is added into 1.5ml EP pipe, 37 DEG C of 200 revs/min of jog culture 1h；

(5) it takes 100 μ l to be coated on the LB plate containing Amp, IPTG, X-gal after shaking up above-mentioned culture solution, faces up 30min is placed, after bacterium solution is cultured base absorption completely, is inverted 37 DEG C of insulating box overnight incubations of culture dish；

(6) next day is observed, and picks from the plate white monoclonal colonies in the 100 μ L LB liquid medium (moulds of benzyl containing ammonia Element) PCR pipe in, 37 DEG C shaken cultivation 2-3 hours.2 μ L are drawn as template and carry out PCR identification, remaining bacterium solution is added to It carries out expanding in the LB liquid medium of 20ml and shake；

(7) above-mentioned dilution bacterium solution is expanded with carrier universal primer RV-M/M13-47, PCR product uses 1% Ago-Gel Electrophoresis identifies positive transformant by detection PCR product size.

Primer RV-M/M13-47 sequence is as follows:

Primer RV-M:5 '-GAGCGGATAACAATTTCACACAGG-3 '；

Primer M13-47:5 '-CGCCAGGGTTTTCCCAGTCACGAC-3 '.

PCR reaction system is as follows:

10×PCR buffer 2μL

DNTP(2.5 μM) 2 μ L

1.5 μ L of primers F (5 μM)

R(5 μM of primer) 1.5 μ L

2 μ L of template

0.5 μ L of Taq enzyme (5U)

ddH₂O 10.5μL。

Amplification program/reaction condition: 94 DEG C of initial denaturation 5min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 35 are followed Ring；72℃ 10min.

(8) positive recombinant plasmid is extracted using the Plasmid Preparation kit of hundred Imtech production, measures concentration and purity, Drawing a part of plasmid purification simultaneously send supreme marine growth Engineering Co., Ltd to be sequenced, and determines the gene order of Insert Fragment It is consistent with aim sequence.

Four, the acquisition of standard items and quantitative

1, the 100 μ L transferred species of bacillus coli DH 5 alpha containing recombinant plasmid pMD18-T-SE-Aap for obtaining step 3 in The LB liquid medium of 5mL, 37 DEG C of 200rpm shake training overnight；

2, take the bacterium solution transferred species of 1ml overnight incubation in 30ml LB liquid medium, 200rpm Zengjing Granule 2-3 hours, Then plasmid is extracted using the Plasmid Preparation kit of hundred Imtech of Beijing production.

3, the ultramicron ultraviolet-uisible spectrophotometer (ND5000) using hundred Tyke Biotechnology Co., Ltd of Beijing is right The plasmid of extraction is measured, and measures A₂₆₀、A₂₈₀, according to A₂₆₀/A₂₈₀Judge the purity of plasmid；

4, plasmid pMD18-T-SE-Aap concentration (copy number) calculates

(1) average molecular weight of each pair of base of molecular weight=2886bp × 660(of plasmid)

(2) measuring plasmid concentration is 103.36ng/ μ l, plasmid purity A₂₆₀/A₂₈₀=2.05, A₂₆₀/A₂₃₀=2.01.Because into When row real-time fluorescence quantitative PCR, need with " copy number " for unit, it is therefore desirable to by Conversion of measurement unit at copies/ml.

Plasmid copies/ml=Avgadro constant × plasmid molal quantity

Wherein Avgadro constant=6.02 × 10²³copies/mol。

Therefore plasmid concentration copies/ml=103 × 10 extracted^-9g/ml×6.02×10²³copies/mol÷ (2886bp × 660g/bp mol)=3.3 × 10¹⁰copies/ml

It is 1.00 × 10 that the sterile water of+23 μ l of 10 μ l plasmid, which just obtains concentration,¹⁰The plasmid of copies/ml, then by the plasmid It carries out 10 doubling dilutions just and a series of plasmid of concentration can be obtained, and saved backup in -20 DEG C.

The foundation of 2 real-time fluorescence quantitative PCR of embodiment detection staphylococcus epidermis method

One, the design and synthesis of specific primer and probe

Using the conservative fragments of the Aap gene of the staphylococcus epidermis of above-mentioned selection as target, using Primer 7 software of 3 software of express, 5 software of Primer Premier and Oligo devises one group of real-time fluorescence quantitative PCR and draws Object and probe.

As core of the invention, one group of primer and probe nucleosides for staphylococcus epidermis real-time fluorescence PCR detection Acid sequence is as follows:

Upstream primer: SE-Aap-189F 5'- TCGGATCTCCATCAACT -3'

Downstream primer: SE-Aap-298R 5'- TCAAACAACGCAACCAG -3'

Probe: SE-Aap-214P 5'- TTTTGTCACATCATCCACTGG -3'

The fluorescent reporter group that probe 5 ' is held is FAM, and 3 ' end labels are fluorescent quenching group TAMRA, while can also be with Mark others fluorescent reporter group such as TET, JOE, HEX, VIC and fluorescent quenching group such as DABCYL, BHQ etc..

The Target Nucleotide Sequence of primer and probe amplification are as follows:

5’-TCGGATCTCCATCAACTGGACCATATTTTGTCACATCATCCACTGGTGGTGTGACTACTTCGCCTG TATCAGGATTTTTAACTCCTGGCTTACCTGGTTGCGTTGTTTGA-3’。

Two, the preparation of sample to be tested

A, the preparation of sample total serum IgE

(1) tissue is put into mortar, liquid nitrogen is added and grinds, 1ml Trizol is added in every 50-100mg tissue, with homogenate Device carries out homogenized；

(2) homogenised sample is placed 5 minutes at (15~30 DEG C) of room temperature, clear homogenate is taken to carry out next step operation；

(3) 0.2ml chloroform is added in every 1ml Trizol, and acutely oscillation 15 seconds, are placed at room temperature for 3 minutes；

(4) 4 DEG C 10000 revs/min are centrifuged 15 minutes, and water phase is transferred in new pipe, and every 1ml Trizol is added 0.5ml isopropanol or 1ml ice ethyl alcohol, -80 DEG C of refrigerator-freezers freeze or freeze 30 minutes or more in -80 DEG C of refrigerator-freezers；

(5) 4 DEG C 10000 revs/min are centrifuged 10 minutes, discard supernatant；

(6) every 1ml Trizol at least adds 75% ethyl alcohol of 1ml.4 DEG C are no more than 7500 revs/min and are centrifuged 5 minutes, in abandoning Clearly；

(7) it being placed at room temperature for dry or vacuum and drains RNA precipitating, 25~200 water of the μ l without RNase are added, piping and druming mixes, Placing 10 minutes for 55~60 DEG C dissolves RNA, -70 DEG C of preservations.

B, post transcription cloning

Reverse transcription system and amplification condition: 10 1 μ l of μ l, Oligo (dT) of total mRNA (10pg-1 μ g), 70 DEG C of denaturation 10 Minute, cooling 10 minutes in ice are inserted into, 1 μ l of dNTP is added, 0.5 μ l, 5 × RT Buffer of reverse transcriptase, 0.5 μ l, PCR is dedicated 7 μ l of water, 42 DEG C are reacted 2 hours.- 20 DEG C of preservations.

Three, real-time fluorescence quantitative PCR condition optimizing

With concentration for 1.00 × 10⁶The positive plasmid of copies/ml be template, using hundred Imtech production 2 × Real-time PCR Premixture(probe) real-time fluorescence quantitative PCR kit tested, test the real-time glimmering of use Fluorescent Quantitative PCR instrument is the FTC-3000 real-time fluorescence quantitative PCR instrument of Shanghai Fengling Biological Technology Co. Ltd.'s production.Reaction system Using 20ul system, wherein the amount of primer and probe is reacted using the combination of table 1.

Primer and probe different amounts in 1 PCR reaction system of table

Reaction condition: 94 DEG C initial denaturation 2 minutes, 94 DEG C 15 seconds, 60 DEG C of 40s and collect fluorescence signal, 40 circulations. As a result referring to Fig.1, when data show 20 μ l system, when primer (5 μM) and probe (5 μM) are separately added into 1.6 μ l, Ct value is minimum, Fluorescence signal is most strong.

Four, method is assessed

1, sensitivity experiment

The above-mentioned plasmid being prepared is subjected to 10 doubling dilutions and obtains a series of plasmid of concentration, choosing concentration is 1.00 ×10⁸copies/ml、1.00×10⁷copies/ml、1.00×10⁶copies/ml、1.00×10⁵copies/ml、1.00× 10⁴copies/ml、1.00×10³copies/ml、1.00×10²Copies/ml etc. is used as gradient template.Reaction system is as follows:

2×premix 10μl

Primer F(5 μM) 1.6 μ l

Primer R(5 μM) 1.6 μ l

Probe(5 μM) 1.6 μ l

2 μ l of template

ddH₂O is mended to 20 μ l.

Reaction condition: 94 DEG C initial denaturation 2 minutes, 94 DEG C 15 seconds, 60 DEG C of 40s and collect fluorescence signal, 40 circulations. The fluorescence signal according to detected by instrument handles through software and obtains fluorescence curve, observes the signal of fluorescence curve, as a result reference Fig. 2, data are shown when plasmid concentration reaches 1.00 × 10³Still there is fluorescence signal when copies/ml, but plasmid concentration reaches 1.00×10²Fluorescence signal is not detected when copies/ml, therefore the sensitivity of this method is 1.00 × 10³copies/ml。

2, specificity experiments

In order to confirm the present invention to the specificity of staphylococcus epidermis detection, it is micro- that we have chosen the infection of other common clinicals Biologic specificity experiment, the microorganism of selection includes: staphylococcus aureus, staphylococcus saprophyticus, streptococcus pyogenes, large intestine angstrom Uncommon bacterium, proteus mirabilis, pseudomonas aeruginosa, haemophilus influenzae, Neisseria gonorrhoeae, clostridium perfringen, enterococcus faecalis, Beta hemolytic streptococcus, shigella flexneri, Neisseria meningitidis, staphylococcus epidermis.

It is test that template carries out that the specific test, which includes with the genomic DNA of above-mentioned sample and cDNA,.Above-mentioned micro- life The DNA extraction of object is all made of hundred Tyke genome DNA extracting reagent kit (DP2001) of Beijing, and RNA extraction is all made of hundred Tykes The high-purity total serum IgE Rapid extraction kit (RP1202) of RNApure, cDNA synthesis are all made of hundred Tyke Super RT Kit cDNA First chain synthetic agent box, the 2 × real-time PCR Premixture(probe produced using hundred Imtech) it is glimmering in real time Fluorescent Quantitative PCR kit is tested, and reaction system is as follows:

2×premix 10μl

Primer F(5 μM) 1.5 μ l

Primer R(5 μM) 1.5 μ l

Probe(5 μM) 1.5 μ l

2 μ l of template

ddH₂O is mended to 20 μ l.

Reaction condition: 94 DEG C initial denaturation 2 minutes, 94 DEG C 15 seconds, 60 DEG C of 40s and collect fluorescence signal, 40 circulations. The fluorescence signal according to detected by instrument handles through software and obtains fluorescence curve, observes the signal of fluorescence curve, and analysis is special Property.As a result Fig. 3 is referred to, as can be seen from Figure 3: regardless of using cDNA being template still using genomic DNA as template, only epidermis grape Coccus test positive, remaining microorganism are feminine gender, show that the present invention has specificity well.Testing result is as shown in table 2.

2 specificity experiments result of table

3, prepared by standard curve

Using the logarithm of the positive template of different copy numbers as abscissa, to reach the first of fluorescence threshold in PCR reaction process Beginning recurring number (Ct) is ordinate, obtains the standard curve of staphylococcus epidermis, the reference mark as sample to be tested quantitative determination Standard, as a result referring to fig. 4.

4, the quantitative detection of sample to be tested

Using the DNA of sample to be tested cDNA and standard items as template, with the specific primer of staphylococcus epidermis, with identical System carries out fluorescent quantitative PCR simultaneously.

PCR reaction system is as follows:

2×premix 10μl

Primer F(5 μM) 1.6 μ l

Primer R(5 μM) 1.6 μ l

Probe(5 μM) 1.6 μ l

2 μ l of template

ddH₂O is mended to 20 μ l.

Reaction condition: 94 DEG C initial denaturation 2 minutes, 94 DEG C 15 seconds, 60 DEG C of 40s and collect fluorescence signal, 40 circulations.

The rapid quantitative detection of staphylococcus epidermis is carried out by the Ct value of standard curve and sample to be tested.

5, the qualitative detection of sample to be tested

Using sample to be tested cDNA as template, with the specific primer of staphylococcus epidermis, sample to be tested cDNA is carried out glimmering Fluorescent Quantitative PCR amplification.

PCR reaction system is as follows:

2×premix 10μl

Primer F(5 μM) 1.6 μ l

Primer R(5 μM) 1.6 μ l

Probe(5 μM) 1.6 μ l

2 μ l of template

ddH₂O is mended to 20 μ l.

Staphylococcus epidermis is qualitatively judged by the Ct value of sample to be tested: when Ct value is less than 38, for the positive；It is no It then, is feminine gender.

Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention, Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features. All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention Within protection scope.

Sequence table

<110>Suzhou Bai Yuan gene technology Co., Ltd

<120>one groups of specific primers and probe for staphylococcus epidermis real-time fluorescence PCR detection

<170> PatentIn version 3.5

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<211> 194

<212> DNA

<213>artificial sequence

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tttaacacgc tcttcacctg gttttaaatc aggattgaat tcacgtttct tgtcgaatgg 60

aatttcttcc gttgacgtaa tcggatctcc atcaactgga ccatattttg tcacatcatc 120

cactggtggt gtgactactt cgcctgtatc aggattttta actcctggct tacctggttg 180

cgttgtttga ctac 194

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<211> 110

<212> DNA

<213>artificial sequence

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tcggatctcc atcaactgga ccatattttg tcacatcatc cactggtggt gtgactactt 60

cgcctgtatc aggattttta actcctggct tacctggttg cgttgtttga 110

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<211> 20

<212> DNA

<213>artificial sequence

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tttaacacgc tcttcacctg 20

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<212> DNA

<213>artificial sequence

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gtagtcaaac aacgcaacca 20

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<212> DNA

<213>artificial sequence

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gagcggataa caatttcaca cagg 24

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<212> DNA

<213>artificial sequence

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cgccagggtt ttcccagtca cgac 24

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<212> DNA

<213>artificial sequence

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tcggatctcc atcaact 17

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<212> DNA

<213>artificial sequence

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tcaaacaacg caaccag 17

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<212> DNA

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ttttgtcaca tcatccactg g

Claims

1. one group of specific primer and probe for staphylococcus epidermis real-time fluorescence PCR detection, it is characterised in that: the spy The nucleotides sequence of specific primer and probe is classified as one of (1) or (2):

(1), upstream primer: 5'- TCGGATCTCCATCAACT -3'

Downstream primer: 5'- TCAAACAACGCAACCAG -3'

Probe: 5'- TTTTGTCACATCATCCACTGG -3'

(2), the sequence complementary with (1) sequence.

2. primer and probe according to claim 1, it is characterised in that: the probe 5 ' end fluorescent reporter groups be FAM, TET, JOE, HEX or VIC, 3 ' end labels are fluorescent quenching group TAMRA, DABCYL or BHQ.

3. the PCR detection kit of staphylococcus epidermis, it is characterised in that: the kit be staphylococcus epidermis qualitative or Immue quantitative detection reagent box；The kit includes primer and probe described in claim 1.

4. the PCR detection kit of staphylococcus epidermis according to claim 3, it is characterised in that: the quantitative detection Kit further includes standard items, the standard items the preparation method comprises the following steps: staphylococcus epidermis conservative gene segment is inserted into load In body pMD18-T, conversion carries out inducing expression into Escherichia coli, obtains the weight containing staphylococcus epidermis conservative gene segment Group plasmid, in this, as standard items；The staphylococcus epidermis conservative gene segment is SEQ ID No.1 in sequence table.

5. the PCR detection kit of staphylococcus epidermis according to claim 4, it is characterised in that: the epidermis grape Coccus conservative gene segment is to carry out PCR amplification using the DNA of staphylococcus epidermis reference culture as template using following primer and obtain :

Upstream primer: 5'-TTTAACACGCTCTTCACCTG-3'

Downstream primer: 5'- GTAGTCAAACAACGCAACCA -3'.

6. the PCR detection kit of staphylococcus epidermis according to claim 5, it is characterised in that: the PCR amplification Condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 35 circulations；72℃ 10min.