CN107746879A - Detect RPA primers, probe, kit and the detection method of staphylococcus aureus - Google Patents
Detect RPA primers, probe, kit and the detection method of staphylococcus aureus Download PDFInfo
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Abstract
The present invention provides RPA primers, probe, kit and the detection method of detection staphylococcus aureus.The RPA primers of present invention detection staphylococcus aureus include being directed toclfASense primer Seq ID No.1 and anti-sense primer Seq the ID No.2 of gene.Relative to prior art, kit of the invention and detection method, staphylococcus aureus nucleic acid can be whether there is in quick detection sample.It is simple to operate, without the instrument of special expensive, there is important application value in fields such as disease surveillance, clinical diagnosis and food safety monitorings.
Description
Technical field
The invention belongs to biological technical field, and in particular to detect RPA primer and probes, the reagent of staphylococcus aureus
Box and RPA isothermal quick determination methods.
Background technology
Staphylococcus aureus (staphylococcus aureus) is under the jurisdiction of staphylococcus, gram-positive bacteria,
It is one of most important food-borne pathogens, often causes skin to suppurate after infection, can also causes pneumonia, pericarditis etc., or even meeting
Cause the general infections such as septicemia, pyemia.It is widely present in nature, its caused enterotoxin can contaminated food, from
And cause to poison by food, therefore, it is very heavy to develop prevention and detection of the quick determination method of staphylococcus aureus to the bacterium
Will.
The detection method of staphylococcus aureus is mainly classical culture protocols and PCR method at present.Although tradition culture
Method is to the less demanding of experimental facilities, and operability is stronger, but the cycle detected is longer, and detection efficiency is low, can not meet
The requirement being used for quickly detecting in current detection work to sample.And with the development of molecular Biological Detection technology, PCR detections
Method also occurs therewith, but PCR method is that amplified reaction is influenceed by many factors, it is necessary to put into instrument costly
, easily there is non-specific amplification in device, and detection time is relatively long.These methods are unfavorable for field quick detection.To ensure to eat
The fast development of product safety, is badly in need of the quick determination method about staphylococcus aureus.
Recombinase polymerase isothermal amplification technique (recombinase polymerase amplification, RPA)
It is a kind of new isothermal amplification technique that developed recently gets up, its principle is recombinase and Oligonucleolide primers at a constant temperature
With reference to, the complex of formation enzyme and primer, enzymatic makes primer navigate on the homologous target sequence of DNA double-stranded templates, and single-stranded
Under the protein-bonded assistance of DNA, unwind template DNA, then in the presence of DNA polymerases, forms new DNA complementary strands.
The main feature of this method under isothermal conditions can efficiently, quick, high special, highly sensitive amplification target gene.RPA skills at present
Art is had been applied in the quick detection of virus, bacterium, mycoplasma, parasite etc., but is not applied at present golden yellow
In the staphylococcic detection application of color.And for RPA methods, the specificity of primed probe is that its detection is specific and sensitive
The basis of property.
The content of the invention
In order to solve the above technical problems, the present invention provides a kind of RPA for detecting staphylococcus aureus easily and fast
Primer, probe, kit and detection method.
A kind of RPA primers for detecting staphylococcus aureus, forclfAGene includes
Sense primer Seq ID No.1:5 '-CCTTGTGGTTTTATTTCAAGTTTAGATGAG-3 ' and anti-sense primer Seq ID
No.2：5’-CCCATCATTAAGCAATAATTATACAAACCC-3’.
Preferably, the anti-sense primer Seq ID No.2 carry out biotin modifications at 5 ' ends.
A kind of RPA primers for detecting staphylococcus aureus, using in the sense primer in described primer, anti-sense primer
At least one, or sense primer, downstream primer sequence or sense primer in described primer, downstream primer sequence
Wall scroll sequence homology is the base sequence of 50% and the above in complementary strand sequence.
A kind of RPA probes for detecting staphylococcus aureus, forclfAGene, it includes probe sequence Seq ID
No.3：5’- GTGGTTTTATTTCAAGTTTAGATGAGCACTCAAGACCTTCTAA -3’
5 ' the end 30bp of probe sequence Seq ID No.3 opening position is modified using dSpacer, dSpacer molecules two
The thymidine of side is substituted by fluorophor and quenching group respectively, and the 3 ' ends of the probe sequence Seq ID No.3 pass through
Blockage group C3Spacer or phosphorylation are modified.
Preferably, the fluorogene uses FAM-dT fluorescence marker groups, and the quenching group uses BHQ1-dT fluorescence
Labelling groups.
A kind of RPA probes for detecting staphylococcus aureus, forclfAGene, including probe sequence Seq ID No.3：
5 '-GTGGTTTTATTTCAAGTTTAGATGAGCACTCAAGACCTTCTAA -3 ', the probe sequence are glimmering in 5 ' end marks
Light reporter group, C3Spacer or phosphorylation modification are carried out at 3 ' ends.
A kind of RPA probes for detecting staphylococcus aureus, including list in described probe sequence or its complementary strand sequence
Bar sequence homology is the base sequence of 50% and the above.
A kind of kit for the RPA for detecting staphylococcus aureus, including containing described primer and described probe with
RPA reactive components, the RPA reaction buffers of freeze-dried powder form.
A kind of method for the RPA for detecting staphylococcus aureus, comprises the following steps：
（1）Extract testing sample genomic DNA；
（2）The testing sample genomic DNA and magnesium acetate extracted using the kit described in claim 6, step (1), is matched somebody with somebody
RPA reaction systems are made；
（3）By step（2）The reaction system of configuration carries out amplified reaction at a constant temperature, and whole course of reaction collects fluorescence signal；
（4）Determine whether contain staphylococcus aureus in testing sample genomic DNA according to amplified reaction result.
Preferably, the step（2）In RPA reaction systems be calculated as with 50uL：
| DNA profiling | 2uL |
| Sense primer, anti-sense primer | 420 nmoL/L |
| Probe | 420 nmoL/L |
| Enzyme reaction freeze-dried powder | 10mg |
| 1 × reaction buffer | 45.5 uL |
| 280mmol/L Mg2+Buffer solution | 2.5uL |
Preferably, the reaction constant temperature of the RPA reaction systems is 37 DEG C -42 DEG C, and the reaction time is 15-20 minutes.
Preferably, the RPA reaction systems further comprise positive quality control product and negative quality-control product.
Relative to prior art, technical scheme overcomes the existing detection technique about staphylococcus aureus to deposit
Grown in detection cycle, the problems such as poor specificity, make full use of the related information of molecular biology and database, design is specific
RPA primers and probe, establish the RPA detection methods of staphylococcus aureus, and provide a kind of high sensitivity on this basis, high
Special RPA detection kits.
The present invention draws for the special target sequence design RPA of the conservative region of the clfA gene orders of staphylococcus aureus
Thing and probe, kit of the invention, it can be used for whether there is in quick detection sample the nucleic acid of staphylococcus aureus, can
Just to obtain amplification at 15 minutes, so as to overcome limitation of the prior art to staphylococcus aureus quick detection.Can
Experimental basis is provided for the disease surveillance of staphylococcus aureus, clinical diagnosis and food safety monitoring etc..
Brief description of the drawings
Fig. 1 is the testing result figure for the RPA reaction systems established using RPA primed probes of the present invention.
Fig. 2 is special to Staphylococcus species using the RPA reaction systems that RPA primed probes of the present invention are established
Different in nature experimental result picture.
Fig. 3 is specific to common pathogen using the RPA reaction systems that RPA primed probes of the present invention are established
Confirmatory experiment result figure.
The sensitivity experiment analysis result for the RPA reaction systems that Fig. 4 is established by RPA primed probes of the present invention
Figure.
Embodiment
Below in conjunction with the accompanying drawings, the preferably embodiment of the present invention is described in further detail：
Embodiment 1：The selection of target gene and RPA primed probes
Gained knowledge using biological information and related analysis software, the virulence gene common to staphylococcus aureus divide
Analysis, such asnuc、coa、clfADeng, it is compared by these genes to staphylococcus aureus with other microorganisms,
WillclfAAs target gene to be selected, and select the higher sequence fragment of specificity.Will according to designs of the RPA to primer and probe
Ask, design specific primer and probe, it is again that the sequence of primer and probe and staphylococcus aureus homology is high afterwards
Species are compared, therefrom selection specificity high primer and probe.Primer to be selected and probe are tested excessively same afterwards
Comparative analysis, therefrom selection specificity is high, a high combination of amplification efficiency.Primer and probe in the present embodiment is by by Shanghai
Bioengineering Co., Ltd synthesizes.
The primer of the present invention is specific as follows：
The primer includes being directed to clfAThe sense primer Seq ID No.1 of gene: 5’-CCTTGTGGTTTTATTTCAAGTT
TAGATGAG-3 ' and anti-sense primer Seq ID No.2：5’-CCCATCATTAAGCAATAATTATACAAACCC-3’.Preferably,
The anti-sense primer Seq ID No.2：5 '-CCCATCATTAAGCAATAATTATACAAACCC-3 ' carry out biotin at 5 ' ends
Modification.
In alternate embodiment, the primer can draw at least one of the sense primer, anti-sense primer, or upstream
Thing, downstream primer sequence or sense primer, downstream primer sequence complementary strand sequence in wall scroll sequence homology be 50% and more than
Base sequence.
The probe of the present invention is specific as follows：
ForclfAGene, it includes probe sequence Seq ID No.3：5’- GTGGTTTTATTTCAAGTTTAGATGAGCAC
TCAAGACCTTCTAA -3’。
In the present embodiment, 5 ' the end 30bp of probe sequence Seq ID No.3 opening position is repaiied using dSpacer
Decorations, the thymidine of dSpacer molecules both sides are substituted by fluorophor FAM and quenching group BHQ1 respectively, and in probe sequence
Row Seq ID No.3 3 ' ends are modified also by blockage group C3Spacer or phosphorylation etc..Probe sequence after modification
Row are expressed as:
5’-GTGGTTTTATTTCAAGTTTAGATGAGC-(FAM-dT)-AC-(dSpacer)-T-(BHQ1-dT)-
CAAGACCTTCTAA-C3Spacer-3’
In the present embodiment, the fluorogene uses FAM-dT fluorescence marker groups, and the quenching group uses BHQ1-dT fluorescence
Labelling groups.
In alternate embodiment, the probe sequence 5 ' hold mark fluorescent reporter groups, 3 ' end carry out C3Spacer or
Phosphorylation etc. is modified.
In alternate embodiment, the probe sequence can also use wall scroll in described probe sequence or its complementary strand sequence
Sequence homology is 50% and the base sequence of the above and other fluorescence marker groups.
Embodiment 2：Detect the RPA of staphylococcus aureus kit
A kind of kit for the RPA for detecting staphylococcus aureus of the present embodiment offer, including forclfAThe primer of gene,
Probe and RPA reaction buffers.The sense primer Seq ID No.1 that the primer can include: 5’-
CCTTGTGGTTTTATTTCAAGTTTAGATGAG-3 ' and anti-sense primer Seq ID No.2：5’-
One kind or sense primer Seq ID No.1 in CCCATCATTAAGCAATAATTATACAAACCC-3 ', anti-sense primer Seq ID
Wall scroll sequence homology is the base sequence of 50% and the above in the complementary strand sequence of No.2 sequences.The probe sequence is Seq
ID No.3：It is single in 5 '-GTGGTTTTATTTCAAGTTTAGATGAGCACTCAAGACCTTCTAA -3 ' or its complementary strand sequence
Bar sequence homology is the base sequence of 50% and the above.Preferably, 5 ' the end 30bp of the probe sequence Seq ID No.3
Opening position is modified using dSpacer, and the thymidine of dSpacer molecules both sides is substituted by fluorophor and quenching group respectively,
3 ' the ends of the probe sequence Seq ID No.3 are modified by blockage group C3Spacer.The fluorogene uses
FAM-dT fluorescence marker groups or other fluorescence marker groups, the quenching group using BHQ1-dT fluorescence marker groups or its
His fluorescence marker groups.In the present embodiment, the primer and probe of kit is the RPA reactive components of freeze-dried powder form.It is preferably real
Apply in mode, the RPA reactive components of the kit kind can also include enzyme, positive quality control product and the negative matter of freeze-dried powder form
Control product.
Embodiment 3：The RPA methods of staphylococcus aureus
（1）Extract testing sample genomic DNA
Staphylococcus aureus ATCC 27217 is inoculated into nutrient broth by the present embodiment, is placed on shaking table, according to every kind of
Bacterium optimum growth temperature overnight incubation, draw 1 mL cultures bacterium solution and be added drop-wise in 1.5 mL centrifuge tubes, 12000 revs/min
Centrifugation 2 minutes, abandoning supernatant add 500 μ L sterile salines, and fully suspending mixes, 12000 revs/min of centrifugations 1
Minute, supernatant is abandoned, repeats to add sterile saline, then centrifuge, abandon supernatant.Add 50 μ L physiological saline, 100 DEG C of water-baths
10-15 minutes, 12000 revs/min of room temperature to be cooled centrifuge 1 minute, supernatant as testing sample genomic DNA in-
20 DEG C save backup.
DNA is extracted after grown cultures being carried out to sample in other embodiment or directly extracts NDA.Sample requires：
Type of clinical specimen includes patient's diarrhoea sample and isolated culture；It should be transported after clinical sample collection with ice, -20 DEG C of preservations,
Multigelation is avoided, DNA is extracted from clinical sample, it is proposed that uses commercial kit stable and reliable for performance, specific method ginseng
Commercial kit specification is answered in photograph, and the DNA of extraction should be detected immediately, otherwise, -80 DEG C be deposited in after should DNA be dispensed
To -20 DEG C of preservations.
（2）The testing sample genomic DNA and acetic acid extracted using the kit described in the 2nd embodiment, step (1)
Magnesium, RPA reaction systems are configured to, a preferred embodiment reaction system, are configured in terms of 50uL as follows：
| DNA profiling | 2uL |
| Sense primer, anti-sense primer | 420 nmoL/L |
| Probe | 420 nmoL/L |
| Enzyme | 10mg |
| 1 × reaction buffer | 45.5 uL |
| 280mmol/L Mg2+Buffer solution | 2.5uL |
The template of the testing sample genomic DNA of the present embodiment is staphylococcus aureus ATCC 27217, concrete configuration
To add the uL of rehydration buffer solution 45.5, the uL of DNA profiling 2, most into the RPA reaction tubes containing lyophilized enzyme powder and primed probe
After add the uL of magnesium acetate solution 2.5 and fully mix, the present embodiment sets blank control reaction system simultaneously.
（3）By step（2）The reaction system of configuration carries out amplified reaction at a constant temperature, and whole course of reaction collects fluorescence letter
Number, the reaction constant temperature of the present embodiment RPA reaction systems is 37 DEG C-42 DEG C, and the reaction time is 15-20 minutes.
（4）Determine whether contain staphylococcus aureus in testing sample genomic DNA according to amplified reaction result.Preferably
In embodiment, experiment every time should set up positive and negative control, and the increase of negative control unstressed configuration value, positive control has fluorescent value
Increase, otherwise experimental result is invalid.The present embodiment result interpretation is suspicious including positive, negative and suspicious three kinds of situations, tool
Body is defined as follows.
It is positive：There is " S " type amplification curve, and fluorescence increment is more than 400；
It is negative：There is not " S " type amplification curve or fluorescence increment within 300；
It is suspicious：There is the amplification of " S " type, and fluorescence increase is between 300-400；To suspect results, experiment should be repeated, if
Experiment or appearance " S " type amplification, and fluorescence increase, between 300-400, negative control does not pollute, and can determine whether as sun
Property.
The present embodiment, testing sample genomic DNA testing result is as shown in Figure 1, and wherein curve 1 is testing sample gene
Group DNA；Curve 2 is blank control.Fig. 1 results show testing sample genomic DNA test positive, contain Staphylococcus aureus
Bacterium.
Embodiment 4：Staphylococcus aureus detects Evaluation on specificity
Evaluation on specificity is detected in one embodiment and uses bacterial strain in table 1 below, accepted standard bacterial strain has extensively in the present embodiment
Zhou Huankai microorganisms Co., Ltd is on behalf of purchase.
Table 1 is used for the bacterial strain of RPA specificity analyses
| Strain name | Strain number | Strain source |
| Staphylococcus aureus | ATCC 21217 | American Type Culture collection |
| Staphylococcus albus | GIM 1.247 | Guangdong Province's Culture Collection |
| MRSE | CMCC 26069 | Chinese medicine Microbiological Culture Collection administrative center |
| Staphylococcus xylosus | CICC 20237 | Chinese industrial Microbiological Culture Collection administrative center |
| False Staphylococcus intermedius | CICC 10499 | Chinese industrial Microbiological Culture Collection administrative center |
| Head staphylococcus | CICC 10290 | Chinese industrial Microbiological Culture Collection administrative center |
| Staphylococcus caprae | CICC 21722 | Chinese industrial Microbiological Culture Collection administrative center |
| Walsh staphylococcus | CICC 23992 | Chinese industrial Microbiological Culture Collection administrative center |
| Silver color staphylococcus | DSM 28299 | German living resources collection |
| Large intestine O157 | ATCC 3895 | American Type Culture collection |
| Vibrio parahemolyticus | ATCC 21617 | American Type Culture collection |
| Shigella | CMCC 51572 | Chinese medicine Microbiological Culture Collection administrative center |
| Salmonella | CMCC 50115 | Chinese medicine Microbiological Culture Collection administrative center |
| Slope qi enterobacteria | ATCC 29544 | American Type Culture collection |
| Listeria monocytogenes | ATCC 19115 | American Type Culture collection |
Using bacterial strain DNA in table 1 as RPA reaction templates, RPA amplifications are carried out using the kit of the RPA in embodiment 2.
The present embodiment RPA amplification systems are：Rehydration is added into the RPA reaction tubes containing lyophilized enzyme powder and primed probe
The uL of buffer solution 45.5, the uL of template 2, finally add the uL of magnesium acetate solution 2.5.
RPA reaction condition：Above-mentioned RPA reaction systems are fully mixed, under the conditions of 39 DEG C, expanded 15 minutes.
As shown in Figures 2 and 3, curve 1 is staphylococcus aureus ATCC 27217, curve 2-10 to amplification in Fig. 2
Respectively silvery white staphylococcus, staphylococcus albus, Staphylococcus caprae, MRSE, head staphylococcus, xylose
The amplification of staphylococcus, walsh staphylococcus, false Staphylococcus intermedius and blank control.Curve 1 is golden yellow in Fig. 3
Staphylococcus A TCC 27217, curve 2-8 are respectively Listeria monocytogenes, large intestine O157, Shigella, slope qi enterobacteria, husky
The amplification of door Salmonella, vibrio parahemolyticus and blank control.It is recognised that only positive control is golden yellow from result
Staphylococcus A TCC 27217 amplification is positive, other bacterium of staphylococcus and the detection of other common pathogens
As a result with the amplification indifference of blank control, present negative.Fig. 2 and Fig. 3 result shows, RPA primers of the present invention, RPA
The specificity of probe, kit and RPA methods is good.
Embodiment 5：The RPA method Monitoring lower-cuts of staphylococcus aureus
It is DNA profiling with staphylococcus aureus ATCC 27217 in the present embodiment, utilizes the RPA kits in embodiment 2
Expanded, wherein in differential responses, the total content of DNA profiling is respectively equivalent to 1.8 × 105cfu/reaction,、1.8×
104cfu/reaction、1.8×103cfu/reaction、1.8×102cfu/reaction、18cfu/reaction。
The experimental result of sensitivity is as shown in figure 4, curve 1：Staphylococcus aureus 1.8 × 105Cfu/reaction, it is bent
Line 2：Staphylococcus aureus 1.8 × 104Cfu/reaction, curve 3：Staphylococcus aureus 1.8 × 103cfu/
Reaction, curve 4：Staphylococcus aureus 180cfu/reaction, curve 5：Staphylococcus aureus gold 18cfu/
Reaction, curve 6：Blank control.From Fig. 4 results, the Monitoring lower-cut of RPA methods of the present invention can reach 18cfu/
reaction。
Relative to prior art, technical scheme overcomes the existing detection technique about staphylococcus aureus to deposit
Grown in detection cycle, the problems such as poor specificity, make full use of the related information of molecular biology and database, design is specific
RPA primers and probe, establish the RPA detection methods of staphylococcus aureus, and provide a kind of high sensitivity on this basis, high
Special RPA detection kits.
The present invention is for staphylococcus aureusclfAThe target sequence design RPA that the conservative region of gene order is special draws
Thing and probe, kit of the invention, it can be used for whether there is in quick detection sample the nucleic acid of staphylococcus aureus, can
Just to obtain amplification at 15 minutes, so as to overcome limitation of the prior art to staphylococcus aureus quick detection.Can
Experimental basis is provided for the disease surveillance of staphylococcus aureus, clinical diagnosis and food safety monitoring etc..
Above content is to combine specific preferred embodiment further description made for the present invention, it is impossible to is assert
The specific implementation of the present invention is confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of not departing from present inventive concept, some simple deduction or replace can also be made, should all be considered as belonging to the present invention's
Protection domain.
Sequence table
<110>Shenzhen Academy of Metrology ＆ Quality Inspection, Shenzhen HYK Gene Technology Co., Ltd.
<120>Detect RPA primers, probe, kit and the detection method of staphylococcus aureus
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ccttgtggtt ttatttcaag tttagatgag 30
<210> 2
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
cccatcatta agcaataatt atacaaaccc 30
<210> 3
<211> 43
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gtggttttat ttcaagttta gatgagcact caagaccttc taa 43
Claims (12)
1. a kind of RPA primers for detecting staphylococcus aureus, it is characterised in that be directed toclfAGene includes
Sense primer Seq ID No.1:5 '-CCTTGTGGTTTTATTTCAAGTTTAGATGAG-3 ' and anti-sense primer Seq ID
No.2：5’-CCCATCATTAAGCAATAATTATACAAACCC-3’.
2. the RPA primers of detection staphylococcus aureus as claimed in claim 1, it is characterised in that the anti-sense primer Seq
ID No.2 carry out biotin modifications at 5 ' ends.
3. a kind of RPA primers for detecting staphylococcus aureus, it is characterised in that using in the primer described in claim 1 or 2
Sense primer, at least one of anti-sense primer, or sense primer in the primer described in claim 1 or 2, downstream
Primer sequence or sense primer, downstream primer sequence complementary strand sequence in wall scroll sequence homology be 50% and the above base
Sequence.
4. a kind of RPA probes for detecting staphylococcus aureus, it is characterised in that be directed toclfAGene, it includes probe sequence
Seq ID No.3：5’- GTGGTTTTATTTCAAGTTTAGATGAGCACTCAAGACCTTCTAA -3’
5 ' the end 30bp of probe sequence Seq ID No.3 opening position is modified using dSpacer, dSpacer molecules two
The thymidine of side is substituted by fluorophor and quenching group respectively, and the 3 ' ends of the probe sequence Seq ID No.3 pass through
Blockage group C3Spacer or phosphorylation are modified.
5. the RPA probes of detection staphylococcus aureus as claimed in claim 4, it is characterised in that the fluorogene is adopted
With FAM-dT fluorescence marker groups, the quenching group uses BHQ1-dT fluorescence marker groups.
6. a kind of RPA probes for detecting staphylococcus aureus, it is characterised in that be directed toclfAGene, it is characterised in that including
Probe sequence Seq ID No.3：5 '-GTGGTTTTATTTCAAGTTTAGATGAGCACTCAAGACCTTCTAA -3 ', it is described
Probe sequence holds mark fluorescent reporter group 5 ', and C3Spacer or phosphorylation modification are carried out at 3 ' ends.
7. a kind of RPA probes for detecting staphylococcus aureus, it is characterised in that using the probe sequence described in claim 4 or 6
Wall scroll sequence homology is the base sequence of 50% and the above in row or its complementary strand sequence.
8. a kind of kit for the RPA for detecting staphylococcus aureus, it is characterised in that including containing as claimed in claim 1
Primer and RPA reactive component, RPA reaction buffer of the probe in the form of freeze-dried powder as described in claim 4 or 6.
A kind of 9. method for the RPA for detecting staphylococcus aureus, it is characterised in that comprise the following steps：
（1）Extract testing sample genomic DNA；
（2）The testing sample genomic DNA and magnesium acetate extracted using the kit described in claim 6, step (1), is matched somebody with somebody
RPA reaction systems are made；
（3）By step（2）The reaction system of configuration carries out amplified reaction at a constant temperature, and whole course of reaction collects fluorescence signal；
（4）Determine whether contain staphylococcus aureus in testing sample genomic DNA according to amplified reaction result.
10. method as claimed in claim 9, it is characterised in that the step（2）In RPA reaction systems be calculated as with 50uL：
。
11. the RPA of detection staphylococcus aureus method as claimed in claim 10, it is characterised in that the RPA reactions
The reaction constant temperature of system is 37 DEG C -42 DEG C, and the reaction time is 15-20 minutes.
12. the RPA of detection staphylococcus aureus method as claimed in claim 10, it is characterised in that the RPA reactions
System further comprises positive quality control product and negative quality-control product.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108359717A (en) * | 2018-05-11 | 2018-08-03 | 上海市农业科学院 | A kind of direct expansion RPA visualization of presence detection methods |
| CN108374052A (en) * | 2018-03-20 | 2018-08-07 | 徐州工程学院 | Probe set sequences for biochip test staphylococcus aureus |
| CN108531633A (en) * | 2018-06-21 | 2018-09-14 | 宁波国际旅行卫生保健中心 | One kind is for detecting the active fluorescence RAA primers of staphylococcus aureus, probe and detection method |
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| CN108374052A (en) * | 2018-03-20 | 2018-08-07 | 徐州工程学院 | Probe set sequences for biochip test staphylococcus aureus |
| CN108359717A (en) * | 2018-05-11 | 2018-08-03 | 上海市农业科学院 | A kind of direct expansion RPA visualization of presence detection methods |
| CN108531633A (en) * | 2018-06-21 | 2018-09-14 | 宁波国际旅行卫生保健中心 | One kind is for detecting the active fluorescence RAA primers of staphylococcus aureus, probe and detection method |
| CN110079586A (en) * | 2019-05-06 | 2019-08-02 | 浙江大学 | A kind of kit for staphylococcus aureus detection |
| CN110195119A (en) * | 2019-06-05 | 2019-09-03 | 中国农业科学院上海兽医研究所 | A kind of kit for detecting staphylococcus aureus, primer pair, probe and method |
| CN111793696B (en) * | 2020-06-18 | 2023-01-10 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | RPA primer, probe and detection method for detecting bare-cover fish |
| CN111793696A (en) * | 2020-06-18 | 2020-10-20 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | RPA primer, probe and detection method for detecting naked gesso |
| CN111893198A (en) * | 2020-07-16 | 2020-11-06 | 广东省微生物研究所（广东省微生物分析检测中心） | Specific molecular target, detection primer set and rapid detection method for identification of Staphylococcus aureus |
| CN111893198B (en) * | 2020-07-16 | 2021-04-20 | 广东省微生物研究所（广东省微生物分析检测中心） | Specific molecular target, detection primer set and rapid detection method for identification of Staphylococcus aureus |
| CN116144660B (en) * | 2020-12-19 | 2024-06-14 | 郑州大学 | MRSA detection based on new CRISPR targets |
| CN116144660A (en) * | 2020-12-19 | 2023-05-23 | 郑州大学 | MRSA bacterial detection based on novel CRISPR target |
| CN112782145A (en) * | 2021-02-05 | 2021-05-11 | 江南大学 | Aspergillus tubingensis absolute quantitative probe and application thereof |
| CN112924429B (en) * | 2021-02-05 | 2022-04-29 | 江南大学 | A probe and kit for absolute quantification of Lactobacillus jinshani |
| CN112782145B (en) * | 2021-02-05 | 2022-04-29 | 江南大学 | Aspergillus tubingensis absolute quantitative probe and application thereof |
| CN112924429A (en) * | 2021-02-05 | 2021-06-08 | 江南大学 | Absolute quantitative probe and kit for Lactobacillus jin Shini |
| CN113699259B (en) * | 2021-09-23 | 2022-06-28 | 中国人民解放军军事科学院军事医学研究院 | Detection method of Staphylococcus aureus based on exo-RPA technology and its complete set of reagents |
| CN113699259A (en) * | 2021-09-23 | 2021-11-26 | 中国人民解放军军事科学院军事医学研究院 | Exo-RPA technology-based staphylococcus aureus detection method and complete set of reagents thereof |
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