CN104328171B - Loop-mediated isothermal amplification primer, kit and method for detecting rat staphylococcus aureus - Google Patents
Loop-mediated isothermal amplification primer, kit and method for detecting rat staphylococcus aureus Download PDFInfo
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- CN104328171B CN104328171B CN201410582652.0A CN201410582652A CN104328171B CN 104328171 B CN104328171 B CN 104328171B CN 201410582652 A CN201410582652 A CN 201410582652A CN 104328171 B CN104328171 B CN 104328171B
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Abstract
The invention discloses a loop-mediated isothermal amplification primer, a kit and a method for detecting rat staphylococcus aureus. 4 LAMP specific primers are designed according to a conserved aroE protein gene of the staphylococcus aureus, and a rat staphylococcus aureus visual detecting method which is suitable for clinical sample detection, strong in specificity and high in flexibility is established; the existence of the staphylococcus aureus can be quickly, visually and accurately detected by observing the existence of green fluorescence, and the lowest limit of detection is 5.1 copy number/reaction, and therefore, the rat staphylococcus aureus visual detecting method is suitable for being applied to a primary-level site.
Description
Technical field
The invention belongs to medical microbial detection technique field, it is related to a kind of lamp of detection mouse staphylococcus aureus and draws
Thing and detection method.
Background technology
Staphylococcus aureus is Gram-positive, no gemma, flagellum, most of no pod membrane, lactic fermentation, amphimicrobian
Spherical bacteria.Staphylococcus aureus is common in skin surface and upper respiratory tract mucous membrane.As opportunist, golden yellow Portugal
Grape coccus be epidermis normal flora, often result in opportunistic infection, and the persister of this bacterium antibiotic therapy is brought bigger
Difficulty, for experiment with one of rodentine common health monitor control index.
The detection methods of staphylococcus aureus commonly used at present is included using monitoring swab direct bed board method and colour developing culture
Base method and conventional pcr method/fluorescent quantitation pcr method, all can detect effectively.But bacterial cultivation is difficult to compared with additive method presence
Standardization, time-consuming, culture medium, operating procedure or condition of culture the shortcomings of different experiments room difference is larger, therefore result may
The false Yin/Yang of inconsistent or appearance.Through denaturation, annealing, extension, conventional pcr method and fluorescent quantitation pcr method, need to add that dna carries
Take whole process probably to need 2～4 hours, and need accurate expensive instrument and reagent, fast and convenient to development at the basic level
Molecular diagnosis unfavorable.
Notomi et al. in invention ring mediated isothermal amplification (lamp) technology in 2000, through progressively improving, borrow by this technology
Help 4 or 6 specific primers and a kind of dna polymerase with strand displacement characteristic, can quick under isothermal conditions, Gao Te
Different and sensitive amplification target sequence.
Content of the invention
It is an object of the invention to provide a kind of loop-mediated isothermal amplification (LAMP) primer for detecting mouse staphylococcus aureus,
Kit and method.
For reaching above-mentioned purpose, present invention employs technical scheme below:
A kind of loop-mediated isothermal amplification (LAMP) primer for detecting mouse staphylococcus aureus, including following 2 pairs of primers: draw outward
Thing f3 and b3 and inner primer fip and bip；, as shown in seq.id.no.1, the nucleotide sequence of bip is such as the nucleotide sequence of fip
Shown in seq.id.no.2, the nucleotide sequence of f3 as shown in seq.id.no.3, the nucleotide sequence such as seq.id.no.4 institute of b3
Show.
A kind of loop-mediated isothermal amplification kit for detecting mouse staphylococcus aureus, this kit includes 40 μ
mol·l-1Fip 1.0 μ l, 40 μm of ol l-1Bip 1.0 μ l, 10 μm of ol l-1F30.5 μ l and 10 μm of ol l-1's
b30.5μl；F3 and b3 be outer primer, fip and bip be inner primer, the nucleotide sequence of fip as shown in seq.id.no.1, bip
Nucleotide sequence as shown in seq.id.no.2, the nucleotide sequence of f3 as shown in seq.id.no.3, the nucleotide sequence of b3
As shown in seq.id.no.4.
Described kit also includes 2 × reaction buffer 12.5 μ l, 8u/ μ l bst polymerase 1 μ l, 25mm dntp 1.5 μ
L and fluorescent dye 1 μ l.
Described fluorescent dye is the calcein aqueous solution of mass fraction 0.5%.
A kind of loop-mediated isothermal amplification method for detecting mouse staphylococcus aureus, comprises the following steps:
1) extract the dna of object to be detected (such as rat, mouse)；
2) following component is mixed, build includes 2 to primer f3 and b3 and fip and bip 25 μ l rings mediate etc.
Isothermal amplification reaction system:
2 × reaction buffer 12.5 μ l, 40 μm of ol l-1Fip 1.0 μ l, 40 μm of ol l-1Bip 1.0 μ l, 10 μ
mol·l-1F30.5 μ l, 10 μm of ol l-1B30.5 μ l, 8u/ μ l bst polymerase 1 μ l, 25mm dntp 1.5 μ l, to be checked
Survey dna 2 μ l, the fluorescent dye 1 μ l of object, ultra-pure water complements to 25 μ l；The nucleotide sequence of fip such as seq.id.no.1 institute
Show, the nucleotide sequence of bip as shown in seq.id.no.2, the nucleotide sequence of f3 as shown in seq.id.no.3, the nucleosides of b3
Acid sequence is as shown in seq.id.no.4；
3) loop-mediated isothermal amplification system is heated 40min at 63 DEG C, then heat 5min at 85 DEG C, reacted
Become；
4) carry out the judgement of result after the completion of question response in the following manner:
The whether coloured change of loop-mediated isothermal amplification system is observed, if ring mediated isothermal amplification after the completion of reaction
It is then positive reaction that reaction system color changes, and shows staphylococcus aureus is detected in object to be detected；If ring
It is then negative reaction that mediated isothermal amplification system color does not change, and shows to be not detected by object to be detected golden yellow
Color staphylococcus.
The extracting method of the dna of described object to be detected is:
The blood of object to be detected, tissue fluid or suppuration liquid are inoculated in broth bouillon, then cultivate 24h at 37 DEG C
Obtain bacterium solution, take bacterium solution to be centrifuged, the supernatant obtaining dna kit will be centrifuged and extract dna.
Also set up positive control and negative control in detection respectively, with staphylococcus aureus gene group dna as the positive
Comparison, with ultra-pure water as negative control；When carrying out the judgement of result, also compare with positive control and negative control, if to be checked
Survey the loop-mediated isothermal amplification system corresponding to object and the loop-mediated isothermal amplification body corresponding to positive control
System is in all green, then judge the testing result of object to be detected as positive reaction, if the ring corresponding to object to be detected mediates etc.
Loop-mediated isothermal amplification system corresponding to isothermal amplification reaction system and negative control all in crocus, then judges to be checked
The testing result surveying object is negative reaction.
Compared with prior art, the present invention has a following beneficial technique effect:
The present invention becomes lamp primer it is achieved that to gold using staphylococcus aureus aroe gene as detection Object combine
Staphylococcus aureus are easy, quick, accurately detection；After this gene is searched for through ncbi blast, in staphylococcus aureus kind
Between genus, aberration rate is low and takes into account conserved sequence between different serotypes, very low with the homology of other kind bacteriums, therefore
Can be used as versatility genetic test staphylococcus aureus.
The present invention provide detection mouse staphylococcus aureus method, its high specificity: the negative control being detected and
The big all no positive result of mouse genome.
The method of the detection mouse staphylococcus aureus that the present invention provides, its detection sensitivity is high: conventional pcr detection method
Sensitivity is 100pg/reaction (100copy number/reaction) order of magnitude, using detection method, detects
Lower limit reacts (5.1 copy numbers/reaction) for 3.6fg/.
The method of the detection mouse staphylococcus aureus that the present invention provides, detection is rapid for it, it is convenient to go out result: conventional pcr
Whole process just can go out result in 2～4 hours, and fluorescent quantitation pcr is also required to 1～1.5 hour, the detection that the present invention provides
Method may occur in which positive findings at about 50 minutes.And easy and simple to handle, low to instrument requirements it is only necessary to common water-bath, and
The direct observed result of dyestuff, the step eliminating electrophoresis can be passed through, detect in the practice of mouse staphylococcus aureus in basic unit
Have wide practical use, be a kind of for easy, quick, accurately detection mouse staphylococcus aureus side of animal used as test department
Method, and it is suitable for basic unit's use.
Brief description
Fig. 1 is the color-distinctive schematic diagram of positive reaction and negative reaction；
Fig. 2 is lamp specificity experiments result schematic diagram；
Fig. 3 is the lamp testing result schematic diagram after gradient dilution；
In figure: pos represents the testing result of positive control, and s.a represents object to be detected (infection staphylococcus aureus)
Testing result, h2O represents the testing result of negative control, and p.a represents the testing result of Pseudomonas aeruginosa genome dna, rat table
Showing the testing result of rat gene group dna, mouse represents the testing result of mouse genome dna, a represents 5.1 ×
106The testing result of copies/ μ l, b represents 5.1 × 105The testing result of copies/ μ l, c represents 5.1 × 104copies/μl
Testing result, d represents 5.1 × 103The testing result of copies/ μ l, e represents 5.1 × 102The testing result of copies/ μ l, f
Represent the testing result of 5.1 × 10copies/ μ l, g represents the testing result of 5.1copies/ μ l, and h represents 5.1 × 10- 1The testing result of copies/ μ l.
Specific embodiment
With reference to the accompanying drawings and examples the present invention is elaborated, described is explanation of the invention rather than limit
Fixed.
The invention discloses a kind of test experience lamp method of mouse staphylococcus aureus, this detection method is based on ring
Mediated isothermality amplification is carrying out.
The present invention is directed to staphylococcus aureus conservative gene aroe specific base sequence, devises including outer primer, interior draws
The specific primer of thing.Only sample need to be added fast by observing the presence or absence of green fluorescence in the lamp reaction system set up
Fast, the intuitive and accurate presence detecting staphylococcus aureus, lowest detection is limited to 5.1 copy numbers/reaction, examines for quick
Survey the convenient method that mouse staphylococcus aureus provides a suitable basic unit.
1st, design of primers and synthesis:
Specificity in view of detection and accuracy, according to document report, select staphylococcus aureus conservative gene
Aroe specific base sequence, as detection target, designs and synthesizes lamp primer, aroe gene is between staphylococcus aureus kind
Aberration rate is low and takes into account conserved sequence between different serotypes, very low with the homology of other kind bacteriums, therefore permissible
As versatility genetic test staphylococcus aureus.
Using primer-design software primer explorer 4.0, conservative for staphylococcus aureus aroe gene
Area devises 4 lamp primers, including forwards/reverse inner primer fip/bip, forwards/reverse outer primer f3/b3, described primer
A part or its complementary strand thereof with aroe (gene accession number: nc_002745.2) the gene nucleotide sequence of 459～688.
Specific primer sequence is shown in Table 1.
Table 1lamp method detects mouse staphylococcus aureus primer sequence
The extraction of bacterial genomes dna template the 2.1st, to be detected
For the ease of the explanation to detection method, specifically used bacterial genomes extracts kit extracts Staphylococcus aureus
Bacterium and (Pseudomonas aeruginosa, Friedlander's bacillus, sramana Li Ding Salmonella) strain gene group dna as comparison, through nucleic acid-protein
Standby after quantitative instrument quantitation.
2.2nd, mouse to be checked first obtains blood sample using the method cutting tail, is inoculated in fresh broth bouillon, 37
DEG C culture 24h obtains bacterium solution, takes bacterium solution to be centrifuged, the supernatant that obtains of centrifugation is utilized Genomic Purification Kit side to specifications
Method extracts dna.
3rd, build lamp reaction system (25 μ l)
Fluorescent dye is the calcein aqueous solution of mass fraction 0.5%.Template is the dna extracting.2 × reaction buffering
Liquid and bst polymerase are purchased from new england biolabs..
4th, the foundation of staphylococcus aureus lamp detection method and the detection of primer availability
The dna extracting from described mouse to be checked is added in reaction system as template, course of reaction is as follows: reaction temperature
63 DEG C of degree, 40 minutes time, then heats 5 minutes at 85 DEG C, reaction terminates.
Press above-mentioned reaction system using positive control (staphylococcus aureus gene group dna) and negative control (ultra-pure water)
Carry out with condition.
5th, the identification of staphylococcus aureus lamp product
Due to adding the fluorescent dye of 1 μ l before reaction in lamp reaction system, detect by an unaided eye lamp reaction system face
Color change, and compare with negative and positive, reaction system is in green is positive reaction (referring to pos and s.a in Fig. 1), in orange
Color is negative reaction (referring to h in Fig. 12o).
Substantial amounts of pyrophosphate ion can be formed in amplification process.Manganese ion in fluorescent dye is tied with calcein
Conjunction leads to fluorescent quenching, and dye colour is crocus simultaneously.When amplified reaction forms pyrophosphate ion, manganese ion and burnt phosphorus
Acid ion combines to form manganese pyrophosphate, causes the generation of fluorescence signal, and dye colour is changed into green simultaneously.I.e. after lamp reaction
Expand a large amount of targets dna, added calcein to may react and assume green.
6th, the specificity experiments of lamp
Non- staphylococcus aureus (Pseudomonas aeruginosa, the e coil k 1 pneumonia that staphylococcus aureus is often sent out with experimental mouse
Bacterium, sramana Li Ding Salmonella) genome dna and big mouse genome dna, set up lamp according to above-mentioned reaction system and condition
Detection method, carries out specific test.Setting staphylococcus aureus gene group dna is positive control, and ultra-pure water is negative right
According to.Result shows, the only reaction system color of s.a is consistent with positive control, and remaining all positive reaction, is negative anti-
Answer (referring to Fig. 2).
7th, the sensitivity tests of lamp
Staphylococcus aureus gene group dna is carried out after 10 times of gradient dilutions, setting negative control (ultra-pure water) according to
Above-mentioned reaction system and condition set up lamp detection method, to determine the sensitiveness of detection method.
Referring to Fig. 3, result shows: the mouse staphylococcus aureus lamp detection method of foundation 5.1 can be copied in detection sample
The staphylococcus aureus dna of shellfish number/reaction.
From the point of view of the present invention, the more conventional pcr of lamp method and fluorescence pcr method have:
High specificity: only by whether amplification can determine that the presence or absence of genes of interest, thus completing bacterium and virus
Qualitative detection.
Sensitivity is high: conventional pcr detection method sensitivity reacts (100 copy numbers/reaction) order of magnitude for 100pg/, adopts
Detection method, Monitoring lower-cut reacts (5.1 copy numbers/reaction) for 3.6fg/.
Operation and identification are simple and efficient: conventional pcr whole process just can go out result, fluorescent quantitation pcr in 2～4 hours
It is also required to 1～1.5 hour, the detection method that the present invention provides may occur in which positive findings at about 50 minutes.And operate letter
Just, low to instrument requirements it is only necessary to common water-bath is it is possible to pass through the direct observed result of dyestuff, the step eliminating electrophoresis,
Detect in the practice of mouse staphylococcus aureus in basic unit and have wide practical use.
Claims (4)
1. a kind of loop-mediated isothermal amplification (LAMP) primer for detecting mouse staphylococcus aureus it is characterised in that: include following 2 right
Primer: outer primer f3 and b3 and inner primer fip and bip；The nucleotide sequence of fip as shown in seq.id.no.1, the core of bip
, as shown in seq.id.no.2, as shown in seq.id.no.3, the nucleotide sequence of b3 is such as the nucleotide sequence of f3 for nucleotide sequence
Shown in seq.id.no.4,4 lamp primers are directed to the conserved regions design of staphylococcus aureus aroe gene.
2. a kind of loop-mediated isothermal amplification kit for detecting mouse staphylococcus aureus it is characterised in that: this kit
Including 40 μm of ol l-1Fip 1.0 μ l, 40 μm of ol l-1Bip 1.0 μ l, 10 μm of ol l-1F3 0.5 μ l and 10 μ
mol·l-1B3 0.5 μ l；F3 and b3 is outer primer, fip and bip is inner primer, and the nucleotide sequence of fip is such as
Shown in seq.id.no.1, the nucleotide sequence of bip as shown in seq.id.no.2, the nucleotide sequence such as seq.id.no.3 of f3
Shown, as shown in seq.id.no.4,4 lamp primers are directed to staphylococcus aureus aroe gene to the nucleotide sequence of b3
Conserved regions design.
3. a kind of loop-mediated isothermal amplification kit for detecting mouse staphylococcus aureus as claimed in claim 2, it is special
Levy and be: described kit also includes 2 × reaction buffer 12.5 μ l, 8u/ μ l bst polymerase 1 μ l, 25mm dntp 1.5 μ l
And fluorescent dye 1 μ l.
4. a kind of loop-mediated isothermal amplification kit for detecting mouse staphylococcus aureus as claimed in claim 3, it is special
Levy and be: described fluorescent dye is the calcein aqueous solution of mass fraction 0.5%.
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| CN108866223A (en) * | 2017-05-15 | 2018-11-23 | 吉林出入境检验检疫局检验检疫技术中心 | It is a kind of for identifying the LAMP primer group and its application of staphylococcus aureus |
| CN108103213A (en) * | 2018-02-10 | 2018-06-01 | 上海实验动物研究中心 | The primer and its kit of one group of detection Corynebacterium bovis |
| CN109735636A (en) * | 2018-12-25 | 2019-05-10 | 华南理工大学 | A kind of primer, kit and the method for PSR detection staphylococcus aureus leucocytic toxin |
| CN110863061A (en) * | 2019-12-30 | 2020-03-06 | 宁夏大学 | Specific LAMP primer, kit and method for detecting staphylococcus aureus |
| CN114540516B (en) * | 2022-03-08 | 2023-06-20 | 河南中检食安生物科技有限公司 | LAMP double-strand detection probe, kit and detection method for staphylococcus aureus |
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| Title |
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| Sequence and mapping of the aroA gene of Staphylococcus aureus 8325-4;CATHERINE O’CONNELL，et al;《Journal of General Microbiology》;19931231;1449-1460页 * |
| 实验动物金黄色葡萄球菌LAMP检测方法的建立;荣蓉等;《中国比较医学杂志》;20121231;第22卷(第4期);68-72页 * |
| 应用LAMP快速检测金黄色葡萄球菌的研究;唐梦君等;《现代食品科技》;20111231;第27卷(第6期);719-722页 * |
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