CN104328174A - LAMP (loop-mediated isothermal amplification) primers, kit and method for detecting murine klebsiella pneumoniae - Google Patents
LAMP (loop-mediated isothermal amplification) primers, kit and method for detecting murine klebsiella pneumoniae Download PDFInfo
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Abstract
The invention discloses LAMP (loop-mediated isothermal amplification) primers, a kit and a method for detecting murine klebsiella pneumoniae. A visual detection method of murine klebsiella pneumonia which is suitable for clinical sample detection and has strong specificity and high sensitivity is established by designing the LAMP primers by virtue of a klebsiella pneumoniae tyrB gene sequence. Whether klebsiella pneumoniae exists or not can be detected quickly and accurately by observing whether green fluorescence exists or not, and the lowest detectable limit is 5.6 copy number/ reaction. According to identification of a specific sequence region of a target sequence by four primers designed and screened, high specificity of LAMP diffusion is guaranteed, so that the LAMP primers are suitable for field application at basic level.
Description
Technical field
The invention belongs to medical microbial detection technique field, relate to a kind of LAMP primer and the detection method that detect mouse Klebsiella pneumonia.
Background technology
Klebsiella pneumonia is Gram-negative, not sports type, has pod membrane, lactic fermentation, amphimicrobian rod-shaped bacterium.Klebsiella pneumonia resides in the enteron aisle of large mouse and other animals usually.As opportunistic pathogen, Klebsiella pneumonia, once enter respiratory tract, can infect large mouse, causes severe lung to damage, and this bacterium has the stronger growth tendency replacing other bacteriums in antibiotics resistant nude mice, it is experiment one of rodentine common health monitor control index.
At present conventional determination of Klebsiella pneumoniae method comprises and uses the monitoring direct bed board method of swab and color developing culture medium method and Standard PCR method/fluorescence quantitative PCR method, all can effectively detect.But bacterial cultivation is difficult to stdn, length consuming time compared with additive method existence, and substratum, operation steps or culture condition are in shortcomings such as different experiments room difference are larger, therefore the possibility of result is inconsistent or occur false Yin/Yang.Standard PCR method and fluorescence quantitative PCR method, through sex change, annealing, extension, need add that the whole process of DNA extraction probably needs 2 ~ 4 hours just can go out result, and need accurate expensive instrument and reagent, and the molecular diagnosis fast and convenient to development at the basic level is unfavorable.
The people such as Notomi were in invention ring mediated isothermal amplification (LAMP) technology in 2000, through progressively improving, this technology by 6 Auele Specific Primers and a kind of archaeal dna polymerase with strand displacement characteristic, can under isothermal conditions fast, high special and sensitive amplified target sequence.
Summary of the invention
The object of the present invention is to provide a kind of LAMP primer, test kit and the method that detect mouse Klebsiella pneumonia.
For achieving the above object, present invention employs following technical scheme:
Detect a LAMP primer for mouse Klebsiella pneumonia, comprise following 2 pairs of primers: outer primer F3 and B3 and inner primer FIP and BIP; The nucleotide sequence of FIP is as shown in SEQ.ID.NO.1, and the nucleotide sequence of BIP is as shown in SEQ.ID.NO.2, and the nucleotide sequence of F3 is as shown in SEQ.ID.NO.3, and the nucleotide sequence of B3 is as shown in SEQ.ID.NO.4.
Detect a LAMP kit for mouse Klebsiella pneumonia, this test kit comprises 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
-1f30.5 μ L and 10 μm ol.L
-1b30.5 μ L; F3 and B3 is outer primer, FIP and BIP is inner primer, and the nucleotide sequence of FIP is as shown in SEQ.ID.NO.1, and the nucleotide sequence of BIP is as shown in SEQ.ID.NO.2, the nucleotide sequence of F3 is as shown in SEQ.ID.NO.3, and the nucleotide sequence of B3 is as shown in SEQ.ID.NO.4.
Described test kit also comprises 2 × reaction buffer 12.5 μ L, 8U/ μ L Bst polysaccharase 1 μ L, 25mM dNTP 1.5 μ L and fluorescence dye 1 μ L.
Described fluorescence dye is the fluorexon aqueous solution of massfraction 0.5%.
Detect a LAMP method for mouse Klebsiella pneumonia, comprise the following steps:
1) DNA of object to be detected (such as rat, mouse) is extracted;
2) following component is mixed, builds the 25 μ L LAMP reaction systems comprising 2 pairs of primers F 3 and B3 and FIP and BIP:
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 μm of ol.L
-1f30.5 μ L, 10 μm of ol.L
-1b30.5 μ L, 8U/ μ L Bst polysaccharase 1 μ L, 25mM dNTP 1.5 μ L, the DNA 2 μ L of object to be detected, fluorescence dye 1 μ L, ultrapure water complements to 25 μ L; The nucleotide sequence of FIP is as shown in SEQ.ID.NO.1, and the nucleotide sequence of BIP is as shown in SEQ.ID.NO.2, and the nucleotide sequence of F3 is as shown in SEQ.ID.NO.3, and the nucleotide sequence of B3 is as shown in SEQ.ID.NO.4;
3) LAMP reaction system is heated 40min at 63 DEG C, then at 85 DEG C of heating 5min, reaction completes;
4) question response carries out the judgement of result in the following manner after completing:
React the whether coloured change of rear observation LAMP reaction system, if LAMP reaction system color changes, be positive reaction, show to detect Klebsiella pneumonia in object to be detected; If LAMP reaction system color does not change, and is negative reaction, show Klebsiella pneumonia not detected in object to be detected.
The extracting method of the DNA of described object to be detected is:
The blood of object to be detected, tissue juice or suppuration liquid are inoculated in broth culture, under 37 DEG C of conditions, then cultivate 24h obtain bacterium liquid, get bacterium liquid centrifugal, the centrifugal supernatant DNA test kit obtained is extracted DNA.
Also setting up positive control and negative control respectively when detecting, with Klebsiella pneumonia genomic dna for positive control, take ultrapure water as negative control; When carrying out the judgement of result, also compare with positive control and negative control, if the LAMP reaction system corresponding to object to be detected and the LAMP reaction system corresponding to positive control are all in green, then judge that the detected result of object to be detected is as positive reaction, if the LAMP reaction system corresponding to object to be detected and the LAMP reaction system corresponding to negative control are all in safran, then judge that the detected result of object to be detected is as negative reaction.
Compared with prior art, the present invention has following useful technique effect:
The present invention adopt Klebsiella pneumonia tyrB gene as detection Object combine become LAMP primer, achieve easy to Klebsiella pneumonia, fast, accurately detect; This gene is after NCBI blast search, and between Klebsiella pneumonia kind, the low and conserved sequence taken into account between different serotypes of aberration rate, very low with the homology of other kind bacteriums, therefore can as versatility gene test Klebsiella pneumonia.
The LAMP method of detection mouse Klebsiella pneumonia provided by the invention, its high specificity: the negative control detected and all no positive result of large mouse genome.
The LAMP method of detection mouse Klebsiella pneumonia provided by the invention, its detection sensitivity is high: the sensitivity of Standard PCR detection method is 100pg/reaction (100copy number/reaction) order of magnitude, adopt detection method, Monitoring lower-cut is 13.5fg/ reaction (5.6 copy numbers/reaction)
The LAMP method of detection mouse Klebsiella pneumonia provided by the invention, it detects rapidly, it is convenient to go out result: the whole process of Standard PCR just can go out result at 2 ~ 4 hours, quantitative fluorescent PCR also needs 1 ~ 1.5 hour, and detection method provided by the invention can occur positive findings at about 50 minutes.And it is easy and simple to handle, low to instrument requirements, only need ortho-water bath, and can the direct observed result of dyestuff be passed through, eliminate the step of electrophoresis, can detect in the practice of mouse Klebsiella pneumonia in basic unit and have wide practical use, be a kind of for laboratory animal department is easy, fast, detect the method for mouse Klebsiella pneumonia accurately, and be applicable to basic unit and use.
Accompanying drawing explanation
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 detected result schematic diagram after gradient dilution;
In figure: POS represents the detected result of positive control, K.p represents the detected result of object to be detected (pneumonia infection klebsiella), Kp represents the detected result of Klebsiella pneumonia genomic dna, and Sa represents the detected result of staphylococcus aureus gene group DNA, H
2o represents the detected result of negative control, and P.a represents the detected result of Pseudomonas aeruginosa genomic dna, and Rat represents the detected result of rat genomic dna, and Mouse represents the detected result of mouse gene group DNA, and a represents 5.6 × 10
6the detected result of copies/ μ L, b represents 5.6 × 10
5the detected result of copies/ μ L, c represents 5.6 × 10
4the detected result of copies/ μ L, d represents 5.6 × 10
3the detected result of copies/ μ L, e represents 5.6 × 10
2the detected result of copies/ μ L, f represents the detected result of 5.6 × 10copies/ μ L, and g represents the detected result of 5.6copies/ μ L, and h represents 5.6 × 10
-1the detected result of copies/ μ L.
Embodiment
Elaborate to the present invention below in conjunction with drawings and Examples, the explanation of the invention is not limited.
The invention discloses the LAMP method of a kind of test experience with mouse Klebsiella pneumonia, this detection method is carried out based on ring mediated isothermal amplification.
The present invention is directed to Klebsiella pneumonia tyrB gene (gene accession number AF074934), devise the Auele Specific Primer comprising outer primer, inner primer.Only need add the existence of sample namely by observing the presence or absence of green fluorescence detect Klebsiella pneumonia fast and accurately in the LAMP reaction system set up, lowest detection is limited to 5.6 copy numbers/reaction, for rapid detection large mouse Klebsiella pneumonia provides the convenient method of an applicable basic unit.
1, design of primers and synthesis:
Consider specificity and the accuracy of detection, Klebsiella pneumonia tyrB gene is adopted to become LAMP primer as detection Object combine, this is because tyrB gene is after NCBI blast search, low and the conserved sequence taken into account between different serotypes of aberration rate between Klebsiella pneumonia kind, very low with the homology of other kind bacteriums, therefore can as versatility gene test Klebsiella pneumonia.
Adopt primer-design software Primer Explorer 4.0, for conserved regions design 4 LAMP primer of Klebsiella pneumonia tyrB gene, comprise forwards/reverse inner primer FIP/BIP, forwards/reverse outer primer F3/B3, a part for the nucleotide sequence of described primer and tyrB (gene accession number: AF074934) gene 150 ~ 333 or its complementary strand thereof.Concrete primer sequence is in table 1.
Table 1 LAMP method detects mouse Klebsiella pneumonia primer sequence
The extraction of bacterial genomes DNA profiling 2.1, to be detected
For the ease of the explanation to detection method, concrete use bacterial genomes is extracted test kit and is extracted Klebsiella pneumonia and (Pseudomonas aeruginosa, streptococcus aureus, sramana Li Ding Salmonella) strain gene group DNA in contrast, for subsequent use after nucleic acid-protein quantitative instrument is quantitative.
2.2, mouse to be checked adopts the method for cutting tail first to obtain blood sample, be inoculated in fresh broth culture, cultivate 24h under 37 DEG C of conditions and obtain bacterium liquid, get bacterium liquid centrifugal, utilized by the centrifugal supernatant obtained Genomic Purification Kit method to specifications to extract DNA.
3, LAMP reaction system (25 μ L) is built
Fluorescence dye is the fluorexon aqueous solution of massfraction 0.5%.Template is the DNA extracted.2 × reaction buffer and Bst polysaccharase are purchased from New England Biolabs..
4, the foundation of Klebsiella pneumonia LAMP detection method and primer operability detect
Join in reaction system from the DNA of described mouse to be checked as template using extraction, reaction process is as follows: temperature of reaction 63 DEG C, 40 minutes time, and then heating 5 minutes at 85 DEG C, reaction terminates.
Positive control (Klebsiella pneumonia genomic dna) and negative control (ultrapure water) is used to be undertaken by above-mentioned reaction system and condition.
5, the qualification of Klebsiella pneumonia LAMP reaction product
Owing to reacting the front fluorescence dye adding 1 μ L in LAMP reaction system, the LAMP reaction system that detects by an unaided eye colour-change, and compare with negative and positive, reaction solution is positive reaction (see POS and K.p in Fig. 1) in green, is negative reaction (see H2O in Fig. 1) in safran.
A large amount of pyrophosphate ions can be formed in amplification process.Mn ion in fluorescence dye is combined with fluorexon and causes fluorescent quenching, and dye colour is safran simultaneously.When amplified reaction forms pyrophosphate ion, mn ion is combined with pyrophosphate ion and forms manganese pyrophosphate, causes the generation of fluorescent signal, and dye colour becomes green simultaneously.Namely to have increased a large amount of target dna after LAMP reaction, added fluorexon and can react with it and present green.
6, the specificity experiments of LAMP
Non-Klebsiella pneumonia (the Pseudomonas aeruginosa that Klebsiella pneumonia and experimental mouse are often sent out, streptococcus aureus, sramana Li Ding Salmonella) genomic dna and large mouse gene group DNA, set up LAMP detection method according to above-mentioned reaction system and condition, carry out specific test.Arranging Klebsiella pneumonia genomic dna is positive control, and ultrapure water is negative control.Result shows, only has Klebsiella pneumonia genome to occur positive reaction, all the other reactions (see Fig. 2) that are all negative.
7, LAMP sensitivity test
After Klebsiella pneumonia genomic dna being carried out 10 times of gradient dilutions, negative control (ultrapure water) is set, sets up LAMP detection method according to above-mentioned reaction system and condition, to determine the susceptibility of detection method.
See Fig. 3, result shows: the mouse Klebsiella pneumonia LAMP detection method of foundation can detect the Klebsiella pneumonia DNA of 5.6 copy number/reactions in sample.
From the present invention, LAMP amplification method comparatively Standard PCR and fluorescence PCR method has:
High specificity: whether the existence that can judge goal gene by means of only whether increasing, thus complete the qualitative detection of bacterium and virus.
Highly sensitive: the sensitivity of Standard PCR detection method is that 100pg/ reacts (100 copy numbers/reaction) order of magnitude, adopts detection method, and Monitoring lower-cut is 13.5fg/ reaction (5.6 copy numbers/reaction).
Operate and identify simple and efficient: the whole process of Standard PCR just can go out result at 2 ~ 4 hours, and quantitative fluorescent PCR also needs 1 ~ 1.5 hour, and detection method provided by the invention can occur positive findings at 50 minutes.And easy and simple to handle, low to instrument requirements, only need ortho-water bath, and can the direct observed result of dyestuff be passed through, eliminate the step of electrophoresis, detect in basic unit in the practice of mouse kerekou pneumonia uncle and have wide practical use.
Claims (7)
1. detect a LAMP primer for mouse Klebsiella pneumonia, it is characterized in that: comprise following 2 pairs of primers: outer primer F3 and B3 and inner primer FIP and BIP; The nucleotide sequence of FIP is as shown in SEQ.ID.NO.1, and the nucleotide sequence of BIP is as shown in SEQ.ID.NO.2, and the nucleotide sequence of F3 is as shown in SEQ.ID.NO.3, and the nucleotide sequence of B3 is as shown in SEQ.ID.NO.4.
2. detect a LAMP kit for mouse Klebsiella pneumonia, it is characterized in that: this test kit comprises 40 μm of olL
-1fIP 1.0 μ L, 40 μm of olL
-1bIP 1.0 μ L, 10 μm of olL
-1f3 0.5 μ L and 10 μm olL
-1b3 0.5 μ L; F3 and B3 is outer primer, FIP and BIP is inner primer, and the nucleotide sequence of FIP is as shown in SEQ.ID.NO.1, and the nucleotide sequence of BIP is as shown in SEQ.ID.NO.2, the nucleotide sequence of F3 is as shown in SEQ.ID.NO.3, and the nucleotide sequence of B3 is as shown in SEQ.ID.NO.4.
3. a kind of LAMP kit detecting mouse Klebsiella pneumonia as claimed in claim 2, is characterized in that: described test kit also comprises 2 × reaction buffer 12.5 μ L, 8U/ μ L Bst polysaccharase 1 μ L, 25mM dNTP 1.5 μ L and fluorescence dye 1 μ L.
4. a kind of LAMP kit detecting mouse Klebsiella pneumonia as claimed in claim 3, is characterized in that: described fluorescence dye is the fluorexon aqueous solution of massfraction 0.5%.
5. detect a LAMP method for mouse Klebsiella pneumonia, it is characterized in that: comprise the following steps:
1) DNA of object to be detected is extracted;
2) following component is mixed, builds the 25 μ L LAMP reaction systems comprising 2 pairs of primers F 3 and B3 and FIP and BIP:
2 × reaction buffer 12.5 μ L, 40 μm of olL
-1fIP 1.0 μ L, 40 μm of olL
-1bIP 1.0 μ L, 10 μm of olL
-1f3 0.5 μ L, 10 μm of olL
-1b3 0.5 μ L, 8U/ μ L Bst polysaccharase 1 μ L, 25mMdNTP 1.5 μ L, the DNA 2 μ L of object to be detected, fluorescence dye 1 μ L, ultrapure water complements to 25 μ L; The nucleotide sequence of FIP is as shown in SEQ.ID.NO.1, and the nucleotide sequence of BIP is as shown in SEQ.ID.NO.2, and the nucleotide sequence of F3 is as shown in SEQ.ID.NO.3, and the nucleotide sequence of B3 is as shown in SEQ.ID.NO.4;
3) LAMP reaction system is heated 40min at 63 DEG C, then at 85 DEG C of heating 5min, reaction completes;
4) question response carries out the judgement of result in the following manner after completing:
React the whether coloured change of rear observation LAMP reaction system, if LAMP reaction system color changes, be positive reaction, show to detect Klebsiella pneumonia in object to be detected; If LAMP reaction system color does not change, and is negative reaction, show Klebsiella pneumonia not detected in object to be detected.
6. a kind of LAMP method detecting mouse Klebsiella pneumonia as claimed in claim 5, is characterized in that: the extracting method of the DNA of described object to be detected is:
The blood of object to be detected, tissue juice or suppuration liquid are inoculated in broth culture, under 37 DEG C of conditions, then cultivate 24h obtain bacterium liquid, get bacterium liquid centrifugal, the centrifugal supernatant DNA test kit obtained is extracted DNA.
7. a kind of LAMP method detecting mouse Klebsiella pneumonia as claimed in claim 5, is characterized in that: also setting up positive control and negative control respectively when detecting, with Klebsiella pneumonia genomic dna for positive control, take ultrapure water as negative control; When carrying out the judgement of result, also compare with positive control and negative control, if the LAMP reaction system corresponding to object to be detected and the LAMP reaction system corresponding to positive control are all in green, then judge that the detected result of object to be detected is as positive reaction, if the LAMP reaction system corresponding to object to be detected and the LAMP reaction system corresponding to negative control are all in safran, then judge that the detected result of object to be detected is as negative reaction.
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Cited By (4)
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| CN108796104A (en) * | 2018-07-06 | 2018-11-13 | 云南科耀生物科技有限公司 | A kind of purposes of CelB genes |
| CN109628621A (en) * | 2019-02-02 | 2019-04-16 | 广西壮族自治区兽医研究所 | A kind of real-time quantitative LAMP primer group and kit detecting Friedlander's bacillus |
| CN111500751A (en) * | 2020-04-10 | 2020-08-07 | 刘洋 | Detection method and kit for rapidly detecting high-toxicity Klebsiella pneumoniae |
| CN114898800A (en) * | 2022-07-14 | 2022-08-12 | 中国医学科学院北京协和医院 | Method and system for predicting sensitivity of klebsiella pneumoniae to ceftriaxone |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108796104A (en) * | 2018-07-06 | 2018-11-13 | 云南科耀生物科技有限公司 | A kind of purposes of CelB genes |
| CN108796104B (en) * | 2018-07-06 | 2021-07-02 | 云南科耀生物科技有限公司 | Application of CelB gene |
| CN109628621A (en) * | 2019-02-02 | 2019-04-16 | 广西壮族自治区兽医研究所 | A kind of real-time quantitative LAMP primer group and kit detecting Friedlander's bacillus |
| CN109628621B (en) * | 2019-02-02 | 2022-02-15 | 广西壮族自治区兽医研究所 | Real-time quantitative LAMP primer group and kit for detecting Klebsiella pneumoniae |
| CN111500751A (en) * | 2020-04-10 | 2020-08-07 | 刘洋 | Detection method and kit for rapidly detecting high-toxicity Klebsiella pneumoniae |
| CN111500751B (en) * | 2020-04-10 | 2023-10-27 | 刘洋 | Detection method and kit for rapidly detecting high-virulence klebsiella pneumoniae |
| CN114898800A (en) * | 2022-07-14 | 2022-08-12 | 中国医学科学院北京协和医院 | Method and system for predicting sensitivity of klebsiella pneumoniae to ceftriaxone |
| CN114898800B (en) * | 2022-07-14 | 2022-09-16 | 中国医学科学院北京协和医院 | Method and system for predicting sensitivity of klebsiella pneumoniae to ceftriaxone |
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