CN104450942A - LAMP (loop-mediated isothermal amplification) kit for detecting acinetobacter baumannii and special primers of LAMP kit - Google Patents
LAMP (loop-mediated isothermal amplification) kit for detecting acinetobacter baumannii and special primers of LAMP kit Download PDFInfo
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Abstract
The invention discloses an LAMP (Loop-mediated isothermal amplification) kit for detecting acinetobacter baumannii, special primers of the LAMP kit and an application of the LAMP kit to detection of the acinetobacter baumannii. The nucleotide sequence of the five special primers for performing LAMP detection for the acinetobacter baumannii in a sequence table is SEQ ID NO: 2(F3), SEQ ID NO: 3(B3), SEQ ID NO: 4(FIP), SEQ ID NO: 5(BIP) and SEQ ID NO: 6(LF). The acinetobacter baumannii can be rapidly, conveniently, efficiently, high-specifically and high-sensitively detected at the equal temperature, complicated instruments are omitted, and a novel technical platform for detecting the acinetobacter baumannii is provided. The LAMP kit can be used for screening and detecting the acinetobacter baumannii in livestock production units, basic medical and health units and disease prevention and control centers, has wide market prospects and high economic and social benefits and is suitable for wide-range popularization and application.
Description
Technical field
The invention belongs to the molecular biology for detection of bacterium in biological technical field, particularly relate to a kind of LAMP kit of Acinetobacter bauamnnii and primer special and its thereof and detecting the application in Acinetobacter bauamnnii.
Background technology
Acinetobacter bauamnnii clinical infection and current treatment status
Acinetobacter bauamnnii (Acinetobacter baumannii, Ab) is a kind of non-fermented, aerobic gram negative bacilli, belongs to acinetobacter (Acinetobacter).Acinetobacter bauamnnii is mainly present in hospital environment, it is one of Main Pathogenic Bacteria of Nosocomial Pneumonia and Ventilator Associated Pneumonia, the particularly patient of intensive care unit(ICU), infect the probability of Acinetobacter bauamnnii higher, this and patients with low immunity, suffer from serious underlying disease, accept the invasives such as respirator and treat and use extensive pedigree antibiotic in close relations in a large number.In addition, there are some researches show, the pollution of the pollution of hospital's communal facility, particularly respirator, ductus venosus etc., by the cross infection of medical personnel's hand, is the principal element causing Nosocomial Acinetobacter bauamnnii to infect.
The fearful part of Acinetobacter bauamnnii is that it not only once caused worldwide repeatedly eruption and prevalence, more because its ever-increasing resistance makes numerous clinical workers feel simply helpless.Acinetobacter bauamnnii is once to penicillin, cynnematin, aminoglycoside, quinolone antibiotics etc. are all responsive, but along with the reason such as to widely use of Broad spectrum antibiotics and immunosuppressor, the general resistant organism of Acinetobacter bauamnnii (pan-drug resistant Acinetobacterbaumannii all over the world, or multi-drug resistant bacteria (multi-drug resistant Acinetobacter baumannii PDR-AB), MDR-Ab) report emerges in an endless stream, even to the imipenum (Imipenem having better result for the treatment of before always, and meropenem (Meropenem IP), etc. MP) bacterial strain of carbapenem antibiotic resistance is also of common occurrence.Also there is resistance report in some newer antibacterials such as polymyxin, Tigecycline etc.
Therefore, quick and precisely diagnosing pathogenic bacteria, give the antibiotic therapy of normative and reasonable, is the effective measure controlling Acinetobacter bauamnnii infection.
LAMP technology is introduced
Ring mediated constant temperature nucleic acid amplification technology (1oop-mediated isothermal amplification, LAMP) be by Notomi (Notomi T, et al.Loop-mediated isothermal amplification of DNA.Nucleic Acids Res 2000; 28 (12): 63.) a kind of novel gene amplification technique invented.Concrete grammar is 6 regions for target gene (DNA or cDNA), design 4-6 species-specific primers, utilize strand displacement archaeal dna polymerase, carry out gene amplification reaction under isothermal conditions, result directly judges by the precipitation turbidity of amplification by product magnesium pyrophosphate.LAMP technology has rapidly and efficiently, high specificity, highly sensitive, simple to operate, detect the features such as directly perceived and equipment requirements is low.
Since invention in 2000, LAMP technology is by the rapid molecular Biological Detection field for fields such as the pathogenic microorganism examination, genetic diseases diagnosis, food safeties.In recent years, this technology is widely used in pathogen detection abroad.Nakauchi M in 2011 etc. establish H1N1 laboratory fast diagnosis method (NakauchiM, the et al.Evaluation of reverse transcription loop-mediated isothermalamplification assays for rapid diagnosis of pandemic influenza A/H1N1 2009virus.J Med Virol.2011 Jan based on LAMP technology; 83 (1): 10-5.).At present, LAMP technology has been widely used in mycoplasma pneumoniae (Gotoh K, et al.Assessment of the loop-mediated isothermalamplification assay for rapid diagnosis of Mycoplasma pneumoniae in pediatriccommunity-acquired pneumonia.Jpn J Infect Dis.2013, 66 (6): 539-42.), tubercule bacillus (Kumar P, et al.Loop-mediated isothermal amplification assay for rapidand sensitive diagnosis of tuberculosis.Pandya D, Singh N, J Infect.2014Sep 9. [Epub ahead of print]), SARS (Poon LL, et al.Rapid detection of thesevere acute respiratory syndrome (SARS) coronavirus by a loop-mediatedisothermalamplification assay.Clin Chem.2004 Jun, 50 (6): 1050-2.), HIV (CurtisKA, et al.Real-Time Detection of HIV-2 by Reverse Transcription-Loop-Mediated Isothermal Amplification.J Clin Microbiol.2014 Jul, 52 (7): 2674-6.) etc. in the detection of pathogenic agent and the diagnosis of relative disease.At present, the LAMP kit for detecting Acinetobacter bauamnnii and primer special thereof is showed no both at home and abroad.
Summary of the invention
The invention provides the primer for carrying out LAMP detection to Acinetobacter bauamnnii, to realize the batch detection of Acinetobacter bauamnnii, improve the specificity and sensitivity that detect.
LAMP primer for detecting Acinetobacter bauamnnii provided by the present invention is according to Acinetobacter bauamnnii β-lactamase OXA-51 gene (bla
oXA-51) specific and conserved sequence design, in order to the Acinetobacter bauamnnii in the samples such as the pure bacterium of qualitative detection, patient's sputum, blood and other secretory product, described LAMP primer is made up of five primers, comprises the combination of outer primer F3 and B3, inner primer FIP and BIP and ring primer LF; Described Acinetobacter bauamnnii bla
oXA-51specific conservative's target sequence of gene is as shown in SEQ ID NO:1 in sequence table.
Specifically, the nucleotide sequence of described five primers for carrying out LAMP detection to Acinetobacter bauamnnii is as shown in SEQ ID NO:2 (F3), SEQ ID NO:3 (B3), SEQ ID NO:4 (FIP), SEQ IDNO:5 (BIP) and SEQ ID NO:6 (LF) in sequence table.
Described LAMP primer is the composition of F3 and B3, FIP and BIP and LF 5:40:20 in molar ratio.
Second object of the present invention is to provide a kind of test kit for carrying out LAMP detection to Acinetobacter bauamnnii.
Test kit provided by the present invention, comprises the above-mentioned primer for carrying out LAMP detection to Acinetobacter bauamnnii.
Specifically, described test kit comprises the following reagent for 25 μ l reaction systems: 20mM TrisHCl (pH8.8), 10mM KCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mM MgSO
4, 1.4mM dNTP each, 8U Bst DNA polymerase, primer add-on is: 5pmol primers F 3 and B3,40pmol primers F IP and BIP, 20pmol primer LF.
For convenience of detecting, also can comprise positive control and negative control in described test kit, described positive control is Acinetobacter bauamnnii type strain genomic dna, and described negative control is not containing the LAMP amplification system of DNA, as distilled water.
Above-mentioned LAMP primer or the application of test kit in Acinetobacter bauamnnii LAMP detects also belong to protection scope of the present invention.
3rd object of the present invention is to provide a kind of LAMP detection method of Acinetobacter bauamnnii.
Detection method provided by the present invention, can comprise the following steps:
1) with testing sample genomic dna for template, under the guiding of above-mentioned primer, carry out LAMP amplification, 25 μ l LAMP reaction systems comprise: testing sample genomic dna 2 μ l, 20mM TrisHCl (pH 8.8), 10mM KCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mM MgSO
4, 1.4mM dNTPeach, 8U Bst DNA polymerase, primer add-on is: 5pmol primers F 3 and B3,40pmol primers F IP and BIP, 20pmol primer LF; LAMP amplification condition is: put 60-65 DEG C of constant temperature 40-50min;
2) reaction carries out result judgement after terminating: in reaction solution, add fluorexon indicator, according to the colour-change judged result of reaction solution, there is Acinetobacter bauamnnii in green expression testing sample, in orange expression testing sample, there is not Acinetobacter bauamnnii; Or do not add fluorexon indicator and directly carry out judged result with the turbidity change of reaction solution before and after turbidimeter detection reaction, turbidity rises and represents in testing sample to there is Acinetobacter bauamnnii, there is not Acinetobacter bauamnnii in the unchanged expression testing sample of turbidity.
In above-mentioned detection method, described step 1) in LAMP reaction system in be also provided with positive control and negative control, described positive control is Acinetobacter bauamnnii type strain genomic dna, and described negative control is not containing the LAMP amplification system of DNA, as distilled water; Described LAMP amplification condition is preferably: put 65 DEG C of constant temperature 50min.
Described step 2) in the addition of fluorexon indicator can be 1 μ l (end reaction system is 26 μ l), containing 0.5mM fluorexon and 10mM Manganous chloride tetrahydrate.
The invention provides a kind of LAMP detection method of Acinetobacter bauamnnii and primer special thereof and test kit.The Acinetobacter bauamnnii principle that the present invention detects adopts LAMP technology, detects Acinetobacter bauamnnii genome blaOXA-51 gene.BlaOXA-5 is natural that carry, that coding has carbapenem antibiotic hydrolysis ability D class β-lactamase (the Carbapenem hydrolyzing class D β-lactamase of Acinetobacter bauamnnii, CHDL), can be used as one of Acinetobacter bauamnnii marker gene.
The present invention has the following advantages:
1, simple to operate, equipment requirements is low.Detection sample and detection reaction liquid only need be put into after 60-65 DEG C of thermostat water bath hatches 40-50 minute and get final product judged result by the present invention.Detected result judges, without the need to complex instrument by visual inspection (fluorexon colour developing) or turbidimeter.
2, specificity is good, highly sensitive.The identification of five primer pair Acinetobacter bauamnnii target sequence specific regions of the present invention's design ensure that the high degree of specificity that LAMP increase, and namely LAMP can find out corresponding target sequence and increases from the gene sample differing only 1 Nucleotide; Sensitivity comparable regular-PCR height 10-100 doubly.
3, detection time is short, amplification efficiency is high.Whole LAMP amplified reaction can complete in one hour, and goal gene productive rate can reach 0.5mg/mL.
In sum, to present invention can be implemented under isothermal condition fast, convenient, efficient, high special, detect Acinetobacter bauamnnii with sensitivity, without the need to complex instrument, the detection for Acinetobacter bauamnnii provides new technology platform.The present invention can be used for Animal husbandry production unit, primary care health unit and the examination of each disease prevention and control center and detects Acinetobacter bauamnnii, has wide market outlook and larger economical, societal benefits, is suitable for applying on a large scale.
Below in conjunction with specific embodiment, the present invention is described in further details.
Accompanying drawing explanation
Fig. 1 is the LAMP response curve of five cover primers;
Fig. 2 is the specific turbidimeter detected result of Acinetobacter bauamnnii LAMP detection method;
Fig. 3 is the turbidimeter detected result of Acinetobacter bauamnnii LAMP detection method sensitivity;
Fig. 4 is the fluorexon indicator detected result of Acinetobacter bauamnnii LAMP detection method sensitivity;
Fig. 5 is the detected result of Acinetobacter bauamnnii regular-PCR detection method sensitivity.
Embodiment
In following embodiment, method therefor is ordinary method if no special instructions, concrete steps can be see: " Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, David W., Molecular Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold SpringHarbor).
Described percentage concentration is mass/mass (W/W if no special instructions, unit g/100g) percentage concentration, mass/volume (W/V, unit g/100mL) percentage concentration or volume/volume (V/V, Unit/mL/100mL) percentage concentration.
The approach that obtains of the various biomaterials be described in embodiment is only to provide a kind of approach of testing acquisition to reach concrete disclosed object, should not become the restriction to biological material source of the present invention.In fact, the source of used biomaterial is widely, and any biomaterial that can obtain with moral ethics that keeps on the right side of the law can replace use according to the prompting in embodiment.
The primer synthesizes by Beijing Sheng Gong biotech firm.
Embodiment is implemented under premised on technical solution of the present invention, gives detailed embodiment and concrete operating process, and embodiment will contribute to understanding the present invention, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1, for carrying out the design of primers of LAMP detection to Acinetobacter bauamnnii
The β-lactamase OXA-51 gene (bla of Acinetobacter bauamnnii is obtained from nucleic acid database GenBank (http://www.ncbi.nlm.nih.gov) retrieval of NCBI
oXA-51) sequence (No. GenBank: DQ385606.1), carry out homology analysis by BLAST software, obtain Acinetobacter bauamnnii β-lactamase OXA-51 gene (bla
oXA-51) specific and conserved sequence (in sequence table SEQ ID NO:1), again according to this specific and conserved sequence, primer Acinetobacter bauamnnii being carried out to LAMP detection is designed for software Primer design V4, design five cover primers, through Experimental comparison, finally have chosen combination of primers OXA-4, primer sequence is as shown in table 1.
Table 1 is for carrying out the combination of primers OXA-4 of LAMP detection to Acinetobacter bauamnnii
The foundation of the LAMP detection method of embodiment 2, Acinetobacter bauamnnii
One, the best combination of primers of Acinetobacter bauamnnii LAMP detection method is determined
The five cover combination of primers designed by embodiment 1 carry out LAMP detection to Acinetobacter bauamnnii (deriving from affiliated hospital of Military Medical Science Institute to breathe and critical care medicine section), and to obtain best combination of primers, concrete grammar is as follows:
1) under the guiding of above-mentioned five cover primers, carry out LAMP amplification respectively, 25 μ l LAMP reaction systems comprise: Acinetobacter bauamnnii genomic dna (Promega
genomic DNA Purification Kit A1125 extracts the nucleic acid in testing sample) 2 μ l, 20mM TrisHCl (pH 8.8), 10mM KCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mM MgSO
4, 1.4mM dNTP each, 8U Bst DNApolymerase, primer add-on is: 5pmol primers F 3 and B3,40pmol primers F IP and BIP, 20pmol primer LF; LAMP amplification condition is: put 65 DEG C of constant temperature 50min;
2) reaction is carried out result and is judged after terminating: in reaction solution, add the fluorexon indicator of 1 μ l (end reaction system as 26 μ l) containing 0.5mM fluorexon and 10mM Manganous chloride tetrahydrate, according to colour-change judged result (principle: fluorexon is Metal ion indicator, the Mg in energy Indicator Reaction liquid of reaction solution
2+change), green represent in testing sample to there is Acinetobacter bauamnnii (positive), in orange expression testing sample, there is not Acinetobacter bauamnnii (feminine gender), or do not add fluorexon indicator directly to carry out judged result with the change of the turbidity of reaction solution before and after turbidimeter detection reaction and (in the process that principle: LAMP reacts, can magnesium pyrophosphate be produced, magnesium pyrophosphate is a kind of white precipitate, according to the change of turbidity, turbidimeter can judge that LAMP reacts), turbidity rises and represents in testing sample to there is Acinetobacter bauamnnii (positive), Acinetobacter bauamnnii (feminine gender) is there is not in the unchanged expression testing sample of turbidity, (X-coordinate is the turbidity value of reaction product at 650nm to LAMP response curve such as Fig. 1 of five cover primers, ordinate zou is the reaction times) shown in, as can be seen from the figure the reaction effect of combination of primers OXA-4 is best.
The LAMP detection system of best Acinetobacter bauamnnii is decided to be (25 μ l): testing sample genomic dna 2 μ l, 20mM TrisHCl (pH 8.8), 10mM KCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mM MgSO
41.4mM dNTP each, 8U Bst DNA polymerase, primer add-on is: 5pmol primers F 3 (in sequence table SEQ ID NO:2) and B3 (in sequence table SEQ ID NO:3), 40pmol primers F IP (in sequence table SEQ ID NO:4) and BIP (in sequence table SEQ ID NO:5), 20pmol primer LF (in sequence table SEQ ID NO:6).
Two, the optimum reaction condition of Acinetobacter bauamnnii LAMP detection method is determined
Carry out LAMP detection with combination of primers OXA-4 to Acinetobacter bauamnnii, to obtain optimum reaction condition, concrete grammar is as follows:
1) under the guiding of upper combination of primers OXA-4, carry out LAMP amplification, 25 μ l LAMP reaction systems comprise: identical with step one; LAMP amplification condition is: put 60-65 DEG C of constant temperature 40-50min;
2) reaction carries out result judgement after terminating: identical with step one.
Result is under the LAMP amplification condition of 60-65 DEG C of constant temperature 40-50min, and combination of primers OXA-4 all achieves good reaction effect, and particularly under the LAMP amplification condition of 65 DEG C of constant temperature 50min, reaction effect is best.
The LAMP reaction conditions of best Acinetobacter bauamnnii is: put 60-65 DEG C of constant temperature 40-50min, preferably put 65 DEG C of constant temperature 50min.
Specificity, the susceptibility of the LAMP detection method of embodiment 3, Acinetobacter bauamnnii detect
One, the specific detection of the LAMP detection method of Acinetobacter bauamnnii
Respectively with Acinetobacter bauamnnii (deriving from affiliated hospital of Military Medical Science Institute to breathe and critical care medicine section), acinetobacter haemolyticus is (purchased from Chinese microorganism strain preservation administrative center (China General MicrobiologicalCulture Collection Center, CGMCC)), hospital's acinetobacter calcoaceticus (acinetobacter calcoaceticus gene kind TU13, purchased from CGMCC), Acinetobacter johnsonii (purchased from CGMCC), Acinetobacter junii (purchased from CGMCC), Pittii (acinetobacter calcoaceticus gene kind 3, purchased from Chinese industrial Microbiological Culture Collection administrative center (China Center ofIndustrial Culture Collection, CICC), Acinetobacter calcoaceticus (purchased from CGMCC), the genomic dna of Acinetobacter lwoffii (by the keeping of Military Medical Science Institute's prevention and control of diseases institute's infectious disease control center) is template, take distilled water as negative control (NC), with Acinetobacter bauamnnii type strain (PC, purchased from CGMCC and CICC) be positive control, detect the specificity of the LAMP detection method of the Acinetobacter bauamnnii of the best that embodiment 2 obtains.
Detected result as Fig. 2 (bacterium english abbreviation and Chinese as follows: A.baumannii is Acinetobacter bauamnnii, be called for short A.b-1, A.b-2; A.calcoaceticus is Acinetobacter calcoaceticus; A.haemolytius is acinetobacter haemolyticus; A.nosocomialis is hospital's acinetobacter calcoaceticus (acinetobacter calcoaceticus gene kind TU13); Pittii is acinetobacter calcoaceticus gene kind 3; A.johnsonii is Acinetobacter johnsonii; A.junii is Acinetobacter junii; A.lwoffii is Acinetobacter lwoffii; NC is negative control (distilled water); PC is Acinetobacter bauamnnii type strain) shown in, can find out, within the 40min reaction times, A.b-1, A.b-2 (Acinetobacter bauamnnii) and PC (Acinetobacter bauamnnii type strain) is only had to there occurs LAMP reaction, all the other acinetobacter calcoaceticus bacterial strains all do not occur, show that the LAMP detection method of Acinetobacter bauamnnii has higher specificity, Acinetobacter bauamnnii can be detected specifically.
Two, the sensitivity technique of the LAMP detection method of Acinetobacter bauamnnii
Detection LAMP detection method and regular-PCR detection method detect the sensitivity of Acinetobacter bauamnnii, method is: extract Acinetobacter bauamnnii (deriving from affiliated hospital of Military Medical Science Institute to breathe and critical care medicine section) genomic dna, then with 10 times of gradients (1 times, 10 times, 10
2doubly, 10
3doubly, 10
4doubly, 10
5doubly, 10
6doubly, 10
7doubly) dilute, again with the Acinetobacter bauamnnii genomic dna through gradient dilution for template, the concentration of Acinetobacter bauamnnii genomic dna in 1-8 template is made to be respectively 500ng/ μ l, 50ng/ μ l, 5ng/ μ l, 500pg/ μ l, 50pg/ μ l, 5pg/ μ l, 0.5pg/ μ l, 0.05pg/ μ l, take distilled water as negative control (NC), use LAMP detection method and regular-PCR method (primer sequence is F3 and B3) to carry out sensitivity technique respectively.
(template concentrations of 1-8 is respectively the specific turbidimeter detected result of Acinetobacter bauamnnii LAMP detection method such as Fig. 3: 500ng/ μ l, 50ng/ μ l, 5ng/ μ l, 500pg/ μ l, 50pg/ μ l, 5pg/ μ l, 0.5pg/ μ l, 0.05pg/ μ l, 8 is negative control) shown in, (template concentrations of 1-8 is respectively specific fluorexon indicator detected result such as the Fig. 4 of Acinetobacter bauamnnii LAMP detection method: 500ng/ μ l, 50ng/ μ l, 5ng/ μ l, 500pg/ μ l, 50pg/ μ l, 5pg/ μ l, 0.5pg/ μ l, 0.05pg/ μ l; "+" represents the result positive (green), "-" represents result feminine gender (orange)) shown in, the detected result of regular-PCR is as shown in Fig. 5 (template concentrations of swimming lane 1-8 is respectively: 500ng/ μ l, 50ng/ μ l, 5ng/ μ l, 500pg/ μ l, 50pg/ μ l, 5pg/ μ l, 0.5pg/ μ l, 0.05pg/ μ l), target product clip size is 570bp, can find out, 50pg/ μ l Acinetobacter bauamnnii genomic dna (10 can be detected by the LAMP detection method of Acinetobacter bauamnnii
4times weaker concn), and regular-PCR method only can detect 500pg/ μ l Acinetobacter bauamnnii genomic dna (10
3times weaker concn), and fluorexon indicator detection method is consistent with the result of turbidimeter detection method, shows that the LAMP detection method of Acinetobacter bauamnnii is than highly sensitive 10 times of regular-PCR detection method.
Embodiment 4, the test kit detected for the LAMP of Acinetobacter bauamnnii
By 20mM TrisHCl (pH 8.8), 10mM KCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mM MgSO
4, 1.4mM dNTP each, 8U Bst DNA polymerase, primer add-on is 5pmol primers F 3 and B3 (shown in SEQ ID NO:2 and SEQ ID NO:3 primer), 40pmol primers F IP and BIP (shown in SEQ ID NO:4 and SEQ ID NO:5 primer), 20pmol primer LF (shown in SEQ IDNO:6 primer), as the Acinetobacter bauamnnii type strain genomic dna 2 μ l of positive control, and jointly pack as the distilled water 2 μ l of negative control, obtain the test kit detected for the LAMP of the Acinetobacter bauamnnii of 25 μ l reaction systems.
Claims (10)
1., for detecting the LAMP primer of Acinetobacter bauamnnii, be according to Acinetobacter bauamnnii β-lactamase OXA-51 gene (bla
oXA-51) specific and conserved sequence design, in order to the Acinetobacter bauamnnii in the samples such as the pure bacterium of qualitative detection, patient's sputum, blood and other secretory product, described LAMP primer is made up of five primers, comprises the combination of outer primer F3 and B3, inner primer FIP and BIP and ring primer LF; Described Acinetobacter bauamnnii β-lactamase OXA-51 gene (bla
oXA-51) specific and conserved sequence as shown in SEQ ID NO:1 in sequence table.
2. LAMP primer according to claim 1, is characterized in that: the nucleotide sequence of described five primers for carrying out LAMP detection to Acinetobacter bauamnnii is as shown in SEQ ID NO:2 (F3), SEQ ID NO:3 (B3), SEQ ID NO:4 (FIP), SEQ ID NO:5 (BIP) and SEQ ID NO:6 (LF) in sequence table.
3. LAMP primer according to claim 2, is characterized in that: described LAMP primer, is the composition of F3 and B3, FIP and BIP and LF 5:40:20 in molar ratio.
4., for carrying out a test kit for LAMP detection to Acinetobacter bauamnnii, comprise described in claim 1 or 2 or 3 for carrying out the primer of LAMP detection to Acinetobacter bauamnnii.
5. test kit according to claim 1, is characterized in that: described test kit comprises the following reagent for 25 μ l reaction systems: 20mM TrisHCl (pH8.8), 10mM KCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mM MgSO
4, 1.4mM dNTP each, 8U Bst DNA polymerase, primer add-on is: 5pmol primers F 3 and B3,40pmol primers F IP and BIP, 20pmol primer LF.
6. LAMP primer according to claim 5, it is characterized in that: in described test kit, also can comprise positive control and negative control, described positive control is Acinetobacter bauamnnii type strain genomic dna, and described negative control is not containing the LAMP amplification system of DNA, as distilled water.
7. claim 1 or the LAMP primer described in 2 or 3 or claim 4 or the application of the test kit described in 5 or 6 in the LAMP of Acinetobacter bauamnnii detects.
8. application according to claim 7, is characterized in that: a kind of LAMP detection method of Acinetobacter bauamnnii, comprises the following steps:
1) with testing sample genomic dna for template, under the guiding of primer described in claim 1 or 2 or 3, carry out LAMP amplification, 25 μ l LAMP reaction systems comprise: testing sample genomic dna 2 μ l, 20mM TrisHCl (pH8.8), 10mM KCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mM MgSO
4, 1.4mM dNTP each, 8U Bst DNA polymerase, primer add-on is: 5pmol primers F 3 and B3,40pmol primers F IP and BIP, 20pmol primer LF; LAMP amplification condition is: put 60-65 DEG C of constant temperature 40-50min;
2) reaction carries out result judgement after terminating: in reaction solution, add fluorexon indicator, according to the colour-change judged result of reaction solution, there is Acinetobacter bauamnnii in green expression testing sample, in orange expression testing sample, there is not Acinetobacter bauamnnii; Or do not add fluorexon indicator and directly carry out judged result with the turbidity change of reaction solution before and after turbidimeter detection reaction, turbidity rises and represents in testing sample to there is Acinetobacter bauamnnii, there is not Acinetobacter bauamnnii in the unchanged expression testing sample of turbidity.
9. application according to claim 8, it is characterized in that: described step 1) in LAMP reaction system in be also provided with positive control and negative control, described positive control is Acinetobacter bauamnnii type strain genomic dna, described negative control is not containing the LAMP amplification system of DNA, as distilled water; Described LAMP amplification condition is preferably: put 65 DEG C of constant temperature 50min.
10. application according to claim 8 or claim 9, is characterized in that: described step 2) in the addition of fluorexon indicator can be 1 μ l (end reaction system is 26 μ l), containing 0.5mM fluorexon and 10mM Manganous chloride tetrahydrate.
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| CN109266658A (en) * | 2018-10-17 | 2019-01-25 | 昆明理工大学 | The specific gene and its primer of a kind of Acinetobacter bauamnnii and application |
| CN110129316A (en) * | 2018-02-05 | 2019-08-16 | 北京智德医学检验所有限公司 | It is a kind of for detecting the LAMP primer composition and application of 4 kinds of gramnegative bacteriums in intraocular liquid |
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| CN111996239A (en) * | 2019-05-27 | 2020-11-27 | 复旦大学附属中山医院 | Primers and probes for detecting acinetobacter baumannii and detection method thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754906A (en) * | 2017-01-11 | 2017-05-31 | 博奥生物集团有限公司 | LAMP primer composition and its application for detecting seven kinds of drug resistant genes of Carbapenem-resistant class antibiotic |
| CN110129316A (en) * | 2018-02-05 | 2019-08-16 | 北京智德医学检验所有限公司 | It is a kind of for detecting the LAMP primer composition and application of 4 kinds of gramnegative bacteriums in intraocular liquid |
| CN109266658A (en) * | 2018-10-17 | 2019-01-25 | 昆明理工大学 | The specific gene and its primer of a kind of Acinetobacter bauamnnii and application |
| CN109266658B (en) * | 2018-10-17 | 2022-08-19 | 昆明理工大学 | Acinetobacter baumannii specific gene and primer and application thereof |
| CN111996239A (en) * | 2019-05-27 | 2020-11-27 | 复旦大学附属中山医院 | Primers and probes for detecting acinetobacter baumannii and detection method thereof |
| CN110283745A (en) * | 2019-06-27 | 2019-09-27 | 浙江工业大学 | Hospital acinetobacter calcoaceticus FK2 and its application in degradable organic pollutant |
| CN110283745B (en) * | 2019-06-27 | 2021-05-11 | 浙江工业大学 | Acinetobacter hospital FK2 and application thereof in degrading organic pollutants |
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