CN103966342B - Streptococcus iniae rapid detection primer and method thereof - Google Patents
Streptococcus iniae rapid detection primer and method thereof Download PDFInfo
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- CN103966342B CN103966342B CN201410226035.7A CN201410226035A CN103966342B CN 103966342 B CN103966342 B CN 103966342B CN 201410226035 A CN201410226035 A CN 201410226035A CN 103966342 B CN103966342 B CN 103966342B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses Streptococcus iniae rapid detection primer and method thereof, Streptococcus iniae rapid detection primer, being made up of outer primer and inner primer, is outer primer by SEQ? ID? outer primer upstream primer shown in NO.1 and SEQ? ID? outer primer downstream primer composition shown in NO.2; Is inner primer by SEQ? ID? inner primer upstream primer shown in NO.3 and SEQ? ID? inner primer downstream primer composition shown in NO.4.The invention solves prior art detect the Streptococcus iniae cycle long, testing cost is high, can not be applied to the problems such as Site Detection, simple and quick.Accuracy is high, highly sensitive, and the minimum concentration detecting Streptococcus iniae is 3 × 10
2cfu/ml.Testing cost is low, without the need to the special instrument equipment of PCR instrument and so on.Do not need through microbial culture, detect required sample size few, achieve Wicresoft's sampling, be applicable to the ornamental fish that economic worth is higher.
Description
Technical field
The present invention relates to a kind of Streptococcus iniae rapid detection primer and method thereof, belong to aquarium fish pathogenic bacteria field of fast detection.
Background technology
Streptococcus iniae is under the jurisdiction of genus bacillus guiding principle (Bacilli), Bacterium lacticum order (Lactobacillales), Streptococcaceae (Streptococcaceae), streptococcus (Streptococcus), for a kind of gram-positive cocci of tool hemolytic, it is one of important pathogenic bacteria of cultured fishes, the infection of tilapia, barramunda, rainbow trout, channel catfish, hybridMoron Saxatilis fish, lefteye flounder and filefish etc. can be caused, cause very high mortality ratio.Streptococcus iniae is repeatedly found in the aquarium fish of cultivation in recent years, and cause the symptoms such as the beautiful fish of infected figure, Pseudorabora parva eyes projection, the gill cover are hemorrhage, ascites, have very strong infectivity, mortality ratio is more than 30%.The value of aquarium fish is its sight, once there is the infection of Streptococcus iniae, even if the life of part fish is retrieved in treatment in time, its ornamental value is also greatly affected, and the financial loss caused thus is huge.Therefore sick for aquarium fish Streptococcus iniae, the early stage detection of cause of disease with take effective prophylactic treatment measure particularly important in time.At present for the detection of Streptococcus iniae, carry out mainly through bacteria distribution, biochemical identification, serological reaction and round pcr, length consuming time, cost is high, mainly with killing premised on aquatic animal, complicated operation, needs plant and instrument and the technician of specialty, is not suitable for the Real-Time Monitoring of cause of disease in actual production.Need badly in breeding production a kind of can be applied to cultivation site, to fish bulk damage little, quick and precisely, Streptococcus iniae detection technique easy and simple to handle.
Loop-mediated isothermal amplification technique (Loop-mediated Isothermal Amplification, LAMP) be a kind of novel nucleic acids amplification technique set up for 2000, it is characterized in that 6 zone design, 4 Auele Specific Primers for target gene, under a kind of effect of strand displacement archaeal dna polymerase, isothermal condition (60 ~ 65 DEG C) 30 ~ 60min is kept to complete nucleic acid amplification reaction, add nucleic acid dye SYBRGreen I or fluorexon, there is nucleic acid amplification to react by Show Color, immediately obtain detected result.Not yet there is the report about Streptococcus iniae LAMP detection technique at present.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of Streptococcus iniae rapid detection primer is provided.
Second object of the present invention be to provide a kind of to fish bulk damage little, quick and precisely, Streptococcus iniae method for quick easy and simple to handle.
Technical scheme of the present invention is summarized as follows:
A kind of Streptococcus iniae rapid detection primer, it is characterized in that being made up of outer primer and inner primer, described outer primer is made up of the outer primer downstream primer shown in the outer primer upstream primer shown in SEQ ID NO.1 and SEQ ID NO.2; Described inner primer is made up of the inner primer downstream primer shown in the inner primer upstream primer shown in SEQ ID NO.3 and SEQ ID NO.4.
A kind of Streptococcus iniae method for quick, is characterized in that comprising the steps:
(1) measuring samples DNA is extracted;
(2) in the 200 μ l PCR reaction tubess that 19 μ l LAMP reaction basal liquids are housed, add 4 μ l primer mixed solutions, 1 μ l BstDNA polysaccharase, 1 μ l measuring samples DNA profiling, is made into the LAMP reaction system of 25 μ l; Place 40-60min in 60-65 DEG C, then obtain detecting liquid in 85 DEG C of placement 15min;
(3) adopt the agarose electrophoresis of mass concentration 1% to detect liquid and whether produce the stair-stepping DNA band of continuous print, then for positive, or 1 μ l fluorescence dye SYBR Green I working fluid is added in detection liquid, visual color reacts, in green be the positive, in orange red be negative, positively to represent in measuring samples containing Streptococcus iniae; Negative expression does not contain Streptococcus iniae;
Described primer mixed solution is that isopyknic concentration is for 5 μm of ol/L are by the outer primer upstream primer shown in SEQ ID NO.1, concentration for 5 μm of ol/L are by the outer primer downstream primer shown in SEQ ID NO.2, concentration for 40 μm of ol/L by the inner primer upstream primer shown in SEQ ID NO.3 and concentration for 40 μm of ol/L are made up of the inner primer downstream primer shown in SEQ ID NO.4.
Measuring samples is preferably fish blood, fish spleen, fish nephridial tissue or bacterium.
The present invention compared with prior art, has the following advantages:
(1) the invention solves prior art detect the Streptococcus iniae cycle long, testing cost is high, can not be applied to the problems such as Site Detection, the present invention can complete in 2h all detection operate, simple and quick.
(2) the present invention is based on the lyse gene co-hemolysin gene detecting Streptococcus iniae and have, can be used for the detection of Streptococcus iniae in breeding production, accuracy is high, PCR height 2-3 order of magnitude that sensitivity is more conventional, the minimum concentration that test in laboratory can detect Streptococcus iniae is 3 × 10
2cfu/ml.
(3) testing cost is low, without the need to the special instrument equipment of PCR instrument and so on, only needs temperature control water-bath accurately, easy and simple to handle, is applicable to Site Detection.
(4) do not need through microbial culture, detect required sample size few, only need to take a morsel Fish Blood or tissue can detect, and achieve Wicresoft's sampling, are particularly useful for the ornamental fish that economic worth is higher.
(5) the present invention can also be used for the detection of Streptococcus iniae in water body.
Accompanying drawing explanation
Fig. 1 is the Streptococcus iniae phylogenetic tree built based on 16S rDNA.
Fig. 2 is a kind of Streptococcus iniae method for quick detected result schematic diagram.
Fig. 3 is a kind of Streptococcus iniae method for quick susceptibility detected result schematic diagram.
Fig. 4 is Streptococcus iniae method for quick specific detection result schematic diagram.
Embodiment
The present invention is further illustrated by specific embodiment below in conjunction with accompanying drawing, the scheme of embodiment described here, do not limit the present invention, one of skill in the art can make improvements and change according to spirit of the present invention, these described improvement and change all should be considered as within the scope of the invention, and scope of the present invention and essence are limited by claim.
Instrument of the present invention and reagent:
Electric-heated thermostatic water bath (purchased from Beijing 3 sixteen scientific instrument company limited), BHI substratum (purchased from American BD company), high speed freezing centrifuge (SIGMA company), Bst archaeal dna polymerase, trimethyl-glycine, dNTP (purchased from NEB company), primer stin-F3, stin-B3, stin-FIP, stin-BIP synthesize by Shanghai Sheng Gong biotechnology company limited; SYBR Green I concentrated solution (purchased from SIGMA company), Tris, ammonium sulfate, Triton X-100 are purchased from Shanghai Sheng Gong biotechnology company limited, and Repone K, magnesium sulfate are domestic analytical pure.
10 × reaction buffer formula: Tris-HCl, 100mmol/L Repone K (KCl) of 200mmol/L pH8.8,100mmol/L ammonium sulfate ((NH4)
2sO
4), 20mmol/L magnesium sulfate (MgSO
4) and 1% triton x-100 (Triton X-100).
SYBR Green I working fluid, for commercialization SYBR Green I concentrated solution DEPC water dilutes 1000 times of acquisitions.
Bst archaeal dna polymerase, every microlitre is containing 8 activity units (8U/ μ l).
The Isolation and ldentification of embodiment 1. figure beautiful source of fish Streptococcus iniae
(1) the beautiful flake ball of the ill figure of picking and spleen, nephridial tissue are in the line of BHI culture medium flat plate, and be placed in 28 DEG C of incubator overnight incubation, picking dominant colony carries out pure culture afterwards;
(2) adopt gramstaining to observe institute's isolated strains form, carry out bacteria bio CHARACTERISTICS IDENTIFICATION with reference to " conventional Bacteria Identification handbook "; The 16S rDNA of amplification bacterium, compares after order-checking, the phylogenetic tree that to build with 16S rDNA be genetic marker;
(3) get institute's isolated strains and carry out mass propgation, after collection bacterium liquid is centrifugal, adjustment bacterial concentration is 3 × 10
7cfu/ml, take the mode of abdominal injection to scheme beautiful fish (the long 12 ± 2cm of body) to health and carry out artificial liver support, every endnote penetrates 100 μ l, injects 10 tails altogether, the 10 tail health figure beautiful fish injection equivalent stroke-physiological saline solution of control group.Experimental session keeps water temperature 28 DEG C, observes every day and records fish morbidity and death condition, and again carrying out bacteria distribution to dying sick fish.
(4) experimental result: the dominant colony 0523 be separated in the beautiful fish body of ill figure is shown as gram-positive cocci through gramstaining, diameter 0.2 ~ 0.8 μm, severally be arranged in chain to tens spherical bacterium, biochemical characteristic and Streptococcus iniae are comparatively close, with 16S rDNA for genetic marker, it is one that the phylogenetic tree result display bacterial strain 0523 built based on N-J method and Streptococcus iniae (DQ303187.1, AB593340.1) gather, and degree of confidence is 96%, sees Fig. 1.The 0523 bacterium liquid cultivated is injected into the beautiful fish belly chamber of healthy figure, testing the 3rd day experiment fish starts dead successively, all dead to the 11st day experimental group fish, show the infection symptom of obvious Streptococcus iniae, and none example of control group is dead, confirm that institute's isolated strains 0523 has pathogenic to the beautiful fishing gear of figure.
The foundation of embodiment 2. Streptococcus iniae method for quick
(1) Template preparation:
DNA extraction kit (the Fast DNA extraction detection kit KG203 of sky root) is adopted to extract the genomic dna of bacterial strain 0523, as reaction template.
(2) design of primers synthesis
Adopt blast software analysis Streptococcus iniae gene order, filter out virulence gene---the nucleotide sequence of lyse gene co-hemolysin gene (GenBank accession number: KC132870.1) that Streptococcus iniae is total, according to LAMP technology design of primers principle, design LAMP primer for this fragment and synthesize
stin-F3：5’-TGCTGTTCAAGCCATTGT-3’(SEQ ID NO.1)
stin-B3：5’-GATGACTTCACATAAATTGTAGC-3’(SEQ ID NO.2)
stinFIP：5’-TGGATCAGCAATTCGGATAACTAATTTTTAACTCAATTAAGCCACAAAGT-3’(SEQID NO.3)
stinBIP：5’-GATGCAATAAAAGCACAAGTCCAAGGATCCTCTATCACTAGCTTTTAAATCTGC-3’(SEQ ID NO.4)
(3) preparation of LAMP reaction system
In the 200 μ l PCR reaction tubess that 19 μ l LAMP reaction basal liquids are housed, add 4 μ l primer mixed solutions, 1 μ l Bst archaeal dna polymerase, 1 μ l measuring samples DNA profiling, is made into the LAMP reaction system of 25 μ l;
19 μ l LAMP react basal liquid and comprise 2.5 μ l10 × reaction buffers, and 3 μ l concentration are the dNTP of 2.5mmol/L, the trimethyl-glycine of 4 μ l1.6mol/L and 9.5 μ l molecular biology grade ultrapure waters.
10 × reaction buffer formula: Tris-HCl, 100mmol/L Repone K (KCl) of 200mmol/L pH8.8,100mmol/L ammonium sulfate ((NH4)
2sO
4), 20mmol/L magnesium sulfate MgSO
4with 1% triton x-100 (Triton X-100), solvent is molecular biology grade ultrapure water.
4 μ l primer mixed solutions are made up of following component: 1 μ l concentration is the outer primer upstream primer of 5 μm of ol/L, 1 μ l concentration is the outer primer downstream primer of 5 μm of ol/L, 1 μ l concentration is the inner primer upstream primer of 40 μm of ol/L, and 1 μ l concentration is the inner primer downstream primer of 40 μm of ol/L.
Above-mentioned Bst archaeal dna polymerase, every microlitre is containing 8 activity units (8U/ μ l).
(4) LAMP reaction conditions and optimization
Setting outer primer and inner primer concentration ratio are respectively 1:1,1:2,1:4,1:6,1:8,1:10,1:12, reaction times is from 20min, 25min, 30min, 40min, 50min, 60min, temperature of reaction is 54 DEG C, 57 DEG C, 60 DEG C, 63 DEG C, 65 DEG C, 68 DEG C, selects the LAMP detection technique of optimum response parameter Erecting and improving.The reaction parameter finally determined is as follows:
The concentration ratio of outer primer and inner primer is 1:8, namely outer primer stin-F3, stin-B3 concentration is 5 μm of ol/L, inner primer stinFIP, stinBIP primer concentration is 40 μm of ol/L, the LAMP reaction system of the 25 μ l prepared is placed 50min in 63 DEG C, 15min is placed afterwards in 85 DEG C of water-baths, obtaining detecting liquid, adopt the agarose electrophoresis of mass concentration 1% to detect liquid and whether produce the stair-stepping DNA band of continuous print, is then for positive; Or 1 μ l fluorescence dye SYBR Green I working fluid (SYBRGreen I concentrated solution DEPC water 1:1000 dilutes) is added in detection liquid, visual color reacts, be positive in green, in orange red be negative, contain Streptococcus iniae in positive expression measuring samples; Negative expression does not contain Streptococcus iniae.
Embodiment 3. 1 kinds of Streptococcus iniae method for quick, comprise the steps:
(1) measuring samples DNA is extracted:
Aseptically extract fish blood 100 μ l to be checked, adopt business-like DNA extraction agent box to extract the DNA of measuring samples.
(2) the LAMP reaction system of 25 μ l in embodiment 2 step (3) is adopted; Using the complete genome DNA of Streptococcus iniae 0523 as the template of positive control, using aseptic molecular biology grade ultrapure water as the template of negative control, place 50min in 62 DEG C, then obtain detecting liquid in 85 DEG C of placement 15min;
(3) adopting the agarose electrophoresis of massfraction 1% to detect liquid and whether produce the stair-stepping DNA band of continuous print, is then for positive;
Use step (1) (2) to obtain another part again and detect liquid, in detection liquid, add 1 μ l fluorescence dye SYBR Green I working fluid, visual color reacts, and is positive in green, in orange red be negative, positively to represent in measuring samples containing Streptococcus iniae; Negative expression does not contain Streptococcus iniae, the results are shown in Figure 2.(in Fig. 2, M is molecular weight Marker DL2000, and 1 is negative control, and it is orange red for detecting liquid, and electrophoresis is without the stair-stepping DNA band of continuous print; The 2 LAMP detected results that are be separated Streptococcus iniae, present obvious green, also there is the stair-stepping DNA band of obvious continuous print in electrophoresis, is the positive)
Embodiment 4. 1 kinds of Streptococcus iniae method for quick, comprise the steps:
(1) measuring samples DNA is extracted:
Aseptically get fish spleen 100mg, add the PBS of 100 μ l, after homogenate, boil 10min, get supernatant as measuring samples DNA profiling;
(2) the LAMP reaction system of 25 μ l in embodiment 2 step (3) is adopted; Using the complete genome DNA of Streptococcus iniae 0523 as the template of positive control, using aseptic molecular biology grade ultrapure water as the template of negative control, place 60min in 60 DEG C, then obtain detecting liquid in 85 DEG C of placement 15min;
(3) in detection liquid, add 1 μ l fluorescence dye SYBR Green I working fluid, visual color reacts, and in green, represents in measuring samples containing Streptococcus iniae.
Embodiment 5. 1 kinds of Streptococcus iniae method for quick, comprise the steps:
(1) measuring samples DNA is extracted:
Get the bacterium liquid 100 μ l of pure culture, extract the DNA of measuring samples with business-like DNA extraction agent box; (also directly bacterium liquid can be boiled 10min, get supernatant as measuring samples DNA profiling)
(2) the LAMP reaction system of 25 μ l in embodiment 2 step (3) is adopted; Using the complete genome DNA of Streptococcus iniae 0523 as the template of positive control, using aseptic molecular biology grade ultrapure water as the template of negative control.Place 40min in 65 DEG C, then obtain detecting liquid in 85 DEG C of placement 15min;
(3) adopting the agarose electrophoresis of 1% to detect liquid and produce the stair-stepping DNA band of continuous print, is the positive.
The detection sensitivity of embodiment 6. 1 kinds of Streptococcus iniae method for quick measures
Accessed by be separated Streptococcus iniae bacterial strain 0523 after cultivating 24h in BHI liquid nutrient medium, wash 3 times with stroke-physiological saline solution, adjustment bacterial concentration is 3 × 10
8cfu/ml, 10 multiple proportions gradient dilutions, get the template that supernatant reacts as LAMP after boiling 10min.According to method described in embodiment 3, detect the Streptococcus iniae bacterium liquid of gradient dilution.Result: the minimum Streptococcus iniae concentration that can detect is 3 × 10
2cfu/ml, is shown in that (M is molecular weight Marker DL2000 to Fig. 3, and sample 1-9 is respectively 3 × 10 as the bacterial concentration of template
1cfu/ml, 3 × 10
2cfu/ml, 3 × 10
3cfu/ml, 3 × 10
4cfu/ml, 3 × 10
5cfu/ml, 3 × 10
6cfu/ml, 3 × 10
7cfu/ml, 3 × 10
8cfu/ml, positive control.It is 3 × 10 that bacterial concentration is worked as in detected result display
2start there is amplified band during cfu/ml, the Streptococcus iniae minimum concentration that namely this detection method can detect is 3 × 10
2cfu/ml).
The detection specificity test of embodiment 7. 1 kinds of Streptococcus iniae method for quick
Get respectively pure culture and qualification after Streptococcus iniae, streptococcus agalactiae, Mermaid luminous bacillus, flavobacterium columnare, Vibrio harveyi, Aeromonas hydrophila, Nocardia bacteria, tarda, get the template that supernatant reacts as LAMP after boiling 10min, detect above sample with the Streptococcus iniae method for quick set up.Detected result: the detection liquid of sample 1 Streptococcus iniae, in green, is the positive; Sample 9 is negative control, is negative; All the other samples are feminine gender, and (1-8 bacterium is respectively Streptococcus iniae, streptococcus agalactiae, Mermaid luminous bacillus, flavobacterium columnare, Kazakhstan Vickers bacterium, Aeromonas hydrophila, Nocardia bacteria, tarda, and 9 is negative control to see Fig. 4.Result shows only Streptococcus iniae and occurs specific amplification, and reaction solution presents green, and other bacterium and negative control are feminine gender).
Those of ordinary skill in the art can understand, and in protection scope of the present invention, modifies for above-described embodiment, and it is all possible for adding and replacing, and it does not all exceed protection scope of the present invention.
Claims (1)
1. a Streptococcus iniae rapid detection primer, is characterized in that being made up of outer primer and inner primer, and described outer primer is made up of the outer primer downstream primer shown in the outer primer upstream primer shown in SEQ IDNO.1 and SEQ ID NO.2; Described inner primer is made up of the inner primer downstream primer shown in the inner primer upstream primer shown in SEQ IDNO.3 and SEQ ID NO.4.
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| CN104651518A (en) * | 2015-03-04 | 2015-05-27 | 广西壮族自治区兽医研究所 | Streptococcus iniae loop-mediated isothermal amplification kit and application thereof |
| CN108342499B (en) * | 2018-04-26 | 2019-04-05 | 暨南大学 | A pair of while quickly detection Streptococcusagalactiae and Streptococcus iniae primer and its application |
Citations (1)
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|---|---|---|---|---|
| CN101586157A (en) * | 2008-12-18 | 2009-11-25 | 中山大学 | Diagnostic kit for Streptococcus iniae molecule and detection method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101586157A (en) * | 2008-12-18 | 2009-11-25 | 中山大学 | Diagnostic kit for Streptococcus iniae molecule and detection method |
Non-Patent Citations (5)
| Title |
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| Evaluation of colorimetric loop-mediated isothermal amplification assay for visual detection of Streptococcus agalactiae and Streptococcus iniae in tilapia;R.Suebsing，et al;《Letters in Applied Microbiology》;20131231;317-324页 * |
| Rapid and sensitive detection of Streptococcus iniae by loop-mediated isothermal amplification (LAMP);H-J Han，et al;《Journal of Fish Diseases》;20111231;395-398页 * |
| Shelly Bolotin，et al.The two-component systemsivS/R regulatesvirulence in Streptococcus iniae.《FEMS Immunol Med Microbiol》.2007,547-554页. * |
| Shuang-Hu Cai，et al.Development of Loop-mediated isothermal amplification method for rapid detection of Streptococcus iniae,the causative agent of streptococcicosis in fish.《Journal of Basic Microbiology》.2012,116-122页. * |
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