CN103966341A - Streptococcus agalactiae rapid detection primer and method thereof - Google Patents
Streptococcus agalactiae rapid detection primer and method thereof Download PDFInfo
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Abstract
The invention discloses a streptococcus agalactiae rapid detection primer and a method thereof. The streptococcus agalactiae rapid detection primer is composed of an outer primer and an inner primer, wherein the outer primer is composed of an outer primer upstream primer shown in SEQ ID NO.1 and an outer primer downstream primer shown in SEQ ID NO.2; the inner primer is composed of an inner primer upstream primer shown in SEQ ID NO.3 and an inner primer downstream primer shown in SEQ ID NO.4. The streptococcus agalactiae rapid detection primer disclosed by the invention solves problems that detection for treptococcus agalactiae in the prior art is long in period, high in detection cost, incapable of being applied to site detection, and the like, is simple and quick, does not need special apparatus equipment of a PCR (polymerase chain reaction) apparatus, and the like, is high in accuracy, high in sensitivity, so that lowest concentration of the detected streptococcus agalactiae is 3*102 cfu/ml. Moreover, samples needed for detection are less, minimally invasive sampling can be adopted, and therefore, the streptococcus agalactiae rapid detection primer is suitable for ornamental fishes with a higher economic value.
Description
Technical field
The present invention relates to a kind of streptococcus agalactiae rapid detection primer and method thereof, belong to aquarium fish pathogenic bacteria rapid detection field.
Background technology
Family Cichlidae fish are as arhat, parrot, map, the important culture of ornamental fish kind of imperial crown Deng Shi China, there is higher economic worth, in recent years under high-density breeding pattern, disease occurs frequent, the bacterial infection disease wherein being caused by streptococcus agalactiae is one of the most serious disease, cause very high infection rate and mortality ratio, bring huge financial loss to raiser.Streptococcus agalactiae is gram-positive microorganism, the aquatic products microbiotic that majority is directed to Gram-negative bacteria is insensitive, and this bacterium can survive in white corpuscle, belong to so-called " intracellular parasitic bacteria ", need to suitably extend medication program, further increase disease and controlled cost, also often fallen flat.Therefore for the prevention and control of streptococcus agalactiae, early detection finds the cause of disease early, adopts an effective measure in time most important.The current detection for streptococcus agalactiae, traditional bacterium separation and Culture technology and the PCR detection technique of many employings, length consuming time, cost is high, need technical professional and plant and instrument, urgently a kind of in breeding production can be for the streptococcus agalactiae of cultivation site detection technique fast and effectively.
Loop-mediated isothermal amplification technique (Loop-mediated Isothermal Amplification, LAMP) be a kind of novel nucleic acid isothermal amplification technique, its principle is that strand displacement type polysaccharase displaces former chain in synthetic new chain, thereby be next round amplification preparation template, therefore amplification does not need denaturing step, can under isothermal condition, carry out.Through the development of more than ten years, LAMP detection technique has demonstrated good application prospect, set up in recent years the LAMP detection technique of multiple causal organism both at home and abroad, 60～90min can complete whole testing process, by adding nucleic acid dye SYBR Green I or fluorexon, has nucleic acid amplification to react by Show Color, immediately obtain detected result, the practicality that tool is stronger, is more suitable in the quarantine that is applied to cultivation site and batch samples, and sensitivity can be compared with real-time quantitative PCR.Not yet there is at present the report about streptococcus agalactiae LAMP detection technique.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of streptococcus agalactiae rapid detection primer is provided.
Second object of the present invention be to provide a kind of to fish body damage little, quick and precisely, streptococcus agalactiae method for quick easy and simple to handle.
Technical scheme of the present invention is summarized as follows:
A kind of streptococcus agalactiae rapid detection primer, is 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 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 agalactiae method for quick, comprises the steps:
(1) extract sample DNA to be checked;
(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 sample DNA template to be checked, is made into the LAMP reaction system of 25 μ l; In 60-65 DEG C of placement 40-60min, then obtain detecting liquid in 85 DEG C of placement 15min;
(3) adopt the agarose electrophoresis detection liquid of mass concentration 1% whether to produce continuous stair-stepping DNA band, positive, or add 1 μ l fluorescence dye SYBR Green I working fluid in detection liquid, visual inspection color reaction, be green positive, be orange red negative, positive expression contained streptococcus agalactiae in sample to be checked; Negative expression does not contain streptococcus agalactiae;
Described primer mixed solution is that isopyknic concentration is that 5 μ mol/L are by the outer primer upstream primer shown in SEQ ID NO.1, concentration be 5 μ mol/L by the outer primer downstream primer shown in SEQ ID NO.2, concentration is that 40 μ mol/L are that 40 μ mol/L are made up of the inner primer downstream primer shown in SEQ ID NO.4 by the inner primer upstream primer shown in SEQ ID NO.3 and concentration.
19 μ l LAMP reaction basal liquid compositions: 4 μ l dNTP, 2.5 μ l10 × Thermopol Roaction Buffer and 12.5 μ l DEPC water.
Sample to be checked 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 and detect that the streptococcus agalactiae cycle is long, testing cost is high, can not be applied to the problems such as Site Detection, the present invention can complete all detections operation in 2h, simple and quick.
(2) the present invention is based on and detect streptococcus agalactiae conservative gene---CAMP factor gene order, can be used for the detection of streptococcus agalactiae in breeding production, accuracy is high, the high 2-3 of the PCR order of magnitude that sensitivity is more conventional, the minimum concentration that laboratory detection can detect streptococcus agalactiae is 3 × 10
2cfu/ml.
(3) testing cost is low, without the professional plant and instrument of PCR instrument and so on, only needs temperature control water-bath or constant-temperature metal 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 have realized 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 water body streptococcus agalactiae.
Brief description of the drawings
Fig. 1 is a kind of streptococcus agalactiae method for quick detected result figure, visual inspection, and 1 negative contrast, it is orange red detecting liquid, 2 is the detected result of streptococcus agalactiae DNA, detects liquid and presents obvious green, positive.
Fig. 2 is a kind of streptococcus agalactiae method for quick practical application detected result figure.
Fig. 3 is a kind of streptococcus agalactiae method for quick susceptibility detected result schematic diagram.
Fig. 4 is streptococcus agalactiae method for quick specific detection result schematic diagram.
Embodiment
Further illustrate the present invention below in conjunction with accompanying drawing and by specific embodiment, 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 variation 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 limiteds), nutrient agar medium (purchased from sky, Hangzhou and microorganism reagent company limited), high speed freezing centrifuge (SIGMA company), Bst archaeal dna polymerase, 10 × Thermopol Roaction Buffer, dNTP (purchased from NEB company), primer cfbF3, cfbB3, cfbFIP, cfbBIP synthesize by Shanghai Sheng Gong biotechnology company limited; SYBR Green I concentrated solution (purchased from SIGMA company), DEPC water is purchased from Shanghai Sheng Gong biotechnology company limited, and to be our station obtain from separating in ill Pseudorabora parva body streptococcus agalactiae.
SYBR Green I working fluid is 1000 times of acquisitions of DEPC water dilution for commercialization SYBR Green I concentrated solution.
Bst archaeal dna polymerase, (8U/ μ l) containing 8 activity units for every microlitre.
The foundation of embodiment 1. streptococcus agalactiae method for quick
(1) template preparation:
Get the streptococcus agalactiae bacterium liquid of pure culture, adopt DNA extraction test kit (the Fast DNA extraction detection kit KG203 of day root) to extract its genomic dna, as reaction template.
(2) design of primers is synthetic
Adopt blast software analysis streptococcus agalactiae gene order, filter out the nucleotide sequence of streptococcus agalactiae CAMP factor gene order (GenBank accession number: JQ289586.1), according to LAMP technology design of primers principle, design LAMP primer synthetic for this fragment
cfbB3：5’-TTGCTTCAATCACATCTGTT-3’(SEQ ID NO.1)
cfbF3：5’-ATCAAGCCCAGCAAATGG-3’(SEQ ID NO.2)
cfbBIP：5’-TAAAGACTTCATTGCGTGCCAGGATCCGCTTCTACACGACTACCAAT-3’(SEQ ID NO.3)
cfbFIP：5’-CGGTTTTTCATAATCTGTTCCCTGATTTTCTCAAAAGCTTGATCAAGATAGC-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 streptococcus agalactiae sample DNA to be checked template, is made into the LAMP reaction system of 25 μ l;
19 μ l LAMP reaction basal liquid compositions: 4 μ l dNTP, 2.5 μ l10 × Thermopol Roaction Buffer and 12.5 μ l DEPC 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 μ mol/L, 1 μ l concentration is the outer primer downstream primer of 5 μ mol/L, 1 μ l concentration is the inner primer upstream primer of 40 μ mol/L, and 1 μ l concentration is the inner primer downstream primer of 40 μ mol/L.
Above-mentioned Bst archaeal dna polymerase, (8U/ μ l) containing 8 activity units for every microlitre.
(4) LAMP reaction conditions and optimization
Set outer primer and inner primer concentration ratio and be 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.Final definite reaction parameter is as follows:
The concentration ratio of outer primer and inner primer is 1:8, be outer primer cfbF3, cfbB3 concentration is 5 μ mol/L, inner primer cfbFIP, cfbBIP primer concentration is 40 μ mol/L, the LAMP reaction system of the 25 μ l that prepare is placed to 50min in 63 DEG C, in 85 DEG C of water-baths, place 15min afterwards, obtain detecting liquid, in detection liquid, add 1 μ l fluorescence dye SYBR Green I working fluid (SYBRGreen I concentrated solution DEPC water 1:1000 dilution), visual inspection color reaction, be green positive, be orange red negative, positive expression contained streptococcus agalactiae in sample to be checked, negative expression do not contained streptococcus agalactiae, sees Fig. 1.
2. 1 kinds of streptococcus agalactiae method for quick of embodiment, comprise the steps:
(1) extract sample DNA to be checked:
Under aseptic condition, extract fish blood 100 μ l to be checked, adopt business-like DNA extraction agent box to extract its DNA, as sample to be checked, or in blood, add the physiological saline of 100 μ l0.85%, boil rear standing 10min, get supernatant as sample to be checked.
(2) the LAMP reaction system of 25 μ l in employing embodiment 1 step (3); Template using streptococcus agalactiae DNA as positive control, the template using DEPC water as negative control.In 63 DEG C of placement 50min, then obtain detecting liquid in 85 DEG C of placement 15min; In detection liquid, add 1 μ l fluorescence dye SYBR Green I working fluid, visual inspection color reaction, is green positive, represents to contain streptococcus agalactiae in sample to be checked.
3. 1 kinds of streptococcus agalactiae method for quick of embodiment, comprise the steps:
(1) extract sample DNA to be checked:
Under aseptic condition, get the ill Pseudorabora parva spleen 100mg of artificial challenge streptococcus agalactiae, the ill Pseudorabora parva kidney 100mg of artificial challenge streptococcus agalactiae, adds respectively the PBS of 100 μ l, boils 10min after homogenate, gets supernatant as sample DNA template to be checked;
(2) adopt respectively the LAMP reaction system of 25 μ l in embodiment 1 step (3); Template using streptococcus agalactiae DNA as positive control, the template using DEPC water as negative control.In 60 DEG C of placement 60min, then obtain detecting liquid in 85 DEG C of placement 15min; In detection liquid, add 1 μ l fluorescence dye SYBR Green I working fluid, visual inspection color reaction, the results are shown in Figure 2.
1,2 ill Pseudorabora parva spleen, the nephridial tissue detected results that are respectively artificial challenge streptococcus agalactiae in Fig. 2, detect liquid and present obvious green, positive, represent to contain streptococcus agalactiae in sample; 3 negative contrasts, detect liquid and are orange red; 4 is streptococcus agalactiae DNA, as positive control, is green.
4. 1 kinds of streptococcus agalactiae method for quick of embodiment, comprise the steps:
(1) extract sample DNA to be checked:
Get the bacterium liquid 100 μ l of pure culture, extract the DNA of sample to be checked with business-like DNA extraction agent box; Also can directly bacterium liquid be boiled to 10min, get supernatant as sample DNA template to be checked.
(2) the LAMP reaction system of 25 μ l in employing embodiment 1 step (3); Template using streptococcus agalactiae DNA as positive control, the template using DEPC water as negative control.In 65 DEG C of placement 40min, then obtain detecting liquid in 85 DEG C of placement 15min; In detection liquid, add 1 μ l fluorescence dye SYBR Green I working fluid, visual inspection color reaction, is green positive.The detection sensitivity of 5. 1 kinds of streptococcus agalactiae method for quick of embodiment is measured
Get the streptococcus agalactiae bacterium liquid of pure culture, with stroke-physiological saline solution washing 3 times, adjusting bacterial concentration is 3 × 10
8cfu/ml, 10 multiple proportions gradient dilutions, boil and get the template of supernatant as LAMP reaction after 10min.Adopt the LAMP reaction system of 25 μ l in embodiment 1 step (3), reaction solution is placed in to 63 DEG C and places 50min, then obtain detecting liquid in 85 DEG C of placement 15min; Adopting the agarose electrophoresis detection liquid of mass concentration 1% whether to produce continuous stair-stepping DNA band, is positive; Positive expression contained streptococcus agalactiae in sample to be checked; Negative expression does not contain streptococcus agalactiae.Result: the minimum streptococcus agalactiae concentration that can detect is 3 × 10
2cfu/ml, is shown in Fig. 3.
Fig. 3 is for adopting 1% agarose gel electrophoresis to observe susceptibility detected result.M is molecular weight Marker DL2000, and sample 1-8 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.Detected result shows that working as bacterial concentration is 3 × 10
2when cfu/ml, start to have amplified band, the streptococcus agalactiae minimum concentration that this detection method can detect is 3 × 10
2cfu/ml.
The detection specificity test of 6. 1 kinds of streptococcus agalactiae method for quick of embodiment
Get respectively streptococcus agalactiae, Mermaid luminous bacillus, flavobacterium columnare, Vibrio harveyi, Aeromonas hydrophila, Nocardia bacteria, tarda, Aeromonas veronii, Vibrio anguillarum, Vibrio vulnificus after pure culture qualification, after boiling 10min, get the template of supernatant as LAMP reaction, according to method described in embodiment 4, use the streptococcus agalactiae method for quick of having set up to detect above sample.Detected result: sample 1 streptococcus agalactiae is and occurs nucleic acid amplification, detection liquid presents green positive, and all the other samples are all negative, see Fig. 4.
Sample 1-9 bacterium is respectively streptococcus agalactiae, Mermaid luminous bacillus, flavobacterium columnare, Vibrio harveyi, Aeromonas hydrophila, Nocardia bacteria, tarda, Aeromonas veronii, Vibrio anguillarum, 10 negative contrasts, result shows that only sample 1 streptococcus agalactiae detection liquid is green, positive, the detection liquid of other bacterium and negative control is all orange red, negative.
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 adding and replacing, and it does not all exceed protection scope of the present invention.
Claims (3)
1. a streptococcus agalactiae 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.
2. a streptococcus agalactiae method for quick, is characterized in that comprising the steps:
(1) extract sample DNA to be checked;
(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 sample DNA template to be checked, is made into the LAMP reaction system of 25 μ l; In 60-65 DEG C of placement 40-60min, then obtain detecting liquid in 85 DEG C of placement 15min;
(3) adopt the agarose electrophoresis detection liquid of mass concentration 1% whether to produce continuous stair-stepping DNA band, positive, or add 1 μ l fluorescence dye SYBR Green I working fluid in detection liquid, visual inspection color reaction, be green positive, be orange red negative, positive expression contained streptococcus agalactiae in sample to be checked; Negative expression does not contain streptococcus agalactiae;
Described primer mixed solution is that isopyknic concentration is that 5 μ mol/L are by the outer primer upstream primer shown in SEQ ID NO.1, concentration be 5 μ mol/L by the outer primer downstream primer shown in SEQ ID NO.2, concentration is that 40 μ mol/L are that 40 μ mol/L are made up of the inner primer downstream primer shown in SEQ ID NO.4 by the inner primer upstream primer shown in SEQ ID NO.3 and concentration.
3. a kind of streptococcus agalactiae method for quick according to claim 2, is characterized in that described sample to be checked is fish blood, fish spleen, fish nephridial tissue or bacterium.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104593509A (en) * | 2015-01-29 | 2015-05-06 | 中国水产科学研究院珠江水产研究所 | Specific-nucleic-acid-probe dot-blot-hybridization detection kit for tilapia streptococcus agalactiae |
| CN106435000A (en) * | 2016-12-06 | 2017-02-22 | 广东海大畜牧兽医研究院有限公司 | Constant-temperature heat insulation type PCR (polymerase chain reaction) detection method for streptococcus agalactiae |
| CN107557457A (en) * | 2017-09-28 | 2018-01-09 | 广州和实生物技术有限公司 | A kind of digital pcr detection kit and detection method for being used to detect GBS |
| CN109321666A (en) * | 2018-09-06 | 2019-02-12 | 新疆农业科学院农业质量标准与检测技术研究所 | The method of Streptococcusagalactiae viable bacteria in SDS-PMA-qPCR method detection cream |
| CN110846428A (en) * | 2019-12-30 | 2020-02-28 | 宁夏大学 | Specific LAMP primer, kit and method for detecting streptococcus agalactiae |
-
2014
- 2014-05-26 CN CN201410226034.2A patent/CN103966341B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| KOUJI KIMURA ET AL: "Rapid and Reliable Loop-Mediated Isothermal Amplification Method for Detecting Streptococcus agalactiae", 《J.INFECT.DIS》, vol. 66, 31 December 2013 (2013-12-31), pages 546 - 548 * |
| 王永等: "LAMP 检测无乳链球菌方法的建立及应用", 《华北农业学报》, vol. 24, no. 5, 31 December 2009 (2009-12-31) * |
| 郑磊等: "罗非鱼无乳链球菌LAMP快速诊断方法的建立", 《中国水产学会鱼病专业委员会2013年学术研讨会论文摘要汇编》, 31 December 2013 (2013-12-31) * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104593509A (en) * | 2015-01-29 | 2015-05-06 | 中国水产科学研究院珠江水产研究所 | Specific-nucleic-acid-probe dot-blot-hybridization detection kit for tilapia streptococcus agalactiae |
| CN106435000A (en) * | 2016-12-06 | 2017-02-22 | 广东海大畜牧兽医研究院有限公司 | Constant-temperature heat insulation type PCR (polymerase chain reaction) detection method for streptococcus agalactiae |
| CN107557457A (en) * | 2017-09-28 | 2018-01-09 | 广州和实生物技术有限公司 | A kind of digital pcr detection kit and detection method for being used to detect GBS |
| CN109321666A (en) * | 2018-09-06 | 2019-02-12 | 新疆农业科学院农业质量标准与检测技术研究所 | The method of Streptococcusagalactiae viable bacteria in SDS-PMA-qPCR method detection cream |
| CN110846428A (en) * | 2019-12-30 | 2020-02-28 | 宁夏大学 | Specific LAMP primer, kit and method for detecting streptococcus agalactiae |
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