CN103966341B - Streptococcus agalactiae rapid detection primer and method thereof - Google Patents

Streptococcus agalactiae rapid detection primer and method thereof Download PDF

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CN103966341B
CN103966341B CN201410226034.2A CN201410226034A CN103966341B CN 103966341 B CN103966341 B CN 103966341B CN 201410226034 A CN201410226034 A CN 201410226034A CN 103966341 B CN103966341 B CN 103966341B
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primer
streptococcus agalactiae
detection
seq
rapid detection
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CN103966341A (en
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姚学良
徐晓丽
李贺密
崔宽宽
钟文慧
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Aquatic Products Technical Advice Station Tianjin
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6844—Nucleic acid amplification reactions

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Abstract

The invention discloses streptococcus agalactiae rapid detection primer and method thereof, streptococcus agalactiae 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 agalactiae cycle long, testing cost is high, can not be applied to the problems such as Site Detection, simple and quick, without the need to the special instrument equipment of PCR instrument and so on, accuracy is high, highly sensitive, the minimum concentration detecting streptococcus agalactiae is 3 × 10 2cfu/ml.Detect required sample size few, Wicresoft can be taked to sample, be applicable to the ornamental fish that economic worth is higher.

Description

Streptococcus agalactiae rapid detection primer and method thereof
Technical field
The present invention relates to a kind of streptococcus agalactiae rapid detection primer and method thereof, belong to aquarium fish pathogenic bacteria field of fast detection.
Background technology
The culture of ornamental fish kind that Family Cichlidae fish are as important in arhat, parrot, map, 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 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, aquatic products microbiotic majority being directed to Gram-negative bacteria is insensitive, and this bacterium can survive in white corpuscle, belong to so-called " intracellular parasitic bacteria ", need suitably to extend medication program, further increase Disease epizootic cost, also often fall 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.At present for the detection of streptococcus agalactiae, the bacteria distribution culture technique that many employings are traditional and PCR detection technique, length consuming time, cost is high, need technical professional and plant and instrument, urgently a kind of streptococcus agalactiae detection technique fast and effectively that may be used for cultivation site in breeding production.
Loop-mediated isothermal amplification technique (Loop-mediatedIsothermalAmplification, LAMP) be a kind of novel nucleic acid isothermal amplification technology, its principle is that strand displacement type polysaccharase displaces former chain synthesizing new chain while, thus be next round amplification preparation template, therefore increase and do not need denaturing step, can carry out under isothermal conditions.Through the development of more than ten years, LAMP detection technique has demonstrated good application prospect, establish the LAMP detection technique of multiple causal organism both at home and abroad in recent years, 60 ~ 90min can complete whole testing process, by adding nucleic acid dye SYBRGreenI or fluorexon, has nucleic acid amplification to react by Show Color, immediately detected result is obtained, the practicality that tool is stronger, be more suitable for the quarantine being applied to cultivation site and batch samples, sensitivity can compared with real-time quantitative PCR.Not yet there is the report about streptococcus agalactiae 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 agalactiae 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 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, be 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 SEQIDNO.1 and SEQIDNO.2; Described inner primer is made up of the inner primer downstream primer shown in the inner primer upstream primer shown in SEQIDNO.3 and SEQIDNO.4.
A kind of streptococcus agalactiae method for quick, comprises the steps:
(1) measuring samples DNA is extracted;
(2) in the 200 μ lPCR reaction tubess that 19 μ lLAMP reaction basal liquids are housed, add 4 μ l primer mixed solutions, 1 μ lBstDNA 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 SYBRGreenI 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 agalactiae; Negative expression does not contain streptococcus agalactiae;
Described primer mixed solution is that isopyknic concentration is for 5 μm of ol/L are by the outer primer upstream primer shown in SEQIDNO.1, concentration for 5 μm of ol/L are by the outer primer downstream primer shown in SEQIDNO.2, concentration for 40 μm of ol/L by the inner primer upstream primer shown in SEQIDNO.3 and concentration for 40 μm of ol/L are made up of the inner primer downstream primer shown in SEQIDNO.4.
19 μ lLAMP react basal liquid composition: 4 μ ldNTP, 2.5 μ l10 × ThermopolRoactionBuffer and 12.5 μ lDEPC water.
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 agalactiae 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 detection streptococcus agalactiae conservative gene---CAMP factor gene order, can be used for the detection of streptococcus agalactiae 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 agalactiae 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 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 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 agalactiae in water body.
Accompanying drawing explanation
Fig. 1 is a kind of streptococcus agalactiae method for quick detected result figure, visual inspection, and 1 is negative control, and it is orange red for detecting liquid, and 2 be the detected result of streptococcus agalactiae DNA, and detection liquid presents obvious green, is the 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
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), nutrient agar medium (purchased from sky, Hangzhou and microorganism reagent company limited), high speed freezing centrifuge (SIGMA company), BstDNA polysaccharase, 10 × ThermopolRoactionBuffer, dNTP (purchased from NEB company), primer cfbF3, cfbB3, cfbFIP, cfbBIP synthesize by Shanghai Sheng Gong biotechnology company limited; SYBRGreenI concentrated solution (purchased from SIGMA company), DEPC water is purchased from Shanghai Sheng Gong biotechnology company limited, and streptococcus agalactiae is that our station is separated and obtains in ill Pseudorabora parva body.
SYBRGreenI working fluid, for commercialization SYBRGreenI concentrated solution DEPC water dilutes 1000 times of acquisitions.
BstDNA polysaccharase, every microlitre is containing 8 activity units (8U/ μ l).
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 kit (the Fast DNA extraction detection kit KG203 of sky root) to extract its genomic dna, as reaction template.
(2) design of primers synthesis
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 for this fragment and synthesize
cfbB3：5’-TTGCTTCAATCACATCTGTT-3’(SEQIDNO.1)
cfbF3：5’-ATCAAGCCCAGCAAATGG-3’(SEQIDNO.2)
cfbBIP：5’-TAAAGACTTCATTGCGTGCCAGGATCCGCTTCTACACGACTACCAAT-3’(SEQIDNO.3)
cfbFIP：5’-CGGTTTTTCATAATCTGTTCCCTGATTTTCTCAAAAGCTTGATCAAGATAGC-3’(SEQIDNO.4)
(3) preparation of LAMP reaction system
In the 200 μ lPCR reaction tubess that 19 μ lLAMP reaction basal liquids are housed, add 4 μ l primer mixed solutions, 1 μ lBstDNA polysaccharase, 1 μ l streptococcus agalactiae sample DNA templates to be checked, is made into the LAMP reaction system of 25 μ l;
19 μ lLAMP react basal liquid composition: 4 μ ldNTP, 2.5 μ l10 × ThermopolRoactionBuffer and 12.5 μ lDEPC 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 BstDNA polysaccharase, 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, i.e. outer primer cfbF3, cfbB3 concentration is 5 μm of ol/L, inner primer cfbFIP, cfbBIP 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, obtain detecting liquid, 1 μ l fluorescence dye SYBRGreenI working fluid (SYBRGreenI concentrated solution DEPC water 1:1000 dilutes) is added in detection liquid, visual color reacts, in green be the positive, in orange red be negative, positive expression in measuring samples contains streptococcus agalactiae, negative expression does not contain streptococcus agalactiae, sees Fig. 1.
Embodiment 2. 1 kinds of streptococcus agalactiae 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 its DNA, as measuring samples, or add the physiological saline of 100 μ l0.85% in blood, boil rear standing 10min, get supernatant as measuring samples.
(2) the LAMP reaction system of 25 μ l in embodiment 1 step (3) is adopted; Using streptococcus agalactiae DNA as the template of positive control, using DEPC water as the template of negative control.Place 50min in 63 DEG C, then obtain detecting liquid in 85 DEG C of placement 15min; In detection liquid, add 1 μ l fluorescence dye SYBRGreenI working fluid, visual color reacts, and is positive in green, represents in measuring samples containing streptococcus agalactiae.
Embodiment 3. 1 kinds of streptococcus agalactiae method for quick, comprise the steps:
(1) measuring samples DNA is extracted:
Aseptically get the ill Pseudorabora parva spleen 100mg of artificial challenge streptococcus agalactiae, the ill Pseudorabora parva kidney 100mg of artificial challenge streptococcus agalactiae, adds the PBS of 100 μ l respectively, boils 10min after homogenate, get supernatant as measuring samples DNA profiling;
(2) the LAMP reaction system of 25 μ l in embodiment 1 step (3) is adopted respectively; Using streptococcus agalactiae DNA as the template of positive control, using DEPC water as the template of negative control.Place 60min in 60 DEG C, then obtain detecting liquid in 85 DEG C of placement 15min; In detection liquid, add 1 μ l fluorescence dye SYBRGreenI working fluid, visual color reacts, and the results are shown in Figure 2.
1,2 ill Pseudorabora parva spleen, the nephridial tissue detected results being respectively artificial challenge streptococcus agalactiae in Fig. 2, detecting liquid and present obvious green, is the positive, represents in sample containing streptococcus agalactiae; 3 is negative control, and it is orange red for detecting liquid; 4 is streptococcus agalactiae DNA, as positive control, in green.
Embodiment 4. 1 kinds of streptococcus agalactiae 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 1 step (3) is adopted; Using streptococcus agalactiae DNA as the template of positive control, using DEPC water as the template of negative control.Place 40min in 65 DEG C, then obtain detecting liquid in 85 DEG C of placement 15min; In detection liquid, add 1 μ l fluorescence dye SYBRGreenI working fluid, visual color reacts, and is positive in green.The detection sensitivity of embodiment 5. 1 kinds of streptococcus agalactiae method for quick measures
Get the streptococcus agalactiae bacterium liquid of pure culture, 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.Adopt the LAMP reaction system of 25 μ l in embodiment 1 step (3), reaction solution is placed in 63 DEG C and places 50min, then obtain detecting liquid in 85 DEG C of placement 15min; Adopting 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; Positive expression in measuring samples contains streptococcus agalactiae; 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 that employing 1% agarose gel electrophoresis observes susceptibility detected result.M is molecular weight MarkerDL2000, 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.It is 3 × 10 that bacterial concentration is worked as in detected result display 2start there is amplified band during cfu/ml, the streptococcus agalactiae minimum concentration that namely this detection method can detect is 3 × 10 2cfu/ml.
The detection specificity test of embodiment 6. 1 kinds of streptococcus agalactiae method for quick
Get respectively pure culture and qualification after streptococcus agalactiae, Mermaid luminous bacillus, flavobacterium columnare, Vibrio harveyi, Aeromonas hydrophila, Nocardia bacteria, tarda, Aeromonas veronii, Vibrio anguillarum, Vibrio vulnificus, the template that supernatant reacts as LAMP is got after boiling 10min, according to method described in embodiment 4, detect above sample with the streptococcus agalactiae method for quick set up.Detected result: sample 1 streptococcus agalactiae is in occurring nucleic acid amplification, and detect liquid and present green for positive, all the other samples are feminine gender, 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 is negative control, result shows only sample 1 streptococcus agalactiae and detects liquid in green, for the positive, the detection liquid of other bacterium and negative control, all in orange red, is 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 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 SEQIDNO.1 and SEQIDNO.2; Described inner primer is made up of the inner primer downstream primer shown in the inner primer upstream primer shown in SEQIDNO.3 and SEQIDNO.4.
CN201410226034.2A 2014-05-26 2014-05-26 Streptococcus agalactiae rapid detection primer and method thereof Expired - Fee Related CN103966341B (en)

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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
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