CN109811073A - Double PCR early stage quickly detects primer and its application of Streptococcusagalactiae and Streptococcus iniae - Google Patents
Double PCR early stage quickly detects primer and its application of Streptococcusagalactiae and Streptococcus iniae Download PDFInfo
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- CN109811073A CN109811073A CN201910241560.9A CN201910241560A CN109811073A CN 109811073 A CN109811073 A CN 109811073A CN 201910241560 A CN201910241560 A CN 201910241560A CN 109811073 A CN109811073 A CN 109811073A
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Abstract
The present invention discloses primer and its application of a kind of double PCR early stage quick detection Streptococcusagalactiae and Streptococcus iniae.Streptococcusagalactiae and Streptococcus iniae, it can be achieved that in disposable specific detection sample simultaneously are detected using primer pair sample of the invention.It is respectively 9.84 × 10 that genome content, which can be detected, in the detection method that the present invention establishes‑5The Streptococcusagalactiae and 9.30 × 10 of ng/ μ L‑ 5The Streptococcus iniae and bacterial concentration of ng/ μ L is 2.76 × 102The Streptococcusagalactiae of cfu/mL and 2.51 × 102The Streptococcus iniae of cfu/mL.This method quick and precisely specificity, sensitivity, repeatability and stability with higher in tilapia mossambica samples detection process, the early molecule early warning of Tilapia mossambica streptococcosis can be achieved, it has a extensive future, provides accurate and effective means for quick diagnosis, the epidemiological survey etc. of Tilapia mossambica streptococcosis.
Description
Technical field
The invention belongs to microorganism detection fields, and in particular to a kind of double PCR early stage quickly detection Streptococcusagalactiae and
The primer of Streptococcus iniae and its application.
Background technique
Tilapia mossambica is one of the aquatic products cultivated extensively in world wide, according to incompletely statistics, 2014, Chinese Tilapia mossambica
Cultured output reaches 169.85 ten thousand tons, and 32% or so of Zhan Quanqiu cultivation total output.In recent years, Tilapia mossambica streptococcosis is increasingly tight
Weight, causes serious economic loss to Tilapia mossambica aquaculture.Research shows that the main pathogenic bacteria of Tilapia mossambica streptococcosis is nothing
Streptococcus lactis and Streptococcus iniae were endangered in recent years the most serious is Streptococcusagalactiae disease, Streptococcusagalactiae be a kind of people, animal and
The pathogenic bacteria that fish suffers from altogether can infect a variety of sea water and fresh water fish morbidities such as sea bream, Tilapia mossambica, cause the higher death rate.Dolphin
Streptococcus is equally a kind of pathogen that can cause fish that infection and death occurs.Streptococcus iniae, Streptococcusagalactiae can be drawn
The illnesss such as septicemia, the meningitis of fish are played, it is accurate to be all difficult from external symptom and the bacterium colony of two kinds of pathogens, thalli morphology
Judgement is that this, which will seriously affect, is effectively treated disease as caused by any streptococcus.
The streptococcic routine diagnostic method of Tilapia mossambica is all difficult to meet the requirement to infection fish sample quick diagnosis at present, together
When some physiological reaction phenomenons the occurrence of may changing with the variation of external environment, also easily leading to erroneous judgement.Regular-PCR
It is once only capable of 1 gene of detection, is likely to result in missing inspection or erroneous detection.Fluorescent antibody technics and ELISA somewhat expensive and relatively needs
Masterful technique operation；Then there is false positive phenomenon in LAMP；Quantitative PCR then needs expensive instrument, professional operation and deposits
The deficiencies of at high cost.Therefore, to reach the quick diagnosis to illness fish, need to establish high special, sensitivity, quick Rofe
Fish streptococcus early molecule diagnostic techniques.
Summary of the invention
In order to overcome the disadvantages and deficiencies of the prior art, the purpose of the present invention is to provide a kind of double PCR early stage is quick
Detect the primer of Streptococcusagalactiae and Streptococcus iniae.
Another object of the present invention is to provide a kind of double PCR early stages quickly to detect Streptococcusagalactiae and Streptococcus iniae
Kit.
Another object of the present invention is to provide the applications of above-mentioned primer or kit.
A further object of the present invention is to provide a kind of double PCR early stages quickly to detect Streptococcusagalactiae and Streptococcus iniae
Method.
The present invention utilizes the specific sldh gene of Streptococcusagalactiae and Streptococcus iniae, has separately designed specificity and has drawn
Object is used for quickly detecting two kinds of streptococcus using double PCR technology.By the optimization to double PCR detection architecture, develop
For the kit of Streptococcusagalactiae and Streptococcus iniae double PCR detection technique, meanwhile, establish detection accurately, efficiently
Detection method.
The purpose of the invention is achieved by the following technical solution:
A kind of double PCR early stage quickly detects the primer of Streptococcusagalactiae and Streptococcus iniae, including following primer sequence:
For the PCR primer of Streptococcusagalactiae 16S rDNA gene:
P-1:5'-ATACCGCATAAGAGTGATT-3'；
P-2:5'-ACCACCTGTCACTTCTGCT-3'；
For the PCR primer of Streptococcus iniae Sip gene:
P-3:5'-TGAATAAGAAATTGTTTTTAGCG-3'；
P-4:5'-ACTTGTAATCGTTGGTTCTGCC-3'.
The present invention provides a kind of kit of double PCR early stage quick detection Streptococcusagalactiae and Streptococcus iniae, including
Above-mentioned primer P-1, P-2, P-3 and P-4.
The kit further includes PCR reaction reagent.
The present invention provides above-mentioned primer or kit and identifies in Streptococcusagalactiae and Streptococcus iniae in non-disease diagnosis
Application.
The present invention also provides a kind of methods that double PCR early stage quickly detects Streptococcusagalactiae and Streptococcus iniae, including
Following steps: the method is non-disease diagnostic method；
(1) measuring samples are taken；
(2) reaction mixture and measuring samples for containing above-mentioned primer P-1, P-2, P-3 and P-4 is added, is made into double PCR
Reaction system mixed liquor；It carries out double PCR and reacts on a response procedures；
(3) whether there is special DNA band using agarose gel electrophoresis detection detection liquid, appearance is then determined as sun
Property, the positive indicates to contain Streptococcusagalactiae or/and Streptococcus iniae in measuring samples；There is not the then judgement of specific band
For feminine gender, feminine gender indicates not containing Streptococcusagalactiae and Streptococcus iniae；Wherein, 870bp segment occur is Streptococcusagalactiae,
614bp segment occur is then Streptococcus iniae.
The double PCR reaction system are as follows: the measuring samples of 1 μ L, each 2 μM of primer P-1, P-2, each 1.6 μM draw
Object P-3, P-4,10 × buffer (no Mg2+) buffer 2.5 μ L, 1.5mM MgCl2, the rTaqDNA of the dNTP of 2.5mM, 0.5U
Polymerase, DEPC are supplemented to 25 μ L.
The double PCR response procedures are as follows: 95 DEG C of initial denaturation 5min, 94 DEG C of 30sec, 54 DEG C of 30sec and 72 DEG C of 30sec
30 circulation, last 72 DEG C of extensions 10min；
The measuring samples include but is not limited to the genomic DNA of Streptococcusagalactiae and Streptococcus iniae, bacterium colony and contain
There is the tissue sample of Streptococcusagalactiae and Streptococcus iniae；
The measuring samples can also be blood, brain, spleen, liver, kidney, the muscle of fish.
The homology of the 16S rDNA gene of Streptococcusagalactiae and Streptococcus iniae reaches 96.6%~96.8%, homology
It is higher, specific primer being designed in region of variability, it is distinguished and is identified.Surface immunogenic protein Sip (Surface
Immunogenic protein, Sip) belong to the surface protein gene monoid for encoding B group streptococcus, in serotype not of the same race
There is presence in bacterial strain.The present invention is quasi- to select Sip gene and 16S rDNA Gene Partial sequence, is established by primer Combinatorial Optimization
It is a kind of quickly, efficiently, stablize, good, high sensitivity the Streptococcusagalactiae of specificity and Streptococcus iniae dual PCR detection method,
It is intended that the quick detection and early warning of Streptococcusagalactiae and Streptococcus iniae provide a kind of accurately and efficiently detection technique,
Theoretical foundation is provided for the Accurate Diagnosis of streptococcosis, quarantine.
The present invention has the following advantages and effects with respect to the prior art:
(1) present invention is most short all detections can be completed in 1.5h operates, simple and quick, solves prior art detection
The problems such as period existing for Streptococcusagalactiae and Streptococcus iniae is long, complicated for operation, testing cost is high；
(2) testing cost is low, only needs two pairs of primers, easy to operate, is suitble to detect while multisample；
(3) sample size needed for detecting is few, it is only necessary to take a small amount of fish blood or tissue that can be detected.
(4) present invention can be also used for the detection and identification of Streptococcusagalactiae and Streptococcus iniae in water body.
(5) two pairs of specific primers used in the present invention, are the 16S rDNA gene and dolphin chain according to Streptococcusagalactiae
The Sip gene order design of coccus.The method according to the invention can realize the nothing in disposable specific detection sample simultaneously
Streptococcus lactis and Streptococcus iniae can divide Streptococcusagalactiae, Streptococcus iniae, Streptococcusagalactiae and Streptococcus iniae mixture
The specific band of 870bp, 614bp, 870bp and 614bp are not amplified.To 6 plants of fish body encountered pathogenic bacterias and one plant of large intestine bar
Bacterium carries out specific analysis, and only Streptococcusagalactiae and Streptococcus iniae can amplify two specificity of 16S rDNA and Sip respectively
Band, other 7 plants of bacterium are without specific fragment.Detection method that the present invention establishes can be detected genome content be respectively 9.84 ×
10-5The Streptococcusagalactiae and 9.30 × 10 of ng/ μ L-5The Streptococcus iniae and bacterial concentration of ng/ μ L is 2.76 × 102cfu/
The Streptococcusagalactiae of mL and 2.51 × 102The Streptococcus iniae of cfu/mL；With the dual PCR detection method established to artificial same
When infection Streptococcusagalactiae and the kidney of Tilapia mossambica of Streptococcus iniae carry out PCR detection, can get the 870, specificity of 614bp
Segment, recall rate 100%.This method in tilapia mossambica samples detection process quick and precisely specificity with higher, sensitivity,
Repeatability is with stability, it can be achieved that the early molecule early warning of Tilapia mossambica streptococcosis, has a extensive future, for Tilapia mossambica streptococcus
Quick diagnosis, the epidemiological survey etc. of disease provide accurate and effective means.
Detailed description of the invention
Fig. 1 is the specific agarose gel electrophoresis of double PCR detection Streptococcusagalactiae and Streptococcus iniae；Wherein, M generation
The big tick marks of table DNA2000bp；Swimming lane 1 and swimming lane 2 have respectively represented Streptococcusagalactiae ATCC13813 and Streptococcus iniae
ATCC29178 reference strain, swimming lane 3 contain the two bacteriums simultaneously；Swimming lane 4~10 is respectively Aeromonas hydrophila, slow love
Moral Fahrenheit bacterium, Aeromonas veronii, Vibrio vulnificus, Aeromonas sobria, vibrio alginolyticus, Escherichia coli genomic DNA.
Fig. 2 is the DNA sensitivity of primer pair Streptococcusagalactiae and Streptococcus iniae；Wherein, M representation DNA 2000bp
Size marker, N are negative control；1~5 respectively represents Streptococcusagalactiae (S.agalactiae) DNA concentration 9.84 × 10-2、
9.84×10-3、9.84×10-4、9.84×10-5、9.84×10-6The DNA concentration of ng/ μ L and Streptococcus iniae (S.iniae)
9.30×10-2、9.30×10-3、9.30×10-4、9.30×10-5、9.30×10-6ng/μL。
Fig. 3 is the bacterium colony sensitivity of primer pair Streptococcusagalactiae and Streptococcus iniae；Wherein, M representation DNA 2000bp
Size marker, N are negative control；1~5 respectively represents concentration of streptococcus agalactiae bacteria solution 2.76 × 106、2.76×105、2.76
×104、2.76×103、2.76×102, 27.6cfu/mL and Streptococcus iniae bacterial concentration 2.51 × 106、2.51×105、
2.51×104、2.51×103、2.51×102、25.1cfu/mL。
Fig. 4 is the agarose gel electrophoresis of repeatability detection of the double PCR to Streptococcusagalactiae and Streptococcus iniae；Its
In, the big tick marks of M representation DNA 2000bp, 1~5: template DNA is the DNA sample of Streptococcusagalactiae and Streptococcus iniae；6: mould
Plate DNA is reference culture DNA sample.
Fig. 5 is that the PCR of the renal tissue amplification of Tilapia mossambica that is artificial while infecting Streptococcusagalactiae and Streptococcus iniae is produced
Object；Wherein, the marker of M representation DNA 2000bp, 1 and 2,3 and 4,5 and 6 respectively represent 6 artificial challenge Streptococcusagalactiaes and
The PCR result of the Tilapia mossambica renal tissue amplification of Streptococcus iniae.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
The test method of specific experiment condition is not specified in the following example, usually according to conventional laboratory conditions or according to system
Make experiment condition proposed by factory.Used material, reagent etc., unless otherwise specified, for the reagent obtained from commercial channels
And material.
Main agents used in the present invention and instrument:
DNA extracts reagent, dNTP, 10 × PCR Buffer, Mg2+, the molecules reagent purchase such as Gold View and TaqDNA in
Beijing Tiangeng biochemistry Co., Ltd；Brain-heart infusion medium (BHI), nutrient agar powder are bought in the triumphant microorganism science and technology of Guangdong ring
Co., Ltd.PCR instrument (ABI company, the U.S.), gel imager (Bio-Rad company, the U.S.), nucleic acid electrophoresis apparatus (Bio-Rad
Company, the U.S.), NanoDrop 2000C ultramicrospectrophotometer (the silent science and technology of match, the U.S.).
(Streptococcus agalactiae) ATCC13813 of Streptococcusagalactiae used in embodiment is purchased from upper Haikang
Bright Biotechnology Co., Ltd, Streptococcus iniae (Strepstococcus iniae) ATCC29178 are purchased from China Microbiological bacterium
Kind collection, Aeromonas hydrophila (Aeromonas hydrophila) BNCC186123, Vibrio vulnificus (Vibrio
Vulnificus) BNCC186281, vibrio alginolyticus (Vibrio alginolyticus) BNCC185983 and Escherichia coli
(Escherichia coli) BNCC336902 receives biology purchased from north, Edwardsiella tarda (Edwardsiellatarda)
ATCC15947 is purchased from Shanghai North Connaught biological technology CO., LTD., Aeromonas veronii (Aeromonas veronii)
ATCC35624 is purchased from Shanghai Hu Zheng Biotechnology Co., Ltd, Aeromonas sobria (Aeromonas sobria) ATCC43979
Purchased from Chinese microorganism strain collection.
1 double PCR of embodiment quickly detects the foundation of Streptococcusagalactiae and Streptococcus iniae method
(1) prepared by template: picking Streptococcusagalactiae, Streptococcus iniae, thermophilic aqueous vapor unit cell in the strain saved from -80 DEG C
Bacterium, Edwardsiella tarda, Aeromonas veronii, Vibrio vulnificus, Aeromonas sobria, vibrio alginolyticus, Escherichia coli are sterile
Under the conditions of be inoculated on brain heart infusion agar culture medium, in 28 DEG C of progress constant temperature incubations, for 24 hours afterwards respectively collect culture bacterium thallus,
The genomic DNA that various bacteriums are extracted with reference to gram positive microbes DNA extracting method, using DNA extraction kit, (Dalian treasured is raw
Object Engineering Co., Ltd) its genomic DNA is extracted, it is used as reaction template；
(2) design of primers synthesizes: according to Streptococcusagalactiae 16S rDNA partial sequence design primer P-1 in GenBank and
P-2, it is contemplated that amplification target fragment size is 870bp.The sequence of Streptococcus iniae specific gene Sip, design primer P-3 and P-
4, it is contemplated that amplified fragments size is 614bp.
Primer sequence is as follows:
P-1:5'-ATACCGCATAAGAGTGATT-3'；
P-2:5'-ACCACCTGTCACTTCTGCT-3'；
P-3:5'-TGAATAAGAAATTGTTTTTAGCG-3'；
P-4:5'-ACTTGTAATCGTTGGTTCTGCC-3'.
(3) double PCR reaction system: 25 μ L reaction system mixed liquors are the templates of 1 μ L, primer P-1, P-2 (100 μM),
It is (0.5 μ L, 0.5 μ L) and (0.4 μ L, 0.4 μ L), 10 × buffer that primer P-3, P-4 primer (100 μM), which respectively correspond volume,
(no Mg2+) buffer 2.5 μ L, MgCl22.5 μ L, rTaqDNA Polymerase (5U/ μ L) of (25mM) 1.5 μ L, dNTP (25mM)
0.1 μ L, DEPC is supplemented to 25 μ L；
(4) double PCR response procedures: 95 DEG C of initial denaturations 5min, 94 DEG C of 30sec), 54 DEG C (30sec) and 72 DEG C (30sec)
30 circulation, last 72 DEG C of extensions 10min；
(5) specific detection of double PCR: with the Streptococcusagalactiae in step (1), Streptococcus iniae, thermophilic aqueous vapor unit cell
Bacterium, Edwardsiella tarda, Aeromonas veronii, Vibrio vulnificus, Aeromonas sobria, vibrio alginolyticus and Escherichia coli DNA
For template, the specificity of double PCR technology, 1.2% agarose gel electrophoresis results such as Fig. 1, Streptococcusagalactiae genome are examined
The band of DNA sample amplification is 870bp；Streptococcus iniae genomic DNA amplification goes out the specific band of 614bp；Streptococcusagalactiae
Then amplify the band of 870bp and 614bp simultaneously with Streptococcus iniae genomic DNA mixing sample；And Aeromonas hydrophila, late
Slow tarda, Aeromonas veronii, Vibrio vulnificus, Aeromonas sobria, vibrio alginolyticus, Escherichia coli DNA be template
It is expanded, is not occurred any band.
2 double PCR of embodiment detects the DNA sensitivity of Streptococcusagalactiae and Streptococcus iniae, includes the following steps:
(1) Streptococcusagalactiae of activation and 2~3mL of bacterium solution of Streptococcus iniae are taken respectively, are extracted and are tried with commercialized DNA
Agent box extracts DNA, with its concentration of spectrophotometric determination；
(2) Streptococcusagalactiae genomic DNA is diluted to 9.84 × 10 respectively-2、9.84×10-3、9.84×10-4、9.84
×10-5、9.84×10-6The genomic DNA of Streptococcus iniae is diluted to 9.30 × 10 by ng/ μ L respectively-2、9.30×10-3、
9.30×10-4、9.30×10-5、9.30×10-6ng/μL。
(3) as shown in Fig. 2, the DNA sensitivity of Streptococcusagalactiae and Streptococcus iniae is respectively 9.84 × 10-5Ng/ μ L and
9.30×10-5ng/μL。
3 double PCR of embodiment detects the single bacteria sensitivity of Streptococcusagalactiae and Streptococcus iniae, includes the following steps:
(1) Streptococcusagalactiae and the Streptococcus iniae bacterium solution for taking activation respectively are washed twice with 0.9% physiology salt, are pressed
Ten times of gradient dilutions, coated plate count；
(2) concentration of streptococcus agalactiae bacteria solution is diluted to 2.76 × 10 respectively6、2.76×105、2.76×104、2.76×
103、2.76×102, 27.6cfu/mL, Streptococcus iniae bacterial concentration is diluted to 2.51 × 10 respectively6、2.51×105、
2.51×104、2.51×103、2.51×102、25.1cfu/mL。
(3) system and program for using embodiment 1, boil 10min for the bacterium solution of above-mentioned preparation, take supernatant as to be checked
The template of sample；
(4) as shown in figure 3, the sensitivity of the pure fungus of Streptococcusagalactiae and Streptococcus iniae be respectively 2.76 ×
102Cfu/mL and 2.51 × 102cfu/mL。
4 double PCR of embodiment detects the repeatability of Streptococcusagalactiae and Streptococcus iniae method, includes the following steps:
(1) Streptococcusagalactiae is had been identified as to 5 plants and 5 plants of bacterial strains for having been identified as Streptococcus iniae carry out strain gene group
DNA is extracted；
(2) system and program of embodiment 1 are used, each positive DNA sample repeats detection 3 times, with reference culture
The DNA sample of ATCC13813 and ATCC29178 is set as positive control as template DNA.
(3) as shown in figure 4,5 parts of different positive DNA samples are that template can amplify two specific bands simultaneously,
Identical as the amplification of standard control result, the result of repeatability detection is completely the same three times.
5 double PCR of embodiment detects the application of Streptococcusagalactiae and Streptococcus iniae artificial challenge, includes the following steps:
(1) it takes 6 tails manually while infecting the renal tissue of the Tilapia mossambica of Streptococcusagalactiae and Streptococcus iniae；
(2) system and program for using embodiment 1, respectively using the kidney of this 6 tail fish as template；
(4) result is as shown in figure 5,6 tails manually infect the Tilapia mossambica kidney sample of Streptococcusagalactiae and Streptococcus iniae simultaneously
This, double PCR all amplifies the specific band of 870bp and 614bp, positive rate 100% simultaneously.
Two pairs of primers of the invention are obtained by screening, express ring according to Streptococcusagalactiae and Streptococcus iniae first
Cfb (Cyclic Adenosine monophosphate factor) gene, Surface immunogenic protein of the adenosine phosphate factor
Sip (Surface immunogenic protein) gene, hly (Hemolysin) gene of hemolysin, vaccine antigen A
Htra (the high temperature for the serine protease that VaA (Vaccine antigen A) gene, heat shock are lured
Reguirement A) gene, fibrinogen binding protein fbp (fibrinogen bingding protein) gene and
Seven genes of the 16S rDNA (16S ribosomal DNA) of 16S rRNA are expressed in Streptococcusagalactiae
(S.agalactiae) 70 pairs of primers are devised and are filtered out by PCR with the otherness in Streptococcus iniae (S.iniae)
14 pairs of primers can expand band, then according to the annealing temperature, clip size and gene specific of 14 pairs of primers, to agalasisa chain
Coccus (S.agalactiae) selects 16S rDNA gene and selects sip gene to Streptococcus iniae (S.iniae).
The sequence of the 14 pairs of primers filtered out is as follows:
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
<110>Ji'nan University
<120>double PCR early stage quickly detects primer and its application of Streptococcusagalactiae and Streptococcus iniae
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Claims (10)
1. the primer that a kind of double PCR early stage quickly detects Streptococcusagalactiae and Streptococcus iniae, it is characterised in that including as follows
Primer sequence:
For the PCR primer of Streptococcusagalactiae 16S rDNA gene:
P-1:5'-ATACCGCATAAGAGTGATT-3'；
P-2:5'-ACCACCTGTCACTTCTGCT-3'；
For the PCR primer of Streptococcus iniae Sip gene:
P-3:5'-TGAATAAGAAATTGTTTTTAGCG-3'；
P-4:5'-ACTTGTAATCGTTGGTTCTGCC-3'.
2. double PCR early stage described in claim 1 quickly detects the primer of Streptococcusagalactiae and Streptococcus iniae in non-disease
The application in Streptococcusagalactiae and Streptococcus iniae is identified in diagnosis.
3. the kit that a kind of double PCR early stage quickly detects Streptococcusagalactiae and Streptococcus iniae, it is characterised in that including power
Benefit require 1 described in primer P-1, P-2, P-3 and P-4.
4. kit according to claim 3, it is characterised in that further include PCR reaction reagent.
5. kit described in claim 3 or 4 identifies answering in Streptococcusagalactiae and Streptococcus iniae in non-disease diagnosis
With.
6. a kind of method that double PCR early stage quickly detects Streptococcusagalactiae and Streptococcus iniae, it is characterised in that including as follows
Step: the method is non-disease diagnostic method；
(1) measuring samples are taken；
(2) reaction mixture and measuring samples containing primer P-1, P-2, P-3 and P-4 described in claim 1 is added, in pairs
Weight PCR reaction system mixed liquor；It carries out double PCR and reacts on a response procedures；
(3) whether there is special DNA band using agarose gel electrophoresis detection detection liquid, appearance is then determined as the positive, sun
Property indicate measuring samples in contain Streptococcusagalactiae or/and Streptococcus iniae；Specific band do not occur is then determined as yin
Property, feminine gender indicates not containing Streptococcusagalactiae and Streptococcus iniae；Wherein, 870bp segment occur is Streptococcusagalactiae, is occurred
614bp segment is then Streptococcus iniae.
7. according to the method described in claim 6, it is characterized by:
The double PCR reaction system are as follows: the measuring samples of 1 μ L, each 2 μM of primer P-1, P-2, each 1.6 μM of primer P-
3, P-4, no Mg2+10 × buffer buffer 2.5 μ L, 1.5mM MgCl2, the rTaqDNA of the dNTP of 2.5mM, 0.5U
Polymerase, DEPC are supplemented to 25 μ L.
8. method according to claim 6 or 7, it is characterised in that:
The double PCR response procedures are as follows: 95 DEG C of initial denaturations 5min, 94 DEG C of 30sec, the 30 of 54 DEG C of 30sec and 72 DEG C of 30sec
A circulation, last 72 DEG C of extensions 10min.
9. method according to claim 6 or 7, it is characterised in that:
The measuring samples include but is not limited to the genomic DNA of Streptococcusagalactiae and Streptococcus iniae, bacterium colony and containing whether there is or not
The tissue sample of streptococcus lactis and Streptococcus iniae.
10. method according to claim 6 or 7, it is characterised in that:
The measuring samples are blood, brain, spleen, liver, kidney, the muscle of fish.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114686612A (en) * | 2022-04-26 | 2022-07-01 | 广东海大畜牧兽医研究院有限公司 | Dual-fluorescence PCR detection primer probe group for tilapia streptococcicosis and freeze-dried kit |
| CN115747361A (en) * | 2022-12-28 | 2023-03-07 | 中国海洋大学 | Real-time fluorescent MIRA and MIRA-LFD primer group for detecting streptococcus iniae and detection method |
-
2019
- 2019-03-27 CN CN201910241560.9A patent/CN109811073B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| 黎炯等: ""双重PCR快速鉴别无乳链球菌和海豚链球菌"", 《湖南农业大学学报(自然科学版)》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114686612A (en) * | 2022-04-26 | 2022-07-01 | 广东海大畜牧兽医研究院有限公司 | Dual-fluorescence PCR detection primer probe group for tilapia streptococcicosis and freeze-dried kit |
| CN114686612B (en) * | 2022-04-26 | 2024-05-07 | 广东海大畜牧兽医研究院有限公司 | Primer probe group for dual fluorescence PCR detection of streptococcicosis of tilapia and freeze-drying type kit |
| CN115747361A (en) * | 2022-12-28 | 2023-03-07 | 中国海洋大学 | Real-time fluorescent MIRA and MIRA-LFD primer group for detecting streptococcus iniae and detection method |
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