CN109055584A - Primer and its kit and method based on digital LAMP technology detection Streptococcus suis - Google Patents
Primer and its kit and method based on digital LAMP technology detection Streptococcus suis Download PDFInfo
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- CN109055584A CN109055584A CN201810979599.6A CN201810979599A CN109055584A CN 109055584 A CN109055584 A CN 109055584A CN 201810979599 A CN201810979599 A CN 201810979599A CN 109055584 A CN109055584 A CN 109055584A
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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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- 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
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Abstract
The invention discloses a kind of primer based on digital LAMP technology detection Streptococcus suis and kit and methods.Kit includes: 2 × ddLAMP Supermix, primer combination, the ultrapure water without RNA enzyme, bacteria total DNA extraction reagent, negative control and positive control.Its detection method is to configure LAMP reaction system by extracting measuring samples DNA, after carrying out droplet and LAMP reaction, by detecting fluorescence signal, judges whether measuring samples contain Streptococcus suis, and measure its content.The present invention has many advantages, such as that quick, accurate, sensitive, applicability is wide, without relying on alternating temperature augmentation apparatus, it can be achieved that absolute quantitation is, it can be achieved that early discovery, early treatment and the early prevention that Streptococcus suis diagnoses, can be effectively controlled Epidemic outbreak of disease.
Description
Technical field
The present invention relates to a kind of Streptococcus suis test equipments, and in particular to one kind detects pig hammer based on digital LAMP technology
The kit of bacterium, belongs to technical field of molecular biology.
Background technique
Streptococcus suis (Streptococcus suis, SS) is a kind of common conditioned pathogen in swinery, is usually existed
The upper respiratory tract (especially tonsillotome and nasal cavity), genital tract or the alimentary canal of pig are interior to settle down, and pig is infected when serious will appear brain
The illnesss such as film inflammation, septicemia, pneumonia and arthritis can lead to young pig sudden death.According to streptococcus thallus capsular antigen characteristic
Difference, Streptococcus suis can be divided into 35 serotypes (1~34 type and 1/2 type), wherein 1,2,7,9 type is the pathogenic bacteria of pig.Pig
Streptococcus can also infect the mankind, also will appear after infection meningitis, septicemia, pneumonia, arthritis, endocarditis, peritonitis and
The serious conditions such as entophthamia, or even death can be caused, seriously threaten human health and public health.Nineteen sixty-eight, Denmark reported for the first time
Also there is the report of Human Streptococcus suis infection in succession in road Human Streptococcus suis infection, the countries and regions such as subsequent South Asia, Northern Europe.Away from
Until the present, which has been reported more than 700, and there is the report of Streptococcus suis in nearly all pig-raising countries and area in global range
Road, wherein being mainly distributed on Asia.According to " People's Republic of China's animal epidemic prevention method " and its relevant regulations, Streptococcus suis
Disease is two class animal epidemic prevention diseases.The propagation of the disease is to China's food safety, Animal husbandry production safety and related practitioner
Bring huge threat.Therefore, reinforce the research to Streptococcus suis detection technique, carry out the detection of Streptococcus suis, prevent pig chain
The incoming outflow of coccus, is of great significance to the technical guarantee of foreign trade.
Currently, the method that Streptococcus suis is commonly detected in laboratory has bacterium separation, serodiagnosis (agglutinating reaction
With enzyme-linked immunosorbent assay etc.) and molecular Biological Detection technology (Standard PCR, real-time fluorescence quantitative PCR and ring mediated isothermal
Amplification technique etc.).Traditional bacterium separation and Serology test, have that time-consuming, behaviour high to testing staff's technical requirements
Make many deficiencies such as complicated and sensibility difference.PCR is complicated for operation, need to carry out running glue analysis, and sensitivity is low；Real-time fluorescence
PCR, which need to use expensive instrument and equipment and rely on standard curve, carries out quantitative detection, and there are still certain limitations for sensitivity；Ring
Mediated isothermal amplification technology is also easy to produce false positive results due to the non-specific amplification between primer.
Digital loop-mediated isothermal amplification technique (digital loop-mediated isothermal
Amplification, dLAMP) it is to be carried out the LAMP system of premix at droplet based on third generation digital pcr technical principle
Reason is formed in tens of thousands of liquefaction droplets to hundreds of thousands Water-In-Oil, is guaranteed in each droplet containing one or without containing nucleic acid mould
Plate, after LAMP is expanded, by the positive droplet number and negative droplet number in each droplet of fluorescence detection, according to Poisson distribution
Principle can calculate nucleic acid-templated initial copy number.This method has the advantage that 1. high specificity: for target gene
Different zones design multipair special primer, not will receive the influence of other non-target sequences present in reaction mixture, while can
Identify non-specific amplification according to melting curve；2. sensitivity is high: dLAMP detection is fluorescence signal, can detect regular-PCR
1/10-1/100 copy number；3. constant temperature: the technology reaction system only needs energy amplified reaction at a constant temperature, does not need
The denaturation of double-strand is carried out in advance；4. quickly: LAMP reaction can be completed in 15-60min；5. equipment is simple: it is special not need
Alternating temperature equipment.
In order to solve above-mentioned traditional technique in measuring Streptococcus suis problems faced, there is an urgent need to develop it is a kind of quickly, efficiently,
The detection technique of low cost.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of kit of digital LAMP technology detection Streptococcus suis, energy
Enough it is applied to quick, the accurate detection of Streptococcus suis.
First technical purpose of the invention is to provide the specific primer for detecting Streptococcus suis, nucleotide sequence
Respectively:
Outer primer F3:CGTCTTGACTGGTCTTCC (SEQ ID NO:1)；
Outer primer B3:TTACCAGAACCTGAGATAAGGA (SEQ ID NO:2)；
Inner primer FIP:AACCAATCTCACGACCACCGACACATCGGACCTTCACT (SEQ ID NO:3)；
Inner primer BIP:AACGCCTCCGCCAGTTCGGATCAATGAACCACCAA (SEQ ID NO:4)；
Ring primer LF:CCGATGTCACCAGCAGG (SEQ ID NO:5)；
Ring primer LB:GCAGGTGTCTTGACTGGTAA (SEQ ID NO:6)；
Preferably, when amplified reaction, inner primer FIP and BIP, ring primer LF and LB, the molar ratio with outer primer F3 and B3
For 8:4:1.
The present invention provides above-mentioned specific primer answering in preparation Streptococcus suis detection kit or detection reagent
With.
The present invention provides a kind of kits containing above-mentioned specific primer.The kit is preferably droplet number
LAMP absolute quantitation detection kit.
Another technical object of the present invention is to provide a kind of to have high sensitivity, high specific, high accuracy and accurately
The droplet number LAMP absolute quantitation detection kit of degree, easy to operate detection Streptococcus suis, which is characterized in that kit
Include the primer described in claim 1.
The kit for being preferably based on digital LAMP technology detection Streptococcus suis further includes the part or complete of following component
Portion: (1) 2 × ddLAMP Supermix；(2) without the distilled water of RNA enzyme；(3) positive control and negative control；(4) bacterium is total
DNA extracts reagent.
Preferably, each 160 μm of ol/L of inner primer FIP and BIP, outer primer F3 and B3 each 80 μm of ol/L, ring primer LF and LB
Each 160 μm of ol/L, 1 × TE, 50mmol/L Tris-HCl (pH8.8), 250mmol/L KCl, 200mmol/L MgSO4,
250mmol/L(NH4)2SO4, 2.8%Tween-20,7.14mol/L glycine betaine, the big segment DNA of 10mmol/L dNTP, Bst is poly-
Synthase 160U, 20 × EvaGreen, RNase Cocktail.
Preferably, positive control is Streptococcus suis DNA, and negative control is ultrapure water.
Preferably, it includes lysate A, washing lotion B, washing lotion C, eluent D, adsorption column and collection that bacteria total DNA, which extracts reagent,
Pipe.
Further more, the invention also discloses a kind of methods based on digital LAMP technology detection Streptococcus suis, including walk as follows
It is rapid:
(1) extraction of bacteria total DNA
1) measuring samples 1g is taken, after 600 μ L lysate A grinding is added, is vortexed and mixes 20 seconds, be stored at room temperature 10 minutes；
2) mixed liquor is moved in adsorption column, 12,000 × g is centrifuged 30~60 seconds；
3) liquid in collecting pipe is abandoned, 500 μ L washing lotion B are added into adsorption column, 12,000 × g is centrifuged 30~60 seconds；
4) liquid in collecting pipe is abandoned, 500 μ L washing lotion C are added into adsorption column, 12,000 × g is centrifuged 30~60 seconds；
5) liquid in collecting pipe is abandoned, 12,000 × g is centrifuged 2 minutes to dry pillar；
6) adsorption column is moved into new 1.5ml centrifuge tube, 50 μ L of eluent is added to pillar center, is stored at room temperature 2 points
Clock, 12,000 × g are centrifuged 1 minute, and liquid is template DNA in centrifuge tube；
(2) number LAMP is operated
1) 19 μ L of number LAMP reaction solution is respectively added in measuring samples, negative control and each reaction tube of positive control；Respectively
1 μ L template DNA is taken, is added in corresponding reaction tube, 20 μ L number LAMP reaction systems are configured to；The template DNA include to
Sample product, negative control and positive control；
2) 20 μ L sample reaction solutions droplet is added to occur to prepare droplet in card；
3) droplet is transferred in PCR plate, is put into water-bath or PCR instrument and is expanded；
4) PCR plate that amplification is completed is put into droplet analyzer, detects the fluorescence signal in droplet, analyze data, shown
Show testing result；
5) result judgement and description: positive control: 40 ± 2 copy, negative control: when 1 copy of <, experimental result at
It is vertical；It is the positive when sample to be tested result >=1 copies；It is feminine gender when 1 copy of sample to be tested result <.
Preferably, digital LAMP reaction system is 20 μ L reaction systems, contains 2 × ddLAMP Supermix, 10 μ L,
9 μ L of ultrapure water is added after 1 μ L template as 20 μ L.
Preferably, amplification program are as follows: 63 DEG C of reaction 35min, and in 80 DEG C of lasting 2min.
The present invention generates system with LAMP and digital pcr droplet and detection system is to rely on, and it is exhausted to develop digital LAMP technology
To the kit and detection method of quantitative detection Streptococcus suis.There has been no droplet number LAMP technology is applied to detection pig at present
Streptococcic kit.This kit can realize the quick, precisely quantitative of Streptococcus suis in a short time, be Streptococcus suis
Early diagnosis provides strong technical support, has broad application prospects and industrialization prospect.
Therefore, compared to other existing detection techniques, the technology of the present invention have quick, accurate, sensitive, applicability is wide,
Simple operation and other advantages:
1. high specificity: designing multipair special primer for the different zones of target gene, not will receive in reaction mixture
The influence of other existing non-target sequences, while non-specific amplification can be identified according to melting curve；2. sensitivity is high: dLAMP
Detection is fluorescence signal, can detect the copy number of the 1/10-1/100 of regular-PCR；3. constant temperature: the technology reaction system is only
Energy amplified reaction at a constant temperature is needed, the denaturation for carrying out double-strand in advance is not needed；4. quickly: LAMP is reacted in 15-60min
It can be completed；5. equipment is simple: not needing special alternating temperature equipment.
Detailed description of the invention
Fig. 1 is the testing result figure of specificity experiments in the embodiment of the present invention 3.In Fig. 1,1: positive control；2: pseudo- mad dog
Virus；3: porcine circovirus 2 type；4: Actinobacillus pleuropneumoniae；5: haemophilus parasuis；6: Escherichia coli；7:
Salmonella；8: negative control.
Specific embodiment
The present invention is further illustrated with reference to embodiments, and however, it is not limited to this.
The foundation of kit of the embodiment 1 based on digital LAMP technology absolute quantitation detection Streptococcus suis
Based on the kit of digital LAMP technology absolute quantitation detection Streptococcus suis, including primer sets, 2 × ddLAMP
Supermix, the ultrapure water without RNA enzyme, bacteria total DNA extract reagent, positive control and negative control.
(1) number LAMP amplimer designs: being protected with 35 serotypes of Streptococcus suis (1~34 type and 1/2 type) specificity
Keeping sequence is the design that target gene carries out primer.Primer sequence is shown in Table 1.
1 primer sequence table of table
(2) when amplified reaction, the molar ratio of inner primer FIP and BIP, ring primer LF and LB and outer primer F3 and B3 are 8:4:
1。
(3) 2 × ddLAMP Supermix include following reagent: inner primer FIP and BIP each 160 μm of ol/L, outer primer F3
With each 80 μm of ol/L of B3, ring primer LF and LB each 160 μm of ol/L, 1 × TE, 50mmol/L Tris-HCl (pH8.8),
250mmol/L KCl, 200mmol/L MgSO4, 250mmol/L (NH4)2SO4, 2.8%Tween-20,7.14mol/L beet
Alkali, big segment archaeal dna polymerase 160U, 20 × EvaGreen, the RNase Cocktail of 10mmol/L dNTP, Bst.
(4) positive control is Streptococcus suis DNA, and negative control is ultrapure water.
(5) it includes lysate A, washing lotion B, washing lotion C, eluent D, adsorption column and collecting pipe that bacteria total DNA, which extracts reagent,.
Detection method of the embodiment 2 based on digital LAMP technology absolute quantitation detection Streptococcus suis
Using the method for the kit detection Streptococcus suis of embodiment 1, include the following steps:
(1) extraction of DNA of bacteria:
1) measuring samples 1g is taken, after 600 μ L lysate A grinding is added, is vortexed and mixes 20 seconds, be stored at room temperature 10 minutes；
2) mixed liquor is moved in adsorption column, 12,000 × g is centrifuged 30~60 seconds；
3) liquid in collecting pipe is abandoned, 500 μ L washing lotion B are added into adsorption column, 12,000 × g is centrifuged 30~60 seconds；
4) liquid in collecting pipe is abandoned, 500 μ L washing lotion C are added into adsorption column, 12,000 × g is centrifuged 30~60 seconds；
5) liquid in collecting pipe is abandoned, 12,000 × g is centrifuged 2 minutes to dry pillar；
6) adsorption column is moved into new 1.5mL centrifuge tube, 50 μ L of eluent is added to pillar center, is stored at room temperature 2 points
Clock, 12,000 × g are centrifuged 1 minute, and liquid is template DNA in centrifuge tube.
(2) number LAMP is operated
1) measuring samples, negative control and positive control sample number summation are set as N, reaction system configuration is as follows:
2 number LAMP reaction system of table
| Title | System |
| 2×ddLAMP Supermix | 14×(N+1)μL |
| Distilled water without RNA enzyme | 5×(N+1)μL |
After the reaction system configured above is mixed well, packing to each 19 μ L of each reaction tube.
Wherein, 2 × ddLAMP Supermix includes following reagent: 160 μm of ol/L inner primer FIP and BIP each 0.2 μ L, and 80
μm ol/L outer primer F3 and B3 each 0.05 μ L, 160 μm of ol/L ring primers LF and LB each 0.1 μ L, TE 0.3 μ L, 50mmol/L
0.8 μ L, 250mmol/L KCl of Tris-HCl (pH8.8), 0.8 μ L, 200mmol/L MgSO40.8 μ L, 250mmol/L (NH4)2SO40.8 3.2 μ L, Bst of μ L, 2.5%Tween-20 0.8 μ L, 7.14mol/L glycine betaine, 2.8 μ L, 10mmol/L dNTP are big
11 μ L, RNase Cocktail of μ L, 20 × EvaGreen of segment archaeal dna polymerase, 1 μ L.
2) 1 μ L template DNA (including measuring samples, negative control and positive control) is taken respectively, and corresponding reaction tube is added
In, final volume is 20 μ L.
3) card is occurred for a new droplet to be put into Kato, 20 μ L sample reaction solutions is added to droplet and are occurred among card
It in 8 holes of one row, is supplied when less than 8 samples with 20 μ L deionized waters, it is proposed that use the 8 channel volley of rifle fires, pipette tips connect when sample-adding
One side bottom of adjacent pores is presented about 15 ° of angles with side wall, slowly gets liquid, slowly promote pipette tips position again after getting a part
Remaining drop is got, not turn rifle to the first file location in order to avoid introducing bubble；
4) occur that 70 μ L droplets generation oil is added in card most 8 holes of bottom next row in droplet, cannot equally there is empty hole；
5) rubber mat is covered, notices that the aperture on both sides will hook jail；
6) Kato is lightly steadily placed in droplet to generate in instrument, starts to generate droplet, pays attention to indicator light shape on instrument
State is completed within general 2 minutes；
7) droplet is created on droplet and occurs in one round of card the top, it is proposed that is carefully slowly drawn, is adjusted using the 8 channel volley of rifle fires
Whole volume aspirated is 40 μ L, and Kato is laid flat, and pipette tips touch bottom hole, about 5 seconds 40 μ of absorption to be put into hole wall in 30~45° angle
L, then equally slowly squeeze into 96 orifice plate corresponding position holes (about 5 seconds), pipette tips close to hole wall access hole bottom, pay attention to sealing up lid with
Grease proofing volatilization discards used droplet every time and card and rubber mat occurs；
8) after the completion of transfer oil droplet enters in 96 orifice plates, sealer (red line court is carried out to it with preheated PX1 heat-sealing instrument
On), run program are as follows: 180 DEG C, 5s, be not necessarily to the secondary sealer of inverted orientation；
9) LAMP reaction should be carried out later in 30 minutes by sealing film, or is put within 4 DEG C of refrigerators 4 hours and is carried out
LAMP.Amplification program are as follows: 63 DEG C of reaction 35min, and in 80 DEG C of lasting 2min.
10) 96 plates that PCR amplification is completed are put into the plate holder of droplet analyzer, pay attention to plate oblique angle orientation,
Droplet is lightly steadily put into after assembling to read in instrument.
11) open QuantaSoft software, it is proposed that every time experiment before be a Flush System, if one week or more not
A Prime, which is first, using suggestion is Flush System again.Then sample message in 96 orifice plates is configured, is after the completion
Can be detected, after result can be analyzed automatically, it is artificial verify after save result.
12) result judgement and description: positive control: 40 ± 2 copies, negative control: when 1 copy of <, experimental result
It sets up；It is positive for Streptococcus suis when sample to be tested result >=1 copies；It is pig chain when 1 copy of sample to be tested result <
Coccus is negative.
3 specificity verification of embodiment
Detect clinical obtained Pseudorabies virus, porcine circovirus 2 type, pig transmissible pleura respectively with kit of the present invention
Actinobacillus, haemophilus parasuis, Escherichia coli and health pig serum sample, and set positive control and negative control sample
Product, totally 8 parts of samples, all samples pass through PCR sequencing PCR verifying, and testing result is shown in Fig. 1.
1: positive control；2: Pseudorabies virus；3: porcine circovirus 2 type；4: Actinobacillus pleuropneumoniae；5:
Haemophilus parasuis；6: Escherichia coli；7: the serum sample of health pig；8: negative control.
Experimental result shows that positive control is after digital LAMP amplification, and threshold line appears above positive drop, and copy number is
40 ± 2 copies, that is, have amplification.And Pseudorabies virus, porcine circovirus 2 type, Actinobacillus pleuropneumoniae, secondary pig
Haemophilus, the serum sample of Escherichia coli and health pig and negative control copy number < 1, i.e., without amplification (see Fig. 1).Thus may be used
See, shows good specificity using digital pcr kit detection tuberculosis Streptococcus suis of the invention.
The verifying of 4 actual sample of embodiment
Actual sample is detected with kit of the present invention, clinical 70 parts obtained of the doubtful pathological material of disease of Streptococcus suis of detection,
Verified simultaneously with PCR sequencing PCR, wherein kit and PCR sequencing PCR of the present invention detection be it is positive have 35, detection method is yin
Property has 35, shown in testing result table 3:
Table 3
The experimental result of upper table can illustrate that this kit and method accuracy are high, and high specificity has very high with PCR sequencing PCR
Consistency.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
SEQUENCE LISTING
<110>Ji'nan University, South China Science & Engineering University
<120>primer and its kit and method based on digital LAMP technology detection Streptococcus suis
<130> 2018
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 1
cgtcttgact ggtcttcc
<210> 2
<211> 22
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 2
ttaccagaac ctgagataag ga
<210> 3
<211> 38
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 3
aaccaatctc acgaccaccg acacatcgga ccttcact
<210> 4
<211> 35
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 4
aacgcctccg ccagttcgga tcaatgaacc accaa
<210> 5
<211> 17
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 5
ccgatgtcac cagcagg
<210> 6
<211> 20
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 6
gcaggtgtct tgactggtaa
Claims (9)
1. a kind of primer based on digital LAMP technology detection Streptococcus suis, including inner primer FIP and BIP, outer primer F3 and B3,
Ring primer LF and LB, nucleotide sequence difference are as follows:
Outer primer F3:CGTCTTGACTGGTCTTCC
Outer primer B3:TTACCAGAACCTGAGATAAGGA
Inner primer FIP:AACCAATCTCACGACCACCGACACATCGGACCTTCACT
Inner primer BIP:AACGCCTCCGCCAGTTCGGATCAATGAACCACCAA
Ring primer LF:CCGATGTCACCAGCAGG
Ring primer LB:GCAGGTGTCTTGACTGGTAA.
2. the primer as described in claim 1 based on digital LAMP technology detection Streptococcus suis, it is characterised in that: amplified reaction
When, inner primer FIP and BIP, the molar ratio of ring primer LF and LB and outer primer F3 and B3 are 8:4:1.
3. a kind of kit based on digital LAMP technology detection Streptococcus suis, which is characterized in that kit includes claim 1
Or primer described in 2.
4. the kit as claimed in claim 3 based on digital LAMP technology detection Streptococcus suis, which is characterized in that further include
Following component it is some or all of: (1) 2 × ddLAMP Supermix；(2) without the ultrapure water of RNA enzyme；(3) positive control and
Negative control；(4) bacteria total DNA extracts reagent.
5. the kit as claimed in claim 4 based on digital LAMP technology detection Streptococcus suis, it is characterised in that: described 2
× ddLAMP Supermix includes following reagent: each 80 μm of ol/ of inner primer FIP and BIP each 160 μm of ol/L, outer primer F3 and B3
L, ring primer LF and LB each 160 μm of ol/L, 1 × TE, 50mmol/L Tris-HCl (pH8.8), 250mmol/L KCl,
200mmol/L MgSO4, 250mmol/L (NH4)2SO4, 2.8%Tween-20,7.14mol/L glycine betaine, 10mmol/L
Big segment archaeal dna polymerase 160U, 20 × EvaGreen, the RNase Cocktail of dNTP, Bst.
6. the kit as claimed in claim 4 based on digital LAMP technology detection Streptococcus suis, it is characterised in that: positive right
According to for pig Streptococcus suis DNA, negative control is deionized water.
7. a kind of method based on digital LAMP technology detection Streptococcus suis, which comprises the steps of:
(1) extraction of DNA of bacteria
1) measuring samples 1g is taken, after 600 μ L lysate A grinding is added, is vortexed and mixes 20 seconds, be stored at room temperature 10 minutes；
2) mixed liquor is moved in adsorption column, 12,000 × g is centrifuged 30~60 seconds；
3) liquid in collecting pipe is abandoned, 500 μ L washing lotion B are added into adsorption column, 12,000 × g is centrifuged 30~60 seconds；
4) liquid in collecting pipe is abandoned, 500 μ L washing lotion C are added into adsorption column, 12,000 × g is centrifuged 30~60 seconds；
5) liquid in collecting pipe is abandoned, 12,000 × g is centrifuged 2 minutes to dry pillar；
6) adsorption column is moved into new 1.5mL centrifuge tube, 50 μ L of eluent is added to pillar center, is stored at room temperature 2 minutes,
12,000 × g is centrifuged 1 minute, and liquid is template DNA in centrifuge tube；
(2) number LAMP is operated
1) 19 μ L of number LAMP reaction solution is respectively added in measuring samples, negative control and each reaction tube of positive control；1 μ L is taken respectively
Template DNA is added in corresponding reaction tube, is configured to 20 μ L number LAMP reaction systems；The template DNA includes to sample
Product, negative control and positive control；
2) 20 μ L sample reaction solutions droplet is added to occur to prepare droplet in card；
3) droplet is transferred in PCR plate, is put into PCR instrument and is expanded；
4) PCR plate that amplification is completed is put into droplet analyzer, detects the fluorescence signal in droplet, analyze data, display inspection
Survey result；
5) result judgement and description: positive control: 40 ± 2 copies, negative control: when 1 copy of <, experimental result is set up；
It is the positive when sample to be tested result >=1 copies；It is feminine gender when 1 copy of sample to be tested result <.
8. the detection method of the kit as claimed in claim 8 based on digital LAMP technology detection Streptococcus suis, feature
Be: 19 μ L of number LAMP reaction solution contains 9 μ L of 2 × ddLAMP Supermix, 10 μ L and ultrapure water in (2), and 1 μ is added
20 μ L number LAMP reaction systems are constituted after L template.
9. the detection method of the kit as claimed in claim 7 based on digital LAMP technology detection Streptococcus suis, feature
It is: amplification program are as follows: 63 DEG C of reaction 35min, and in 80 DEG C of lasting 2min.
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| CN201810979599.6A Pending CN109055584A (en) | 2018-08-24 | 2018-08-24 | Primer and its kit and method based on digital LAMP technology detection Streptococcus suis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113943825A (en) * | 2021-11-16 | 2022-01-18 | 暨南大学 | Primer probe set for detecting pathogenic streptococcus of fish based on fluorescent quantitative PCR technology, kit and application thereof |
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| CN105063172A (en) * | 2015-07-30 | 2015-11-18 | 山东省滨州畜牧兽医研究院 | Method for rapidly screening and identifying streptococcus suis |
| CN105779625A (en) * | 2016-04-21 | 2016-07-20 | 韶关学院 | Dual-fluorescence quantitative PCR (Polymerase Chain Reaction) primer, kit and method for simultaneously detecting general type and type 2 Streptococcus suis |
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| CN105063172A (en) * | 2015-07-30 | 2015-11-18 | 山东省滨州畜牧兽医研究院 | Method for rapidly screening and identifying streptococcus suis |
| CN105779625A (en) * | 2016-04-21 | 2016-07-20 | 韶关学院 | Dual-fluorescence quantitative PCR (Polymerase Chain Reaction) primer, kit and method for simultaneously detecting general type and type 2 Streptococcus suis |
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| CN113943825A (en) * | 2021-11-16 | 2022-01-18 | 暨南大学 | Primer probe set for detecting pathogenic streptococcus of fish based on fluorescent quantitative PCR technology, kit and application thereof |
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Application publication date: 20181221 |