CN108977512A - Primer and probe and its kit and method based on digital pcr technology detection haemophilus parasuis - Google Patents
Primer and probe and its kit and method based on digital pcr technology detection haemophilus parasuis Download PDFInfo
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Abstract
The invention discloses a kind of primer and probe based on digital pcr technology detection haemophilus parasuis and its kit and methods.Primer and probe, including upstream primer FP, downstream primer BP, probe Probe；Detection kit includes 2 × ddPCR Supermix, the ultrapure water without RNA enzyme, bacteria total DNA extraction reagent, negative control and positive control.Its detection method is to carry out quantitative detection by extracting measuring samples DNA to measuring samples DNA using droplet digital pcr technology, judge whether measuring samples contain haemophilus parasuis by the copy number that Amplification Analysis obtains, and measure its content.The present invention has many advantages, such as that accurate, sensitive, applicability is wide, without relying on standard curve, it can be achieved that absolute quantitation is, it can be achieved that early discovery, early treatment and the early prevention that Haemophilus parasuis diagnoses, can be effectively controlled Epidemic outbreak of disease.
Description
Technical field
The invention belongs to technical field of molecular biology, are related to a kind of based on digital pcr technology detection haemophilus parasuis
Primer and probe and its kit and method.
Background technique
Haemophilus parasuis (Haemophilus parasuis, HPS) is one kind of the pig upper respiratory tract often in bacterium.Certain
Under the conditions of can cause serious systemic disease, using cellulosic polyserositis, arthritis and meningitis as main feature,
Disease caused by the cause of disease is also known as Graves disease.Haemophilus parasuis has 15 or more serotypes, 1,5,10,12,13 and of serum
14 types are to cause pig morbidity and dead virulent serotype；Serotype 2,4 and 15 be can cause polyserositis it is medium solely
Vertical type；Serotype8 belongs to weak virulence type, and serotype 3,6,7,9 and 11 is not cause the avirulence type of clinical symptoms, and China is with blood
Clear 4 and 5 types are popular at most.The Haemophilus parasuis death rate is higher, is not easy to cure after disease hair, case fatality rate is up to 50%.The disease
Infectiousness is strong, and mostly seriously endangers with the mixed infections such as other cause of diseases such as porcine reproductive and respiratory syndrome virus, pig circular ring virus
The health of piglet and young pig.The disease has showed fashion trend in China, has brought tremendous economic losses to pig breeding industry.For
This, establishes prevention that early stage rapid detection method infects haemophilus parasuis and control has a very important significance.
Currently, the method that haemophilus parasuis is commonly detected in laboratory has bacterium separation, serodiagnosis (complement
In conjunction with test, indirect hemagglutination test and enzyme-linked immunosorbent assay etc.) and molecular Biological Detection technology (Standard PCR, it is glimmering in real time
Fluorescent Quantitative PCR and loop-mediated isothermal amplification technique etc.).Traditional bacterium separation and Serology test, have that time-consuming, right
Many deficiencies such as testing staff's technical requirements are high, complicated for operation and sensibility is poor.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, sensitivity
There are still certain limitations；Loop-mediated isothermal amplification technique is also easy to produce false positive knot due to the non-specific amplification between primer
Fruit.
Digital pcr (digital PCR, dPCR) is the third generation round pcr risen nearly ten years, and principle is will to premix
PCR reaction system carry out droplet processing, formed in tens of thousands of liquefaction droplets to hundreds of thousands Water-In-Oil, guarantee each droplet
In containing one or without containing nucleic acid-templated, after PCR amplification, by each droplet of fluorescence detection positive droplet number and
Negative droplet number can calculate nucleic acid-templated initial copy number according to Poisson distribution principle.Compared with real-time fluorescence PCR,
This method has many advantages, such as accurate, sensitivity superelevation and does not influence vulnerable to PCR mortifier.And there is no utilize number for the prior art
Round pcr for haemophilus parasuis carry out absolute quantitation detection correlative study and whether feasible relevant programme and be suitble to
The finished product kit of industrialization.Therefore, the related method that haemophilus parasuis is detected using digital pcr technology of exploitation with
Reagent is industry technical problem urgently to be resolved.
Summary of the invention
In view of this, technical problem solved by the invention is to provide, a kind of based on digital pcr technology to detect secondary pig thermophilic
The primer and probe of blood bacillus.
Technical problem solved by the invention also reside in provide it is a kind of based on digital pcr technology detection haemophilus parasuis
Kit.
Technical problem solved by the invention also reside in provide it is a kind of based on digital pcr technology detection haemophilus parasuis
Method.
In order to solve the above-mentioned technical problem, the technical solution adopted in the present invention is as follows:
On the one hand, the present invention provides a kind of primer and probe based on digital pcr technology detection haemophilus parasuis, packets
Upstream primer FP, downstream primer BP, probe Probe are included, nucleotide sequence difference is as follows:
FP:5 '-TCGCCTTGGCCGTAATTCTA-3 ' (SEQ ID NO:1)；
BP:5 '-ACGTAAGCCTTTTCTGTTGTAACATC-3 ' (SEQ ID NO:2)；
Probe:5 '-FAM-AAATGATGCAGGATGGG-BHQ1-3 ' (SEQ ID NO:3), wherein 5 ends label is glimmering
Light group is FAM, and the quenching group of 3 ends label is BHQ1.
Preferably, when amplified reaction, FP primer, BP primer and probe molar ratio be 9:9:2.
On the other hand, the invention also discloses it is a kind of based on digital pcr technology detection haemophilus parasuis kit,
It is characterized in that, kit includes primer and probe described in claims 1 or 2.
Be preferably based on digital pcr technology detection haemophilus parasuis kit further include following component part or
Person is whole: (1) 2 × ddPCR Supermix；(2) without the distilled water of RNA enzyme；(3) positive control and negative control；(4) bacterium
Genome DNA extraction reagent.
Preferably, 2 × ddPCR Supermix contains: 2 × PCR Buffer, 2 × Taq DNA
Polymerase, 36 μM of FP primers, 20 μM of BP primers, 400nM probe, 400 μM of dNTP mix and 2 × RNase
Cocktail(ThermoFisher)。
Preferably, 2 × PCR Buffer contains 20mM Tris-HCl (pH 8.3), 50mM MgCl2, 20mM
KCl and 2mg/mL BSA.
Preferably, positive control is haemophilus parasuis 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 method based on digital pcr technology detection haemophilus parasuis, including it is as follows
Step:
(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) digital pcr operates
1) 19 μ L of digital pcr 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 digital pcr 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 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 pcr reaction system is 20 μ L reaction systems, contains 2 × ddPCR Supermix, 10 μ L, is surpassed
9 μ L of pure water is added after 1 μ L template as 20 μ L.
Preferably, amplification program are as follows: 60 DEG C of reaction 30min；95 DEG C of reaction 5min；94 DEG C of reaction 30sec, 60 DEG C of reactions
60sec, 40 circulations；98 DEG C of reaction 10min；The warming and cooling rate of 2.5 DEG C/sec is all arranged in every step.
The present invention is to rely on droplet digital pcr (droplet digital PCR, ddPCR) technology, and exploitation is based on number
The kit and detection method of round pcr absolute quantitation detection haemophilus parasuis.There has been no by droplet digital pcr technology at present
Kit applied to detection haemophilus parasuis.This kit can realize in a short time haemophilus parasuis it is quick,
It is precisely quantitative, strong technical support is provided for the early diagnosis of Haemophilus parasuis, has broad application prospects and produces
Industry prospect.
Therefore, compared to other existing detection techniques, the technology of the present invention has many advantages, such as that accurate, sensitive, applicability is wide:
(1) result is accurate: realizing absolute quantitation, does not depend on Ct value, be not necessarily to standard curve, more accurately detects virus and contain
Amount provides accurate diagnosis basis for swine disease diagnosis；
(2) high sensitivity: detectable single copy template；
(3) applicability is wide: being applicable in complex sample detection, does not influence vulnerable to PCR mortifier.
Therefore, this method overcomes existing real-time fluorescence PCR and relies on the limitation of standard curve, it can be achieved that accurately absolutely fixed
Amount, without relying on standard curve；Sensitivity superelevation, detection limit is down to single copy, it can be achieved that the morning of Haemophilus parasuis diagnosis
It was found that, early treatment, can effectively avoid large area outburst schweineseuche disease and lead to economic loss.
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: secondary pig is thermophilic
Blood bacillus；3: the serum sample of health pig；4: Actinobacillus pleuropneumoniae；5: Streptococcus suis；6: pig source more killing property bar
Family name bacillus；7: Escherichia coli；8: salmonella；9: staphylococcus aureus.
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 pcr technology absolute quantitation detection haemophilus parasuis
Based on the kit of digital pcr technology absolute quantitation detection haemophilus parasuis, including primer sets, 2 × ddPCR
Supermix, the ultrapure water without RNA enzyme, bacteria total DNA extract reagent, positive control and negative control.
(1) digital pcr amplimer designs: carrying out by target gene of haemophilus parasuis 1-15 type specificity conserved sequence
The design of primer.Primer sequence is shown in Table 1.
1 primer sequence table of table
(2) FP primer in primer sets, BP primer and probe molar ratio be 9:9:2.
(3) 2 × ddPCR Supermix contain: 2 × PCR Buffer, 2 × Taq DNA Polymerase, 36 μM of FP
With BP primer, 20 μM of probes, 400 μM of dNTP mix and 2 × RNase Cocktail (ThermoFisher).
(4) positive control is haemophilus parasuis 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 pcr technology absolute quantitation detection haemophilus parasuis
Using the method for the kit detection haemophilus parasuis 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) digital pcr operates
1) measuring samples, negative control and positive control sample number summation are set as N, reaction system configuration is as follows:
2 digital pcr reaction system of table
| Title | System |
| 2×ddPCR Supermix | 11×(N+1)μL |
| Distilled water without RNA enzyme | 8×(N+1)μL |
After the reaction system configured above is mixed well, packing to each 19 μ L of each reaction tube.
Wherein, 2 × ddPCR Supermix contains 2 × PCR Buffer, 5 μ L, 2 × Taq DNA Polymerase 0.5
μ L, each 0.5 μ L of 36 μM of FP and BP primers, 20 μM of probes 0.2 μ L, 400 μM of dNTP mix 4 μ L and 2 × RNase
Cocktail(ThermoFisher)0.3μL。
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 (about 5 seconds) are equally slowly squeezed into 96 orifice plate corresponding position holes, pipette tips pay attention to sealing up lid close to hole wall access hole bottom
With grease proofing volatilization, used droplet is discarded every time, 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, 5sec, be not necessarily to the secondary sealer of inverted orientation；
9) PCR 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
PCR.Amplification program are as follows: 60 DEG C of reaction 30min；95 DEG C of reaction 5min；94 DEG C of reaction 30sec, 60 DEG C of reaction 60sec, 40 are followed
Ring；98 DEG C of reaction 10min.The warming and cooling rate of 2.5 DEG C/sec is all arranged in every step.
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 haemophilus parasuis when sample to be tested result >=1 copies；It is when 1 copy of sample to be tested result <
Haemophilus parasuis is negative.
3 specificity verification of embodiment
Detect clinical obtained haemophilus parasuis, porcine contagious pleuropneumonia unwrapping wire bar respectively with kit of the present invention
Bacterium, Streptococcus suis, swine Pasteurella multocida, Escherichia coli, salmonella, staphylococcus aureus sample and health pig
Serum sample, and positive control sample is set, totally 9 parts of samples, all samples pass through PCR sequencing PCR verifying, and testing result is shown in Fig. 1.
Experimental result shows that after the amplification of haemophilus parasuis clinical sample digital pcr, threshold line appears above positive liquid
Drop, that is, have amplification.And Actinobacillus pleuropneumoniae, Streptococcus suis, swine Pasteurella multocida, Escherichia coli,
The serum sample of salmonella, staphylococcus aureus sample and health pig is in threshold line hereinafter, i.e. without amplification (see Fig. 1).
It can be seen that showing good specificity using digital pcr kit detection haemophilus parasuis of the invention.
The verifying of 4 actual sample of embodiment
Actual sample is detected with kit of the present invention, the clinical obtained doubtful pathological material of disease 50 of haemophilus parasuis of detection
Part, while being verified with PCR sequencing PCR, wherein kit and PCR sequencing PCR detection of the present invention be it is positive have 35, detection method is equal
There are 15, shown in testing result table 3 for negative:
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
<120>primer and probe and its kit and method based on digital pcr technology detection haemophilus parasuis
<130> 2018
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 1
tcgccttggc cgtaattcta
<210> 2
<211> 24
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 2
acgtaagcct tttctgttgt aacatc
<210> 3
<211> 15
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 3
aaatgatgca ggatggg
Claims (10)
1. a kind of primer and probe based on digital pcr technology detection haemophilus parasuis, including upstream primer FP, downstream primer
BP, probe Probe, nucleotide sequence difference are as follows:
FP:5 '-TCGCCTTGGCCGTAATTCTA-3 '；
BP:5 '-ACGTAAGCCTTTTCTGTTGTAACATC-3 '；
Probe:5 '-FAM-AAATGATGCAGGATGGG-BHQ1-3 ', wherein the fluorophor of 5 ends label is FAM, 3 ends label
Quenching group be BHQ1.
2. the primer and probe as described in claim 1 based on digital pcr technology detection haemophilus parasuis, feature exist
In: when amplified reaction, FP primer, BP primer and probe molar ratio be 9:9:2.
3. a kind of kit based on digital pcr technology detection haemophilus parasuis, which is characterized in that kit is wanted comprising right
Seek primer and probe described in 1 or 2.
4. the kit as claimed in claim 3 based on digital pcr technology detection haemophilus parasuis, which is characterized in that also
Including some or all of of following component: (1) 2 × ddPCR Supermix；(2) without the ultrapure water of RNA enzyme；(3) positive right
According to and negative control；(4) bacteria total DNA extracts reagent.
5. the kit as claimed in claim 4 based on digital pcr technology detection haemophilus parasuis, it is characterised in that: institute
2 × ddPCR the Supermix stated contains: 2 × PCR Buffer, 2 × Taq DNA Polymerase, 36 μM of FP primers and BP
Primer, 20 μM of probes, 400 μM of dNTP mix and 2 × RNase Cocktail (ThermoFisher).
6. the kit based on digital pcr technology detection haemophilus parasuis as described in right 5, it is characterised in that: described 2
× PCR Buffer contains 20mM Tris-HCl (pH 8.3), 50mM MgCl2, 20mM KCl and 2mg/mL BSA.
7. the kit as claimed in claim 4 based on digital pcr technology detection haemophilus parasuis, it is characterised in that: sun
Property control be haemophilus parasuis DNA, negative control is deionized water.
8. a kind of method based on digital pcr technology detection haemophilus parasuis, 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) digital pcr operates
1) 19 μ L of digital pcr 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 digital pcr reaction systems；The template DNA include measuring samples,
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 <.
9. the method as claimed in claim 8 based on digital pcr technology detection haemophilus parasuis, it is characterised in that: described
(2) 19 μ L of digital pcr reaction solution contains 9 μ L of 2 × ddPCR Supermix, 10 μ L and ultrapure water in, constitutes after 1 μ L template is added
20 μ L digital pcr reaction systems.
10. the method as claimed in claim 8 based on digital pcr technology detection haemophilus parasuis, it is characterised in that: amplification
Program are as follows: 60 DEG C of reaction 30min；95 DEG C of reaction 5min；94 DEG C of reaction 30sec, 60 DEG C of reaction 60sec, 40 recycle；98 DEG C anti-
Answer 10min；The warming and cooling rate of 2.5 DEG C/sec is all arranged in every step.
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| CN117844956A (en) * | 2024-03-06 | 2024-04-09 | 赛锐思生物技术(吉林)有限公司 | Fluorescent PCR primer probe set, kit and method for quantitatively detecting haemophilus parasuis |
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