CN109593869A - A kind of RPA primer for detecting haemophilus parasuis, RPA probe, kit and nucleic acid detection method - Google Patents
A kind of RPA primer for detecting haemophilus parasuis, RPA probe, kit and nucleic acid detection method Download PDFInfo
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- CN109593869A CN109593869A CN201910118897.0A CN201910118897A CN109593869A CN 109593869 A CN109593869 A CN 109593869A CN 201910118897 A CN201910118897 A CN 201910118897A CN 109593869 A CN109593869 A CN 109593869A
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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 present invention discloses a kind of RPA primer for detecting haemophilus parasuis, RPA probe, kit and nucleic acid detection method, it is infBRPAF that wherein RPA primer, which includes forward primer, its sequence are as follows: CATTGAATCAGCWGGYCCATCAATTCCTGTGGA, reverse primer is infBRPAB, its sequence are as follows: Bio-CACTTTTACTTCTGCCGTTGAAAGCTCGTGTAAA, RPA probe wherein based on the design of the primer amplified region RPA is infBRPAP, sequence are as follows: FITC-ACTTTCAGGCGTACCAG CCGCAGGTGATGAAGHGACAGTGGTGCGTGAT-C3s Pacer.The simply and easy treat of haemophilus parasuis sample, RPA technology and Sidestream chromatography are detected and are organically combined by the present invention, by designing special RPA primer, RPA probe, can easy, fast and accurately carry out the diagnosis and detection of haemophilus parasuis.
Description
Technical field
The invention belongs to livestock product safety field of biotechnology, and in particular to a kind of for detecting haemophilus parasuis
RPA primer, RPA probe, kit and nucleic acid detection method.
Background technique
Haemophilus parasuis is also known as multiple cellulosic scrositis and arthritis, Graves disease (Glasser ' s
It disease), is the infectious disease of a boar as caused by haemophilus parasuis, clinically with body temperature raising, abscess of joint, breathing
The infectious disease that difficult, polyserositis and high mortality are characterized seriously endangers the health of piglet and young pig.Currently, secondary
Haemophilus suis disease has affected the development of pig breeding industry in the world, brings huge economic loss to pig breeding industry.
Haemophilus parasuis category Gram-negative bacillus pumilis, form is changeable, has 15 or more serotypes, wherein serotype
5,4,13 most commonly seen (accounting for 70% or more).The bacterium strictly necessary nicotinamide adenine dinucleotide (NAD) when growing, does not need
The X factor (ferroheme or other porphyrin substances), grows on blood culture medium and chocolate culture-medium, and bacterium colony is small and transparent,
Without haemolysis on blood culture medium；It is good in staphylococcus bacterium platform surrounding growth, form satellitism.It is difficult under general condition
To separate and cultivate, the pathological material of disease of sick pig especially is crossed using antibiotic treatment, thus brings difficulty to the diagnosis of this disease；According to report
Road: the True incidences of haemophilus parasuis infection may be as many as 10 times actually made a definite diagnosis.
The polyserositis due to caused by secondary pig and abscess of joint can also be by swine plague, contagious pleuropneumonia, hammers
The cause of diseases such as bacterium, brickpox cause, and therefore, are difficult to make a definite diagnosis it only according to symptom and lesion energy difficulty, it is also necessary to by laboratory
Detection method.Mainly there is bacterium to be separately cultured the detection method of haemophilus parasuis at present, round pcr etc..But above method
It is required to expensive laboratory condition, instrument and equipment and professional technique, and is takeed a long time, it is difficult to meet scene or field
Between under the conditions of detection need.
The in recent years emerging isothermal nucleic acid detection technique for having very much clinical application potentiality of RPA technology.RPA technology relies on recombination
The collective effect of enzyme, single strand binding protein and strand displacement polymerase can be carried out the amplification of nucleic acid at normal temperature, can be non-
Increase target gene with exponential in the often short time, mating mark probe and Sidestream chromatography technology can be realized and be produced to amplification
The real time monitoring of object is not required to the instrument and equipment of any profession, and easy to be quick, is very suitable for scene or field and uses,
The detection that cause of disease can be carried out in family, pet outpatient service or farm, diagnosis and prevention and control for disease have great face
Bed application value, has broad application prospects.
Haemophilus parasuis is detected using RPA technology and Sidestream chromatography technology not yet both at home and abroad at present.
Summary of the invention
In order to solve the problems, such as that existing haemophilus parasuis makes a definite diagnosis the middle instrument and equipment for needing valuableness, reagent or time-consuming, sheet
Invention provides instant, the easy and quick nucleic acid of a kind of detectable pig joint fluid, fester, peritoneal exudate and lung tissue liquid
Detection method.
The present invention is achieved by the following technical solutions.
It is a kind of for detecting the RPA primer and RPA probe of haemophilus parasuis, the RPA primer includes forward primer
For infBRPAF, sequence are as follows:
CATTGAATCAGCWGGYCCATCAATTCCTGTGGA,
Reverse primer is infBRPAB, sequence are as follows:
Bio-CACTTTTACTTCTGCCGTTGAAAGCTCGTGTAAA,
RPA probe wherein based on the design of the primer amplified region RPA is infBRPAP, sequence are as follows:
FITC-ACTTTCAGGCGTACCAGCCGCAGGTGATGAAGHGACAGTGGTGCGTGAT-C3 spacer。
The present invention also provides a kind of for detecting the RPA kit of haemophilus parasuis comprising above-mentioned RPA primer and
RPA probe, specifically, further include: (1) sample treatment liquid: 0.2M KOH solution；(2) reaction solution is detected: 50mM pH7.9's
Tris-HCl；The potassium acetate of 100mM；The dithiothreitol (DTT) of 2mM；The adenosine triphyosphate of 3mM；The phosphocreatine of 20mM；5
The creatine kinase of μ g；The polyethylene glycol of 5% mass fraction, 200 μM of deoxyribonucleoside triphosphate, RPA primer, the vinegar of 14mM
Sour magnesium；(3) dry powder pipe: the single-stranded combination egg of recombinase uvsY, 30 μ g that recombinase uvsX, 750ng of 3.75 μ g freeze-drying are lyophilized
White, 900ng strand displacement polymerase；(4) lateral flow strip；(5) positive control reaction solution: haemophilus parasuis infB gene piece
Section；The TE buffer of 10mM pH8.0；(6) negative control reaction solution: the TE buffer of 10mM pH8.0；(7) loading treatment fluid:
The PBST buffer that 0.01M pH is 7.3；(8) suction pipe；(9) asepsis injector, the concentration of above-mentioned substance are in each reagent
Final concentration.
The present invention also provides a kind of nucleic acid detection methods for detecting haemophilus parasuis, including following operating procedure:
(1) sample treatment: taking pig joint fluid, fester, peritoneal fluid or lung tissue liquid to be checked with asepsis injector, past
0.05ml is added dropwise in 3ml sample treatment liquid, sufficiently oscillation obtains sample treatment liquid after mixing；
(2) it expands: 0.05ml being added dropwise in dry powder pipe and detects reaction solution, the quantitative sample that then addition is drawn with micropipet
Product treatment fluid 5ul, while positive and negative control is set up, it mixes, dry powder pipe is placed under operator's waistband and is reacted 20 minutes, is obtained
To reaction solution；
(3) determine: being added in 2ml loading treatment fluid mix with the reaction solution that suction pipe draws 0.01ml after the reaction was completed,
Then lateral flow strip is put into progress result measurement in loading treatment fluid, observation determines result after five minutes: such as negative control sample
Only there is band on the position of C in lateral flow strip in product, and the lateral flow strip in positive control sample is in the position of C and T
On all there is band, show that test is set up, the band of the lateral flow strip in test sample occurs as positive control sample
Two bands are the positive, show to contain haemophilus parasuis in pig to be checked；Occurring a band as negative control sample is
Feminine gender shows in pig to be checked without containing haemophilus parasuis.
From the above technical scheme, it can be seen that the beneficial effects of the present invention are:
A kind of RPA primer for detecting haemophilus parasuis, RPA probe, kit and nucleic acid inspection provided by the invention
Survey method first by the simply and easy treat of haemophilus parasuis sample, then by RPA technology and Sidestream chromatography detection combination, is led to
RPA primer, the RPA probe of design are crossed, the diagnosis and detection of haemophilus parasuis can easy, be fast and accurately carried out.In clinic
Not needing any equipment can be detected, and reduced the cost of purchase specialized instrument and equipment, reduced the throwing of lab construction
Enter；Easy to operate, ordinary person can be carried out detecting, and reduce the technical threshold of detection, raiser, farm and base beast
Doctor's outpatient clinician can detect, and great convenience application and promote；Detection quickly, is can be detected in 1 hour as a result, energy
It takes measures to carry out treatment processing at the first time.
Detailed description of the invention
Fig. 1 and Fig. 2 is the testing result of the joint fluid of illness pig, and wherein Fig. 1 is the positive detection knot of first pig sample
Fruit, Fig. 2 are the negative result of second pig sample.
Specific embodiment
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.Reality used in the examples
The condition of applying can be for further adjustments according to the condition of producer, and unaccounted implementation condition is usually conventional laboratory conditions.
Embodiment 1
It is a kind of for detecting the RPA primer and RPA probe of haemophilus parasuis, the RPA primer includes forward primer
For infBRPAF, sequence are as follows:
CATTGAATCAGCWGGYCCATCAATTCCTGTGGA,
Reverse primer is infBRPAB, sequence are as follows:
Bio-CACTTTTACTTCTGCCGTTGAAAGCTCGTGTAAA,
RPA probe wherein based on the design of the primer amplified region RPA is infBRPAP, sequence are as follows:
FITC-ACTTTCAGGCGTACCAGCCGCAGGTGATGAAGHGACAGTGGTGCGTGAT-C3 spacer。
The present invention also provides a kind of for detecting the RPA kit of haemophilus parasuis comprising above-mentioned RPA primer and
RPA probe, specifically, further include: (1) sample treatment liquid: 0.2M KOH solution；(2) reaction solution is detected: 50mM pH7.9's
Tris-HCl；The potassium acetate of 100mM；The dithiothreitol (DTT) of 2mM；The adenosine triphyosphate of 3mM；The phosphocreatine of 20mM；5
The creatine kinase of μ g；The polyethylene glycol of 5% mass fraction, 200 μM of deoxyribonucleoside triphosphate, RPA primer, the vinegar of 14mM
Sour magnesium；(3) dry powder pipe: the single-stranded combination egg of recombinase uvsY, 30 μ g that recombinase uvsX, 750ng of 3.75 μ g freeze-drying are lyophilized
White, 900ng strand displacement polymerase；(4) lateral flow strip；(5) positive control reaction solution: haemophilus parasuis infB gene piece
Section；The TE buffer of 10mM pH8.0；(6) negative control reaction solution: the TE buffer of 10mM pH8.0；(7) loading treatment fluid:
The PBST buffer that 0.01M pH is 7.3；(8) suction pipe；(9) asepsis injector, the concentration of above-mentioned substance are in each reagent
Final concentration.
The present invention also provides a kind of nucleic acid detection methods for detecting haemophilus parasuis, including following operating procedure:
(1) sample treatment: taking pig joint fluid, fester, peritoneal fluid or lung tissue liquid to be checked with asepsis injector, past
0.05ml is added dropwise in 3ml sample treatment liquid, sufficiently oscillation obtains sample treatment liquid after mixing；
(2) it expands: 0.05ml being added dropwise in dry powder pipe and detects reaction solution, the quantitative sample that then addition is drawn with micropipet
Product treatment fluid 5ul, while positive and negative control is set up, it mixes, dry powder pipe is placed under operator's waistband and is reacted 20 minutes, is obtained
To reaction solution；
(3) determine: being added in 2ml loading treatment fluid mix with the reaction solution that suction pipe draws 0.01ml after the reaction was completed,
Then lateral flow strip is put into progress result measurement in loading treatment fluid, observation determines result after five minutes: such as negative control sample
Only there is band on the position of C in lateral flow strip in product, and the lateral flow strip in positive control sample is in the position of C and T
On all there is band, show that test is set up, the band of the lateral flow strip in test sample occurs as positive control sample
Two bands are the positive, show to contain haemophilus parasuis in pig to be checked；Occurring a band as negative control sample is
Feminine gender shows in pig to be checked without containing haemophilus parasuis.
Test:
It is past to fill 3ml with the joint fluid of the sterile illness pig for taking 2 doubtful haemophilus parasuis infection of asepsis injector
0.05ml (drop) is added dropwise in the processing pipe of sample treatment liquid, concussion mixes, and places 5min, obtains sample treatment liquid, then use
Micropipet draws 5ul sample treatment liquid, and dry powder pipe is added in detection reaction solution together, and mixing is placed under operator's waistband anti-
It answers 20 minutes, obtains reaction solution, extract reaction solution 0.01ml after the completion of amplification and be put into loading treatment fluid and mix, by Sidestream chromatography
Test strips are inserted into loading treatment fluid, observe result after five minutes.As a result negative and positive control is all set up, in detection first
Occur two detection lines on the lateral flow strip of pig sample, shows that it has infected haemophilus parasuis.The joint of the pig sample
Liquid is extracted through conventional gene, PCR and electrophoresis detection result are also positive.
Certainly, the above description is not a limitation of the present invention, and the present invention is also not limited to the example above, the art
Those of ordinary skill, within the essential scope of the present invention, variation, change, addition or the replacement made all should belong to the present invention
Protection scope.
Claims (4)
1. a kind of for detecting the RPA primer and RPA probe of haemophilus parasuis, which is characterized in that the RPA primer includes
Forward primer is infBRPAF, sequence are as follows:
CATTGAATCAGCWGGYCCATCAATTCCTGTGGA,
Reverse primer is infBRPAB, sequence are as follows:
Bio-CACTTTTACTTCTGCCGTTGAAAGCTCGTGTAAA,
RPA probe wherein based on the design of the primer amplified region RPA is infBRPAP, sequence are as follows:
FITC-ACTTTCAGGCGTACCAGCCGCAGGTGATGAAGHGACAGTGGTGCGTGAT-C3 spacer。
2. a kind of for detecting the RPA kit of haemophilus parasuis, which is characterized in that it includes RPA described in claim 1
Primer and RPA probe.
3. according to claim 2 a kind of for detecting the RPA kit of haemophilus parasuis, which is characterized in that also wrap
It includes: (1) sample treatment liquid: 0.2M KOH solution；(2) reaction solution: the Tris-HCl of 50mM pH7.9 is detected；The acetic acid of 100mM
Potassium；The dithiothreitol (DTT) of 2mM；The adenosine triphyosphate of 3mM；The phosphocreatine of 20mM；The creatine kinase of 5 μ g；5% mass
The polyethylene glycol of score, 200 μM of deoxyribonucleoside triphosphate, RPA primer, the magnesium acetate of 14mM；(3) dry powder pipe: 3.75 μ
Single strand binding protein, the 900ng strand displacement polymerase of recombinase uvsY, 30 μ g that recombinase uvsX, 750ng of g freeze-drying are lyophilized；
(4) lateral flow strip；(5) positive control reaction solution: haemophilus parasuis infB genetic fragment；The TE of 10mM pH8.0 is buffered
Liquid；(6) negative control reaction solution: the TE buffer of 10mM pH8.0；(7) loading treatment fluid: the PBST that 0.01M pH is 7.3 is slow
Fliud flushing；(8) suction pipe；(9) asepsis injector, the concentration of above-mentioned substance are the final concentration in each reagent.
4. a kind of nucleic acid detection method for detecting haemophilus parasuis, which is characterized in that including following operating procedure:
(1) sample treatment: taking pig joint fluid, fester, peritoneal fluid or lung tissue liquid to be checked with asepsis injector, toward 3ml sample
0.05ml is added dropwise in product treatment fluid, sufficiently oscillation obtains sample treatment liquid after mixing；
(2) it expands: 0.05ml being added dropwise in dry powder pipe and detects reaction solution, at the quantitative sample that then addition is drawn with micropipet
Liquid 5ul is managed, while setting up positive and negative control, mixes, dry powder pipe is placed under operator's waistband and is reacted 20 minutes, is obtained anti-
Answer liquid；
(3) determine: being added in 2ml loading treatment fluid mix with the reaction solution that suction pipe draws 0.01ml after the reaction was completed, then
Lateral flow strip is put into progress result measurement in loading treatment fluid, observation determines result after five minutes: in negative control sample
Lateral flow strip only occur band on the position of C, the lateral flow strip in positive control sample on the position of C and T all
There is band, shows that test is set up, the band of the lateral flow strip in test sample occurs two as positive control sample
Band is the positive, shows to contain haemophilus parasuis in pig to be checked；Occurring a band as negative control sample is feminine gender,
Show in pig to be checked without containing haemophilus parasuis.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110066885A (en) * | 2019-04-30 | 2019-07-30 | 南京林业大学 | A kind of primer and probe combination and its application based on RPA- Sidestream chromatography technology detection chestnut Heisui River phytophthora |
| CN110205395A (en) * | 2019-06-17 | 2019-09-06 | 武汉科前生物股份有限公司 | Serotype method, primer combination and the PCR system of haemophilus parasuis |
| CN111139307A (en) * | 2019-12-18 | 2020-05-12 | 江苏海洋大学 | Specific primer pair for rapidly detecting vibrio alginolyticus by RPA-LFS, kit and application thereof |
| CN115029460A (en) * | 2022-07-05 | 2022-09-09 | 长江大学 | Instant visual detection method for haemophilus parasuis |
| 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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| CN104946749A (en) * | 2015-06-01 | 2015-09-30 | 山东省农业科学院奶牛研究中心 | Universal primers and probe for on-site rapid detection of Brucella and kit |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110066885A (en) * | 2019-04-30 | 2019-07-30 | 南京林业大学 | A kind of primer and probe combination and its application based on RPA- Sidestream chromatography technology detection chestnut Heisui River phytophthora |
| CN110205395A (en) * | 2019-06-17 | 2019-09-06 | 武汉科前生物股份有限公司 | Serotype method, primer combination and the PCR system of haemophilus parasuis |
| CN111139307A (en) * | 2019-12-18 | 2020-05-12 | 江苏海洋大学 | Specific primer pair for rapidly detecting vibrio alginolyticus by RPA-LFS, kit and application thereof |
| CN115029460A (en) * | 2022-07-05 | 2022-09-09 | 长江大学 | Instant visual detection method for haemophilus parasuis |
| 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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Application publication date: 20190409 |