CN107164511A - A kind of method of the type of quick detection haemophilus parasuis serum 4 - Google Patents
A kind of method of the type of quick detection haemophilus parasuis serum 4 Download PDFInfo
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Abstract
The present invention by providing a kind of specific gene fragment of type of haemophilus parasuis serum 4 and with specific primer sets, and its with include above-mentioned primer sets kit detect sample in the presence or absence of the type specificity genetic fragment of haemophilus parasuis serum 4 so that determine sample whether be the type of haemophilus parasuis serum 4.Detection reagent and detection method of the present invention have sensitiveness height, and (detection sensitivity to the type genomic DNA of haemophilus parasuis serum 4 is 1.0pg, and the detection sensitivity to the type bacterium solution of haemophilus parasuis serum 4 is 1.6 × 102Cfu/mL), high specificity, it is convenient and swift, do not need specific apparatus, it is applied widely the advantages of, it is possible to resolve the field quick detection of the type of haemophilus parasuis serum 4 and the problem of basic unit's popularization and application.
Description
Technical field
It is bloodthirsty more particularly, to a kind of secondary pig of quick detection the present invention relates to the quick determination method technical field of pathogen
The method of the type of bacillus serum 4.
Background technology
Haemophilus parasuis (Haemophilus parasuis, HPS) is a kind of important porcine respiratory pathogen, at present
At least identify and there is the significance differences such as high virulence, medium virulence and avirulence between 15 serotypes, different serotypes bacterial strain
Different, identification HPS serotypes are to the sick diagnosis and prevent and treat significant.The type of HPS serum 4 is supper toxic strain, is me again
State is currently a popular at most, one of distribution most wide serotype.The current bacterium routine serum type authentication method is bacteria distribution
On the basis of culture and identification, reacted to judge blood by the positive serum of agar gel diffusion test and all serotypes of HPS
Clear type, it usually needs the time of 3-5 days, waste time and energy, and there are about 30% HPS isolated strains can not be by precise Identification bleeding
Clear type, has a strong impact on quick diagnosis and the detection of Haemophilus parasuis.
Loop-mediated isothermal amplification technique (LAMP, International Patent Publication No. WO 00/28082) is that Notomi in 2000 etc. is opened
A kind of nucleic acid amplification new technology sent, i.e. 6 regions for target gene design 4 specific primers (if desired for can be with
Add ring primer pair), protected using a kind of strand displacement archaeal dna polymerase (Bst DNApolymerase) in 65 DEG C or so constant temperatures
Temperature about 60 minutes, you can complete nucleic acid amplification reaction, amplification can be detected by gel electrophoresis.Expand for LAMP technology
The 2 pairs of primers increased are 6 sections for target-gene sequence, thus with the specificity higher than PCR, are not required to while also possessing
Want thermal cycle, amplification efficiency it is higher, do not need specific apparatus the advantages of.
The epidemiological study of haemophilus parasuis infection is mainly carried out using serological typing method, i.e., separated in HPS
On the basis of culture and identification, reacted to judge blood by the positive serum of agar gel diffusion test and all serotypes of HPS
Clear type, it usually needs the time of 3-5 days, waste time and energy, and there are about 30% HPS isolated strains can not be by precise Identification bleeding
Clear type, has a strong impact on quick diagnosis and the detection of Haemophilus parasuis.
The content of the invention
The technical purpose of the present invention is to set up a kind of easy, quick, HPS blood of high sensitivity, high specific, high accuracy
Clear 4 type LAMP detection method.
Rapidly and accurately to identify the type of HPS serum 4, the haemophilus parasuis SW124 bacterium that the present invention is delivered with reference to GenBank
Lipopolysaccharides synthetic proteins (lipopolysaccharide biosynthesis protein, wciP4) gene order of strain (is stepped on
Record KC795356.1), it is online to carry out LAMP primer design with primer-design software Primer Explorer 4.0.According to
Design of primers principle, 4 primers are designed for its 6 specific regions：Two outer primers be respectively upstream outer primer (F3) and under
Swim outer primer (B3)；Two inner primers are respectively upstream inner primer (FIP) and downstream inner primer (BIP), FIP by F1 complementary sequence
Row and positive sequence F2 are constituted, and BIP is made up of B1 complementary series and positive sequence B 2, and target fragment is upstream outer primer
Gene order between F3 and downstream outer primer B3.According to the primer of the designs of Primer Explorer 4.0, multiple targets can be obtained
Genetic fragment, then carries out sequence alignment with BLAST, filters out the high and highly conserved target gene of wciP4 gene specifics
Fragment (as shown in SEQ ID No.5).Novelty finds that this wciP4 gene-specific fragment is expanded through LAMP method, only in perseverance
(62 DEG C) are reacted 60 minutes under the conditions of temperature, you can expand target DNA to 10 from several copies9Individual copy, passes through electrophoresis detection
Amplified production carrys out result of determination.So a kind of molecular labeling of the type method of utilization LAMP technology quick detection HPS serum 4 can be used as
Use.
On this basis, the present invention further provides one group of loop-mediated isothermal amplification (LAMP) primer, it can specific ring mediation etc.
The described molecular labeling of temperature amplification.According to one kind preferred embodiment, the primer sets include upstream outer primer F3 and downstream
Outer primer B3, its nucleotide sequence is as shown in SEQ ID No.1-2；And upstream inner primer FIP and downstream inner primer BIP, its
Nucleotide sequence is as shown in SEQ ID No.3-4.
Further, the present invention provides a kind of method of the type of quick detection HPS serum 4, with testing sample cellular genome
DNA is template, and loop-mediated isothermal amplification is carried out using above-mentioned specific primer group.The testing sample cellular genome
Testing sample can be extracted bacterial genomes DNA by DNA after conventional Bacteria Culture.
Described method, in addition to step：Negative control template and positive control template are set, and negative control template is super
Pure water, positive control template is the type reference strain HS79 genomic DNAs of HPS serum 4；Ring mediated isothermal is expanded using gel electrophoresis
Volume increase thing is detected that electrophoresis result characteristic gradient shape electrophoretic band occurs and is determined as the positive, if without any amplified production
For feminine gender.
The reaction system of the loop-mediated isothermal amplification includes：10×LAMP bufffer：2.5μL；10mmol/L
DNTPs：3.0μL；0.25mol/L MgSO4：0.2μL；5mol/L Betaine：0.5μL；10 μm of ol/L FIP primers：
2.0μL；10 μm of ol/L BIP primers：2.0μL；10 μm of ol/L F3 primers：0.5μL；10 μm of ol/L B3 primers：0.5μL；
8U/ μ L Bst archaeal dna polymerases：0.7μL；Template DNA：1.0μL；System is supplied with sterilizing ultra-pure water to 25 μ L；
Wherein 10 × LAMP bufffer are constituted：200mmol/L Tris-HCl, 100mmol/L KCl, 20mmol/L
MgSO4, 100mmol/L (NH4)2SO4, 1.0%Tritonx-100.
The reaction condition of the loop-mediated isothermal amplification is as follows：95 DEG C of denaturation 5min, take out sample ice bath, add
Bst archaeal dna polymerases, then 62 DEG C are reacted 60min, last 80 DEG C of reactions 10min terminating reactions.
The reagent preferred fabrication used in methods described is the kit of the type of quick detection HPS serum 4, including described special
Property primer sets.
Preferably include：
10×LAMP bufffer：2.5μL；10mmol/L dNTPs：3.0μL；0.25mol/L MgSO4：0.2μL；
5mol/L Betaine：0.5μL；10 μm of ol/L FIP primers：2.0μL；10 μm of ol/L BIP primers：2.0μL；10μmol/
L F3 primers：0.5μL；10 μm of ol/L B3 primers：0.5μL；8U/ μ L Bst archaeal dna polymerases：0.7μL；Template DNA：1.0
μL；System is supplied to 25 μ L with sterilizing ultra-pure water when using；
Wherein 10 × LAMP bufffer are constituted：200mmol/L Tris-HCl, 100mmol/L KCl, 20mmol/L
MgSO4, 100mmol/L (NH4)2SO4, 1.0%Tritonx-100.
The present invention by providing a kind of specific gene fragment of type of HPS serum 4 and with specific primer sets, and its
Detected with the kit for including above-mentioned primer sets and whether there is the type specificity genetic fragment of HPS serum 4 and then determination in sample
Whether sample is the type of HPS serum 4.Detection reagent and detection method of the present invention have sensitiveness height (to the type genome of HPS serum 4
DNA detection sensitivity is 1.0pg, and the detection sensitivity to the type bacterium solution of haemophilus parasuis serum 4 is 1.6 × 102cfu/
ML), high specificity (only detect that the type reference strain of HPS serum 4 and isolated strains are the positive, and the other serotypes of HPS are referred to and
The Bacteria Detection of isolated strains and other kinds is feminine gender), it is convenient and swift (entirely detect and completed in 3 hours, it is and existing
Some agar gel diffusion tests detection serotype is compared time saving 2-3 days), do not need specific apparatus (can be anti-in thermostat water bath
Should), (be applied to animal doctor's scientific research testing laboratory, inspection and quarantine department, breeding enterprise etc.) applied widely the advantages of, it is possible to resolve
The field quick detection of the type of HPS serum 4 and the problem of basic unit's popularization and application.
Brief description of the drawings
Fig. 1：LAMP detects the specific outcome of HPS different serotypes bacterial strains, wherein M：DL2000DNA Marker；1-17
The template used corresponding bacterial strain in hole is consistent with the bacterial strain sequence number in table 3, wherein there is positive band in the 5th hole, it is the type of HPS serum 4
Reference strain HS79.
Fig. 2：LAMP detects the specific outcome of other kind bacterium bacterial strains, wherein M：DL2000DNA Marker；1-20
The template used corresponding bacterial strain in hole is consistent with the bacterial strain sequence number in table 4, and the 20th hole is the type reference strain HS79 conducts of HPS serum 4
Positive control.
Fig. 3：The susceptibility results of the type genomic DNA of LAMP detection HPS serum 4, wherein M：DL2000DNA Marker；Mould
Plate DNA amount is marked above swimming lane respectively, and the detection of the testing result display present invention is limited to 1.0pg.
Fig. 4：The susceptibility results of the type bacterium solution of LAMP detection HPS serum 4, wherein M：DL2000DNA Marker；On swimming lane
The dilution factor for being labeled as bacterium solution in face, the detection of the testing result display present invention is limited to 4 bacterium, and sensitiveness is 0.16cfu/ μ L
(1.6×102cfu/mL)。
Fig. 5：LAMP detections are clinically separated HPS bacterial strain results, wherein M：DL2000；The template used corresponding bacterial strain in 1-21 holes
Consistent with the bacterial strain sequence number in table 5, as a result display only has 1-14 this type bacterial strain of 14 plants of HPS serum 4 in duct typical scalariform occur
Band, other serological type strains are feminine gender.
Embodiment
Following examples further illustrate present disclosure, but should not be construed as limiting the invention.Without departing substantially from
In the case of spirit and essence of the invention, the modifications or substitutions made to the inventive method, step or condition belong to the present invention
Scope.
Unless otherwise specified, the conventional meanses that technological means used in embodiment is well known to those skilled in the art.
The design and synthesis of the LAMP primer of embodiment 1
With reference to the lipopolysaccharides synthetic proteins of the GenBank haemophilus parasuis SW124 bacterial strains delivered
(lipopolysaccharide biosynthesis protein, wciP4) gene order (accession number KC795356.1), fortune
It is online to carry out LAMP primer design with primer-design software Primer Explorer 4.0.According to design of primers principle, for
Design 4 primers in its 6 specific regions：Two outer primers are respectively upstream outer primer (F3) and downstream outer primer (B3)；Two
Inner primer is respectively upstream inner primer (FIP) and downstream inner primer (BIP), FIP by F1 complementary series and positive sequence F2 structures
Into BIP is made up of B1 complementary series and positive sequence B 2, and target fragment is upstream outer primer F3 and downstream outer primer B3
Between gene order.According to the primer of the designs of Primer Explorer 4.0, multiple target fragments, Ran Houyun can be obtained
Sequence alignment is carried out with BLAST, the high and highly conserved target fragment of wciP4 gene specifics (such as SEQ ID are filtered out
Shown in No.5).Primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd., and Primer and sequence are shown in Table 1.
The LAMP the primers title of table 1 and sequence
The HPS bacterial genomes DNA that embodiment 2 is used as template is extracted
The HPS bacterial strains being separately cultured are taken, line is incubated at addition final concentration 100 μ g/mL NAD tryptic soy agar
(TSA) on flat board, the overnight incubation in 37 DEG C of incubators.From culture plate, picking single bacterium colony is inoculated in 5mL addition final concentrations 100
In μ g/mL NAD tryptic soy broth (TSB) culture medium, overnight incubation is shaken in 37 DEG C of shaking tables.Take the bacterium of incubated overnight
Supernatant is abandoned after liquid 1ml, centrifugation, bacterium precipitation is resuspended in 50 μ L ultra-pure waters, boiled after 5min cracking bacteriums, centrifuging and taking supernatant
1.0 μ L are used for LAMP reaction templates.
The foundation of the LAMP reaction systems of embodiment 3
LAMP reaction systems total amount is 25 μ L, and reactive component is shown in Table 2.
The component and addition of the LAMP reaction systems of table 2
LAMP reactions are carried out in PCR instrument, 95 DEG C first denaturation 5min, are taken out sample ice bath, are added Bst DNA polymerizations
Enzyme, then 62 DEG C are reacted 60min, last 80 DEG C of reactions 10min terminating reactions.LAMP is expanded using agarose gel electrophoresis and produced
Thing is detected that electrophoresis result characteristic gradient shape electrophoretic band occurs and is determined as the positive, is the moon if without any amplified production
Property.
The foundation of the specific detection method of embodiment 4
(1) Bacteria Culture.The HPS isolated strains for intending identification serotype are taken, the 5mL addition μ g/mL cigarettes of final concentration 100 are inoculated in
In tryptic soy broth (TSB) culture medium of acid amides adenine-dinucleotide (NAD), overnight incubation is shaken in 37 DEG C of shaking tables.
(2) bacterial genomes DNA extraction.The bacterium solution 1ml of incubated overnight is taken, supernatant is abandoned after centrifugation, bacterium precipitation is resuspended
In 50 μ L ultra-pure waters, boil after 5min cracking bacteriums, the μ L of centrifuging and taking supernatant 1.0 are used as LAMP reaction templates.
(3) the LAMP detections of HPS bacterial strains.Will be other in addition to Bst archaeal dna polymerases according to the LAMP reaction systems in table 2
Component is added in 0.2mL reaction tubes, is put into PCR instrument, 95 DEG C of denaturation 5min, is taken out sample ice bath, is added Bst DNA polymerizations
Enzyme, then 62 DEG C are reacted 60min, last 80 DEG C of reactions 10min terminating reactions.Negative control and positive control are set simultaneously.
(4) electrophoresis detection of LAMP amplified productions.Detected using agarose gel electrophoresis：Take 9 μ L LAMP amplification productions
Thing adds 1 10 × sample-loading buffers of μ L to mix, using DL2000DNA Marker as Relative molecular weight markers, with containing nucleic acid staining
1.5% Ago-Gel of agent electrophoresis about 30min under 100V voltages, the photographic analysis result in gel imaging system.Electrophoresis
Picture shows LAMP characteristic gradient shape bands, then result is the positive；Result is feminine gender if without any band.
The specificity experiments of embodiment 5
The HPS different serotypes bacterial strains for taking glycerine to preserve, line is incubated at addition final concentration 100 μ g/mL NAD pancreatin
On soy agar (TSA) flat board, the overnight incubation in 37 DEG C of incubators.From culture plate, picking single bacterium colony is inoculated in 5mL additions
In the μ g/mL NAD of final concentration 100 tryptic soy broth (TSB) culture medium, overnight incubation is shaken in 37 DEG C of shaking tables.For with
Other kind bacterium bacterial strains between identification LAMP strains used in specific test, the corresponding bacterium for equally taking qs glycerin to preserve
Strain, line is incubated at TSA flat boards, the overnight incubation in 37 DEG C of incubators.From culture plate, picking single bacterium colony is inoculated in 5mL TSB
In culture medium, overnight incubation is shaken in 37 DEG C of shaking tables.Utilize the bacterium complete genome DNA extracts kit of Promega companies
Bacterial genomes DNA is extracted, operating procedure summary is：2mL is taken to cultivate 12~16h bacterium solution to 1.5mL centrifuge tubes；13,
000rpm centrifuges 2min, abandons net supernatant, leaves and takes bacterial sediment；600 μ L Nuclei Lysis Solution are added, are gently blown and beaten
It is resuspended to precipitation；80 DEG C of culture 5min, make cellular lysate, then allow to cool to room temperature；3 μ L RNase Solution are added,
It is reverse 2~5 times, mix；37 DEG C of culture 30min of water-bath, and it is cooled to room temperature；Add 200 μ L Protein
Precipitation Solution, high speed vortex oscillation 20s；Sample is placed into 5min on ice；13,000rpm is centrifuged
10min；Supernatant containing DNA is transferred to a clean 1.5mL centrifuge tube equipped with 600 μ L isopropanols；Gently run
Mix until there is linear DNA；13,000rpm centrifuges 5min；Supernatant is fallen off, and with clean blotting paper by pipe
Raffinate is blotted；600 μ L70% ethanol are added, are gently overturned several times；13,000rpm centrifugation 2min, carefully suction out ethanol；Will centrifugation
10~the 15min that dries in the air in atmosphere is managed, to ethanol evaporating completely；Add 100 μ L DNA Rehydration Solution, 65 DEG C
1h aquation DNA are cultivated, and periodically beat centrifuge tube, also aquation DNA can be stayed overnight at 4 DEG C；The DNA of extraction is stored in -20 DEG C.Carry
The template DNA taken analyzes its quality and concentration by gel electrophoresis and ultraviolet specrophotometer.Nucleic acid concentration is determined：With ultraviolet point
Light photometer determines ultraviolet absorption value of the nucleic acid at 260nm and 280nm respectively, with the ultraviolet absorption value at 260nm and 280nm
Ratio calculation purity, the OD260/280 of high quality DNA value is 1.8 or so.Bacterial genomes DNA integrity mensuration's：Take 3 μ
The template DNA solution electrophoresis that L is extracted, the running gel of selection is 1.0% Ago-Gel, and DNA is detected under ultraviolet projectoscope
Size, DNA integrality is judged according to its brightness and anemostat degree.
(1) LAMP detects the specificity of HPS different serotypes bacterial strains
Respectively using the reference strain genomic DNA of 17 plants of haemophilus parasuis different serotypes in table 3 as template, profit
Expanded with LAMP reaction systems and program, HPS serotype specificities are identified by agarose gel electrophoresis.Testing result is shown in
Have in table 3 and the type reference strain HS79 of Fig. 1, HPS serum 4 hole in obvious stepped band, other serum type holes without band
Occur, show the LAMP reaction systems of the present invention has high specific to HPS different serotypes bacterial strains.
(2) LAMP detects the specificity of other kind bacterium bacterial strains
It is anti-using the LAMP after optimization respectively using 19 plants of other kind bacterium bacterial strain genomic DNAs in table 4 as template
Answer system and program to be expanded, specificity of the LAMP method to other kind bacteriums is identified by agarose gel electrophoresis.Inspection
Survey, which the results are shown in Table, obvious stepped band, other kind bacteriums in 4 and Fig. 2, the type reference strain HS79 of HPS serum 4 hole
Occur in bacterial strain hole without band, show the LAMP reaction systems of the present invention has high specific to other kind bacterium bacterial strains.
The LAMP of table 3 detects the specificity of HPS different serotypes bacterial strains
Note："-" is feminine gender in testing result；"+" is the positive.
The LAMP of table 4 detects the specificity of other kind bacterium bacterial strains
Note："-" is feminine gender in testing result；"+" is the positive.
The sensitivity experiments of embodiment 6
(1) LAMP detects the sensitiveness of the type genomic DNA of HPS serum 4
The type reference strain HS79 of HPS serum 4 for taking glycerine to preserve, line is incubated at addition final concentration 100 μ g/mL NAD's
On tryptic soy agar (TSA) flat board, the overnight incubation in 37 DEG C of incubators.From culture plate, picking single bacterium colony is inoculated in 5mL
In tryptic soy broth (TSB) culture medium for adding the μ g/mL NAD of final concentration 100, overnight incubation is shaken in 37 DEG C of shaking tables.Profit
Bacterial genomes DNA is extracted with the bacterium complete genome DNA extracts kit of Promega companies, operating procedure summary is：Take
2mL cultivates 12~16h bacterium solution to 1.5mL centrifuge tubes；13,000rpm centrifugation 2min, abandon net supernatant, leave and take bacterial sediment；Plus
Enter 600 μ L Nuclei Lysis Solution, gently piping and druming to precipitation is resuspended；80 DEG C of culture 5min, make cellular lysate, then
Allow to cool to room temperature；3 μ L RNase Solution are added, are overturned 2~5 times, are mixed；37 DEG C of culture 30min of water-bath, and it is cold
But to room temperature；Add 200 μ L Protein Precipitation Solution, high speed vortex oscillation 20s；By sample in ice
Upper placement 5min；13,000rpm centrifuges 10min；By the supernatant containing DNA be transferred to one it is clean different equipped with 600 μ L
The 1.5mL centrifuge tubes of propyl alcohol；Gently overturn and mix until there is linear DNA；13,000rpm centrifuges 5min；Fall off supernatant
Liquid, and blotted raffinate in pipe with clean blotting paper；The ethanol of 600 μ L 70% is added, is gently overturned several times；13,000rpm from
Heart 2min, carefully suctions out ethanol；Centrifuge tube is dried in the air 10~15min in atmosphere, to ethanol evaporating completely；Add 100 μ L DNA
Rehydration Solution, 65 DEG C of culture 1h aquation DNA, and centrifuge tube is periodically beaten, also it can stay overnight aquation DNA at 4 DEG C；
The DNA of extraction is stored in -20 DEG C.The template DNA of extraction by gel electrophoresis and ultraviolet specrophotometer analyze its quality and
Concentration.Nucleic acid concentration is determined：Determine ultraviolet absorption value of the nucleic acid at 260nm and 280nm respectively with ultraviolet specrophotometer, with
The ratio calculation purity of ultraviolet absorption value at 260nm and 280nm, the OD260/280 of high quality DNA value is 1.8 or so.Carefully
Bacterium genomic DNA integrity mensuration'：The template DNA solution electrophoresis for taking 3 μ L to extract, the running gel of selection is 1.0% agarose
Gel, DNA size is detected under ultraviolet projectoscope, DNA integrality is judged according to its brightness and anemostat degree.
Using type reference strain HS79 genomic DNAs of HPS serum 4 that concentration is 100ng/ μ L as original template, respectively with super
Pure water is serially diluted, and each gradient respectively takes 1 μ L to add in reaction tube, bacterial genomes DNA amount difference in reaction system
For 100ng, 10ng, 1ng, 100pg, 10pg, 5pg, 2.5pg, 1.0pg, 0.1pg, 0.05pg, 0.01pg, 0.001pg, with super
Pure water identifies susceptibility results after amplification as negative control by agarose gel electrophoresis.As a result as shown in figure 3, being swum from the 7th
There is the typical stepped electrophoretic band of LAMP reactions in road to the 14th swimming lane, and with the increase of template DNA concentration, band
Brightness gradually strengthens.As a result show, the LAMP method bottom line that the present invention is set up can detect the 1.0pg type of HPS serum 4 ginseng
Bacterial strain HS79 genomic DNAs are examined, and with the increase of template DNA concentration, LAMP amplified production amounts also increase therewith.
(2) LAMP detects the sensitiveness of the type bacterium solution of HPS serum 4
The type reference strain HS79 of HPS serum 4 for taking glycerine to preserve, line is incubated at addition final concentration 100 μ g/mL NAD's
On TSA flat boards, the overnight incubation in 37 DEG C of incubators.From culture plate, picking single bacterium colony is inoculated in 5mL addition final concentrations 100 μ
In g/mL NAD TSB culture mediums, overnight incubation is shaken in 37 DEG C of shaking tables.Bacterial concentration OD600 is measured through spectrophotometer
For 0.6, by bacterium solution ultra-pure water doubling dilution to 10-11, from 10-6、10-7With 10-8Each 100 μ L that take out are coated in TSA respectively in pipe
On flat board, the clump count of bacterium is calculated after incubated overnight.Simultaneously 1 μ L are taken out from each dilution factor pipe to carry out as template DNA
LAMP is expanded, and susceptibility results are identified by agarose gel electrophoresis after amplification.As a result as shown in figure 4, from the 9th swimming lane to the 13rd
Swimming lane has typical scalariform band, and bottom line can at least detect 10-5.Plate count result is shown in 10-7In culture medium
It is 80cfu/mL, 10-6It is 400cfu/mL in culture medium, multiple proportions calculates and obtains 10-5About 4000cfu/mL, template taken amount is 1 μ L,
So the minimum bacteria containing amount of the LAMP reaction detections is 4 bacterium, i.e., sensitivity is 0.16cfu/ μ L (1.6 × 102cfu/mL)。
The LAMP of embodiment 7 detections are clinically separated HPS bacterial strains
21 plants of HPS clinical separation strains (identifying serotype using agar gel diffusion test) in table 5 are taken, respectively incubated overnight
After take supernatant abandoned after bacterium solution 1ml, centrifugation, bacterium precipitation is resuspended in 50 μ L ultra-pure waters, is boiled after 5min cracking bacteriums, centrifuging and taking
The μ L of supernatant 1.0 are used for LAMP reaction template DNAs.Expanded with the LAMP reaction systems and program after optimization, nov nucleic acid
Swimming observation LAMP amplifications.LAMP testing results are as shown in figure 5, in all HPS isolated strains, the only 14 plants types of serum 4
There is typical scalariform band in bacterial strain, and other serological type strains are negative reaction, the LAMP detection sides for pointing out the present invention to set up
Method can be used for the detection and identification of the type of clinic HPS separation strains serum 4.
The LAMP of table 5 detects the result for being clinically separated HPS different serotypes bacterial strains
Note："-" is feminine gender in testing result；"+" is the positive.
Sequence table
<110>Beijing City Agriculture and Forestry Institute
<120>A kind of method of the type of quick detection haemophilus parasuis serum 4
<130> P1710133
<160> 5
<170> PatentIn version 3.5
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<210> 4
<211> 58
<212> DNA
<213>Artificial sequence
<400> 4
gtggaaagat tgtatcatgt acataaaaag acctttgata ctagattgaa tagcaaga 58
<210> 5
<211> 224
<212> DNA
<213>WciP4 genetic fragments
<400> 5
agagtttctt tttcgagaaa atataaaaat tcttaattcc cctttttatg gattcttttt 60
atatcgaacg tgtagttatt tggtttctag agctgttgtg gaaagattgt atcatgtaca 120
taaaaagttt tgttatttag cagatgattg ggggaatctt gctattcaat ctagtatcaa 180
aggttttaca tattgtaaga ttttttctca tcctgaagat ttat 224
Claims (10)
1. type lipopolysaccharides synthetic proteins (wciP4) gene of haemophilus parasuis serum 4 is quickly examined in loop-mediated isothermal amplification technique
The application surveyed in the type method of haemophilus parasuis serum 4.
2. a kind of DNA molecular marker for ring mediated isothermal amplification, its sequence is as shown in SEQ ID No.5.
3. one group of loop-mediated isothermal amplification (LAMP) primer, the molecular labeling that it can be described in specific ring mediated isothermal amplification claim 2.
4. primer sets according to claim 3, including upstream outer primer F3 and downstream outer primer B3, its nucleotide sequence is such as
Shown in SEQ ID No.1-2；And upstream inner primer FIP and downstream inner primer BIP, its nucleotide sequence such as SEQ ID No.3-
Shown in 4.
5. primer sets are quickly examined in loop-mediated isothermal amplification technique described in molecular labeling described in claim 2 or claim 3 or 4
The application surveyed in the type method of haemophilus parasuis serum 4.
6. a kind of method of the type of quick detection haemophilus parasuis serum 4, it is characterised in that with testing sample cellular genome
DNA is template, and loop-mediated isothermal amplification is carried out using the specific primer group described in claim 3 or 4；It is preferred that, also
Including step：Negative control template and positive control template are set, and negative control template is ultra-pure water, and positive control template is pair
The type reference strain HS79 genomic DNAs of haemophilus suis serum 4；Ring mediated isothermal amplification product is examined using gel electrophoresis
Survey, electrophoresis result characteristic gradient shape electrophoretic band occurs and is determined as the positive, be feminine gender if without any amplified production.
7. method according to claim 6, it is characterised in that the reaction system bag of the loop-mediated isothermal amplification
Include：10×LAMP bufffer：2.5μL；10mmol/L dNTPs：3.0μL；0.25mol/L MgSO4：0.2μL；5mol/L
Betaine：0.5μL；10 μm of ol/L FIP primers：2.0μL；10 μm of ol/L BIP primers：2.0μL；10 μm of ol/L F3
Primer：0.5μL；10 μm of ol/L B3 primers：0.5μL；8U/ μ L Bst archaeal dna polymerases：0.7μL；Template DNA：1.0μL；With
Sterilizing ultra-pure water supplies system to 25 μ L；
Wherein 10 × LAMP bufffer are constituted：200mmol/L Tris-HCl, 100mmol/L KCl, 20mmol/L MgSO4,
100mmol/L(NH4)2SO4, 1.0%Tritonx-100.
8. the method according to claim 6 or 7, it is characterised in that the reaction condition of the loop-mediated isothermal amplification
It is as follows：95 DEG C of denaturation 5min, take out sample ice bath, add Bst archaeal dna polymerases, then 62 DEG C of reaction 60min, and last 80 DEG C anti-
Answer 10min terminating reactions.
9. primer sets are quickly examined in loop-mediated isothermal amplification technique described in molecular labeling described in claim 2 or claim 3 or 4
The application surveyed in the type kit of haemophilus parasuis serum 4.
10. a kind of kit of the type of quick detection haemophilus parasuis serum 4, it includes specificity described in claim 3 or 4 and drawn
Thing group, preferably includes：
10×LAMP bufffer：2.5μL；10mmol/L dNTPs：3.0μL；0.25mol/L MgSO4：0.2μL；5mol/L
Betaine：0.5μL；10 μm of ol/L FIP primers：2.0μL；10 μm of ol/L BIP primers：2.0μL；10 μm of ol/L F3
Primer：0.5μL；10 μm of ol/L B3 primers：0.5μL；8U/ μ L Bst archaeal dna polymerases：0.7μL；Template DNA：1.0μL；Make
Used time supplies system with sterilizing ultra-pure water to 25 μ L；
Wherein 10 × LAMP bufffer are constituted：200mmol/L Tris-HCl, 100mmol/L KCl, 20mmol/L MgSO4,
100mmol/L(NH4)2SO4, 1.0%Tritonx-100.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114457177A (en) * | 2022-03-30 | 2022-05-10 | 贵州傲农七环畜牧养殖有限公司 | Haemophilus parasuis nested PCR amplification primer combination, kit and application |
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2017
- 2017-06-21 CN CN201710474421.1A patent/CN107164511B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| KATE J. HOWELL等: "Gene Content and Diversity of the Loci Encoding Biosynthesis of Capsular Polysaccharides of the 15 Serovar Reference Strains of Haemophilus parasuis", 《J BACTERIOL》 * |
| 斯特劳著: "《猪病学（第9版）》", 28 February 2008 * |
| 朱杰仪: "副猪嗜血杆菌环介导等温扩增检测方法的建立", 《中国畜牧兽医》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114457177A (en) * | 2022-03-30 | 2022-05-10 | 贵州傲农七环畜牧养殖有限公司 | Haemophilus parasuis nested PCR amplification primer combination, kit and application |
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