CN108570510B - LAMP primer, kit and detection method for detecting haemophilus parasuis - Google Patents
LAMP primer, kit and detection method for detecting haemophilus parasuis Download PDFInfo
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Abstract
The invention discloses an LAMP primer, a kit and a detection method for detecting haemophilus parasuis, and belongs to the technical field of detection of pathogenic microorganisms. According to the invention, LAMP primers are designed according to 6 different regions of the lipopolysaccharide histone gene of Haemophilus parasuis with GenBank accession number of CP015099, and compared with primers designed only aiming at 2 regions in conventional PCR, the specificity is obviously improved. The LAMP detection method of the haemophilus parasuis has the advantages of simple operation, easy judgment of results, quick detection time, high sensitivity and the like. The common PCR detection is extracted from sample DNA to obtain a detection result, the whole process is at least 5h, and the LAMP detection can be completed within 2h in the invention. Especially when the direct visual method is adopted to judge the infection condition of the sample, the whole operation process only needs simple and small equipment such as a constant-temperature water bath, a micropipettor and the like, does not need electrophoresis, and can accurately distinguish the result under visible light.
Description
Technical Field
The invention relates to an LAMP primer for detecting haemophilus parasuis, a kit containing the primer and a detection method, and belongs to the technical field of pathogenic microorganism detection.
Background
Haemophilus parasuis is an opportunistic pathogen present in the upper respiratory tract of normal pigs and can invade the body under specific conditions to cause diseases characterized primarily by cellulolytic polyarthritis, meningitis and serositis. With the popularization of large-scale cultivation in China, the cultivation density is higher and higher, and the requirement on environmental control is lower and lower. At present, haemophilus parasuis caused by environmental problems becomes an epidemic disease commonly occurring in small and medium-sized pig farms in China, and extremely serious harm is caused to the breeding industry. Particularly, after the high fever disease occurs in 2006, the secondary infection of haemophilus parasuis disease further aggravates the disease of the sick swinery, and the death and culling rate of piglets is up to more than 50%. Haemophilus parasuis has become one of the most important secondary pathogens of affected swine herds. Therefore, the rapid detection and prevention and control of the haemophilus parasuis disease are problems to be solved urgently.
At present, the detection method of haemophilus parasuis mainly comprises the morphological identification, physiological and biochemical identification, serological identification, molecular biological identification and the like of pathogeny. The morphological identification of the pathogen is mainly performed by isolated culture and microscopic morphological observation on haemophilus parasuis in the disease material, but the haemophilus parasuis has poor tolerance to the external environment and is difficult to culture, so the success rate of clinically detecting by isolated culture strains is low. The physiological and biochemical identification of the pathogen needs to be carried out after the suspected strains are successfully separated, but the test results have large difference, so that the strains are difficult to accurately identify. Serological identification is a method which is commonly used for detecting pathogeny, but the serological identification result is not ideal because haemophilus parasuis has cross immune reaction with haemophilus influenzae, actinomyces pleuropneumoniae, shigella, salmonella, pasteurella and listeria. The molecular biological identification is carried out by detecting a certain section of gene of the haemophilus parasuis to judge the result, and the haemophilus parasuis can be accurately identified as long as the specificity of a primer is ensured under general conditions. However, this method is only suitable for detection under laboratory conditions because it requires special instruments such as a PCR instrument and a gel imaging system, which are not available in general basic pig farms and clinics, and requires a high degree of technical expertise for operating these instruments. Therefore, a rapid and simple detection method is urgently needed in clinic.
At present, LAMP technology is used for detecting various bacteria or viruses such as salmonella typhimurium, mycoplasma pneumoniae, porcine infectious pleuropneumonia, enterococcus, staphylococcus, porcine parvovirus and hantavirus, but the application of the LAMP technology to detecting haemophilus parasuis is still less. The invention patent of publication No. CN103205494A discloses a LAMP method for detecting haemophilus parasuis, which uses the transcription initiation factor (infB) gene sequence of haemophilus parasuis as a target gene to design synthetic primers (outer primer F3/B3 and inner primer FIP/BIP), and can be used as a universal gene to detect haemophilus parasuis due to low variation rate of the gene between species and consideration of conserved sequences between different serotypes, so that the method can not distinguish different serotypes or pathogenic and nonpathogenic haemophilus parasuis. The invention patent of publication No. CN102776283A discloses a visual loop-mediated isothermal amplification kit for detecting Haemophilus parasuis, which comprises an inner primer pair (FIP/BIP), an outer primer pair (F3/B3) and an annular primer pair (LF/LB) designed by referring to a conserved region of a peptidoglycan related lipoprotein gene (palA) of Haemophilus parasuis, wherein the inner primer pair, the outer primer pair and the annular primer pair are used for carrying out loop-mediated isothermal amplification reaction on total DNA of a sample, so that whether the sample contains the Haemophilus parasuis can be directly inspected and judged after color development, or the sample is inspected under ultraviolet irradiation after color development, or the sample is inspected and judged by a nucleic acid electrophoresis method. The method provides a detection method based on the peptidoglycan related lipoprotein genes of haemophilus parasuis, but the peptidoglycan related lipoprotein genes of haemophilus parasuis of different serotypes have certain differences, so that the method can cause missed detection when the haemophilus parasuis is detected.
Disclosure of Invention
The invention aims to provide an LAMP primer for detecting haemophilus parasuis, which is designed and synthesized based on lipopolysaccharide histone gene.
Meanwhile, the invention also provides a kit containing the primer.
Finally, the invention further provides an LAMP method for detecting haemophilus parasuis.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
LAMP primers for detecting Haemophilus parasuis were designed from 6 different regions of the Lipopolysaccharide (LPS) histone gene of Haemophilus parasuis with GenBank accession number CP015099 as follows:
the primer comprises an outer primer LPS-F3/LPS-B3 and an inner primer LPS-F1C-F2/LPS-B1C-B2, and the sequences of the primers are shown as follows:
LPS-F3：5’-TGTTGCAGCTTGCTGTAGTC-3’，
LPS-B3：5’-AACGCATCTGTTCGTTATTCT-3’；
LPS-F1C-F2：5’-AGTCTGATCACCTTGTATAATATCCCAGATTTGAGAATGCAATGTTTACA-3’，
LPS-B1C-B2：5’-GTATGGAAATGAATTATCCTTCTGAGGAACGATTACCTTGCTTAACACTA-3’。
the LAMP detection kit for haemophilus parasuis comprises the two pairs of specific LAMP primers (20pM) and 2 times LAMP Buffer (20mM Tri)s-HCL、10mM KCl、10mM(NH4)2SO4、10mM MgSO40.1% Triton X-20), Bst DNA polymerase (8000U/mL), dNTPs (25mM), MgSO4(50mM), a fluorescent color developing agent (10000 XSYBR Green I), sterile ultrapure water, a positive control (standard Haemophilus parasuis DNA) and the like.
An LAMP method for detecting haemophilus parasuis comprises the following steps:
1) taking the sample genome DNA as a template, and carrying out loop-mediated isothermal amplification reaction by using a specific LAMP primer;
2) after the reaction is finished, adding a fluorescent color developing agent into the reaction liquid for dyeing or taking the reaction liquid for agarose gel electrophoresis detection, and judging whether the sample contains haemophilus parasuis or not;
the specific LAMP primers in the step 1) comprise an outer primer LPS-F3/LPS-B3 and an inner primer LPS-F1C-F2/LPS-B1C-B2, and the sequences of the primers are shown as follows:
LPS-F3：5’-TGTTGCAGCTTGCTGTAGTC-3’，
LPS-B3：5’-AACGCATCTGTTCGTTATTCT-3’；
LPS-F1C-F2：5’-AGTCTGATCACCTTGTATAATATCCCAGATTTGAGAATGCAATGTTTACA-3’，
LPS-B1C-B2：5’-GTATGGAAATGAATTATCCTTCTGAGGAACGATTACCTTGCTTAACACTA-3’。
the sample in the step 1) is a suspected haemophilus parasuis infected disease material or an enriched suspected haemophilus parasuis strain.
The sample genome DNA in the step 1) is suspected haemophilus parasuis infected disease material extracted by adopting a bacterial genome DNA extraction kit or enriched genetic material of suspected haemophilus parasuis strain.
The reaction system of the loop-mediated isothermal amplification in the step 1) is as follows: 2 × LAMP Buffer (20mM Tris-HCl, 10mM KCl, 10mM (NH) using 25 μ L system as standard4)2SO4、10mM MgSO4、0.1％Triton X-20)12.5μL，50mM MgSO 41 μ L of 8000U/mL Bst DNA polymerase, 2 μ L of 25mM dNTPs, 0.2 μ L of each of 20pM outer primer LPS-F3/LPS-B3 and 0.8 μ L of each of 20pM inner primer LPS-F1C-F2/LPS-B1C-B2, 10 ℃0.5. mu.L of 50pM DNA template was made up to 25. mu.L in sterile ultrapure water.
The reaction conditions of the loop-mediated isothermal amplification in the step 1) are as follows: the temperature is 60-65 ℃, and the time is 30-50 min; inactivating at 80-85 ℃ for 5-15 min. The above reaction can be carried out in a constant temperature water bath.
10000 times SYBR Green I is adopted as the fluorescent color developing agent in the step 2). Taking the 25 mu L loop-mediated isothermal amplification reaction system as a standard, and adding 1-3 mu L SYBR Green I.
When a fluorescent color reagent SYBR Green I is added for dyeing in the step 2), a direct visual inspection method can be adopted to judge whether the sample contains haemophilus parasuis, and the specific operation is as follows: adding SYBR Green I into the reaction solution, and observing the result after dyeing; under normal sunlight, the reaction solution is green and is a positive reaction, and the reaction solution is reddish orange and is a negative reaction. If the sample tube and the positive control tube are green, and the negative control tube is orange red, the sample contains haemophilus parasuis; if the sample tube and the negative control tube are orange red, the positive control tube is green, which indicates that the sample does not contain haemophilus parasuis.
When the reaction solution is taken for agarose gel electrophoresis detection in the step 2), whether the sample contains haemophilus parasuis can be judged according to the electrophoresis result, and the specific operation is as follows: taking the reaction solution to carry out electrophoresis in agarose gel, and observing the result in an ultraviolet instrument: the positive reaction is shown in the ladder-shaped strip, and the negative reaction is shown in the non ladder-shaped strip. If the sample tube and the positive control tube are in ladder-shaped strip-shaped bands and the negative control tube is not, the sample contains haemophilus parasuis; if the positive control tube is in a ladder-shaped strip shape, and the sample tube and the negative control tube are not in the same shape, the sample does not contain the haemophilus parasuis.
The electrophoresis conditions are as follows: the agarose gel concentration is 1-2% (mass volume ratio), the electrophoresis voltage is 60-90V, and the time is 20-50 min.
The invention has the beneficial effects that:
according to the invention, LAMP primers are designed according to 6 different regions with highly conserved lipopolysaccharide histone genes of Haemophilus parasuis with GenBank accession number of CP015099, and compared with primers designed only aiming at 2 regions by conventional PCR, the specificity is obviously improved.
The LAMP detection method of the haemophilus parasuis has the advantages of simple operation, easy judgment of results, quick detection time, high sensitivity, strong specificity, low cost and the like. The common PCR detection technology is extracted from sample DNA to obtain a detection result, the whole process is at least 5h, and the LAMP detection can be completed within 2 h. Particularly, when the infection condition of the sample is judged by adopting a direct visual inspection method, only simple and small equipment such as a constant-temperature water bath, a micropipette, a small centrifugal machine and the like is needed in the whole operation process, expensive equipment such as a PCR instrument, a gel imaging system, an electrophoresis instrument and the like needed by PCR detection is not needed, electrophoresis is not needed, the result can be accurately distinguished under visible light, and the method is very suitable for rapidly detecting the haemophilus parasuis disease in primary clinic.
Drawings
FIG. 1 is a diagram showing an amplification product of a sample detected by agarose gel in example 3;
FIG. 2 is a diagram showing the reaction solution in example 3 under normal visible light after the addition of a color-developing agent;
FIG. 3 is a graph showing the reaction solution in example 3 under ultraviolet light after the addition of a color-developing agent;
FIG. 4 is a diagram showing an amplification product of a sample detected by agarose gel in an experimental example;
FIG. 5 is a diagram showing the reaction solution in the test example under normal visible light after the color-developing agent is added;
FIG. 6 shows the results of the sensitivity detection by the PCR method in the test examples;
FIG. 7 shows the results of detection of the sensitivity of LAMP method in the test examples.
Detailed Description
The following examples are intended to illustrate the invention in further detail, but are not to be construed as limiting the invention in any way.
Example 1
The LAMP primer for detecting the haemophilus parasuis is designed according to 6 different regions of Lipopolysaccharide (LPS) histone genes of the haemophilus parasuis with GenBank accession number CP015099, and comprises an outer primer LPS-F3/LPS-B3 and an inner primer LPS-F1C-F2/LPS-B1C-B2, wherein the primer sequences are shown as follows:
LPS-F3：5’-TGTTGCAGCTTGCTGTAGTC-3’，
LPS-B3：5’-AACGCATCTGTTCGTTATTCT-3’；
LPS-F1C-F2：5’-AGTCTGATCACCTTGTATAATATCCCAGATTTGAGAATGCAATGTTTACA-3’，
LPS-B1C-B2：5’-GTATGGAAATGAATTATCCTTCTGAGGAACGATTACCTTGCTTAACACTA-3’。
example 2
The LAMP detection kit for the haemophilus parasuis comprises the following components: 20 μ L of 20pM outer primer LPS-F3/LPS-B3 (shown in SEQ ID NO:1, 2), 20 μ L of 20pM inner primer LPS-F1C-F2/LPS-B1C-B2 (shown in SEQ ID NO:3, 4), 2 × LAMP Buffer (20mM Tris-HCl, 10mM KCl, 10mM (NH)4)2SO4、10mM MgSO40.1% Triton X-20)1.5mL, 8000U/mL Bst DNA polymerase 100. mu.L, 25mM dNTPs 250. mu.L, 50mM MgSO41.5mL, 1000 XSSYBR Green I100. mu.L, sterile ultrapure water and positive control (10pM standard Haemophilus parasuis DNA 100. mu.L).
The using method comprises the following steps: preparing a loop-mediated isothermal amplification reaction system according to the following formula, adding different DNA templates into the loop-mediated isothermal amplification reaction system, and reacting for 40min at the temperature of 62 ℃;
the reaction system is as follows: 2 × LAMP Buffer 12.5 μ L, 8000U/mL Bst DNA polymerase 1 μ L, 25mM dNTPs2 μ L, 50mM MgSO41.5 mu L, 0.2 mu L of 20pM outer primer LPS-F3/LPS-B3, 0.8 mu L of 20pM inner primer LPS-F1C-F2/LPS-B1C-B2, 0.5 mu L of 10-50 pM DNA template, and sterile ultrapure water which is complemented to 25 mu L.
Example 3
The LAMP method for detecting the haemophilus parasuis comprises the following steps:
1) treatment of pathological material
Taking 0.5g of suspected haemophilus parasuis infected pig lung disease material aseptically, adding 1mL of PBS buffer solution, grinding, repeatedly freezing and thawing at-20 ℃ for 3 times, centrifuging at 1500r/min, taking supernatant, and extracting with a bacterial genome extraction kit to obtain total genome DNA for later use.
2) LAMP amplification
Taking the sample genome DNA extracted in the step 1) as a template, and performing loop-mediated isothermal amplification reaction by using a specific LAMP primer; the specific LAMP primer comprises an outer primer pair and an inner primer pair, and the primer sequences are as follows:
LPS-F3：5’-TGTTGCAGCTTGCTGTAGTC-3’，
LPS-B3：5’-AACGCATCTGTTCGTTATTCT-3’；
LPS-F1C-F2：5’-AGTCTGATCACCTTGTATAATATCCCAGATTTGAGAATGCAATGTTTACA-3’，
LPS-B1C-B2：5’-GTATGGAAATGAATTATCCTTCTGAGGAACGATTACCTTGCTTAACACTA-3’。
the reaction system of the loop-mediated isothermal amplification is as follows: 2 × LAMP Buffer (20mM Tris-HCl, 10mM KCl, 10mM (NH) using 25 μ L system as standard4)2SO4、10mM MgSO4、0.1％Triton X-20)12.5μL，50mM MgSO4mu.L of 1. mu.L of Bst DNA polymerase, 8000U/mL of 1. mu.L of 25mM dNTPs, 0.2. mu.L of each of 20pM outer primer LPS-F3/LPS-B3, 0.8. mu.L of each of 20pM inner primer LPS-F1C-F2/LPS-B1C-B2, 0.5. mu.L of 20pM sample genomic DNA, and sterile ultrapure water to 25. mu.L.
The above reaction is carried out in a constant temperature water bath kettle, the reaction temperature is set to be 62 ℃, the time is set to be 40min, and then inactivation is carried out for 10min at 80 ℃.
The LAMP amplification reaction of the negative control and the positive control is the same as that of the LAMP amplification reaction, the negative control takes ultrapure water as a template, and the positive control takes the standard haemophilus parasuis genome DNA as a template.
3) Result judgment
And (3) electrophoretic observation: after the reaction is finished, 2 μ L of the reaction solution is subjected to 80V electrophoresis in 2.0% agarose gel for 20min, and then placed in a gel imaging system to observe whether a ladder-shaped band appears, if the ladder-shaped band appears, the ladder-shaped band is a positive reaction, if the ladder-shaped band does not exist, the ladder-shaped band is a negative reaction, the result is shown in figure 1, wherein M is a standard Marker, lane 1 is a negative control, lane 2 is a positive control, and lane 3 is a disease sample.
And (3) color development reaction: after the reaction is finished, adding 2 μ L of 10000 XSYBR Green I into the reaction solution, dyeing for 4min (3-5 min is enough) after fully and uniformly mixing, observing the color of the reaction solution by naked eyes, wherein the Green reaction is positive reaction, the orange reaction is negative reaction, and the result is shown in figure 2, wherein 1 is a disease sample, 2 is a positive control, and 3 is a negative control. The results of the UV light test are shown in FIG. 3, in which 1 is a specimen, 2 is a positive control, and 3 is a negative control.
Test examples
1. Haemophilus parasuis LAMP primer and specificity verification method thereof
The LAMP detection of Haemophilus parasuis HPS 4 type (BNCC176334) was performed simultaneously with the method of example 3 using pathogenic Escherichia coli (ATCC25922), Salmonella typhimurium (BNCC103602), Salmonella enteritidis (BNCC103443), Staphylococcus aureus (BNCC165476), Listeria monocytogenes (BNCC128219), Bacillus subtilis (ATCC6633), Lactobacillus plantarum (CCTCC M2016136) as control bacteria.
And respectively taking the reaction liquid for electrophoresis detection, wherein the result is shown in figure 4, wherein a lane M is a standard Marker, a lane 1 is haemophilus parasuis, a lane 2 is pathogenic escherichia coli, a lane 3 is salmonella typhimurium, a lane 4 is salmonella enteritidis, a lane 5 is staphylococcus aureus, a lane 6 is listeria monocytogenes, a lane 7 is bacillus subtilis, and a lane 8 is lactobacillus plantarum.
As can be seen from FIG. 4, only positive reaction was detected by H.parasuis HPS 4, and no ladder-like condition was observed in the remaining strains, indicating that the LAMP primer of the present invention has high specificity.
The results of the color development detection of the reaction solutions are shown in fig. 5, in which 1 is haemophilus parasuis, 2 is pathogenic escherichia coli, 3 is salmonella typhimurium, 4 is salmonella enteritidis, 5 is staphylococcus aureus, 6 is listeria monocytogenes, 7 is bacillus subtilis, and 8 is lactobacillus plantarum.
As can be seen from FIG. 5, only positive reaction was detected by H.parasuis HPS 4, and no color turning green was observed in any of the other strains, indicating that the LAMP primer of the present invention has high specificity.
2. Haemophilus parasuis LAMP primer and sensitivity verification method thereof
The concentration of extracted Hps genomic nucleic acid was measured to be 241 ng/. mu.L with a nucleic acid analyzer, and diluted with a 10-fold gradient. Respectively carrying out LAMP reaction and PCR reaction by taking genomes with different concentrations as templatesCompared with the sensitivity of the two reactions. The LAMP reaction was the same as in example 3. The PCR reaction system is as follows: 5U/. mu.L rTaq DNA polymerase 0.5. mu.L, 10 XPCR buffer (Mg)2+) mu.L of 2.5. mu.L, 2.5mM dNTPs 2. mu.L, 1. mu.L of 10pM of upstream and downstream primers P1 and P2, respectively, and sterile water to make up to 25. mu.L. The conditions of the PCR reaction were: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 59 ℃ for 30s, and extension at 72 ℃ for 50s for 30 cycles; extension at 72 ℃ for 10 min.
Primers P1, P2 are shown below:
upstream primer P1: 5'-GTGATGAGGAAGGGTGGTGT-3' the flow of the air in the air conditioner,
the downstream primer P2: 5'-GGCTTCGTCACCCTCTGT-3' are provided.
Respectively taking the reaction solution to carry out electrophoresis detection, wherein the PCR sensitivity detection result is shown in figure 6, M is DL 2000DNA marker, and 1-9 are template dilution times of 10 in sequence0、10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8The detection result of (3); negative control 10. The LAMP sensitivity detection result is shown in figure 7, wherein M is DL 2000DNA marker, and 1-9 are template dilution times of 10 in sequence0、10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-810 is a negative control.
As is clear from FIGS. 6 and 7, the PCR method can detect 10 or less-4Doubling the amount of template, 10 detectable by LAMP-6The amount of template multiplied is 100 times the sensitivity of the PCR method. Through calculation, the lowest detection amount of Hps detected by the LAMP method is 0.241 pg/mu L.
Sequence listing
SEQUENCE LISTING
<110> university of Henan science and technology
<120> LAMP primer, kit and detection method for detecting haemophilus parasuis
<170> PatentIn version 3.5
<211> 20
<212> DNA
<213> Artificial sequence
<221> primer LPS-F3
<222> (1)..(20)
<400> 1
TGTTGCAGCT TGCTGTAGTC 20
<211> 21
<212> DNA
<213> Artificial sequence
<221> primer LPS-B3
<222> (1)..(21)
<400> 2
AACGCATCTG TTCGTTATTC T 21
<211> 50
<212> DNA
<213> Artificial sequence
<221> primer LPS-F1C-F2
<222> (1)..(50)
<400> 3
AGTCTGATCA CCTTGTATAA TATCCCAGAT TTGAGAATGC AATGTTTACA 50
<211> 50
<212> DNA
<213> Artificial sequence
<221> primer LPS-B1C-B2
<222> (1)..(50)
<400> 4
GTATGGAAAT GAATTATCCT TCTGAGGAAC GATTACCTTG CTTAACACTA 50
<211> 2334
<212> DNA
<213> sequence
<221> lipopolysaccharide histone gene
<222> (1)..(2334)
<400> 5
ATGAAACCAT ATCGCTATAG TTTACTTTCT ATTGCTATTG TTGCAGCTTG CTGTAGTCAA 60
TATGTAATAG CAGATTTGAG AATGCAATGT TTACAAGGAA TTCCTACTTT TCAGGGGGAT 120
ATTATACAAG GTGATCAGAC TCTTCTACCT GTGTATATTG AGTCAGATCG TATGGAAATG 180
AATTATCCTT CTGAGGCAAC ATATTTTGGT GAAGTTAGTG TTAAGCAAGG TAATCGTTCT 240
CTTATTACAG AAAAAGCTAA GGTAGAACAG AATAACGAAC AGATGCGTTT TGCTTATTTA 300
ATCGGTAAAT TCGATTATCG GGATAATTTC ATTAATGCGA TAGGTAATAA TGCAAAGATT 360
AATTTGAACA ACTATGATGC TCAGTTAGGC AGGGTCGATT ACCATTTAGT TGATCGACAA 420
GGAAGAGGTA TTGCTGATGA AGTCATGATA ACTGAACAAG CCCGTCGAAT GAAAAATGCA 480
ACTTTTACAG CTTGTTTACC TCATGATAAC TCCTGGGCTA TTGAAGCTAA TGAAATGGTT 540
CAGCATGTTG AAGAGGAATA TGCTGAAGTA TGGCATGCGC GTTTTAAAGT ATTGGGTATA 600
CCTATTTTCT ATTCTCCTTA TTTGCAATTT CCAATAGGGG ATCGTCGTCG TTCTGGTTTA 660
TTGGCTCCGA GTGTTGGTCA TTCAAGACGA GATGGTTATA TATATGCACA GCCAATATAT 720
TGGAATATTG CTTCTAACAT GGATATGACA TTTGTACCAA CATACTATTC AAAACGTGGT 780
TGGCAGATTG CACCTGAACT GCGTTATTTA ACTTATTTAG GAGAAGGTAA AATAGCCGGT 840
GAATATTTAG GTAAGGATCG ACTTGAAAAT TGGGCAAATC ATAGTAAATC TCGTTATATG 900
CTATTTTGGC AACATGATAT GAGTTTTTTT AATAATTGGA GATTAGCTGT TGATTATAAA 960
AGAGTTAGTG ATAGTCGGTA CTTTTCTGAT TTTAATTCTA TGTACGGGAA TAGTACTGAT 1020
GGTTATGCGG TACAGCAGTT TAAATTAGGG TACTATCATT CAGATTATAA TTTTTCTATT 1080
TCAGGAAAAA AATTTCAGTC ATTTTATGTA GAAAATGATA GCACTACCTA TCATGTGTTA 1140
CCAAAGATTG AGCTTAATTA CTATAAAAAT AATTTAGTTT CTGATGTTGA TTTCAAGATG 1200
TTTGGGCAGA TGGCGCATTT TGTTAATCAA AATAAACTCA TGCCATCAGC ATGGCGTTTT 1260
CATTTAGAAC CTAGTTTAAA TGTTCCTTTT GTTAACCGGT TAGGAAGTTT AGCTTTAGAA 1320
ACTAAGCTAT ATACCACCTA TTATTTACAG AAACTGGGGC AATCACAAGT AATACCGATA 1380
GAGTCACATA TACTCCGGGT ATTACCTCAA TTTAAGTTGG ATTTTAAAAC TGTCTTGGAA 1440
AAGGAAAGAC CTTTGTTTAA TGGGTTTTCA CAAGTTTTTG AGCCAACAAT ACAATATTTA 1500
TATCGTCCAT ATAAAAATCA AGGAAATATT GGAGTTAGAA ATCCTAAATA TTTAGGTTTA 1560
GGTTATGACT CCGTTTTATT GCAGCAGGAT TATTACTCTT TATTTAATGA GCGTAGTTAT 1620
AGTGGGTTGG ACCGCATTGC CTCTGCGAAT CAGGTTACGT TAGGCGGTAC TACACGATTT 1680
TTTAGCGACA AAACAGGAAA AGAAGTATTT AACTTTAGTG CAGGACAGAT TCAATATTTA 1740
TCTCCTTCAA GAGTTGATAG TGAGTCGTTA AATAGCGATA CAAAACACTC ATCTTCTTGG 1800
TCATTAGAAT CTAACTGGAA ATTTCATTCA AACTGGAATT GGCATGGAAG TTATCAATAT 1860
GATACACGTC TAAACGAAAC ATCGTTAGCA AATGTTTCAT TACAATTTAA GCCTAAAAAT 1920
AATAACGTGA TTCAGCTTAA TTATCGTTAT GCAAGCAAAA ACTATATAGA TCAAAATTTA 1980
AGTGATAATC GCTATGGGCA GGATATTAAG CAAGTTGGAG GTATTATAGC TTGGTCATTG 2040
AGCAATAATT TATCAATAGT AATGAGCCAC TATCAAGATA TTGCTTTAAA AAAACCGGTT 2100
GAGAGTCAAT TGGGTTTAAA TTATAGCACT TGTTGTTGGA GTGCAAAATT TTATACTTCT 2160
AGACATTTGA TTTCAACTCC GATGGGGAAA AGCGATACGA TAAACGATTT CTACTATGAT 2220
AATCGTTTTG GTATTAATTT TGAAATTAGA TTTAATGGAA ATTATGAACA TACCGTGCCT 2280
AAGATGCTTA AAAAAGGAAT TCTCCCTTAT ATTGAAGCGT TTAATATCAA TTAA 2334
<211> 20
<212> DNA
<213> sequence
<221> upstream primer P1
<222> (1)..(20)
<400> 6
GTGATGAGGA AGGGTGGTGT 20
<211> 18
<212> DNA
<213> sequence
<221> downstream primer P2
<222> (1)..(18)
<400> 7
GGCTTCGTCA CCCTCTGT 18
Claims (8)
1. The LAMP primer for detecting the HPS 4 type of the haemophilus parasuis is characterized by comprising the following steps of: comprises an outer primer LPS-F3/LPS-B3 and an inner primer LPS-F1C-F2/LPS-B1C-B2, and the primer sequences are shown as follows:
LPS-F3：5’-TGTTGCAGCTTGCTGTAGTC-3’，
LPS-B3：5’-AACGCATCTGTTCGTTATTCT-3’；
LPS-F1C-F2：
5’-AGTCTGATCACCTTGTATAATATCCCAGATTTGAGAATGCAATGTTTACA-3’，
LPS-B1C-B2：
5’-GTATGGAAATGAATTATCCTTCTGAGGAACGATTACCTTGCTTAACACTA-3’。
2. a kit comprising the LAMP primer of claim 1.
3. The kit of claim 2, wherein: also comprises 2 × LAMP Buffer, Bst DNA polymerase, dNTPs and MgSO4And sterile ultrapure water.
4. An LAMP method for detecting HPS 4 type haemophilus parasuis for non-diagnostic purposes, which is characterized in that: the method comprises the following steps:
1) taking the sample genome DNA as a template, and carrying out loop-mediated isothermal amplification reaction by using a specific LAMP primer;
2) after the reaction is finished, adding a fluorescent color developing agent into the reaction liquid for dyeing or taking the reaction liquid for agarose gel electrophoresis detection, and judging whether the sample contains the haemophilus parasuis HPS 4 type;
the specific LAMP primers in the step 1) comprise an outer primer LPS-F3/LPS-B3 and an inner primer LPS-F1C-F2/LPS-B1C-B2, and the sequences of the primers are shown as follows:
LPS-F3：5’-TGTTGCAGCTTGCTGTAGTC-3’，
LPS-B3：5’-AACGCATCTGTTCGTTATTCT-3’；
LPS-F1C-F2：
5’-AGTCTGATCACCTTGTATAATATCCCAGATTTGAGAATGCAATGTTTACA-3’，
LPS-B1C-B2：
5’-GTATGGAAATGAATTATCCTTCTGAGGAACGATTACCTTGCTTAACACTA-3’。
5. the method of claim 4, wherein: the reaction system of the loop-mediated isothermal amplification in the step 1) is as follows: 2 × LAMP Buffer 12.5 μ L, 50mM MgSO 25 μ L system as standard4mu.L of 1. mu.L of Bst DNA polymerase, 8000U/mL of Bst DNA polymerase, 2. mu.L of 25mM dNTPs, 0.2. mu.L of 20pM outer primer LPS-F3/LPS-B3, 0.8. mu.L of 20pM inner primer LPS-F1C-F2/LPS-B1C-B2 and 0.5. mu.L of 10-50 pM DNA template, and the content of sterile ultrapure water is up to 25. mu.L.
6. The method of claim 5, wherein: the reaction conditions of the loop-mediated isothermal amplification in the step 1) are as follows: the temperature is 62 ℃, and the time is 40 min; inactivating at 80 deg.C for 10 min.
7. The method of claim 4, wherein: when a fluorescent color reagent SYBR Green I is added for dyeing in the step 2), judging whether the sample contains the haemophilus parasuis HPS 4 type by adopting a direct visual inspection method, wherein the specific operation is as follows: adding SYBR Green I into the reaction solution, and observing the result after dyeing; under normal sunlight, the reaction solution is green and is a positive reaction, and the reaction solution is orange and is a negative reaction.
8. The method of claim 4, wherein: when the reaction solution is taken for agarose gel electrophoresis detection in the step 2), whether the sample contains the haemophilus parasuis HPS 4 type is judged according to the electrophoresis result, and the specific operation is as follows: taking the reaction solution to carry out electrophoresis in agarose gel, and observing the result in an ultraviolet instrument: the positive reaction is shown in the ladder-shaped strip, and the negative reaction is shown in the non ladder-shaped strip.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003008583A3 (en) * | 2001-03-02 | 2003-07-03 | Sagres Discovery | Novel compositions and methods for cancer |
| EP2484780A1 (en) * | 2004-07-23 | 2012-08-08 | The University of North Carolina At Chapel Hill | Methods and materials for determining pain sensibility and predicting and treating related disorders |
| CN103890169A (en) * | 2011-08-24 | 2014-06-25 | 诺维信股份有限公司 | Aspergillus fumigatus cellulolytic enzyme compositions and uses thereof |
| CN104630360A (en) * | 2015-02-04 | 2015-05-20 | 重庆出入境检验检疫局检验检疫技术中心 | Rapid HPS (haemophilus parasuis) isothermal amplification detection primers, kit and detecting method |
| CN104630361A (en) * | 2015-02-04 | 2015-05-20 | 重庆出入境检验检疫局检验检疫技术中心 | Rapid APP (actinobacillus pleuropneumoniae) isothermal amplification detection primers, kit and detecting method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102776283B (en) * | 2012-07-05 | 2014-04-09 | 福建省农业科学院畜牧兽医研究所 | Visualized loop-mediated isothermal amplification kit for detecting Haemophilus parasuis |
-
2017
- 2017-03-14 CN CN201710150721.4A patent/CN108570510B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003008583A3 (en) * | 2001-03-02 | 2003-07-03 | Sagres Discovery | Novel compositions and methods for cancer |
| EP2484780A1 (en) * | 2004-07-23 | 2012-08-08 | The University of North Carolina At Chapel Hill | Methods and materials for determining pain sensibility and predicting and treating related disorders |
| CN103890169A (en) * | 2011-08-24 | 2014-06-25 | 诺维信股份有限公司 | Aspergillus fumigatus cellulolytic enzyme compositions and uses thereof |
| CN104630360A (en) * | 2015-02-04 | 2015-05-20 | 重庆出入境检验检疫局检验检疫技术中心 | Rapid HPS (haemophilus parasuis) isothermal amplification detection primers, kit and detecting method |
| CN104630361A (en) * | 2015-02-04 | 2015-05-20 | 重庆出入境检验检疫局检验检疫技术中心 | Rapid APP (actinobacillus pleuropneumoniae) isothermal amplification detection primers, kit and detecting method |
Non-Patent Citations (3)
| Title |
|---|
| [Haemophilus] parasuis strain SC1401, complete genome;Dai,K.等;《GenBank》;20160406;第2页 * |
| Understanding Haemophilus parasuis infection in porcine spleen through a transcriptomics approach;Hongbo Chen等;《BMC Genomics》;20090205;第10卷(第64期);第1-18页 * |
| 副猪嗜血杆菌环介导等温扩增快速检测方法的建立;魏晓媛;《中国优秀硕士学位论文全文数据库农业科技辑（月刊）》;20140315(第03期);D050-296 * |
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