CN110894532A - RAA constant temperature fluorescence detection method and reagent for bacterial septicemia (FBS) - Google Patents
RAA constant temperature fluorescence detection method and reagent for bacterial septicemia (FBS) Download PDFInfo
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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
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- 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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- 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
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses a RAA constant-temperature fluorescence detection method and a detection kit for bacterial septicemia (FBS). The detection kit comprises a forward primer SEQ ID NO.1, a reverse primer SEQ ID NO.2, a specific fluorescent probe SEQ ID NO.3, reaction liquid, recombinant polymerase and a reference substance. The kit has strong specificity; the detection sensitivity is high and can reach 0.1 fg/mu L; the accuracy is high and reliable; the method is simple, convenient and quick to operate, is suitable for field detection, and has wide application scenes.
Description
Technical Field
The invention belongs to the technical field of molecular biology, relates to a detection method for marine aquaculture industry, and particularly relates to an RAA constant-temperature fluorescence detection method and a kit for Aeromonas hydrophila.
Background
In freshwater fish culture, bacterial septicemia (FBS) is a most harmful bacterial infectious disease. The disease is caused by infection of various gram-negative bacilli such as aeromonas hydrophila, aeromonas sobria, yersinia ruckeri, aeromonas caviae, vibrio fluvialis biovar III, pseudomonas alcaligenes and the like. It is mainly caused by Aeromonas hydrophila. The disease is prevalent all over the country and mainly damages silver carps, bighead carps, crucian carps, megalobrama amblycephala, white crucian carps, xenocypris davidi bleekers, dace and the like. In recent years, famous fishes such as mandarin fish and channel catfish have been reported to be ill, mainly endangering fishes more than 1-month old, but recently, the fish species have been expanded to 2-month old.
The disease is the most serious, the prevalence is the most extensive, the period is the longest, the most fish species are affected, the death rate is the highest, the fish suffering from the disease can die greatly in a short period of time from symptom discovery to death of only 3-5 days, and even the fish cannot be produced at all, and the disease is a malignant disease for pond culture. The earliest incidence in pond polyculture was crucian carp, white crucian carp or silver carp, followed by megalobrama amblycephala and bighead carp. The epidemic temperature is 9-36 deg.C, the peak is 28-32 deg.C, and acute outbreak is easy in 6-7 months. The disease is dangerous to various cultured fishes, particularly filter-feeding fishes such as chubs, bighead carps and the like, generally feed on plankton, and feed fed manually is not too much, and the disease generally occurs in large water bodies such as lakes, reservoirs and the like, so that the prevention and treatment work of the drug is difficult. Therefore, it is important to perform preventive tests on fish diseases at a previous stage.
The method mainly comprises microscopic observation, molecular detection and immunological detection. The PCR molecular detection method is one of the most commonly used detection methods, and is sensitive, accurate, rapid and widely applied, but is not suitable for field detection and popularization due to the requirement of expensive instruments and equipment, higher detection cost and higher technical requirements for detection personnel. The method for detecting the bacterial septicemia by RAA constant-temperature fluorescence is established, is rapid, convenient, accurate and reliable, meets the time requirements of rapid port detection and general customs, and plays an important role in promoting the aquaculture and product trade in China.
The Recombinase-aid Amplification (RAA) technique is also a method by which nucleic acids can be rapidly amplified at a constant temperature. Unlike RPA, RAA amplification uses a recombinase obtained from bacteria or fungi, which binds tightly to the primer DNA at a constant temperature of 37 ℃ to form an aggregate of the enzyme and the primer, when the primer searches for a sequence on the template DNA that is completely complementary to the primer, the template DNA is melted with the help of single-strand DNA binding protein (SSB), and a new complementary strand of DNA is formed under the action of DNA polymerase, and the reaction product is exponentially increased, and usually an amplified fragment that can be detected by agarose gel electrophoresis can be obtained within 1 hour. The fluorescent group is added into the RAA reaction system, the whole RAA amplification process is monitored in real time by utilizing the accumulation of fluorescent signals, and the quantitative and qualitative analysis of the initial template can be realized within 20 minutes. The whole reaction is simple and quick, and high-temperature circulation is not needed, so the method is particularly suitable for being used in non-laboratory detection places with a large number of samples, and is suitable for the field of quick detection of foods.
Disclosure of Invention
In view of the above, the present invention aims to provide a RAA isothermal fluorescence nucleic acid detection kit and a detection method for bacterial sepsis (FBS).
In order to achieve the purpose, the invention adopts the following technical scheme:
a kit for detecting bacterial sepsis (FBS) nucleic acid comprising: the kit comprises a forward primer for the bacterial sepsis, a reverse primer and a specific fluorescent probe, wherein the nucleotide sequence of the forward primer for the bacterial sepsis is shown as SEQ ID No.1, the nucleotide sequence of the reverse primer for the bacterial sepsis is shown as SEQ ID No.2, the nucleotide sequence of the specific fluorescent probe is shown as SEQ ID No.3, a fluorescent reporter group is marked at the 5 'end of the specific fluorescent probe, and a fluorescent quencher group is marked at the 3' end of the specific fluorescent probe.
In some embodiments, the fluorescent reporter group of the specific fluorescent probe is selected from one of FAM, VIC, JOE, TET, CY3, CY5, ROX, Texas Red, or LC Red460, and the fluorescence quenching gene is selected from one of BHQ1, BHQ2, BHQ3, Dabcy1, or Tamra.
In some embodiments, the nucleic acid detection kit further comprises a primer mixture, a specific fluorescent probe, a Buffer, a RAA dry powder reagent, a bacterial sepsis standard substance and ddH2At least one of O.
In some embodiments, the kit of (a), wherein the a Buffer is 20% PEG; b Buffer is 280mM MgAc.
In some embodiments, the kit, wherein the composition of the RAA dry powder reagent is as follows: 1mmol/L dNTP, 90ng/μ L SSB protein, 120ng/μ L recA recombinase protein (SC-recA/BS-recA) or 30ng/μ LRad51, 30ng/μ L Bsu DNA polymerase, 100mmol/L Tricine, 20% PEG, 5mmol/L dithiothreitol, 100ng/μ L creatine kinase, Exo exonuclease.
In some embodiments, the nucleic acid detection kit and the bacterial sepsis standard are positive plasmids containing a part of the sequence of a gene of a hydrosphere.
In some embodiments, the kit comprises a positive plasmid containing a gene partial sequence of Aeromonas hydrophila, the sequence of which is shown in SEQ ID No. 4.
The invention also provides an RAA constant-temperature fluorescence detection method for bacterial septicemia, which comprises the steps of extracting DNA of a sample to be detected, taking the DNA of the sample to be detected as a template, and performing reverse primer detection on the bacterial septicemia by using a forward primer, a reverse primer, a specific fluorescent probe, an RAA dry powder reagent, an A Buffer, a B Buffer and a ddH2Carrying out real-time fluorescence RAA reaction in the presence of O, and analyzing a sample to be detected according to a real-time fluorescence RAA amplification curve; wherein the nucleotide sequence of the forward primer of the bacterial sepsis is shown as SEQ ID NO.1, the nucleotide sequence of the reverse primer of the bacterial sepsis is shown as SEQ ID NO.2, the nucleotide sequence of the specific fluorescent probe is shown as SEQ ID NO.3, the 5 'end of the specific fluorescent probe is marked with a fluorescent reporter group, and the 3' end of the specific fluorescent probe is marked with a fluorescent quenching group.
Use in some embodiments, further comprising the steps of:
(1) and (3) purifying and separating the aeromonas hydrophila strain: sampling from tissues of fishes to be detected by using a sterile inoculating loop, streaking on an AHM flat plate for overnight culture, and purifying and separating strains;
(2) preparing an aeromonas hydrophila DNA solution: : adding 50 mu L of sterile TE solution (10mM Tris-HCl, 1mM EDTA, pH 8.0) into a centrifuge tube, selecting a single colony to the TE solution, placing the centrifuge tube on a vortex mixer, fully shaking for 10s, boiling in boiling water for 10min, cooling to room temperature, fully shaking for 10s, then centrifuging at 12000r/min for 2min, and placing the supernatant, namely DNA, at-20 ℃ for later use;
in some embodiments, the performing a fluorescent RAA reaction procedure is: at 37 ℃ for 40 s; at 37 ℃ for 20min, and 40 cycles in total;
according to the detection method, after the real-time fluorescence RAA reaction is required to be finished, the to-be-detected sample is analyzed according to the amplification curve of the real-time fluorescence RAA by using the analysis software of the real-time fluorescence RAA instrument. Preferably, the FAM channel fluorescence curve of the sample to be tested is S-shaped and the CT value is less than or equal to 35, and the sample to be tested is judged to be a positive result of the bacterial septicemia; and when the curve of the sample to be detected does not show an S shape or the CT value is more than 35, judging as a negative result of the bacterial septicemia.
Advantageous effects
1. Fast and efficient: the whole amplification can be completed within 20-30min, and the amplification yield can reach 109-1010A copy;
2. the operation is simple: no special reagent is needed, complicated steps such as deformation of double-stranded DNA and the like are not needed in advance, only a constant-temperature fluorometer is needed, and the conditions are mild;
3. high specificity: the invention does not amplify DNA of other fish diseases GCRV1, CYHV2, CEV, SAV, ISAV and IPNV.
4. High sensitivity: the detection limit of the invention can reach 0.1 fg/reaction
5. The identification is simple: and the amplification result is directly judged according to the real-time fluorescence data, electrophoresis detection is not needed, and the method is suitable for field detection.
Drawings
FIG. 1 is a graph showing RAA amplification curves of 3 pairs of primers involved in the present invention.
FIG. 2 is a graph showing the sensitivity test of the RAA detection method to FBS, and the amplification results of the positive standards are 1 pg/. mu.L, 100 fg/. mu.L, 10 fg/. mu.L, 1 fg/. mu.L, and 0.1 fg/. mu.L, in that order from left to right.
FIG. 3 is a graph showing the specificity of the RAA detection method for FBS.
Detailed description of the invention
The present invention is further illustrated by the following specific examples, but is not limited thereto.
Example 1:
the invention searches gene sequences of aeromonas hydrophila in a Genebank database for bacterial septicemia, and compares the multiple sequences by using DNAMAN 6.0 software to find out conserved segments. 3 sets of primers and probes were designed in conserved regions and BLAST alignments were performed in the NCBI database, with the sequences of the primers and probes as shown in Table 1. The positive sample amplification curve is shown in FIG. 1.
TABLE 1 primer and Probe sequences
As can be seen from the results in FIG. 1, the amplification curves for primers and probes in group 2 are most typical, with distinct exponential and plateau phases, higher fluorescence intensity (ordinate values), and smaller CT values (abscissa corresponding to the intersection of the curve with the threshold line) and the results are analyzed in Table 2. The rise height of other primer probe curves is lower, the CT value is larger, and the plateau period is not obvious; or no amplification occurs and missed detection occurs. The second group of primers and probes are shown to have higher replication speed, more quantity and higher amplification reaction efficiency.
TABLE 2 analysis of primer Probe screening results
| Group \ result | CT value | Intensity of fluorescence |
| First group | 10.61 | 652,500 |
| Second group | 9.81 | 625,000 |
| Third group | 12.39 | 550,000 |
Real-time example 2: the kit is used for treating bacterial septicemia
The nucleic acid detection kit also comprises primer mixed liquor, a specific fluorescent probe, A Buffer, BBbuffer, RAA dry powder reagent, bacterial septicemia standard substance and ddH2O。
The kit of the invention, wherein the A Buffer is 20% PEG; b Buffer is 280mM MgAc.
The kit of the invention, wherein the RAA dry powder reagent comprises the following components: 1mmol/L dNTP, 90ng/μ L SSB protein, 120ng/μ L recA recombinase protein (SC-recA/BS-recA) or 30ng/μ L Rad51, 30ng/μ L Bsu DNA polymerase, 100mmol/L Tricine, 20% PEG, 5mmol/L dithiothreitol, 100ng/μ L creatine kinase, Exo exonuclease.
In the primer mixture, the base sequence of the forward primer is shown as SEQ ID NO.1, the base sequence of the reverse primer is shown as SEQ ID NO.2, and the molar ratio of the forward primer to the reverse primer is SEQ ID NO. 1: SEQ ID NO.2 is 1: 1.
The base sequence of the specific probe for bacterial sepsis is shown in SEQ ID NO.3, the 5 'end of the probe is marked with a FAM fluorescent reporter group, and the 3' end of the probe is marked with a BHQ1 fluorescent quenching group.
The bacterial septicemia standard provided by the invention contains a positive plasmid of a gene sequence of a hydrosphere monad, and the base sequence of the plasmid is shown in SEQ ID NO. 4.
Base sequence of plasmid (SEQ ID NO.4):
TTAATGCTTATTTTTATTGGTGGTTGCACCGTAAATTTTCAGGTTGAAACTACTTCTACAGCACAAAGTGGAAATGGTGAACTTGCGGAAGTCATTGAAACAAAAGAAACAAGCGAAACGGATGCCCAAGTCTTTCCTGTAAAATGAATTAGTTAACATATATTGATCATAAAAATGCATTCTTTTTTACAAAGTAACATTTCATTTATGTTTGTTTTCTTATTGATGCAATACGTTTAACTTAAACGTGTTGCATAATTTTGTGCAATTTAATAGGAGAACATCATGAGTAACAATATAAAACATGAAACTGACTATTCTCACGATTGGACTGTCGAACCAAACGGAGGCGTCACAGAAGTAGACAGCAAACATACACCTATCATCCCGGAAGTCGGTCGTAGTGTAGACATTGAGAATACGGGACGTGGGGAGCTTACCATTCAATACCAATGGGGTGCGCCATTTATGGCTGGCGGCTGGAAAGTGGCTAAATCACATGTGGTACAACGTGATGAAACTTACCATTTACAACGCCCTGATAATGCATTCTATCATCAGCGTATTGTTGTAATTAACAATGGCGCTAGTCGTGGTTTCTGTACAATCTATTACCACTAAGAAGGTGCTCACATGACTAACGAATACGTTGTAACAATGTCATCTTTGACGGAATTTAACCCTAACAATGCTCGTAAAAGTTATTTATTTGATAACTATGAAGTTGATCCTAACTATGCTTTCAAAGCAATGGTTTCATTTGGTCTTTCAAATATTCCTTACGCGGGTGGTTTTTTATCAACGTTATGGAATATCTTTTGGCCAAATACGCCAAATGAGCCAGATATTGAAAACATTTGGGAACAATTACGTGACAGAATCCAAGATTTAGTAGATGAATCGATTATAGATGCCATCAATGGAATATTGGATAGCAAAATCAAAGAGACACGCGATAAAATTCAAGACATTAATGAGACTATCGAAAACTTCGGTTATGCTGCGGCAAAAGATGATTACATTGGTTTAGTTACTCATTACTTGATTGGACTTGAAGAGAACTTTAAGCGCGAGCTAGACGGTGATGAATGGCTTGGTTATGCGATATTGCCTCTATTAGCAACAACTGTAAGTCTTCAAATTACTTACATGGCTTGTGGTCTGGATTATAAGGATGAATTCGGTTTCACCGATTCTGATGTGCATAAGCTAACACGTAATATTGATAAGCTTTATGATGATGTATCGTCTTACATTACAGAACTCGCTGCGTGGGCTGATAACGACTCTTACAATAATGCAAA
example 3: the kit of the invention is used for treating bacterial septicemia
1. Extraction of nucleic acids from Positive samples
1.1, nucleic acid extraction:
(1) and (3) purifying and separating the aeromonas hydrophila strain: sampling from tissues of fishes to be detected by using a sterile inoculating loop, streaking on an AHM flat plate for overnight culture, and purifying and separating strains;
(2) preparing an aeromonas hydrophila DNA solution: : adding 50 mu L of sterile TE solution (10mM Tris-HCl, 1mM EDTA, pH 8.0) into a centrifuge tube, selecting a single colony to the TE solution, placing the centrifuge tube on a vortex mixer, fully shaking for 10s, boiling in boiling water for 10min, cooling to room temperature, fully shaking for 10s, then centrifuging at 12000r/min for 2min, and placing the supernatant, namely DNA, at-20 ℃ for later use;
2. configuration of RAA reaction system: one RAA reaction dry powder tube was used for each test sample, and the reaction components and the added volume in each RAA reaction dry powder tube are shown in Table 3.
Table 3:
| RAA reaction system component | Volume (μ L) |
| A Buffer | 12.5μL |
| B Buffer | 2.5μL |
| Primer mixture | 4μL |
| Specific fluorescent probe | 0.6μL |
| DNA template | 2μL |
| ddH2O | 28.4μL |
| Total volume | 50μL |
A Buffer is 20% PEG; b Buffer is 280mM MgAc
TABLE 3 RAA reaction Dry powder tube reaction Components and addition volumes
3. Placing the RAA reaction tube with the prepared reaction system in an ABI7500 amplification instrument, and carrying out RAA amplification according to the following procedures: at 39 ℃ for 40 s; at 39 ℃ for 20min, for a total of 40 cycles. Fluorescence of FAM channels was collected for each cycle.
4. And after the amplification is finished, judging a positive result or a negative result of the bacterial sepsis according to the fluorescence curve judgment and the CT value. And (4) judging the result: the fluorescence curve of the FAM channel is S-shaped, the CT value is less than or equal to 35, and the positive result of the bacterial septicemia is judged; and when the curve of the sample to be detected does not show an S shape or the CT value is more than 35, judging as a negative result of the bacterial septicemia.
Example 4: evaluation of RAA detection kit of the invention in clinical practical application
The kit is adopted to carry out clinical blind sample experiments, and 30 parts of crucian are detected; the experimental result shows that the second primer pair can distinguish the bacterial septicemia, and the positive detection rate of the second primer pair is the same as that of the experimental result cultured by an actual culture medium. Of 30, laboratory media, 19 were positive results, 11 were negative results, 19 were positive results and 11 were also negative results as measured by the RAA method, and they may correspond one to one.
Test example 5: sensitivity test of the kit of the invention
The bacterial sepsis standard plasmid provided by the kit in the embodiment 2 of the invention is used for extracting a positive plasmid, measuring the concentration of the positive plasmid by using the NanoDrop, and diluting the positive plasmid to 5 concentration gradients of 1 pg/muL, 100 fg/muL, 10 fg/muL, 1 fg/muL and 0.1 fg/muL respectively to perform a sensitivity test.
The detection results are shown in figure 2, and are the amplification results of the positive standard substances of 1 pg/muL, 100 fg/muL, 10 fg/muL, 1 fg/muL and 0.1 fg/muL from left to right in sequence, so that the RAA fluorescence amplification reagent and the detection sensitivity can reach 0.1 fg/muL, the accuracy is superior to that of the common PCR detection method, and the RAA constant temperature fluorescence detection kit and the detection method have high sensitivity to FBS diagnosis.
Test example 6: specificity test of the kit of the present invention
In order to detect the specificity of the kit, the detection methods in example 3 are adopted to respectively detect the viruses GCRV1, CYHV2, CEV, SAV, ISAV and IPNV samples, and the detection conditions of the kit on EHP and other common viruses of fish are analyzed.
The detection result shows that: normal amplification occurred only in FBS samples, negative control (ddH)2O) and none of the GCRV1, CYHV2, CEV, SAV, ISAV and IPNV samples were amplified (as shown in figure 3). The results show that the RAA constant temperature fluorescence detection kit can specifically amplify the target sequence in FBS without cross reaction with other virus nucleic acids. The method and the kit have good specificity and do not generate false negative.
Meanwhile, the same specificity experiment is carried out on the primers 1 and 3 designed by the invention, and the primers can not distinguish different samples well and have poor specificity (the specific experimental data is slight).
The invention shown and described herein may be practiced in the absence of any element or elements, limitation or limitations, which is specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the invention. It should therefore be understood that although the present invention has been specifically disclosed by various embodiments and optional features, modification and variation of the concepts herein described may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
The contents of the articles, patents, patent applications, and all other documents and electronically available information described or cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other documents.
Sequence listing
<110> Hangzhou Zhongzhuang Biotech Co., Ltd
RAA constant temperature fluorescence detection method and reagent for <120> bacterial sepsis (FBS)
<130>18-100070-00006987
<141>2018-09-13
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gatgcccaag tctttcctgt aaaatgaatt agttaacata tattgatcat aaaaatgcat 180
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Claims (9)
1. A kit for detecting bacterial sepsis (FBS) nucleic acid comprising: the kit comprises a forward primer for the bacterial sepsis, a reverse primer and a specific fluorescent probe, wherein the nucleotide sequence of the forward primer for the bacterial sepsis is shown as SEQ ID No.1, the nucleotide sequence of the reverse primer for the bacterial sepsis is shown as SEQ ID No.2, the nucleotide sequence of the specific fluorescent probe is shown as SEQ ID No.3, a fluorescent reporter group is marked at the 5 'end of the specific fluorescent probe, and a fluorescent quencher group is marked at the 3' end of the specific fluorescent probe.
2. The nucleic acid detection kit according to claim 1, wherein the fluorescence reporter group of the specific fluorescent probe is selected from one of FAM, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460, and the fluorescence quenching gene is selected from one of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.
3. The nucleic acid detection kit according to claims 1 and 2, further comprising a primer mixture, a specific fluorescent probe, ABuffer, B Buffer, RAA dry powder reagent, bacterial sepsis standard, and ddH2At least one of O.
4. The kit according to claim 3, wherein the A Buffer is 20% PEG; b Buffer is 280mM MgAc.
5. The kit of claim 5, wherein the RAA dry powder reagent is comprised of: 1mmol/LdNTP, 90 ng/muL SSB protein, 120 ng/muL recA recombinase protein (SC-recA/BS-recA) or 30 ng/muL Rad51, 30 ng/muL Bsu DNA polymerase, 100mmol/L Tricine, 20% PEG, 5mmol/L dithiothreitol, 100 ng/muL creatine kinase, and Exo exonuclease.
6. The nucleic acid detection kit according to any one of claims 1 to 5, wherein the standard for bacterial sepsis is a positive plasmid containing a partial sequence of a gene of Aeromonas hydrophila.
7. The kit according to claim 6, wherein the positive plasmid containing the gene partial sequence of Aeromonas hydrophila has a sequence shown in SEQ ID No. 4.
8. The RAA constant temperature fluorescence detection method for bacterial septicemia comprises the steps of extracting DNA of a sample to be detected, taking the DNA of the sample to be detected as a template, and performing reverse amplification on a forward primer, a reverse primer, a specific fluorescent probe, an RAA dry powder reagent, A Buffer, BBuffer and ddH of the bacterial septicemia2Carrying out real-time fluorescence RAA reaction in the presence of O, and analyzing a sample to be detected according to a real-time fluorescence RAA amplification curve; wherein the nucleotide sequence of the forward primer of the bacterial sepsis is shown as SEQ ID NO.1, the nucleotide sequence of the reverse primer of the bacterial sepsis is shown as SEQ ID NO.2, the nucleotide sequence of the specific fluorescent probe is shown as SEQ ID NO.3, the 5 'end of the specific fluorescent probe is marked with a fluorescent reporter group, and the 3' end of the specific fluorescent probe is marked with a fluorescent quenching group.
9. Use according to claim 8, characterized in that it comprises the following steps:
(1) and (3) purifying and separating the aeromonas hydrophila strain: sampling from tissues of fishes to be detected by using a sterile inoculating loop, streaking on an AHM flat plate for overnight culture, and purifying and separating strains;
(2) preparing an aeromonas hydrophila DNA solution: : adding 50 mu L of sterile TE solution (10mM Tris-HCl, 1mM EDTA, pH 8.0) into a centrifugal tube, picking single bacterial colony to the TE solution, placing the centrifugal tube on a vortex mixer, fully shaking for 10s, boiling in boiling water for 10min, cooling to room temperature, fully shaking for 10s, then centrifuging at 12000r/min for 2min, and placing the supernatant, namely DNA, at-20 ℃ for later use.
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| CN114657271A (en) * | 2022-03-15 | 2022-06-24 | 中国动物卫生与流行病学中心 | Method for rapidly detecting bovine tuberculosis |
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