CN108504764A - A kind of kit and its method of the detection cyanobacteria based on isothermal amplification technique - Google Patents
A kind of kit and its method of the detection cyanobacteria based on isothermal amplification technique Download PDFInfo
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- CN108504764A CN108504764A CN201810535714.0A CN201810535714A CN108504764A CN 108504764 A CN108504764 A CN 108504764A CN 201810535714 A CN201810535714 A CN 201810535714A CN 108504764 A CN108504764 A CN 108504764A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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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 kit and its method for the detection cyanobacteria that the invention discloses a kind of based on isothermal amplification technique, it is characterized in that the isothermal amplification technique based on recombinase-mediated and combining the exploitation of lateral chromatography test paper technical principle.The kit includes RPA freeze-dried powders, and RPA reacts primer, and RPA reacts probe, RPA reaction buffers, Adlerika, lateral chromatography test paper, sample-loading buffer, double steaming sterile waters and cyanobacteria DNA.The present invention utilizes the extraction that DNA is completed in bursting by freezing method or microwave method rapid extraction cyanobacteria DNA, 15min.The amplified reaction of the present invention carries out under 30 DEG C 45 DEG C of constant temperature, can be read as a result, without relying on specific apparatus in 20min.Kit and its detection method of the present invention, high sensitivity, high specificity is easy to operate, and detection time is short, can be applied to the field quick detection of cyanobacteria in water head site and other water bodys.
Description
Technical field
The kit and its method for detecting cyanobacteria based on isothermal amplification technique that the present invention relates to a kind of, it is characterized in that being based on
The isothermal amplification technique of recombinase-mediated, the field condition that can be used for cyanobacteria quickly detect the monitoring, it can be achieved that cyanobacterial bloom
And early warning, belong to water quality monitoring technical field.
Background technology
Cyanobacteria is to lead to one of Main Algae of lake eutrophication.In recent years, cyanobacterial bloom all over the world breaks out thing
Part is commonplace, and the prevention of wawter bloom has become world environments problem.The prevention key of wawter bloom be how to establish effective monitoring and
Early warning technology reduces the harm that wawter bloom is brought to adopt an effective measure before its outburst in due course.
Currently, the monitoring technology of bloom blue algae is broadly divided into following four：The first is microscope detection method, and this method is to adopting
Sample it is more demanding, and the error of method is also larger.Second is conventional index monitoring method, these indexs include dissolved oxygen,
Total nitrogen, total phosphorus, pH, chlorophyll a etc..This method has certain limitation, because the variation of conventional index might not indicate
The generation of wawter bloom.This third be Satellite Remote Sensing technology, disclosure satisfy that the needs monitored on a large scale, can be reduced artificial
The inconvenience of sampling.But the limited resolution of satellite image, easily influenced by weather.4th kind is secreted by detecting cyanobacteria
Algae toxin content quantifies cyanobacteria, and common method includes HPLC methods, LC/MS methods, ELISA methods, PCR methods etc..This
A little methods disclosure satisfy that the needs of highly sensitive detection algae toxin, have important references meaning for the detection of cyanobacteria.But it is above
Method is required to dependent on accurate detecting instrument, can not be applied to field condition and be detected.Therefore, exploitation one kind being applicable to open country
The kit and its method of cyanobacteria are very necessary in outer Site Detection water.
Recombinase polymeric enzymatic amplification reaction (Recombinase Polymerase Amplification, RPA) is a kind of
Isothermal amplification technique based on recombinase-mediated, the reaction can be carried out in 30 DEG C -45 DEG C of temperature range（Human body auxillary temperature is just
It can react）, monomolecular nucleic acid detection can be carried out in 15 minutes.Requirement of the technology to hardware device is very low, applicable
In the quick detection of cyanobacteria.A kind of detection cyanobacteria kit and its method provided by the invention are based on RPA technologies and to combine side
To chromatographic test paper（Lateral flow strips, LF）Technological development, it can be achieved that being detected to the field condition of cyanobacteria.
Invention content
The purpose of the present invention is the deficiencies for existing cyanobacteria detection technique, provide a kind of inspection based on isothermal amplification technique
The kit and its method for surveying cyanobacteria are the isothermal amplification techniques based on recombinase-mediated and combine lateral chromatography test paper technology former
Reason exploitation, it is applicable to Site Detection of the field to cyanobacteria.
A kind of kit of the detection cyanobacteria based on isothermal amplification technique, the kit includes RPA freeze-dried powders, RPA
Primer is reacted, RPA reacts probe, RPA reaction buffers, Adlerika, lateral chromatography test paper, sample-loading buffer, double steaming nothings
Bacterium water and cyanobacteria DNA.
The RPA reaction primers are as shown in table 1, and the designed combination of 4 pairs of primers H1, H2, H3, H4 can be real right
Use Biotin in 5 ' ends of the specific amplification of cyanobacteria, all reverse primers（Biotin）Label.
The mark mode of the reverse primer is not limited to Biotin labels, can also be replaced with other markers, such as
DIG, FITC, FAM etc..
Table 1- primer sequences
The RPA reaction probes are as shown in table 2, and the probe of all designs is marked with FAM at 5 ' ends, uses THF（Tetrahydrofuran
Base）Instead of the 33rd base, with 3 ' end of SpacerC3 closings.
The RPA reactions probe LF-P1 can be adapted for the combination of H1 and H2 primers, and probe LF-P2 can be adapted for H1
It is combined with H2 primers, probe LF-P3 can be adapted for the combination of H1, H2, H3 and H4 primer.Probe and primer combination are according to above-mentioned
Corresponded manner combined use can specifically detect cyanobacteria.
The mark mode of the RPA reaction probes is not limited to FAM labels, can also be replaced with other markers, such as
Biotin, FITC, FAM etc..
The closing mode that the RPA reaction probes 3 ' are held can be not limited to SpacerC3, can also use other closing sides
Formula replaces, such as phosphate, dideoxy nucleotide.
Table 2- probe sequences
| Probe title | Sequence (5'-3') |
| LF-P1 | FAM-cagccgcggtaatacgggggaggcaagcgtta-THF-ccggaattattgggcgt-SpacerC3 |
| LF-P2 | FAM-cgtccgcaggtggtcagccaagtctgccgtca-THF-atcaggttgcttaacga-SpacerC3 |
| LF-P3 | FAM-cggtggaaactggcagactagagagcagtagg-THF-gtagcaggaattcccag-SpacerC3 |
The RPA freeze-dried powders include bacteriophage recombinase UvsX, confactor UvsY, archaeal dna polymerase, single stranded DNA knot
Synthase and dNTPs.
Lateral chromatography test paper front end is coated with the nano Au particle of FAM antibody modifications, is coated in detection line
Biotin antibody, nature controlling line are coated with sessile antibody（Colour developing can be combined with the nano Au particle with antibody）.
The antibody modified on the coated nano Au particle in lateral chromatography test paper front end is not limited to FAM antibody, can be with
It is replaced with the antibody of the marker of other probe modifications.
Coated antibody can be not limited to Biotin antibody in the detection line of the lateral chromatography test paper, can use other
The antibody of the marker of reverse primer modification replaces.
A method of the detection cyanobacteria based on isothermal amplification technique, experimental principle are as follows：
The present invention devises RPA with one section of general conserved genetic sequences of cyanobacteria and reacts primer, is combined, is passed through to primer
RPA reaction experiments filtered out can specific amplification cyanobacteria optimal primer combination, and by many experiments optimization determine most
Excellent RPA reaction systems.The standard cyanobacteria sample of laboratory cultures or practical cyanobacterial bloom sample are passed through into bursting by freezing method or microwave
Method rapid cleavage cell, released dna take supernatant as DNA laboratory samples after high speed centrifugation.DNA sample is utilized and is carried
The primer combination of Biotin labels and the probe of FAM labels carry out RPA reactions, the product amplified simultaneous with Biotin and
FAM is marked.Lateral chromatography test paper front end is coated with the nano Au particle with FAM antibody modifications, and Biotin is coated in detection line
Antibody.When reaction solution enters test strips, it will be combined and be made by antigen-antibody with the amplified production that Biotin and FAM is marked
With, formed and biotin antibody-nucleic acid gold particle composite and developed the color in detection line, on nature controlling line it is coated determine antibody can be straight
It connects and is combined colour developing with the nano Au particle with FAM antibody.When nature controlling line and two band of detection line develop the color simultaneously, there is spy with regard to explanation
Specific amplification product generates, it was demonstrated that there are the DNA of cyanobacteria in the sample of detection.
A method of the detection cyanobacteria based on isothermal amplification technique carries out according to following operating procedure：
（1）Rapid cleavage cyanobacteria DNA（Following two methods can be quickly obtained cyanobacteria DNA）
Bursting by freezing method：It takes 1 mL water samples containing cyanobacteria to move to 1.5 mL centrifuge tubes, passes through high speed centrifugation（13000 rpm/min, 5-10
min）Cells of Blue-green Algae is collected, supernatant is abandoned, retains precipitation, 50 μ L distilled waters or cell pyrolysis liquid is added, Cells of Blue-green Algae is resuspended,
Liquid nitrogen or -80 DEG C of refrigerator 3-5 min are placed on, sample is taken out, constant temperature places 1- in 38 DEG C of -45 DEG C of water-baths or metal bath
2min, multigelation operate 3 times, high speed centrifugation sample（13000 rpm/min, 10 min）, take supernatant as subsequent experimental
DNA sample；
Microwave method：It takes 1 mL water samples containing cyanobacteria to move to 1.5 mL centrifuge tubes, passes through high speed centrifugation（13000 rpm/min, 5-10
min）Cells of Blue-green Algae is collected, supernatant is abandoned, retains precipitation, 50 μ L cell pyrolysis liquids are added, Cells of Blue-green Algae is resuspended, by resuspension
Cyanobacteria sample is placed in a micro-wave oven special box with cover, is closed the lid, and puts 750 W power or high temperature in micro-wave oven into,
Heat 1-3min.Sample is taken out, is put into centrifuge, high speed centrifugation sample（13000 rpm/min, 10 min）, take supernatant
As subsequent experimental DNA sample；
（2）Reaction system：RPA reaction buffers are added in RPA freeze-dried powders（Rehydration Buffer）25-29.5 μL,
10 μm of ol/L forward primer 1-2.4 μ L, 10 μm of ol/L reverse primer 1-2.4 μ L, 10 μm of ol/L probe 0.1-0.6 μ L, it is blue
Algae DNA 0.5-2 μ L add double steaming sterile waters to supply volume and add 280 mmol/ on lid after centrifuging mixing to 47.5 μ L
2.5 μ L of L magnesium acetates are overturned 8-10 times, centrifuge mixing；
（3）Reaction process：Reaction system is placed in the thermostat of 30-45 DEG C of temperature range（Such as water-bath, metal bath, human body oxter
Or other isoperibols）It is incubated 15-20 min.It takes out, reaction product plus sample-loading buffer is diluted by 50-200 times, take 10 μ
L is applied directly to the front end of lateral chromatography test paper, and the sample-loading buffer that lateral chromatography test paper is placed in 100-200 μ L is incubated 2-
8min takes out, you can by visually directly reading result；
（4）Result judgement：When there was only nature controlling line colour developing on lateral chromatography test paper, result is feminine gender, in the sample for illustrating detection
There is no cyanobacterias；When nature controlling line on lateral chromatography test paper and detection line develop the color simultaneously, result is the positive, illustrates the sample of detection
In there are cyanobacterias；When nature controlling line does not develop the color on lateral chromatography test paper, illustrates that this detection is invalid, need to detect again.
Compared with prior art, the present invention has following progress and effect:（1）The present invention utilizes bursting by freezing method or microwave method
The DNA of rapid extraction cyanobacteria is obtained with the DNA sample of cyanobacteria in 15 minutes, shorten the extraction time of DNA；（2）This hair
Bright to carry out visualization quantitative and semi-quantitative detection to the amplified production of RPA using lateral chromatography test paper, entire amplified reaction is 25
It is carried out under the constant temperature of DEG C -45 DEG C of ranges, the band on test paper can be observed by the naked eye in 15-20 minutes, is directly read
As a result, reaction and detection process are not necessarily to rely on specific apparatus.Kit and its detection method of the present invention have sensitive
Degree is high, and high specificity is easy to operate, and detection time is short, can be applied to the scene quickly inspection of cyanobacteria in water head site and other water bodys
It surveys, contributes to the prevention and control to cyanobacterial bloom and monitoring.
Description of the drawings
Fig. 1, positive test result, 1-8 be blue algae strain, 9 for non-blue algae strain as negative control.
Fig. 2, negative experimental result, 1 for blue algae strain be used as positive control, 2-7 be non-cyanobacteria sample.
Fig. 3 is used for the detection of actual water sample, and the concentration of cyanobacteria is respectively 107Cell/mL, 106Cell/mL, 105
Cell/mL, 104Cell/mL, 103Cell/mL, 102Cell/mL, 10 cell/mL, 1 cell/mL, 0.1 cell/mL,
0.01 cell/mL, NC are sterile water, as blank control.
Specific implementation mode
Below in conjunction with the accompanying drawings, the specific implementation mode of the present invention is described in further detail, it is to be understood that this hair
Bright protection domain is not restricted by specific implementation.
Embodiment
1. experiment material：RPA freeze-dried reagents, Adlerika, RPA reaction buffers are purchased from TwistDX companies
TwistAmp nfo kits, lateral chromatography test paper and its sample-loading buffer are purchased from the Milenia of TwistDX companies
1 kits of HybriDetect.
2. the primer and probe selected：The combination of H4 primers and LF-P3 probes is selected to carry out RPA reactions.
3. reaction system：RPA reaction buffers are added in RPA freeze-dried powders（Rehydration Buffer）29.5 μ
L, 10 μm of ol/L forward primers 2 μ L, 10 μm of ol/L reverse primers 2 μ L, 10 μm of ol/L probes 0.2 μ L, cyanobacteria DNA 2
μ L add double steaming sterile waters to supply volume and add 280 mmol/L magnesium acetates, 2.5 μ on lid after centrifuging mixing to 47.5 μ L
L is overturned 10 times, centrifuges mixing.
4. reaction process：Reaction system is placed in 37 DEG C of 15 min of metal bath.It takes out, by reaction product plus sample-loading buffer
It is diluted by 200 times, takes 10 μ L to be applied directly to the front end of lateral chromatography test paper, lateral chromatography test paper is placed in the loading of 200 μ L
Buffer solution is incubated 4min, takes out, you can by visually directly reading result.
5. specificity experiments：In order to verify the specificity of this detection kit and its detection method, several plants of different indigo plants have been taken
Algae sample and several plants of non-cyanobacteria samples, are detected, as a result as depicted in figs. 1 and 2.
6. sensitivity experiment：In order to verify the detection limit and sensitivity of this detection kit and its detection method, by cyanobacteria
The reservoir water sample of algal bloom carries out microscopic counting cyanobacteria quantity and is detected by 10 times of gradient dilutions, as a result such as Fig. 3
It is shown.
In Fig. 1, nature controlling line when blue algae strain samples different this 8 plants of 1-8 is detected（Control）And detection line
（Test）Two bands develop the color, and 9 non-blue algae strain of negative control group only has nature controlling line（Control）One band develops the color.In Fig. 2,
Non- cyanobacteria samples different 2-7 this 6 only has nature controlling line when being detected（Control）One band develops the color, and positive controls 1
Wawter bloom Microcystis aeruginosa（Cyanobacteria）Nature controlling line（Control）And detection line（Test）Two bands develop the color.From the result of Fig. 1 and Fig. 2
As can be seen that the kit and its detection method of a kind of detection cyanobacteria based on isothermal amplification technique provided by the invention are detecting
When different cyanobacterias is with non-blue algae strain, there is higher specificity, cyanobacteria and non-cyanobacteria sample can be distinguished well.
From Fig. 3 as can be seen that it is provided by the invention it is a kind of based on isothermal amplification technique detection cyanobacteria kit and its
Detection method can be applied to the detection of practical cyanobacterial bloom water sample, and detection is limited to 0.1 cell/mL.
Claims (5)
1. a kind of kit of the detection cyanobacteria based on isothermal amplification technique, which is characterized in that the kit includes RPA freeze-dryings
Powder, RPA react primer, and RPA reacts probe, RPA reaction buffers, Adlerika, lateral chromatography test paper, loading buffer
Liquid, double steaming sterile waters and cyanobacteria DNA.
2. the kit of detection cyanobacteria according to claim 1, which is characterized in that RPA reaction primers such as 1 institute of table
Show, the combination of designed 4 pairs of primers H1, H2, H3, H4 can reality to the specific amplification of cyanobacteria, all reverse primers
5 ' ends are marked with Biotin；The mark mode of the reverse primer is not limited to Biotin labels, can also be marked with other
Object replaces, such as DIG, FITC, FAM.
3. the kit of detection cyanobacteria according to claim 1, which is characterized in that RPA reaction probes such as 2 institute of table
Show, the probe of all designs is marked with FAM at 5 ' ends, uses THF（Tetrahydrofuran base）Instead of the 33rd base, use
3 ' end of SpacerC3 closings；The described RPA reactions probe LF-P1 can be adapted for the combination of H1 and H2 primers, and probe LF-P2 can be with
It is combined suitable for H1 and H2 primers, probe LF-P3 can be adapted for the combination of H1, H2, H3 and H4 primer；Probe and primer combination are pressed
Cyanobacteria can be specifically detected according to above-mentioned corresponded manner combined use；The mark mode of the probe is not limited to FAM
Label, can also be replaced, such as Biotin with other markers（Biotin）, FITC, FAM etc.；The RPA reaction probes 3 ' are held
Closing mode can be not limited to SpacerC3, can also be replaced with other closing modes, such as phosphate, dideoxy nucleotide
Deng.
4. the kit of detection cyanobacteria according to claim 1, which is characterized in that lateral chromatography test paper front end is coated with
There is the nano Au particle of FAM antibody modifications, Biotin antibody is coated in detection line, nature controlling line is coated with sessile antibody（It can be with
Colour developing is combined with the nano Au particle with antibody）；It is modified on the coated nano Au particle in lateral chromatography test paper front end
Antibody is not limited to FAM antibody, can be replaced with the antibody of the marker of other probe modifications；The inspection of the lateral chromatography test paper
Coated antibody can be not limited to Biotin antibody on survey line, the antibody generation for the marker that can be modified with other reverse primers
It replaces.
5. a kind of method of the detection cyanobacteria based on isothermal amplification technique, which is characterized in that carried out according to following operating procedure：
（1）Rapid cleavage cyanobacteria DNA（Following two methods can be quickly obtained cyanobacteria DNA）
Bursting by freezing method：It takes 1 mL water samples containing cyanobacteria to move to 1.5 mL centrifuge tubes, passes through high speed centrifugation（13000 rpm/min, 5-10
min）Cells of Blue-green Algae is collected, supernatant is abandoned, retains precipitation, 50 μ L distilled waters or cell pyrolysis liquid is added, Cells of Blue-green Algae is resuspended,
Liquid nitrogen or -80 DEG C of refrigerator 3-5 min are placed on, sample is taken out, constant temperature places 1- in 38-45 DEG C of water-bath or metal bath
2min, multigelation operate 3 times, high speed centrifugation sample（13000 rpm/min, 10 min）, take supernatant as subsequent experimental
DNA sample；
Microwave method：It takes 1 mL water samples containing cyanobacteria to move to 1.5 mL centrifuge tubes, passes through high speed centrifugation（13000 rpm/min, 5-10
min）Cells of Blue-green Algae is collected, supernatant is abandoned, retains precipitation, 50 μ L cell pyrolysis liquids are added, Cells of Blue-green Algae is resuspended, by resuspension
Cyanobacteria sample is placed in a micro-wave oven special box with cover, is closed the lid, and puts 750 W power or high temperature in micro-wave oven into,
1-3min is heated, sample is taken out, is put into centrifuge, high speed centrifugation sample（13000 rpm/min, 10 min）, take supernatant
As subsequent experimental DNA sample；
（2）Reaction system：RPA reaction buffers are added in RPA freeze-dried powders（Rehydration Buffer）25-29.5 μL,
10 μm of ol/L forward primer 1-2.4 μ L, 10 μm of ol/L reverse primer 1-2.4 μ L, 10 μm of ol/L probe 0.1-0.6 μ L, it is blue
Algae DNA 0.5-2 μ L add double steaming sterile waters to supply volume and add 280 mmol/ on lid after centrifuging mixing to 47.5 μ L
2.5 μ L of L magnesium acetates are overturned 8-10 times, centrifuge mixing；
（3）Reaction process：Reaction system is placed in the thermostat of 30-45 DEG C of temperature range（Such as water-bath, metal bath, human body oxter
Or other isoperibols）15-20 min are incubated, takes out, reaction product plus sample-loading buffer is diluted by 50-200 times, take 10 μ
L is applied directly to the front end of lateral chromatography test paper, and the sample-loading buffer that lateral chromatography test paper is placed in 100-200 μ L is incubated 2-
8min takes out, you can by visually directly reading result；
（4）Result judgement：When there was only nature controlling line colour developing on lateral chromatography test paper, result is feminine gender, in the sample for illustrating detection
There is no cyanobacterias；When nature controlling line on lateral chromatography test paper and detection line develop the color simultaneously, result is the positive, illustrates the sample of detection
In there are cyanobacterias；When nature controlling line does not develop the color on lateral chromatography test paper, illustrates that this detection is invalid, need to detect again.
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Cited By (3)
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| WO2020208235A1 (en) * | 2019-04-11 | 2020-10-15 | Microbia Environnement | Use of probes for detecting toxigenic cyanobacteria, detection method and corresponding kits |
| CN112301107A (en) * | 2020-11-03 | 2021-02-02 | 中国科学院城市环境研究所 | Kit and method for detecting microcystin synthesis gene mcyG based on isothermal amplification technology |
| CN114703177A (en) * | 2022-03-29 | 2022-07-05 | 河南省农业科学院动物免疫学重点实验室 | Pseudorabies virus detection composition, method and kit based on RPA (reverse transcriptase amplification) isothermal amplification and immunochromatography technology |
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| FR3094989A1 (en) * | 2019-04-11 | 2020-10-16 | Microbia Environnement | USE OF PROBES FOR DETECTION OF TOXINOGENOUS CYANOBACTERIA, DETECTION METHOD AND CORRESPONDING KITS |
| CN112301107A (en) * | 2020-11-03 | 2021-02-02 | 中国科学院城市环境研究所 | Kit and method for detecting microcystin synthesis gene mcyG based on isothermal amplification technology |
| CN114703177A (en) * | 2022-03-29 | 2022-07-05 | 河南省农业科学院动物免疫学重点实验室 | Pseudorabies virus detection composition, method and kit based on RPA (reverse transcriptase amplification) isothermal amplification and immunochromatography technology |
| CN114703177B (en) * | 2022-03-29 | 2023-08-11 | 河南省农业科学院动物免疫学重点实验室 | Pseudorabies virus detection composition, method and kit based on RPA isothermal amplification and immunochromatography technology |
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