CN111593139B - RT-RAA primer, probe and kit for detecting classical swine fever virus and application - Google Patents
RT-RAA primer, probe and kit for detecting classical swine fever virus and application Download PDFInfo
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Abstract
The invention discloses an RT-RAA primer, a Probe and a kit for detecting classical swine fever virus and application thereof, wherein the RT-RAA primer and the Probe for detecting classical swine fever virus comprise a forward primer CSFV-5 'NTR-F (SEQ ID NO.1), a reverse primer CSFV-5' NTR-R (SEQ ID NO.2) and a Probe CSFV-5 'NTR-Probe (SEQ ID NO.3) for identifying a classical swine fever virus 5' NTR gene. The amplification result obtained by RT-RAA nucleic acid amplification through the primers and the probe can be visually judged through a portable blue light instrument, and the CSFV can be quickly, specifically, sensitively and simply detected, so that the defects of the existing detection technology are overcome.
Description
Technical Field
The invention relates to the technical field of animal epidemic disease detection, in particular to an RT-RAA primer, a probe and a kit for detecting classical swine fever virus and application thereof.
Background
Classical Swine Fever (CSF) is an acute, febrile, highly contagious disease of pigs caused by Classical Swine Fever Virus (CSFV), characterized by high fever retention and extensive bleeding, infarction and necrosis caused by small vessel wall degeneration, with high morbidity and mortality, and classified as one of class a official infectious diseases by the world animal health Organization (OIE). Pigs are the only natural host of the disease, and sick pigs and pigs with viruses are the most important infectious sources. The swine fever has serious harm to pigs, and can cause huge economic loss in the pig industry. In recent years, the epidemic and the onset characteristics of swine fever have changed a lot, and the swine fever has the characteristics of atypical swine fever, mild swine fever, sporadic epidemic, mild or unobvious onset characteristics and clinical symptoms, low mortality, uncharacteristic pathological changes and often recessive infection, thereby bringing great difficulty to clinical diagnosis and being capable of confirming the diagnosis only by relying on laboratory diagnosis.
In order to reduce the huge economic loss of swine fever to the swine industry, a rapid and accurate diagnosis technology is needed. At present, a frozen section direct Fluorescent Antibody (FA) test is one of methods for checking CSFV antigens, tonsil is the first choice disease material, and serum neutralization test, ELISA, PCR, animal inoculation test such as rabbit body interactive immunity test and other methods are also the more common detection methods in laboratories at present, can be used for determining the swine fever and play an important role in diagnosis and prevention and control of the swine fever. The conventional diagnosis method has the defects of complex operation, complex instrument and equipment and long required time, is not beneficial to the rapid diagnosis of the swine fever, and is not suitable for the primary layer and the point of care testing (POCT). Therefore, the established method for detecting the hog cholera virus is rapid, simple, convenient, high in specificity and high in sensitivity, and has important significance for diagnosis, prevention and control of the hog cholera virus.
The Recombinase mediated isothermal Amplification (RAA) is a novel isothermal in-vitro nucleic acid Amplification technology, and an Amplification product equivalent to that of the traditional PCR reaction can be obtained within 30min at 37-42 ℃. The RAA technique relies primarily on three enzymes: recombinases that bind single-stranded nucleic acids (oligonucleotide primers), single-stranded DNA binding proteins (SSBs), and strand-displacing DNA polymerases. Mixtures of these three enzymes are also active at ambient temperature. The recombinase, in combination with the primer, forms a protein-DNA complex that is able to search for homologous sequences in double-stranded DNA. Once the primers locate the homologous sequences, the double-stranded structure of the template DNA is opened with the aid of the single-stranded DNA binding protein, and a new complementary strand of DNA is formed under the action of DNA polymerase, thereby exponentially amplifying the target region on the template.
The RAA reaction is simple and quick, the requirement on the instrument is simple, and a temperature control instrument for thermal cycle reaction is not needed. Reverse transcriptase is added in a basic system, so that an additional cDNA synthesis step is omitted, and the RNA template can be amplified in one step. Real-time reading of data can be achieved using exo probe technology and exonuclease III, just like fluorescent quantitative PCR. The exo RT-RAA combining exo probe technology, exonuclease III and reverse transcriptase can realize real-time amplification of an RNA template in one step. The technology has the characteristics of high sensitivity, better specificity and reliability, capability of realizing on-site rapid detection and the like. And then, according to the fact that FAM in the probe is a green fluorophore which can be excited to emit green fluorescence under blue light, visual judgment can be carried out on RAA amplification products by combining a portable blue light instrument, and the method is suitable for instant detection and is an isothermal amplification technology with popularization and application prospects in the aspect of future nucleic acid detection.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Disclosure of Invention
The invention aims to provide a RT-RAA primer and a probe for detecting classical swine fever viruses, wherein Reverse transcription recombinase-induced amplification (RT-RAA) is carried out on the classical swine fever viruses through the primer and the probe so as to realize quick, specific, sensitive, visual and simple detection of the classical swine fever viruses.
In order to achieve the purpose, the invention provides an RT-RAA primer and a Probe for detecting classical swine fever virus, which comprise a forward primer CSFV-5 ' NTR-F, a reverse primer CSFV-5 ' NTR-R and a Probe CSFV-5 ' NTR-Probe for identifying the 5 ' NTR gene of the classical swine fever virus, wherein the sequences of the forward primer CSFV-5 ' NTR-F, the reverse primer CSFV-5 ' NTR-R and the Probe CSFV-5 ' NTR-Probe are as follows: forward primer CSFV-5' NTR-F: 5'-ACTGGGCTAGCCATGCCCACAGTAGGACTAGCAAA-3' (SEQ ID NO.1), reverse primer CSFV-5 ' NTR-R: 5'-GCTTCTGCTCACGTCGAACTACTGACGACTG-3' (SEQ ID NO.2), Probe CSFV-5 ' NTR-Probe5 ' -ACTAGCCGTAGTGGCGAGCTCCCTGGGTGGTCTAAGTCCTGAGTACAG-3 ' (SEQ ID NO. 3).
In one embodiment of the invention, the Probe CSFV-5 ' NTR-Probe is characterized in that the 31 st base T from the 5 ' end marks FAM luminophore, the 32 nd base C is replaced by tetrahydrofuran THF, the 33 rd base T marks BHQ1 quencher, and the 3 ' end is modified by C3-spacer blocking.
The invention also provides a kit for detecting the classical swine fever virus, which comprises the RT-RAA primer and the probe for detecting the classical swine fever virus.
In one embodiment of the invention, the kit further comprises a reagent for reverse transcription recombinase-mediated isothermal amplification.
In one embodiment of the invention, the reagents include reaction buffer, RNase Free ddH 2 O, buffer solution containing magnesium ions and an RT fluorescence basic reaction unit.
In one embodiment of the present invention, the buffer containing magnesium ions is magnesium acetate, the RT fluorescence-based reaction unit comprises a single-stranded DNA binding protein, a recombinase, a reverse transcriptase and a polymerase, and preferably, the RT fluorescence-based reaction unit is in the form of a lyophilized powder.
The invention also provides application of the kit for detecting the classical swine fever virus in preparation of a preparation for detecting the classical swine fever virus, which comprises the following steps: and (3) RNA extraction: extracting RNA of a sample to be detected; amplification of an RT-RAA reaction system: RT-RAA primers and probes for detecting classical swine fever virus by taking extracted RNA as template and adding the extracted RNA into a kit and reverse transcription recombinase mediated isothermal amplificationThe reagents form an RT-RAA reaction system to carry out RT-RAA amplification; and judging the result: quality control standard: if the negative control has no amplification curve or the visual result is colorless, and the positive control has amplification curve or the visual result is green, the experimental data is valid, otherwise the experimental result is invalid; and (3) describing and judging results: the sample to be detected has no amplification curve or the visual result is colorless, and the sample is judged to be negative; the appearance of an amplification curve or a visual result is green, and the sample is judged to be positive; wherein, the template in the negative control is added with RNase Free ddH 2 O, the template in the positive control is an RNA template of a known CSFV strain.
In one embodiment of the present invention, the total amount of the RT-RAA reaction system is 50. mu.L, and the RT-RAA reaction system comprises 29.4. mu.L of reaction buffer, 2. mu.L each of 10 pmol/. mu.L of CSFV-5 ' NTR-F and CSFV-5 ' NTR-R, 0.6. mu.L of 10 pmol/. mu.L of CSFV-5 ' NTR-Probe, and RNase Free ddH 2 O and sample RNA template to be detected are 13.5 mu L, magnesium acetate is 2.5 mu L, and RT fluorescence basic reaction unit (comprising single-stranded DNA binding protein, recombinase, reverse transcriptase and polymerase) is adopted.
In one embodiment of the invention, the reaction condition of the RT-RAA is isothermal amplification at 42 ℃ for 30 min.
In one embodiment of the present invention, the result judgment is performed by a blue light detector for visual judgment of the result, and preferably, the visual judgment is performed directly by naked eyes in a darkroom using a portable TGreen Monitor blue light electrophoresis Monitor (OSE-470M) of skyhoot biochemical technology ltd.
Compared with the prior art, the invention has the following beneficial effects:
firstly, the invention provides an RT-RAA primer, a probe and a kit for detecting classical swine fever virus and application thereof. The detection of the classical swine fever virus by using the kit containing the RT-RAA primer and the probe can be completed only by reacting for 30min at the constant temperature of 37-42 ℃, the method has the advantages of quick reaction, simple requirement on instruments, no need of a temperature control instrument for thermal cycle reaction, greatly shortened reaction time, suitability for instant detection (POCT), and capability of really realizing portable quick nucleic acid detection.
Secondly, the RT-RAA amplification result can be visually judged by a portable blue light instrument so as to realize the rapid, specific, sensitive and simple detection of CSFV, thereby making up the defects of the existing traditional detection technology.
Thirdly, the method detects the swine fever virus by a reverse transcription recombinase mediated isothermal amplification method (RT-RAA), has simple operation, short time, small and exquisite instrument, convenient carrying and mobility, is suitable for rapid detection of the swine fever on a basic level or a field, and strives for precious time for early prevention and control of the swine fever.
Drawings
FIG. 1A is a schematic diagram of the RT-RAA optimized primer screening principle for CSFV according to one embodiment of the present invention;
FIG. 1B is a schematic diagram showing the specific positions of primers and probes for RT-RAA optimization of classical swine fever virus according to one embodiment of the present invention;
FIG. 2A is the real-time RT-RAA amplification result for detecting optimal reaction temperature according to one embodiment of the present invention;
FIG. 2B is the results of RT-RAA visualization to detect optimal reaction temperature according to one embodiment of the present invention;
FIG. 3A is the real-time RT-RAA amplification result specific to the detection primers and probes according to one embodiment of the present invention;
FIG. 3B is the real-time RT-qPCR amplification result specific to the detection primers and probes according to one embodiment of the invention;
FIG. 3C is the result of RT-RAA visualization of the specificity of the detection primers and probes according to one embodiment of the invention;
FIG. 4A is the real-time RT-RAA amplification result of detecting primer and probe sensitivity according to one embodiment of the invention;
FIG. 4B is the real-time RT-qPCR amplification result for detecting primer and probe sensitivity according to one embodiment of the invention;
FIG. 4C is the results of RT-RAA visualization of the sensitivity of detection primers and probes according to one embodiment of the invention.
Description of the main reference numerals:
1: 42 ℃; 2: 41 ℃; 3: 40 ℃; 4: 39 ℃; 5: 38 ℃; 1': porcine pseudorabies virus; 2': porcine reproductive and respiratory syndrome virus; 3': porcine circovirus type 2; 4': porcine epidemic diarrhea virus; 5': porcine delta coronavirus; 6': porcine type a seneca virus; 7': bovine viral diarrhea virus; 8': hog cholera virus; 9': negative control; 1": 10 3 TCID 50 RNA CSFV；2”：10 2 TCID 50 RNA CSFV；3”：10 1 TCID 50 RNA CSFV；4”：10 0 TCID 50 RNA CSFV；5”：10 -1 TCID 50 RNA CSFV; 6": and (5) negative control.
Detailed Description
The following detailed description of the present invention is provided in conjunction with the accompanying drawings, but it should be understood that the scope of the present invention is not limited to the specific embodiments.
Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.
The experimental methods involved in the invention are all conventional methods unless otherwise specified.
Example 1 hog cholera virus RT-RAA detection primer and Probe design and screening
The method is characterized in that the NCBI finds out 51 public CSFV whole genome sequences from countries in the world, Vector NTI software is used for comparing and analyzing conserved regions of the 51 CSFV whole genome sequences, and after homology analysis, the 5 'NTR gene of CSFV is found to be very conserved, so that primers and probes of RT-RAA are designed according to the most conserved region of the 5' NTR gene of CSFV. The design of the RT-RAA primers follows the following basic principles: the length of the primer is more than or equal to 30bp, preferably between 30 and 35 bp; the length of the amplicon is not more than 500bp, preferably between 100 and 200 bp; a GC content of more than 30%, less than 70%, preferably between 40% and 60%; it is desirable to avoid the presence of many repeated short sequences in the primer; avoid the primer to directly form a hairpin structure, or the formation of primer dimer, and the like. In order to ensure the efficiency of RT-RAA amplification, a large amount of primers need to be screened, so that a better primer combination can be obtained. The principle of primer screening is as follows: the first screening is to screen all reverse primers by using a certain forward primer, then to screen all forward primers by selecting the optimal reverse primer, and finally to screen a pair of good primers. For the primer combination needing to obtain high sensitivity, a second primer screening is carried out, and the screening method is improved by using the optimal primer combination screened out in the first time as follows: the position of the primer on the template is moved back and forth at a speed of 1 to 3 bases or bases are added and removed at both ends of the primer to screen a primer combination better than the first primer.
The primer combinations and probes of Table 1 below were designed and screened according to the above primer screening principle.
Primer combinations and probe sequences designed in Table 1
The screening of the primer pair and the probe comprises the following steps:
1) preparation of RT-RAA reaction System
Total 50. mu.L of reaction buffer 29.4. mu.L, 10 pmol/. mu.L of each of CSFV-5 ' NTR-F and CSF V-5 ' NTR-R2. mu.L, 10 pmol/. mu.L of CSFV-5 ' NTR-Probe 0.6. mu.L, RNase Free ddH 2 O and a sample RNA template to be detected are 13.5 mu L together, magnesium acetate is 2.5 mu L, and an RT fluorescence basic reaction unit (containing single-stranded DNA binding protein, recombinase, reverse transcriptase and freeze-dried powder of polymerase) is purchased from Weifang' an ordinary future biotechnology limited company. The negative control template was added with RNase Free ddH 2 O, positive control template is an RNA template of a known CSFV strain.
2) Amplification of RT-RAA reaction systems
The detection reaction conditions were set as: amplification was carried out at a constant temperature of 42 ℃ for 30 min.
The result of visualization was directly judged by naked eyes in a dark room using a portable TGreen Monitor blue-light electrophoresis Monitor (OSE-470M) of Tiangen Biochemical technology Ltd.
Quality control standard: and if the negative control has no amplification curve or the visual result is colorless, and the positive control has amplification curve or the visual result is green, the experimental data is valid, otherwise, the experimental result is invalid.
And (3) describing and judging results: the sample to be detected has no amplification curve or the visual result is colorless, and the sample is judged to be negative; the result of the amplification curve or visualization is green, and the sample is judged to be positive. The early time of positive peak, the fast amplification speed, or the brighter the color of the visual result, the better the primer combination and the probe.
The primer screening steps are as follows: the first screening is to fix a certain upstream primer F4, then to screen 8 downstream primers of R1, R2, R3, R4, R5, R6, R7, and R8, to screen R2 as the optimal downstream primer, then to fix the optimal downstream primer R2, then to screen 6 upstream primers of F1, F2, F3, F4, F5, and F6, to screen F3 as the optimal upstream primer, therefore, the optimal primer combination for the first screening is F3 and R2. For the primer combination needing to obtain high sensitivity, the second primer screening is needed, and the optimal primer combination selected in the first screening needs to be improved as follows: the position of the first selected best primer on the template is moved back and forth at a speed of 1-3 bases or bases are added and removed at both ends of the primer to continue to screen a better primer combination than the first primer. Finally, the optimal primer combination is selected from the combination of the upstream primer F3-5 and the downstream primer R2-2. A schematic of primer screening is shown in FIG. 1A.
The optimal primer combinations and probe sequences were finally screened as follows: forward primer CSFV-5' NTR-F: 5'-ACTGGGCTAGCCATGCCCACAGTAGGACTAGCAAA-3', reverse primer CSFV-5 ' N TR-R: 5'-GCTTCTGCTCACGTCGAACTACTGACGACTG-3', Probe CSFV-5 ' NT R-Probe: 5 ' -ACTAGCCGTAGTGGCGAGCTCCCTGGGTGG (FAM-dT) (THF) (BH Q1-dT) AAGTCCTGAGTACAG [ C3-spacer ], wherein the 31 st base T from the 5 ' end of the probe is used for marking a FAM luminous group, the 32 nd base C is replaced by tetrahydrofuran THF, the 33 rd base T is used for marking a BHQ1 quenching group, and the 3 ' end is subjected to C3-spacer blocking modification.
Example 2 screening of optimum reaction temperature for hog cholera virus RT-RAA
5 sets of reaction conditions (1: 42 ℃, 2: 41 ℃, 3: 40 ℃, 4: 39 ℃ and 5: 38 ℃) with different temperatures are set, and RT-RAA reaction is carried out in the same system by using the optimum primer combination and the same template which are screened out.
The results in FIGS. 2A-2B show that the reaction temperature that produces the strongest fluorescence signal in the shortest time is 42 deg.C, and thus the optimum reaction temperature for the classical swine fever virus RT-RAA of the present invention is 42 deg.C.
EXAMPLE 3 sensitive and specific detection of primer probes
Sensitivity of detection
5 groups of hog cholera virus RNA templates with different concentrations are arranged, and the amplification of nucleic acid is carried out under the optimal condition of RT-RAA.
Extracting RNA of classical swine fever virus with 5x10 according to the instruction of RNA extraction reagent 5 TCID 50 Perml original titer hog cholera cytotoxic, diluted to 10 3 TCID 50 2ul, then 10 times diluted to 10 2 TCID 50 /2ul，10 1 TCID 50 /2ul，10 0 TCID 50 /2ul，10 -1 TCID 50 2ul, 2uL of each sample was used as a reaction template, and a negative control was set, and RT-RAA nucleic acid amplification was performed according to the aforementioned sample addition method. Comparison was also performed using real-time RT-qPCR method in published literature (Haines et al, 2013).
The results in FIGS. 4A-4C show that the primer and probe combination designed by the present invention can ensure the sensitivity in detection, with a detection sensitivity of 10 1 TCID 50 CSFV RNA/response and the visualization sensitivity can reach 10 1 TCID 50 CSFV RNA/reaction.
Detection specificity
Respectively using porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, porcine epidemic diarrhea virus, porcine delta coronavirus, porcine type A seneca virus, bovine viral diarrhea virus and classical swine fever virus as reaction templates, setting negative controls, and carrying out RT-RAA nucleic acid amplification according to the sample adding method. Comparison was also performed with real-time RT-qPCR in published literature (Haines et al, 2013). The detection reaction conditions are as follows: amplification is carried out at a constant temperature of 42 ℃ for 30 min. The results in fig. 3A-3C show that, except for the test group corresponding to the RNA template of the classical swine fever virus showing a normal fluorescence detection curve and a visual green fluorescence result, no amplification curve was shown in the other viruses and the negative control group, and the visual result was colorless. The result shows that the primer and the probe used by the invention can realize the specific detection of the classical swine fever virus and do not have cross reaction with other related viruses.
Example 4 detection application to actual samples
Taking 114 clinical tissue disease material and cytotoxic samples, extracting 114 clinical samples to be detected according to the instruction of an RNA extraction reagent, and storing the extracted RNA at-80 ℃ (repeated freeze thawing is preferably not more than 3 times). RT-RAA nucleic acid amplification was performed according to the loading method described above. This clinical sample was also tested simultaneously for comparison using real-time RT-qPCR method in published literature (Haines et al, 2013).
The results showed that, as shown in Table 2 below, 49 parts (43%) of positive results were detected in 114 clinical samples, 51 parts (44.7%) of positive results were detected in real-time RT-qPCR, and 45 parts (39.5%) of positive results were detected in visual RT-RAA. The Kappa value of Real-time RT-RAA and Real-time RT-qPCR is 0.964, P is less than 0.001, the Kappa value of visual RT-RAA and Real-time RT-qPCR is 0.892, and P is less than 0.001.
TABLE 2 clinical sample test results
In conclusion, the method provided by the invention can realize rapid, specific, sensitive, visual and simple detection of the classical swine fever virus. The method has the advantages of quick reaction, simple requirement on instruments, no need of temperature control instruments for thermal cycle reaction, suitability for the instant detection of the disease on the basis or on the site, and capability of truly realizing portable quick nucleic acid detection.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Sequence listing
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Claims (2)
1. An RT-RAA kit for detecting classical swine fever virus, which is characterized by comprising a forward primer CSFV-5 ' NTR-F, a reverse primer CSFV-5 ' NTR-R and a Probe CSFV-5 ' NTR-Probe for identifying the 5 ' NTR of the classical swine fever virus, wherein the sequences of the forward primer CSFV-5 ' NTR-F, the reverse primer CSFV-5 ' NTR-R and the Probe CSFV-5 ' NTR-Probe are as follows:
forward primer CSFV-5' NTR-F:
5′-ACTGGGCTAGCCATGCCCACAGTAGGACTAGCAAA-3′(SEQ ID NO.1)，
reverse primer CSFV-5' NTR-R:
5'-GCTTCTGCTCACGTCGAACTACTGACGACTG-3' (SEQ ID NO.2), Probe CSFV-5 ' NTR-Probe:
5′-ACTAGCCGTAGTGGCGAGCTCCCTGGGTGGTCTAAGTCCTGAGTACAG-3′(SEQ ID NO.3)；
the 31 st base T of the Probe CSFV-5 ' NTR-Probe is used for marking FAM luminophor from the 5 ' end, the 32 nd base C is replaced by tetrahydrofuran THF, the 33 th base T is used for marking BHQ1 quencher, and the 3 ' end is subjected to C3-spacer blocking modification;
the kit also comprises a reverse transcription recombinase mediated isothermal amplification reagent, wherein the reagent comprises reaction buffer and RNase Free ddH 2 O, buffer solution containing magnesium ions and an RT fluorescence basic reaction unit; the buffer solution containing magnesium ions is magnesium acetate, and the RT fluorescence basic reaction unit mainly comprises single-stranded DNA binding protein, recombinase, reverse transcriptase and polymerase.
2. The use of the reagent kit for detecting classical swine fever virus according to claim 1, for preparing a preparation for detecting classical swine fever virus, wherein the preparation comprises the following steps in performing classical swine fever virus detection:
and (3) RNA extraction: extracting RNA of a sample to be detected;
amplification of RT-RAA reaction system: adding an RT-RAA primer and a probe for detecting the classical swine fever virus in the kit and a reagent containing reverse transcription recombinase mediated isothermal amplification into an RT-RAA reaction system by taking the extracted RNA as a template to carry out RT-RAA amplification; the reaction condition of the RT-RAA is constant temperature amplification for 30min at 42 ℃, and the result judgment is carried out by a blue light detector for visual judgment of the result; if the negative control has no amplification curve or the visual result is colorless, and the positive control has amplification curve or the visual result is green, the experimental data is valid, otherwise the experimental result is invalid; the sample to be detected has no amplification curve or the visual result is colorless, and the sample is judged to be negative; the result of the amplification curve or visualization is green, and the sample is judged to be positive.
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006086377A2 (en) * | 2005-02-09 | 2006-08-17 | Idexx Laboratories | Method of detection of classical swine fever |
| CN105567871A (en) * | 2016-01-15 | 2016-05-11 | 中国农业科学院兰州兽医研究所 | RT-RPA (reverse transcription recombinase polymerase amplification) detection kit for fast detecting high-pathogenicity porcine reproductive and respiratory syndrome virus and application thereof |
| CN106521027A (en) * | 2016-11-03 | 2017-03-22 | 河北出入境检验检疫局检验检疫技术中心 | A real-time isothermal recombinase-polymerase amplification detection kit for African swine fever viruses |
| CN106957926A (en) * | 2017-04-14 | 2017-07-18 | 北京出入境检验检疫局检验检疫技术中心 | A kind of detection kit and primer and probe that can simultaneously detect and differentiate classic swine fever, African swine fever and swine pox |
| CN107435080A (en) * | 2017-04-20 | 2017-12-05 | 华南农业大学 | A kind of recombinase normal temperature amplification of nucleic acid for detecting CSFV（RT‑RPA）Test strips kit and application |
| CN107699635A (en) * | 2017-06-19 | 2018-02-16 | 中国检验检疫科学研究院 | Porcine epidemic diarrhea virus fluorescence RPA detection methods |
| CN109971886A (en) * | 2018-11-22 | 2019-07-05 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | A kind of kit for detecting CPV nucleic acid, RPA primer pair, probe and method |
| CN110373502A (en) * | 2019-08-27 | 2019-10-25 | 中国人民解放军东部战区疾病预防控制中心 | It is a kind of for RPA detection hantaan virus complete nucleic acid, kit and detection method |
| CN110453012A (en) * | 2019-08-05 | 2019-11-15 | 江苏奇天基因生物科技有限公司 | A kind of 24 genotype universal primers of RAA Fluorometric assay African swine fever virus, probe and detection method |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087825B (en) * | 2015-07-01 | 2018-10-02 | 浙江善测禾骑士生物科技有限公司 | Room temperature isothermal quickly detects method, reagent and the primer and probe of Ebola virus |
| CN107619885A (en) * | 2017-10-25 | 2018-01-23 | 宁波国际旅行卫生保健中心 | A kind of fluorescence RT RAA primers, probe and detection method for being used to detect dengue virus |
| CN110241261A (en) * | 2019-06-20 | 2019-09-17 | 中国动物卫生与流行病学中心 | A kind of RT-RAA detection method of avian infectious bronchitis virus |
| CN110358867A (en) * | 2019-08-21 | 2019-10-22 | 郑州中道生物技术有限公司 | African hog cholera virus fluorescent type RAA detection kit |
| CN110699490B (en) * | 2019-11-12 | 2021-01-15 | 南宁众册生物科技有限公司 | RAA constant-temperature fluorescence detection primer probe set, kit and method for African swine fever virus CD2V gene |
| CN111088403A (en) * | 2020-01-21 | 2020-05-01 | 广东省农业科学院动物卫生研究所 | Constant-temperature fluorescent amplification primer group, probe, method and kit for detecting African swine fever virus |
-
2020
- 2020-05-13 CN CN202010402843.XA patent/CN111593139B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006086377A2 (en) * | 2005-02-09 | 2006-08-17 | Idexx Laboratories | Method of detection of classical swine fever |
| CN105567871A (en) * | 2016-01-15 | 2016-05-11 | 中国农业科学院兰州兽医研究所 | RT-RPA (reverse transcription recombinase polymerase amplification) detection kit for fast detecting high-pathogenicity porcine reproductive and respiratory syndrome virus and application thereof |
| CN106521027A (en) * | 2016-11-03 | 2017-03-22 | 河北出入境检验检疫局检验检疫技术中心 | A real-time isothermal recombinase-polymerase amplification detection kit for African swine fever viruses |
| CN106957926A (en) * | 2017-04-14 | 2017-07-18 | 北京出入境检验检疫局检验检疫技术中心 | A kind of detection kit and primer and probe that can simultaneously detect and differentiate classic swine fever, African swine fever and swine pox |
| CN107435080A (en) * | 2017-04-20 | 2017-12-05 | 华南农业大学 | A kind of recombinase normal temperature amplification of nucleic acid for detecting CSFV（RT‑RPA）Test strips kit and application |
| CN107699635A (en) * | 2017-06-19 | 2018-02-16 | 中国检验检疫科学研究院 | Porcine epidemic diarrhea virus fluorescence RPA detection methods |
| CN109971886A (en) * | 2018-11-22 | 2019-07-05 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | A kind of kit for detecting CPV nucleic acid, RPA primer pair, probe and method |
| CN110453012A (en) * | 2019-08-05 | 2019-11-15 | 江苏奇天基因生物科技有限公司 | A kind of 24 genotype universal primers of RAA Fluorometric assay African swine fever virus, probe and detection method |
| CN110373502A (en) * | 2019-08-27 | 2019-10-25 | 中国人民解放军东部战区疾病预防控制中心 | It is a kind of for RPA detection hantaan virus complete nucleic acid, kit and detection method |
Non-Patent Citations (4)
| Title |
|---|
| A recombinase polymerase amplification-based assay for rapid detection of African swine fever virus;Jianchang Wang等;《Can J Vet Res》;20171030;第81卷(第4期);第308-312页 * |
| Phylogenetic analysis of Classical swine fever virus from archival formalin fixed clinical tissues reveals vietnamese origin of the isolates;Vinod Kumar Singh等;《Virusdisease》;20170213;第28卷(第1期);第121-125页 * |
| 猪流行性腹泻病毒实时荧光RPA等温检测方法的建立;吕继洲等;《中国畜牧兽医》;20171231;第44卷(第12期);第3436页 * |
| 猪瘟病毒重组酶聚合酶扩增检测方法的建立及应用;王金凤等;《中国兽医科学》;20190401;第49卷(第6期);第687、689页 * |
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