CN107557496A - A kind of fluorescence RT RAA primers, probe and detection method for being used to detect MERS CoV viruses - Google Patents
A kind of fluorescence RT RAA primers, probe and detection method for being used to detect MERS CoV viruses Download PDFInfo
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Abstract
The invention provides the application in a kind of fluorescence RT RAA primers for reverse transcription recombinase-mediated nucleic acid amplification technologies detection Middle East respiration syndrome coronavirus and fluorescence probe and fluorescence RT RAA primers and fluorescence probe in the detection eastern respiration syndrome coronavirus, fluorescence RT RAA primers and fluorescence probe can be quickly, in qualitative detection throat swab sample Middle East respiration syndrome coronavirus, it can be detected under 39 DEG C of normal temperature, diagnostic result can be gone out in 20 minutes, greatly shorten detection time；Fluorescence RT RAA primers and fluorescence probe specificity is stronger, sensitivity is higher, fully meet epidemic situation quick diagnosis and complete monitoring, for the early diagnosis of epidemic situation, early treatment, reduce case fatality rate and control epidemic situation to race against time.
Description
Technical field
The invention belongs to beyond body nucleic acid detection field, and in particular to it is comprehensive that one kind is used to detect Middle East breathing in Pharyngeal swab samples
Close the fluorescence RT- of disease coronavirus (Middle East Respiratory Syndrome Coronavirus, MERS-CoV)
RAA primers, probe and detection method.
Background technology
Middle East respiration syndrome (Middle East respiratory syndrome, MERS) is to be breathed to integrate by the Middle East
A kind of serious respiratory disease caused by levying coronavirus (MERS coronavirus, MERS-CoV), it is after serious anxious
Property respiration syndrome coronavirus (severe acute respiratory syndrome coronavirus, SARS-CoV)
The newfound transmissibility of latter is strong, case fatality rate is high, can infect the coronavirus of people.From 2012 Saudi Arabia be found first with
To come, cases of infection are continuously increased, according to the World Health Organization (WHO) recent statistics, by July 1st, 2017, Laboratory Diagnosed
2040 MERS-CoV virus cases of infection, wherein dead 722 people, case fatality rate reach 35%, far above dying of illness for SARS-CoV
Rate, there are 27 countries in the area such as the Middle East, north African, America, Europe, Asia infected zone, wherein 82% case is mainly concentrated
In Saudi.In May, 2015, the first Introduced cases MERS-CoV virus infection diseases from South Korea are made a definite diagnosis by China
Example, epidemic prevention and control situation are extremely severe.Therefore, it is viral for timely and effective discoverys MERS-CoV, by the virus prevention in the gateway of a country it
Outside, it is very necessary that quick, sensitive, special MERS-CoV detection methods are established.
With development of globalization, easily and efficiently transport facility makes tourism and country variant and regional animal, animal
The cross-border movement of product is increasingly frequent, and also the propagation to infectious disease and prevalence create advantage.In addition, MERS-CoV early stages
Non-typical symptoms are presented in infection, similar with other respiratory virus infection symptoms, it is difficult to distinguish.And it there is no be directed at present
Effective antiviral therapy medicine of MERS-CoV infection, undoubtedly further increase the prevention and control difficulty of MERS-CoV viruses.Therefore,
The laboratory method for establishing quick diagnosis MERS-CoV infection is particularly important.
Traditional detection method detection cycle length, and separation positive rate is low, is often delayed the control of the course of disease and epidemic situation.Point
Sub- biometric diagnostic method can detect the infection of virus, and rapid sensitive in viremia virusemia period, extensive at present
Be applied to MERS-CoV virus detection.At present, the MERS-CoV detection methods established both at home and abroad have real-time RT-PCR inspection
Survey method, heminested PCR detection method, LAMP detection method etc., but PCR method needs to use the instrument and well-equipped of complexity
Laboratory, and LAMP method primer is more complicated, it is necessary to special Software for Design, and the amplified production obtained with LAMP method
The fragment that some differ in size, can not Direct Cloning and sequencing, be only used for judging the presence of target gene.
The content of the invention
It is described it is an object of the present invention to provide a kind of primer pair for being used to detect Middle East breathing syndrome coronavirus
At least one of primer pair includes following 1) -3)：
1) SEQ ID № in sequence table：SEQ ID № in nucleotide sequence shown in 1 and sequence table：Nucleotide sequence shown in 2；
2) can be with SEQ ID № in sequence table under high high stringency conditions：The nucleotides sequence of 1 nucleotide sequence hybridization limited
Row；With can be with SEQ ID № in sequence table under high high stringency conditions：The nucleotide sequence of 2 nucleotide sequence hybridizations limited；
1) or 2) 3) nucleotide sequence with limiting has more than 90% homology, and has the nucleotides of identical function
Sequence.
Specifically, the homology is more than 95%；Specific again is more than 96%；Specific again is more than 97%；Again
Specific is more than 98%；Specific again is more than 99%.
Above-mentioned high high stringency conditions can be with 6 × SSC, 0.5%SDS solution, hybridize at 65 DEG C, then with 2 × SSC,
0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.
It is described that there is identical function to specifically include the nucleotide sequence that can be used for detecting Middle East breathing syndrome coronavirus.
It is described it is also another object of the present invention to provide a kind of probe for being used to detect Middle East breathing syndrome coronavirus
At least one of probe includes following 1) -3)：
1) nucleotides sequence of the probe is classified as：
5'-GAGGCAATAGTCAATCATCTTCAAGAGCCNMNAGCGTAAGCAGAAAC-3', wherein, the N is represented
Nucleotides after any nucleotides or any modification, the M represent the compound of tool cyclic structure；
2) under high high stringency conditions can with 1) described in probe nucleotide sequence hybridization nucleotide sequence；
3) with 1) or 2) described in the nucleotide sequence of probe there is more than 90% homology, and there is identical function
Nucleotide sequence.
Specifically, the N represents the thymidylic acid FAM-dT for carrying fluorescein base group FAM or carried
Fluorescent quenching group BHQ thymidylic acid BHQ-dT, the M represents tetrahydrofuran.
Specifically, the homology is more than 95%；Specific again is more than 96%；Specific again is more than 97%；Again
Specific is more than 98%；Specific again is more than 99%.
Above-mentioned high high stringency conditions can be with 6 × SSC, 0.5%SDS solution, hybridize at 65 DEG C, then with 2 × SSC,
0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.
It is described that there is identical function to specifically include the nucleotide sequence that can be used for detecting Middle East breathing syndrome coronavirus.
Specifically, the nucleotides sequence of the probe is classified as SEQ ID № in sequence table：Nucleotide sequence shown in 3.
A further object of the present invention is to provide a kind of composition for being used to detect Middle East breathing syndrome coronavirus, institute
State composition include it is following 1) and 2)：
1) primer pair of the present invention；
2) any described probe of the present invention.
A further object is for the present invention provides a kind of kit for being used to detect Middle East breathing syndrome coronavirus, institute
At least one of stating kit includes following 1) -3) described：
1) primer pair of the present invention；
2) any described probe of the present invention；
3) composition of the present invention.
Specifically, when the kit simultaneously include described primer pair and described probe when, described primer pair and
The mol ratio of described probe is 4：4：1.
It is also another object of the present invention to provide a kind of detection method of Middle East breathing syndrome coronavirus, the detection
Method does not include the method for diagnosis or the treatment of disease；The detection method is including the use of following 1) -3) at least one of enter
Row detection：
1) primer pair of the present invention；
2) any described probe of the present invention；
3) composition of the present invention.
At least one of specifically, the detection method also includes following 1) -2)：
1) when the detection method has used described primer pair and described probe simultaneously, described primer pair and institute
The mol ratio for the probe stated is 4：4：1；
2) RNA for extracting detected sample carries out RT-RAA reactions, and the reaction temperature of the RT-RAA reactions includes 39 DEG C；
Reaction time includes more than 20 minutes.
A further object of the present invention is to provide any primer pair of the present invention, any probe of the present invention, this hair
East breathing is comprehensive in the detection for bright any composition, any kit of the present invention, any detection method of the present invention
The application in disease coronavirus is closed, the application does not include the application for being related to the diagnosis or treatment method of disease.
The present invention's a further object is any primer pair of the offer present invention, any probe of the present invention, this hair
Bright any composition, any kit of the present invention, any detection method of the present invention are exhaled in the preparation detection Middle East
Inhale the application in syndrome coronavirus Related product.
Beneficial effects of the present invention include：
It is an object of the present invention to provide one kind to be used for reverse transcription recombinase-mediated nucleic acid amplification technologies detection Middle East breathing synthesis
The fluorescence RT-RAA primers and fluorescence probe and fluorescence RT-RAA primers and fluorescence probe of sign coronavirus are eastern in the detection
Application in respiration syndrome coronavirus, fluorescence RT-RAA primers and fluorescence probe can quickly, qualitative detection throat swab
Middle East respiration syndrome coronavirus in sample.
Fluorescence RT-RAA primers and fluorescence probe provided by the invention, examined using fluorescence RT-RAA primers and fluorescence probe
When surveying Middle East respiration syndrome coronavirus, independent of the PCR instrument device of costliness, and detection time is compared with PCR method or fluorescent PCR
Significantly shorten, and sensitivity even more high suitable with fluorescent PCR.
In RT-RAA courses of reaction, MERS-CoV viral RNAs synthesize cDNA by reverse transcriptase first, recycle restructuring
Enzyme replaces PCR high-temperature denatured, completes to unwind to double-strand, the double-strand untied is combined by single strand binding protein, prevents DNA from answering
Property, then the extension by polymerase completion chain, using cDNA as templated synthesis purpose product.Reaction carried out at 39 DEG C 20 minutes this
Outside, RAA technologies can also complete multi-primerses amplification, coordinate luminoscope, can form a set of RAA multi-fluorescences real-time detecting system,
The fluorescence labeling of different colours is utilized, different target gene is detected in same reaction, this is other constant temperature nucleic acids
What amplification technique or Nested PCR Technique can not be compared.
The present invention can realize single tube scene, quick detection Middle East breath syndrome virus, and other detection techniques are compared to tool
Have the advantage that：
1st, totally-enclosed reaction, monitor fluorescence in real time data, without subsequent treatment, avoid polluting, ensure testing result can
By property；
2nd, it independent of expensive instruments such as PCR, or even can be detected under 37-39 DEG C of normal temperature, diagnosis can be gone out in 20 minutes
As a result, detection time is greatly shortened；
3rd, fluorescence RT-RAA primers provided by the invention and fluorescence probe specificity is stronger, sensitivity is higher, fully meet
Epidemic situation quick diagnosis and complete monitoring, raced against time for the early diagnosis of epidemic situation, early treatment, reduction case fatality rate and control epidemic situation.
Brief description of the drawings
Fig. 1 is sensitivity experiment result figure.
Fig. 2 is specificity experiments result figure.
Fig. 3 is positive clinical sample detection result figure.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Do not make the experimental methods of molecular biology illustrated, equal reference in following embodiments《Molecular Cloning:A Laboratory guide》
Listed specific method is carried out in the book of (third edition) J. Pehanorm Brookers one, or is carried out according to kit and product description.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Embodiment 1, the design of fluorescence RT-RAA primer, probe for detecting Middle East breathing syndrome coronavirus
The special conservative region for being directed to Middle East respiration syndrome coronavirus is targeting regions, design specific primer and
Fluorescence RAA probes, its sequence are respectively：
Forward primer, such as SEQ ID № in sequence table：Nucleotide sequence shown in 1：
5'-TCCACATTGAGGGGACTGGAGGCAATAGTC-3'
Reverse primer, such as SEQ ID № in sequence table：Nucleotide sequence shown in 2：
5'-GATCACCTCCTACTGCTCCGATTCCAGATG-3'
Oligonucleotide probe, such as SEQ ID № in sequence table：Nucleotide sequence shown in 3：
5'-GAGGCAATAGTCAATCATCTTCAAGAGCC(FAM-dT)(THF)(BHQ-dT)AGCGTAAGCAGAAAC-
3'；
Wherein：
FAM-dT represents to carry fluorescein base group FAM (6-Carboxyfluorescein) thymidine
Acid；
THF represents tetrahydrofuran (tetrahydrofuran) connexon；
BHQ-dT represents to carry fluorescent quenching group BHQ (black hole quencher) thymidine deoxidation core
Thuja acid；
Embodiment 2, a kind of foundation for being used to detect the fluorescence RT-RAA methods of Middle East respiration syndrome coronavirus
(1) fluorescence RT-RAA reacts
1) extraction of sample rna：The sample rna can use the viral of German Kai Jie bioengineering Co., Ltd
RNA nucleic acid extraction kits, are extracted according to kit specification.
2) forward primer, reverse primer and the probe designed using embodiment 1, and RAA reaction kit (the present embodiment
The specific RAA basic agents box for employing Jiangsu Qi Tian genes bio tech ltd), prepared with the present embodiment step 1)
Sample rna to be detected is template, carries out amplified reaction, and wherein reaction system is as follows：
25 μ l RAA reaction buffers (offer of Jiangsu Qi Tian genes bio tech ltd RAA basic agents box)；
Forward primer, each 2 μ L of reverse primer (10 μM) of the design of embodiment 1；
The probe (10 μM) of 0.5 μ L embodiments 1 design；
Sample rna template to be detected prepared by 1 μ L steps 1)；
0.5 μ L M-MLV reverse transcriptases (ThermoFisher Products, 200U/ μ L)
Add 16.5 μ L distilled waters to 47.5 μ L, be well mixed, be then added to dry powder (the strange day gene biotechnology in Jiangsu
Co., Ltd RAA basic agents box provide) reaction tube in, mix again.Each pipe adds 2.5 μ L 280mM magnesium acetate solutions simultaneously
Mix.
(2) fluoroscopic examination
Above-mentioned reaction tube is positioned over RAAF-6100 fluorogenes detector (the strange limited public affairs of day gene biotechnology in Jiangsu
Department), 20min is reacted at 39 DEG C, carries out fluoroscopic examination.
Negative control：FAM passages are without amplification curve, and without Tt values；
Positive control：FAM passages have an amplification curve, Tt values≤8min
The above-mentioned requirement to positive control and negative control need to meet that otherwise, this is tested simultaneously in once experiment
It is invalid, it is necessary to re-start experiment.
Sample detection result interpretation：
When the sample F AM passages detected have amplification curve, when quality control is normal, the MERS positives are can determine that；
When the sample F AM passages detected are without amplification curve, and Tt values are shown as Undet or No Tt, and quality control is normal
When, it can determine that MERS feminine genders.
(3) sensitivity experiment
The minimum detection limit of the experimental verification detection method, uses concentration as 1.0 × 106copies/mL、1.0×
105copies/mL、1.0×104copies/mL、1.0×103copies/mL、1.0×102copies/mL、10copies/mL
Deng pseudovirus positive criteria product as test limit reference product, the test limit for showing the detection method by the experiment reaches
10copies/mL, specific experiment result are as shown in Figure 1.
10 in Fig. 16Copy, 105Copy, 104Copy, 103Copy, 102Copy, 10 copy respectively represent concentration be 1.0 ×
106copies/mL、1.0×105copies/mL、1.0×104copies/mL、1.0×103copies/mL、1.0×
102Copies/mL, 10copies/mL positive quality control product amplification curve, feminine gender are negative quality-control product, and abscissa represents reaction
Time, ordinate mv represent fluorescent value.Result shows shown in Fig. 1, and the test limit of the detection method reaches 10copies/mL.
(4) specificity experiments
The experimental selection has the common causative of identical infection site as special with Middle East respiration syndrome coronavirus
Specific reference product.Specific reference material is respectively H1N1virus, influenza B virus, Respiratory Syncytial Virus(RSV), nose
Virus.Carried out in fact as template using the RNA in the Viral viral RNAs extracts kit extraction sample above of Qigen companies
Test.Specificity experiments result is as shown in Figure 2.
Abscissa represents the reaction time in Fig. 2, and ordinate mv represents fluorescent value.Result shows shown in Fig. 2, and this 4 kinds special
Property reference material do not have amplification curve, illustrate that the specificity of the detection method is good.
(5) positive clinical sample detection is tested
The Chinese the first Introduced cases MERS-CoV viral nucleic acids of laboratory selection and Britain QCMD rooms interstitial in 2014 are commented
Sample (inactivation sample) is detected to this method, verified.Specific experiment result is as shown in Figure 3.
Abscissa represents the reaction time in Fig. 3, and ordinate mv represents fluorescent value.Result shows shown in Fig. 3, this 2 parts of samples
There is obvious amplification curve, the detection method expanding effect is excellent, has good performance.
Embodiment described above only expresses embodiments of the present invention, and its description is more specific and detailed, but can not
Therefore the limitation to the scope of the claims of the present invention is interpreted as, as long as the skill obtained using the form of equivalent substitution or equivalent transformation
Art scheme, it all should fall within the scope and spirit of the invention.
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Claims (10)
1. a kind of primer pair for being used to detect Middle East breathing syndrome coronavirus, it is characterised in that under the primer pair includes
At least one of state 1) -3)：
1) SEQ ID № in sequence table：SEQ ID № in nucleotide sequence shown in 1 and sequence table：Nucleotide sequence shown in 2；
2) can be with SEQ ID № in sequence table under high high stringency conditions：The nucleotide sequence of 1 nucleotide sequence hybridization limited；With
Can be with SEQ ID № in sequence table under high high stringency conditions：The nucleotide sequence of 2 nucleotide sequence hybridizations limited；
1) or 2) 3) nucleotide sequence with limiting has more than 90% homology, and has the nucleotide sequence of identical function.
2. a kind of be used to detect the probe of Middle East breathing syndrome coronavirus, it is characterised in that the probe include it is following 1)-
At least one of 3)：
1) nucleotides sequence of the probe is classified as：
5'-GAGGCAATAGTCAATCATCTTCAAGAGCCNMNAGCGTAAGCAGAAAC-3', wherein, the N represents any
Nucleotides after nucleotides or any modification, the M represent the compound of tool cyclic structure；
2) under high high stringency conditions can with 1) described in probe nucleotide sequence hybridization nucleotide sequence；
3) with 1) or 2) described in the nucleotide sequence of probe there is more than 90% homology, and there is the nucleosides of identical function
Acid sequence.
3. probe according to claim 2, it is characterised in that the nucleotides sequence of the probe is classified as SEQ ID in sequence table
№：Nucleotide sequence shown in 3.
4. a kind of composition for being used to detect Middle East breathing syndrome coronavirus, it is characterised in that under the composition includes
1) and 2) state：
1) primer pair described in claim 1；
2) any described probe of Claims 2 or 3.
5. a kind of kit for being used to detect Middle East breathing syndrome coronavirus, it is characterised in that under the kit includes
At least one of state 1) -3) described：
1) primer pair described in claim 1；
2) any described probe of Claims 2 or 3；
3) composition described in claim 4.
6. kit according to claim 5, it is characterised in that when the kit simultaneously including described primer pair and
During described probe, the mol ratio of described primer pair and described probe is 4：4：1.
A kind of 7. detection method of Middle East breathing syndrome coronavirus, it is characterised in that：The detection method does not include disease
Diagnosis or treatment method；The detection method is including the use of following 1) -3) at least one of detected：
1) primer pair described in claim 1；
2) any described probe of Claims 2 or 3；
3) composition described in claim 4.
8. detection method according to claim 7, it is characterised in that：The detection method also includes following 1) -2) in
It is at least one：
1) when the detection method simultaneously used described primer pair and described probe when, described primer pair with it is described
The mol ratio of probe is 4：4：1；
2) RNA for extracting detected sample carries out RT-RAA reactions, and the reaction temperature of the RT-RAA reactions includes 39 DEG C；Reaction
Time includes more than 20 minutes.
9. composition, right will described in any probe of primer pair, Claims 2 or 3 described in claim 1, claim 4
Ask any kits of 5-6, any detection methods of claim 7-8 in the detection in eastern breathing syndrome coronavirus
Application, it is described application not include be related to disease diagnosis or treatment method application.
10. composition, right described in any probe of primer pair, Claims 2 or 3 described in claim 1, claim 4
It is required that any kits of 5-6, any detection methods of claim 7-8 are coronal in preparation detection Middle East breathing syndrome
Application in viral Related product.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315478A (en) * | 2018-02-13 | 2018-07-24 | 吴斌 | The probe and kit of RAA Fluorometric assay hydrophobins |
| CN108531632A (en) * | 2018-06-21 | 2018-09-14 | 宁波国际旅行卫生保健中心 | One kind is for detecting Escherichia coli O 157:The active fluorescence RAA primers of H7, probe and detection method |
| CN110592271A (en) * | 2018-06-13 | 2019-12-20 | 杭州众测生物科技有限公司 | RAA constant temperature fluorescence detection method and reagent for Infectious Hematopoietic Necrosis Virus (IHNV) |
| CN110592267A (en) * | 2018-06-13 | 2019-12-20 | 杭州众测生物科技有限公司 | RAA constant-temperature fluorescence detection method and reagent for Spring Viremia of Carp Virus (SVCV) |
| CN111270021A (en) * | 2020-04-15 | 2020-06-12 | 齐永 | Primer pair, probe, composition, kit and application for detecting novel coronavirus SARS-CoV-2 |
| CN111518951A (en) * | 2020-05-06 | 2020-08-11 | 首都儿科研究所 | Primer, probe, kit and detection method for detecting SARS-CoV-2 by RT-RAA fluorescence method |
| CN112458201A (en) * | 2020-10-20 | 2021-03-09 | 宁波国际旅行卫生保健中心(宁波海关口岸门诊部) | Fluorescent RT-RPA primer, probe and detection method for detecting novel coronavirus |
| CN113151580A (en) * | 2020-10-21 | 2021-07-23 | 宁波国际旅行卫生保健中心(宁波海关口岸门诊部) | Triple real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) primer, probe and detection method for detecting novel coronavirus |
| CN114959120A (en) * | 2022-06-30 | 2022-08-30 | 华南农业大学 | Primer probe set and kit for detecting chicken infectious anemia virus by RAA fluorescence method and application of primer probe set and kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543409A (en) * | 2015-12-11 | 2016-05-04 | 中华人民共和国上海出入境检验检疫局 | Double-target-gene real-time fluorescent PCR detection method for Middle East respiratory syndrome coronavirus |
| CN105624335A (en) * | 2016-03-10 | 2016-06-01 | 中山大学达安基因股份有限公司 | Kit for performing gene detection on middle east respiratory syndrome coronaviruses |
| CN106868220A (en) * | 2017-03-31 | 2017-06-20 | 中国科学院过程工程研究所 | A kind of LAMP primer group and kit for expanding MERS CoV |
-
2017
- 2017-10-13 CN CN201710955224.1A patent/CN107557496A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543409A (en) * | 2015-12-11 | 2016-05-04 | 中华人民共和国上海出入境检验检疫局 | Double-target-gene real-time fluorescent PCR detection method for Middle East respiratory syndrome coronavirus |
| CN105624335A (en) * | 2016-03-10 | 2016-06-01 | 中山大学达安基因股份有限公司 | Kit for performing gene detection on middle east respiratory syndrome coronaviruses |
| CN106868220A (en) * | 2017-03-31 | 2017-06-20 | 中国科学院过程工程研究所 | A kind of LAMP primer group and kit for expanding MERS CoV |
Non-Patent Citations (1)
| Title |
|---|
| ABD EL WAHED ET AL: "Reverse transcription recombinase polymerase amplification assay for the detection of middle East respiratory syndrome coronavirus", 《PLOS CURRENTS》 * |
Cited By (11)
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| CN108315478A (en) * | 2018-02-13 | 2018-07-24 | 吴斌 | The probe and kit of RAA Fluorometric assay hydrophobins |
| CN110592271A (en) * | 2018-06-13 | 2019-12-20 | 杭州众测生物科技有限公司 | RAA constant temperature fluorescence detection method and reagent for Infectious Hematopoietic Necrosis Virus (IHNV) |
| CN110592267A (en) * | 2018-06-13 | 2019-12-20 | 杭州众测生物科技有限公司 | RAA constant-temperature fluorescence detection method and reagent for Spring Viremia of Carp Virus (SVCV) |
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| CN111270021A (en) * | 2020-04-15 | 2020-06-12 | 齐永 | Primer pair, probe, composition, kit and application for detecting novel coronavirus SARS-CoV-2 |
| CN111270021B (en) * | 2020-04-15 | 2024-02-02 | 齐永 | Primer pair, probe, composition, kit and application for detecting novel coronavirus SARS-CoV-2 |
| CN111518951A (en) * | 2020-05-06 | 2020-08-11 | 首都儿科研究所 | Primer, probe, kit and detection method for detecting SARS-CoV-2 by RT-RAA fluorescence method |
| CN112458201A (en) * | 2020-10-20 | 2021-03-09 | 宁波国际旅行卫生保健中心(宁波海关口岸门诊部) | Fluorescent RT-RPA primer, probe and detection method for detecting novel coronavirus |
| CN113151580A (en) * | 2020-10-21 | 2021-07-23 | 宁波国际旅行卫生保健中心(宁波海关口岸门诊部) | Triple real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) primer, probe and detection method for detecting novel coronavirus |
| CN113151580B (en) * | 2020-10-21 | 2022-09-23 | 山东博思源生物技术有限公司 | Triple real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) primer, probe and detection method for detecting novel coronavirus |
| CN114959120A (en) * | 2022-06-30 | 2022-08-30 | 华南农业大学 | Primer probe set and kit for detecting chicken infectious anemia virus by RAA fluorescence method and application of primer probe set and kit |
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