CN107460255A - A kind of RT LAMP primers group, kit and application for detecting pig fourth type coronavirus - Google Patents

A kind of RT LAMP primers group, kit and application for detecting pig fourth type coronavirus Download PDF

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Publication number
CN107460255A
CN107460255A CN201710761229.0A CN201710761229A CN107460255A CN 107460255 A CN107460255 A CN 107460255A CN 201710761229 A CN201710761229 A CN 201710761229A CN 107460255 A CN107460255 A CN 107460255A
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China
Prior art keywords
lamp
pig
detection
type coronavirus
coronavirus
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Inventor
何颖
卢冰霞
陈忠伟
秦毅斌
赵武
段群棚
周英宁
李斌
梁家幸
苏乾莲
蒋冬福
卢敬专
杨思仪
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Guangxi Veterinary Research Institute
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Guangxi Veterinary Research Institute
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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
    • C12Q1/6851—Quantitative amplification
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a kind of RT LAMP primers group, kit and application for detecting pig fourth type coronavirus, the primer sets such as SEQ ID NO：Shown in 1 ~ 6；The kit includes RT LAMP primers group described above, 2 × reaction buffer, EM, ultra-pure water and pig fourth type coronavirus RNA templates.Methods described includes the preparation of material, the design of RT LAMP primers and synthesis, virus genome RNA extraction, the foundation of RT LAMP reaction systems and carries out specific detection, sensitivity Detection and Real_time quantitative detection using RT LAMP detection methods.Specific detection and sensitivity Detection confirm, RT LAMP kits provided by the invention can monitor in real time reacts and pig fourth type coronavirus copy number can be carried out quantitative detection, testing result is fast and accurately obtained, for simplicity, quickly and reliably detection pig fourth type coronavirus offers convenience.

Description

A kind of RT-LAMP primer sets, kit and application for detecting pig fourth type coronavirus
Technical field
The present invention relates to technical field of microbial detection, particularly relates to a kind of quick, visualization and can determine in real time The reverse transcription loop-mediated isothermal amplification kit and application method of amount detection pig fourth type coronavirus.
Background technology
Pig fourth type coronavirus disease (porcine epidemic diarrhea,PDCo) be one kind by the coronal disease of pig fourth type Poison (porcine epidemic diarrhea virus, PDCo V) caused by pig a kind of high degree in contact enteric infection Disease, especially whole section of small intestine of its main infection, jejunum and ileum, causes serious enteritis and the diarrhoea with piggy and vomiting.
The viral PDCo V are reported first in Hong-Kong within 2009~2010 years.Pig fourth type coronavirus infection flows with pig Row is suffered from diarrhoea（PED）And transmissible gastroenteritis of swine（TGE）Clinical onset situation it is more similar, can cause sucking pig suffer from diarrhoea and vomit Tell, morbidity and mortality are up to 50%~100%, and the death rate is low after grower pigs and Adult Pig infection.China is widely present pig stream Row is suffered from diarrhoea, in July, 2015, and PDCo occurs for the age in days of Compact Develop 5 to 15 sucking pig of 2 000 basic sows of Guangdong livestock on hand V, the disease propagate rapid and diarrhoea seriously, the incidence of disease 90%, death and culling rate more than 90%.Because PDCo V morbidity age in days is small, sense Dye rate, the death rate are high, therefore the necessary fast diagnosis method to pig fourth type coronavirus is studied, so as to effective and fast Fast ground prevention and control PDCo.
Quick and precisely diagnosis to pig fourth type coronavirus is clinically to prevent and treat the key of pig fourth type coronavirus disease.At present Laboratory diagnosis PDCo V method is the molecular biology method of conventional RT-PCR and real-time fluorescence quantitative PCR, although PCR side Method compared with Virus Isolation method quick and precisely, but needs expensive instrument and equipment, and cost is higher, needed in result judgement into Row agarose gel electrophoresis, easily causing laboratory pollution causes false positive results occur, is also not suitable for basic unit and Site Detection.
The content of the invention
The invention aims to solve that basic unit detection pig fourth type coronavirus is difficult, time-consuming and expensive equipment etc. is asked Topic, there is provided a kind of kit for easy, the quick pig fourth type coronavirus of detection exactly of basic unit, to realize the object of the invention institute The technical scheme used is：A kind of RT-LAMP primer sets for detecting pig fourth type coronavirus, the primer sets such as SEQ ID NO： Shown in 1 ~ 6.
Further, the primer sets SEQ ID NO：1 ~ 6 sequence is passed through the substitution of one or several nucleotides and/or lacked Lose and/or addition and with sequence SEQ ID NO：1 ~ 6 has the DNA molecular of identical function.
A kind of RT-LAMP kits for detecting pig fourth type coronavirus, the kit include above-described detection pig The RT-LAMP primer sets of fourth type coronavirus.
Further, described kit also includes 2 × reaction buffer, EM, ultra-pure water and pig fourth type coronavirus RNA Template.
Further, the reaction temperature of the kit is 63 DEG C, reacts and was expanded at 15~25 minutes.
Kit is used to detect by a kind of application for the RT-LAMP kits for detecting pig fourth type coronavirus, the application Sample to be tested whether infected pigs fourth type coronavirus, including the preparation of material, the design of RT-LAMP primer sets and synthesis, reaction The extraction of template, RT-LAMP reaction systems are established and carry out specific detection, sensitiveness inspection using RT-LAMP detection methods Survey and quantitatively detect.
Further, the RT-LAMP reaction systems are established in terms of 25 μ L,
The μ L of 2 × reaction buffer 12.5
EM 1 μL
Primer SEQ ID NO：1 5 pmol
Primer SEQ ID NO：2 5 pmol
Primer SEQ ID NO：3 40 pmol
Primer SEQ ID NO：4 40 pmol
Primer SEQ ID NO：5 20pmol
Primer SEQ ID NO：6 20pmol
The μ L of pig fourth type coronavirus RNA 2
Ultra-pure water supplies 25 μ L.
Further, described RT-LAMP detection methods detect using specific detection, sensitivity Detection and quantitatively Method.
Further, described RT-LAMP detection methods use the closed progress of the real-time transmissometers of loopamp, complete monitoring The amplification situation of reaction tube.
The substantive distinguishing features of the present invention and progress are：
1）High specificity
The negative control virus and water of the RT-LAMP detection reagent box specific detection of the present invention compare no positive result and gone out Come, it is consistent with PCR testing results.And it is easy to operate, quickly obtain testing result, without instrument costly.
2）High sensitivity
The sensitivity of regular-PCR detection method is 2.47 × 10-8Ng/ μ L, and the RT-LAMP detection methods of the present invention are used, Test limit is about 2.47 × 10-10Ng/ μ L, it is 100 times of regular-PCR.
3）It is rapid to obtain result
Common RT-PCR whole process can just obtain a result at 24 hours or so, at present most RT-LAMP reaction sides established Method after the completion of reaction, must be imaged come result of determination using agarose gel electrophoresis ultraviolet analysis, carried from virus genome RNA Get and obtain result of the test, it is necessary to 4~5 hours or so.RT-LAMP detection methods reaction provided by the invention was on 15 minutes left sides It is right to expand, it can complete to expand in 60 minutes, and result interpretation mode is easy, under visible light carries out positive and negative pipe Compare, it is transparent in obvious muddiness, negative reaction pipe to be clearly visible positive reaction by naked eyes；After of short duration centrifugation, positive reaction Bottom of the tube has an obvious white magnesium pyrophosphate sediment, and negative reaction bottom of the tube deposit-free；Or fluorescent dye is added, it is positive Reaction tube, with the naked eye can observation experiment result under ultraviolet light in green.It is ultraviolet row agarose gel electrophoresis need not to be entered again Line analysis imaging carrys out result of determination, is extracted from geneome RNA and obtains final result and can be completed in 2~3 hours.
4）Do not pollute
RT-LAMP methods are used for the fluorescent dye directly observed to be added after reaction at present, and the fluorescent dye of the present invention be The calcein commercial dyes added before reaction（Non- syber-green）, detection process need not uncap.In addition, the present invention In result judgement, the turbidity value for directly detecting reaction tube by transmissometer can not enter come result of determination RT-LAMP detection methods Row fluorescent dye determination testing result enters row agarose gel electrophoresis testing result, it is not necessary to uncaps, can effectively avoid polluting.
5）Can real-time quantitative
The present invention analyzes the result of RT-LAMP reactions using Tubidimeter real-time LA-320 transmissometers in real time, The standard curve that the time of turbidity value corresponding to the concentration of different standard samples is depicted as, substitute into calibration curve equation, you can The pig fourth type coronavirus copy number of each time is obtained, reaches the purpose of quantitative detection product.
Brief description of the drawings
Fig. 1 is the RT-LAMP method specific detection results of the present invention, wherein 1：Pig fourth type coronavirus；2：Pig is popular Property diarrhea virus；3：Transmissible gastro-enteritis virus；4：Pig ridge virus；5：Pig bocavirus；6：Pig parvoviral；7：Pig annulus Viral 2 types；8：PRV；9：Blank control（Water）.There is the ascending curve of turbidity in pig fourth type coronavirus reaction tube, 7 plants of comparison virus reaction tubes and water control reaction Guan Junwu amplifications.
Fig. 2 and Fig. 3 is the coronal disease of pig fourth type carried out respectively using RT-LAMP methods of the present invention and conventional RT-PCR method The result of malicious sensitivity Detection.Wherein 1：2.47×101ng/μL；2：2.47 ng/μL；3：2.47×10-1ng/μL； 4： 2.47×10-2ng/μL；5：2.47×10-3ng/μL；6：2.47×10-4ng/μL；7：2.47×10-5ng/μL；8：2.47× 10-6ng/μL；9：2.47×10-7ng/μL；10：2.47×10-8ng/μL；11：2.47×10-9ng/μL；12：2.47×10- 10ng/μL；13：Water（Blank control）.Pig fourth type coronavirus gene group RNA initial concentration is 2.47 × 101Ng/ μ L, warp After 10 times of multiple proportions serial dilutions, RT-LAMP and PCR amplifications are carried out, the RT-LAMP method test limits for as a result showing the present invention are about 2.47×10-10Ng/ μ L, and regular-PCR method test limit is about 2.47 × 10-8ng/μL。
Fig. 4 is to add visual results after fluorescent dye：Zuo Guanwei is using pig fourth type coronavirus gene group RNA as template Response situation, be positive findings, right pipe is the response situation of negative control, is negative findings.
Fig. 5 is pig fourth type coronavirus quantitation curves of the present invention：Using corresponding to the concentration of different standard samples The standard curve that turbidity value is depicted as to the time, substitute into calibration curve equation, you can obtain the pig fourth type coronavirus of each time Copy number.
Embodiment
1st, the preparation of material
Pig fourth type coronavirus, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, pig ridge virus, pig bocavirus, pig Parvovirus, porcine circovirus 2 type and PRV, be commercial available vaccines poison or Guangxi veterinary institute separation identification and Preserve.RT-LAMP RNA amplifications kit is purchased from Beijing Lanpu Biological Technology Co., Ltd., article No. LMP204；Viral genome DNA/RNA extracts kits, purchased from health be century bio tech ltd, article No. CW0548.
2nd, the design and synthesis of RT-LAMP primers
Pig fourth type coronavirus N gene order in GenBank, utilizes RT-LAMP method primer Autocads The a set of RT-LAMP primers of PrimerExplorer V4 Software for Design, wherein F3, B3 are outer primer, and FIP, BIP draw to be interior Thing, LF and LB are ring primer, wherein
F3 CGCAACCCCAACAATCCT
B3 TGCAGATTGGTCGCGTTTC
FIP GCGTTGAAGGGGTCAACTCTGAATCAGCTGTTACCTCTCCGA
BIP TAGAGGAAGACCTCAGGAGCGTGCTGATTGCCTGTGCCTC
LF GCCATCTCCGGTTGGGAA
LB GGAAGTGGCCCAAGATCTCA；
3rd, virus genome RNA extracts
Use virus genom DNA/RNA extracts kits（Health be century bio tech ltd, article No. CW0548）Extraction The DNA/RNA of the intestinal tissue and excrement of pig fourth type coronavirus or doubtful pig fourth type coronavirus infection, and comparison virus Genomic DNA/RNA.
4th, RT-LAMP reaction systems are established
According to kit specification, by 25 μ l system configurations：
The μ L of 2 × reaction buffer 12.5
EM 1 μL
F3 5 pmol
B3 5 pmol
FIP 40 pmol
BIP 40 pmol
LF 20 pmol
LB 20 pmol
The μ L of pig fourth type coronavirus RNA 5
Ultra-pure water supplies 25 μ L.
RT-LAMP reactions are carrying out the shape of closed complete monitoring with real-time transmissometer (LA-320C, Japanese Rong Yan companies) Formula monitors the detection situation of this method, and transmissometer monitors amplification situation in real time, standard curve can be drawn, by obtaining unknown sample Reach time value corresponding to 0.1 turbidity value, you can calculate the starting copy number of the sample from standard curve, reaction temperature with 63 DEG C as reaction temperature.
5th, RT-LAMP detection methods
1）Specific detection
Use virus genom DNA/RNA extracts kits extraction pig fourth type coronavirus and control strain-pig epidemic Diarrhea virus, transmissible gastro-enteritis virus, pig ridge virus, pig bocavirus, pig parvoviral, porcine circovirus 2 type and pig are pseudo- Genomic DNA/RNA of rabies viruses, as the template of RT-LAMP reactions, while using water as blank control, checking R T- The specificity of LAMP method.
2）Sensitivity Detection
The pig fourth type coronavirus gene group RNA of extraction, determines its concentration, with the continuous 10 times of doubling dilutions of RNA-Free Water Into 12 dilution factors, using each RNA dilution factors as template, RT-LAMP amplifications and PCR amplifications are carried out, contrasts the RT- of the present invention LAMP method and regular-PCR method are to detecting the sensitiveness of pig fourth type coronavirus.
3）Fluorescent visual detects
According to the condition of transmissometer monitoring optimization, fluorescent dye is added, fluorescent dye adds before the reaction, and the dyestuff of addition is calcium Yellowish green plain commercial dyes, after reacting 30 minutes at 63 DEG C, observed under uviol lamp, do not use agarose gel electrophoresis ultraviolet point Analysis imaging, avoid Aerosol Pollution laboratory caused by race electrophoresis observation of uncapping.
The specific outcome of the RT-LAMP detection methods of embodiment 1
1 plant of pig fourth type coronavirus, 7 plants of comparison virus and water are compareed and carry out RT-LAMP amplifications, as a result as shown in figure 1, pig fourth There is the ascending curve of turbidity at 15 minutes or so in type coronavirus reaction tube, was positive findings, 7 plants of comparison virus reaction tubes and Water control reaction Guan Junwu amplification situations occur, and are negative findings.
The susceptibility results of the RT-LAMP detection methods of embodiment 2
Pig fourth type coronavirus gene group RNA initial concentration is 2.47 × 101Ng/ μ L, after 10 times of multiple proportions serial dilutions, RT-LAMP and regular-PCR amplification are carried out, as a result as shown in Figures 2 and 3, as a result shows the RT-LAMP methods test limit of the present invention about For 2.47 × 10-10Ng/ μ L, and the detection of regular-PCR method is limited to 2.47 × 10-8ng/μL。
The fluorescent visual testing result of the RT-LAMP detection methods of embodiment 3
According to the condition of transmissometer monitoring optimization, fluorescent dye is added, after 63 DEG C are reacted 60 minutes, is observed under uviol lamp, Fig. 4 To observe result, response situations of the Zuo Guanwei using pig fourth type coronavirus RNA as template is positive findings, and right pipe is negative right According to being negative findings.Result of the test shows that the RT-LAMP methods of foundation can facilitate basic unit to use, and need to only be matched somebody with somebody using kit The RT-LAMP primers of this method design are closed, after adding sample, are kept for 63 DEG C 60 minutes with cheap water-bath, you can be quick Result is observed, and need not be uncapped, avoids pollution.
The drafting of the pig fourth type coronavirus quantitation curves of embodiment 4
Using pig fourth type coronavirus RNA as template, using the outer primer F3 and B3 of this RT-LAMP methods design as pcr amplification primer thing, The PCR target gene fragments for expanding to obtain are connected to carrier pMD18-T, convert Escherichia coli permissive cell DH5a, ammonia benzyl west Woods resistance screening obtains monoclonal bacterium, extracts the plastid rna of restructuring, after sequencing confirms, as standard sample, measure restructuring matter Grain pMD18-T-M initial concentration, carries out continuous 10 times of doubling dilutions, 12 dilution factors with RNA-Free Water, takes each dilution Spend 2 μ L and carry out RT-LAMP amplifications as template.
Control is set：Concentration is 2.474 × 101 ng/μL、2.474 ng/μL、2.474×10-1 ng/μL、2.474× 10-2 ng/μL、2.474×10-3ng/μL、2.474×10-4 ng/μL、2.474×10-5 ng/μL、2.474×10-6ng/μ L、2.474×10-7ng/μL、2.474×10-8Ng/ μ L and 2.474 × 10-9Ng/ μ L standard recombinant plasmid pMD18-T-M samples Each one of product, because the negative logarithm of concentration and the time value that turbidity value is 0.1 are linear, it is possible to which transmissometer is caught The value grasped and time（Such as table 1）Make standard curve, obtain calibration curve equation, y=0.4611x-8.059, as shown in Figure 5. The coefficient R from the point of view of calibration curve equation2For 0.9959, in good linear relationship.Using the time as X values, Y value can be obtained i.e. Negative number formulary of concentration, then concentration is 10-y, multiplied by with radix 2.474, as 2.474 × 10-yng/μL.According to copy number Reduction formula copies/ μ L=（6.02 × 1023× (ng/ul × 10-9)）/ (RNA length × 660), RNA Length is that carrier sequence size add objective gene sequence size, for the bp of 2693+191=2884, is converted into copy number （copies/μL）：6.02 × 1023×（2.474 ×10-y×10-9）/ (2884 × 660), are reduced to：7.82 × 108 ×10-y.As certain test specimen reaches the time that turbidity value is 0.1 be 25 minutes when, bring established standard curve side into Journey, Y is obtained equal to 3.4685, then concentration is 10-3.4685, multiplied by with the concentration 2.474 of radix 2.474, the as test specimen ×10-3.4685Ng/ μ L, copy number are 7.82 × 108 ×10-3.4685, as 7.82 × 104.5315Copies/ μ L, so as to reach To quantitative effect.
The sample concentration negative logarithm of table 1 and PDCO V-LAMP turbidity value linearly relation tables
Time（Point） 15.4 17.3 19.5 21.5 24.2 26.1 28.5 31.5 33 34.5 36.2
Concentration bears logarithm -1 0 1 2 3 4 5 6 7 8 9
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for those skilled in the art For member, the present invention can have various modifications and variations.Any modification for being made within the spirit and principles of the invention, etc. With replacement, improvement etc., should be included within the scope of the present invention.
Sequence table
<110>Veterinary Institute of Guangxi Zhuang Autonomous Region
<120>A kind of RT-LAMP primer sets, kit and application for detecting pig fourth type coronavirus
<130>2017
<160>6
<170>PatentIn version 3.3
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tgcagattggtcgcgtttc
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<223>Description to artificial sequence：Inner primer FIP
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gccatctccggttgggaa
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Claims (9)

  1. A kind of 1. RT-LAMP primer sets for detecting pig fourth type coronavirus, it is characterised in that the primer sets such as SEQ ID NO：Shown in 1 ~ 6.
  2. A kind of 2. RT-LAMP primer sets for detecting pig fourth type coronavirus, it is characterised in that the primer sets SEQ ID NO：1~ 6 sequences by one or several nucleotides substitution and/or missing and/or addition and with sequence SEQ ID NO：1 ~ 6 has phase The DNA molecular of congenerous.
  3. 3. a kind of RT-LAMP kits for detecting pig fourth type coronavirus, it is characterised in that the kit includes claim The RT-LAMP primer sets of detection pig fourth type coronavirus described in 1.
  4. 4. the RT-LAMP kits of pig fourth type coronavirus are detected according to claim 3, it is characterised in that described examination Agent box also includes 2 × reaction buffer, EM, ultra-pure water and pig fourth type coronavirus RNA templates.
  5. 5. according to the RT-LAMP kits of the detection pig fourth type coronavirus of claim 3 or 4, it is characterised in that the examination The reaction temperature of agent box is 63 DEG C, reacts and was expanded at 15~25 minutes.
  6. 6. a kind of application for the RT-LAMP kits for detecting pig fourth type coronavirus, the application treat kit for detection Test sample originally whether infected pigs fourth type coronavirus, it is characterised in that the preparation including material, the design of RT-LAMP primer sets with Synthesis, virus genome RNA extraction, RT-LAMP reaction systems are established and carry out specificity using RT-LAMP detection methods Detection, sensitivity Detection and quantitative detection.
  7. 7. the application of the RT-LAMP kits of pig fourth type coronavirus is detected according to claim 6, it is characterised in that institute RT-LAMP reaction systems are stated to establish in terms of 25 μ L,
    The μ L of 2 × reaction buffer 12.5
    EM 1 μL
    Primer SEQ ID NO：1 5 pmol
    Primer SEQ ID NO：2 5 pmol
    Primer SEQ ID NO：3 40 pmol
    Primer SEQ ID NO：4 40 pmol
    Primer SEQ ID NO：5 20pmol
    Primer SEQ ID NO：6 20pmol
    The μ L of pig fourth type coronavirus RNA 2
    Ultra-pure water supplies 25 μ L.
  8. 8. the application of the RT-LAMP kits of pig fourth type coronavirus is detected according to claim 6, it is characterised in that institute The RT-LAMP detection methods stated are to use specific detection, sensitivity Detection and the method quantitatively detected.
  9. 9. the application of the RT-LAMP kits of pig fourth type coronavirus is detected according to claim 6, it is characterised in that institute The RT-LAMP detection methods stated use the closed progress of the real-time transmissometers of loopamp, the amplification situation of complete monitoring reaction tube.
CN201710761229.0A 2017-08-30 2017-08-30 A kind of RT LAMP primers group, kit and application for detecting pig fourth type coronavirus Pending CN107460255A (en)

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CN110982935A (en) * 2019-12-13 2020-04-10 华南农业大学 LAMP primer composition for detecting porcine delta coronavirus by adopting microfluidic chip technology and kit thereof
CN111057798A (en) * 2020-01-20 2020-04-24 复旦大学附属华山医院 LAMP primer combination and kit for detecting novel coronavirus
CN113981149A (en) * 2021-11-23 2022-01-28 广东省农业科学院动物卫生研究所 Porcine delta coronavirus detection primer group, probe, kit and application

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CN109655621A (en) * 2018-12-21 2019-04-19 广西壮族自治区兽医研究所 Pig fourth type coronavirus N protein indirect ELISA antibody detection method and its kit
CN109655621B (en) * 2018-12-21 2022-06-03 广西壮族自治区兽医研究所 Indirect ELISA antibody detection method for swine T-type coronavirus N protein and kit thereof
CN110982935A (en) * 2019-12-13 2020-04-10 华南农业大学 LAMP primer composition for detecting porcine delta coronavirus by adopting microfluidic chip technology and kit thereof
CN111057798A (en) * 2020-01-20 2020-04-24 复旦大学附属华山医院 LAMP primer combination and kit for detecting novel coronavirus
CN113981149A (en) * 2021-11-23 2022-01-28 广东省农业科学院动物卫生研究所 Porcine delta coronavirus detection primer group, probe, kit and application

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