CN106119416B - A kind of kit and detection method of accurate quantification detection astrovirus - Google Patents

A kind of kit and detection method of accurate quantification detection astrovirus Download PDF

Info

Publication number
CN106119416B
CN106119416B CN201610630403.3A CN201610630403A CN106119416B CN 106119416 B CN106119416 B CN 106119416B CN 201610630403 A CN201610630403 A CN 201610630403A CN 106119416 B CN106119416 B CN 106119416B
Authority
CN
China
Prior art keywords
astrovirus
rna
droplet
detection
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201610630403.3A
Other languages
Chinese (zh)
Other versions
CN106119416A (en
Inventor
魏海燕
马丹
徐蕾蕊
李丹
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Inspection and Quarantine Technology Center Beijing Entry-Exit Inspection and Q
Original Assignee
Inspection and Quarantine Technology Center Beijing Entry-Exit Inspection and Q
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Inspection and Quarantine Technology Center Beijing Entry-Exit Inspection and Q filed Critical Inspection and Quarantine Technology Center Beijing Entry-Exit Inspection and Q
Priority to CN201610630403.3A priority Critical patent/CN106119416B/en
Publication of CN106119416A publication Critical patent/CN106119416A/en
Application granted granted Critical
Publication of CN106119416B publication Critical patent/CN106119416B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • 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

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses the kits and detection method of a kind of accurate quantification detection astrovirus.More particularly to one group for detecting the primer and probe of astrovirus, with nucleotide sequence shown in sequence table SEQ ID No.1 to SEQ ID No.3, and kit and its detection method containing these primer and probes.The present invention provides a kind of detection method using droplet type digital pcr technology accurate quantification detection astrovirus, and certified reference material or other standards product since this method is not necessarily to have higher accuracy, sensitivity and repeatability, are easy to standardize.

Description

A kind of kit and detection method of accurate quantification detection astrovirus
Technical field
The invention belongs to molecular biology fields, and in particular to a kind of kit and oligonucleotides for detecting astrovirus, It more precisely, is a kind of reverse transcription-droplet type digital polymerase chain reaction (droplet for detecting astrovirus Digital reverse transcriptional polymerase chain reaction, RT-ddPCR)) fast quantification Detection kit.
Background technique
Appleton is equal to 1975 finds astrovirus (Human for the first time in the excrement of gastroenteritis patient Astrovirus, HAstV), various countries all astrovirus infection can occur to some extent every year and break out situation.Astrovirus without The single strand plus RNA virus of capsid, 28~35nm of diameter have 5~6 small angles, make the star-shaped structure of entire virion.Disease The long 6.8kb of virus gene, including a 5 ' noncoding region (5 ' non-coding region, NCR), three open reading frames (open reading frams, ORFs) ORF1a, ORF1b and ORF2,3 ' noncoding regions of 80 nucleotide and one about 30 The poly-A tail of a nucleotide).Astrovirus can be divided into 8 serotype HAstV-1~HAstV-8.Currently, starlike disease Poison has been considered as causing one of infant, the elderly and important pathogen of immunocompromised person's acute viral enteritis.
Although food-borne virus is stringent cytozoon, irreproducible in food, micro food in food Borne virus will cause disease.And there may be in the food samples of food-borne virus pollution, virus titer is generally in Relatively low level.Therefore need one kind for food-borne virus precise and quantitative detection method, realization has food-borne virus Effect monitoring.Currently, the quantitative detecting method of astrovirus mainly includes with polymerase chain reaction (Polymerase Chain Reaction, PCR) basis real-time fluorescence quantitative PCR (Real-time Fluorescent Quantitative PCR, RT- ) and reverse transcription-ring mediated constant temperature nucleic acid amplification (Realtime reverse transcription lood- qPCR Mediated isothermal amplification, RT-LAMP) method.Both methods is needed by carefully aligned standard Curve and stable astrovirus standard substance, and detect every time all and need to establish standard curve, therefore is potential in interpretation of result The presence of subjective factor causes the diversity of result between the difference and different experiments room of result between repeated detection, influences starlike The standardization and standardization of Viral Quantification detection.
Digital pcr (Digital PCR, dPCR) technology developed in recent years is a kind of completely new nucleic acid quantification detection Method.Currently, digital pcr includes: micro- reaction chamber/orifice plate digital pcr (Chamber Digital PCR, cdPCR), microfluid Digital pcr (Microfluidic Digital PCR, mdPCR) (large-scale integrated micro-fluidic chip) and droplet type digital pcr (Droplet Digital PCR, ddPCR) three kinds of dPCR systems.Currently, being showed no both at home and abroad starlike using RT-ddPCR progress The relevant report of Viral Quantification detection.
Summary of the invention
The object of the present invention is to provide a group-specifics, and strong, high sensitivity the RT-ddPCR for detecting astrovirus draws Object and probe.
It is a further object to provide a kind of easy to operate, accurate astrovirus RT-ddPCR of result quantitatively to examine Test agent box.
To achieve the above object, the invention adopts the following technical scheme:
The present invention is chosen its conservative fragments ORF2, is designed specificity and drawn by the composition of genome of analysis astrovirus The sequence of object and probe, the primer and probe for detecting astrovirus is shown in Table 1.Wherein, 5 ' flag F AM of probe, 3 ' marks Remember BHQ.
Table 1RT-ddPCR primer probe sequence
The present invention also provides a kind of kit of accurate quantification detection astrovirus, which includes above-mentioned for detecting The primer and probe of astrovirus.Further, which further includes completing RT-ddPCR reaction material requested and reagent, example As astrovirus positive control RNA, one-step RT-ddPCR supermix, acetolase, droplet generate oil, droplet generates Card, 96 orifice plates and aluminium foil heat-sealing film etc., above-mentioned material and reagent are preferably individually packed.
The present invention also provides a kind of astrovirus precisely quantitative detection methods, i.e. RT-ddPCR method comprising with Lower content:
1. extracting sample to be tested RNA, extracting method or commercial kit well known in the prior art can be used and carry out It extracts；
2. preparing RT-ddPCR reaction system, wherein the reaction system includes SEQ ID NO:1 to SEQ in sequence table Primer and probe shown in ID NO:3.In one embodiment of the invention, RT-ddPCR reaction system such as table 2 institute Show；
2 astrovirus ddPCR reaction system of table
3. RT-ddPCR reaction system and droplet that step 2. is prepared generate oil and be added in droplet generation card, it is placed in droplet It generates in instrument and generates droplet；
4. carrying out amplification reaction, reaction condition is shown in Table 3；
3 astrovirus RT-ddPCR reaction condition of table
Note: warming and cooling rate answers≤2.5 DEG C/s
5. result judgement.
96 orifice plates after amplification are placed in droplet to read in instrument (QX200, Bio-Rad, Pleasanton, CA), are utilized QuantaSoft software carries out result reading and analysis.Positive droplet containing amplified production and the feminine gender without amplified production are micro- Drop can show the difference of fluorescence signal intensity, carry out given threshold line as boundary using the highest point of negative droplet cluster fluorescence amplitude.It presses It is calculated according to Poisson distribution principle and obtains astrovirus target fragment content in RT-ddPCR reaction system, and then pass through RNA template Sample-adding amount (2 μ L) and the RNA template body product extracted calculate obtain the accurate quantitative of sample stellate virus as follows.
The invention has the advantages that 1) caused by avoiding the absolute quantitation of nucleic acid molecules by PCR amplification efficiency variance Deviation；2) without relying on certified reference material or other standards product；3) there is higher accuracy, sensitivity and repeatability, easily In standardization；4) it is more suitable for the detection of nucleic acids of low copy number；5) since RT-ddPCR is end point determination, to the tolerance level of inhibitor It is higher, therefore the detection error caused by can reducing by matrix type.
The invention will be further described with specific embodiment with reference to the accompanying drawings of the specification, all to disclose according to the present invention The equivalent replacement of any this field that content is done, all belongs to the scope of protection of the present invention.
Detailed description of the invention
Astrovirus target fragment RT-ddPCR testing result under the conditions of Fig. 1 different annealing temperature.
Fig. 2 RT-ddPCR kit detects astrovirus specificity and analyzes result.
Fig. 3 RT-ddPCR kit detects astrovirus sensitivity analysis result.
Fig. 4 RT-ddPCR kit detects astrovirus repeatability and analyzes result.
Specific embodiment
Embodiment 1RT-ddPCR primer, probe design
To realize that we choose astrovirus open reading frame to the specific detection and absolute quantification analysis of astrovirus 2 (Open Reading Frame, ORF2) encode the target sequence of caspid protein, carry out sequence by NCBI online tool Analysis and comparison, design more than 10 to primer using Prime Express software V4.0 (ABI, Foster City, CA, USA) And probe combinations, by screening final acquisition high specificity, being suitable for each 1 set of RT-ddPCR primer/probe combinations, sequence is shown in Table 1.Its middle probe 5 ' holds flag F AM, 3 ' end label BHQ.Primer/probe leads to Trade Co., Ltd. by Beijing six directions and synthesizes.
The foundation of 2 detection method of embodiment
(1) RNA is extracted: being extracted using commercial kit；Or RNA is extracted using tradition Trizol method, specifically It operates as follows: 1mL Trizol being 1. added in 100 μ L samples, after shaking 30s, be placed at room temperature for 5min；2. 250 μ L chlorine are added It is imitative, 30s is acutely shaken, 5min is placed at room temperature for；4 DEG C, 12000g, it is centrifuged 5min；3. supernatant is transferred in new centrifuge tube, it is added The isopropanol of 500 μ L, which acutely shakes, mixes 30s, is placed at room temperature for 5min；4. 4 DEG C, 5000g, being centrifuged 5min；5. carefully removing Clearly, 12000g after precipitating is cleaned with 70% ethyl alcohol of 1mL, 4 DEG C, centrifugation 5min (siphons away supernatant, centrifuge tube is placed in ultra-clean as far as possible Precipitating is dried up on platform)；(in order to dissolve viral RNA preferably, 60 DEG C of heating are set without RNase water dissolution RNA 6. 50 μ L are added 10min).The RNA of extraction is saved backup in -80 DEG C.
(2) the RT-ddPCR reaction system of 20 μ L is prepared according to table 2
(3) droplet generates
The ddPCR reaction system of 20 μ L and 65 μ L droplets are generated oil to be added separately in 8 hole droplets generation card, are placed in micro- Drop, which generates, generates droplet in instrument (QX200, Bio-Rad, Pleasanton, CA).
(4) amplified reaction
The droplet (40 μ L) of the Water-In-Oil of generation is slowly transferred in 96 orifice plates, sealer is placed on PCR instrument (Veriti Thermal cycler, Applied BioSystems, Foster City, CA) on carry out amplification reaction, amplification condition such as table 3 It is shown.
(5) result judgement
96 orifice plates after amplification are placed in droplet to read in instrument (QX200, Bio-Rad, Pleasanton, CA), are utilized QuantaSoft software carries out result reading and analysis.Positive droplet containing amplified production and the feminine gender without amplified production are micro- Drop can show the difference of fluorescence signal intensity, can the highest point of negative droplet cluster fluorescence amplitude be that boundary carrys out given threshold line. It is calculated according to Poisson distribution principle and obtains astrovirus target fragment content in RT-ddPCR reaction system, and then pass through RNA mould Plate sample-adding amount (2 μ L) and the RNA template body product extracted calculate obtain the accurate fixed of sample stellate virus as follows Amount.
The present invention is also optimized RT-ddPCR testing conditions, such as annealing temperature, takes the astrovirus RNA of extraction As template, is carried out amplification reaction under 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C and 70 DEG C of annealing temperature respectively, compare different condition The testing result of lower RT-ddPCR.As seen from Figure 1, the amplification efficiency of astrovirus target gene fragment is relatively low under the conditions of 50 DEG C, Positive droplet and negative droplet boundary are unobvious；Target gene fragment amplification efficiency is higher under the conditions of 55 DEG C and 60 DEG C, may occur in which Obvious positive droplet cluster, and the fluorescence signal expanded under the conditions of 60 DEG C is slightly above 55 DEG C, therefore, by 60 DEG C as best Most fiery temperature.
3 kit forms of embodiment
1. the composition (being stored in -20 DEG C) of kit
(1) the primer and probe SEQ ID No.1-3 for being used to detect astrovirus that embodiment 1 designs, by Beijing six directions Logical Trade Co., Ltd.'s synthesis；
(2) U.S. BioRad company, article No. 186- one-step RT-ddPCR supermix, 25mM magnesium acetate: are purchased from 3021；
(3) droplet generates oil: being purchased from U.S. BioRad company, article No. 186-3030；
(4) droplet generates card: being purchased from U.S. BioRad company, article No. 186-4007；
(5) U.S. BioRad company, article No. 181-4000 aluminium foil heat-sealing film: are purchased from；
(6) Eppendorf company, Germany, article No. 0,030,128,605 96 orifice plate of Twin Tec Semi-Skirted: are purchased from；
(7) negative control: DEPC water；Purchased from the raw work in Shanghai, article No.: D1005；
(8) positive control: astrovirus RNA standard substance；
(9) DEPC water: purchased from the raw work in Shanghai, article No.: D1005.
2. prepared by astrovirus RNA standard substance
Using astrovirus RNA as template, using primer SEQ ID No.1-2, the specific segment of about 63bp is amplified, is claimed For Ast (comprising RT-ddPCR expand target gene)；(II carrier of pcDNA is purchased from II-Ast of construction recombination plasmid pcDNA Invitrogen company), sequencing is carried out in Shanghai Sangon Biotech Company, and sequencing result and GenBank stellate is viral Gene order carries out homology analysis, and homology is up to 95% or more.Simultaneously plasmid DNA purification is extracted, with Promega company Ribo MAXTMLarge Scale RNAProduction system-T7 kit (article No.: P1300) is transcribed in vitro (carrying out by kit operational manual) is transcribed in vitro product and is digested with DNase, remove DNA therein.Utilize RNeasy RNA is further purified in MiniElute Cleanup kit (being purchased from QIAGEN company, article No. 74204).The RNA of purifying is in Shanghai Sheng Gong company carries out sequencing, and the gene order of sequencing result and GenBank stellate virus is carried out homology analysis, Its homology is up to 95% or more.CRNA is dispensed, is stored in -80 DEG C.The astrovirus RNA standard substance of preparation is carried out uniform Property, stability test, meet related request；Definite value research is carried out to RNA using the laboratory Duo Jia valued methods, it is final to prepare Astrovirus RNA standard substance concentration are as follows: 5.52 × 106copies/μl.- 80 DEG C of preservations, the positive as kit are right According to.
4 detection method of embodiment specificity and sensitivity evaluation
1. detection specificity analysis
(1) material: rotavirus, astrovirus, hepatitis A virus, I type of norovirus and II type RNA of norovirus are in State's disease prevention and control center's virosis is provided.
(2) method: kit of the present invention is used, includes rotavirus, astrovirus, first to common diarrhea virus Hepatovirus, I type of norovirus and II type RNA of norovirus carry out RT-ddPCR augmentation detection, and observing the kit has spy nothing but The generation of opposite sex reaction.
(3) result: rotavirus, astrovirus, hepatitis A virus, I type of norovirus and II type RNA of norovirus are through PCR After instrument (Veriti Thermal cycler, Applied BioSystems, Foster City, CA) amplification, read using droplet It takes instrument (QX200, Bio-Rad, Pleasanton, CA) to detect, carries out result reading and analysis using QuantaSoft software.Knot Fruit shows that only specific amplification occurs in astrovirus, remaining virus is negative through RT-ddPCR detection, it was demonstrated that this method tool There is good specificity.As a result see Fig. 2.
2. detection sensitivity is analyzed
(1) method: astrovirus detection RNA standard substance 10 times of gradient dilutions of progress prepared above to 5.52 are copied Shellfish/μ L respectively takes 2 μ L, RT-ddPCR detection is carried out using kit described in embodiment 3, by analyzing reagent of the present invention The minimum that box can be detected, reaction system are shown in Table 2.
(2) result: discovery utilizes kit of the present invention, and the astrovirus RNA standard of 5.52 copies/μ L can be detected Substance.As a result see Fig. 3.It can be seen that this kit detection astrovirus can reach 5.52 copies/μ L, astrovirus phase is substantially increased The sensitivity for closing detection method, does not depend on standard items, it can be achieved that absolute quantitation, visual result are reliable.
5 detection method accuracy of embodiment and reproducibility
1. detection accuracy is evaluated
Accuracy (truness) refers to the consistency journey between the average value and acceptable reference value of one group of test result Degree.According to the standard of European Union and Codex, the criterion of accuracy is the measured value Ying Can in entirely detection dynamic range It examines in ± 25% range of value.
(1) method: astrovirus RNA standard substance (5.52 × 10 prepared above is utilized6Copies/ μ l) it is diluted to 5.52×103copies/μl、5.52×102copies/μl、5.52×101Copies/ μ l and 5.52copies/ μ l, is expressed as Sample U1-U4 (table 4)
Each sample carries out RT-ddPCR detection respectively, and each sample sets 7 repetitions, while 1 blank control is arranged (to go DEPC water replaces template ribonucleic acid).
(2) result: the RT-ddPCR measured value of astrovirus RNA is very close with predicted value, the equal < of standard deviation 15% (table 4), the criterion less than 25% illustrate that this method has extraordinary accuracy.
The accuracy of 4 astrovirus RT-ddPCR of table detection
2. detection repeatability analysis
(1) method: astrovirus RNA standard substance (5.52 × 10 prepared above is utilized6Copies/ μ l) it is diluted to 5.52×103Copies/ μ l carries out RT-ddPCR detection, if 9 repetitions, while 1 blank control is set (to remove DEPC water Instead of template ribonucleic acid).
(2) result: from fig. 4, it can be seen that being less than to the measurement coefficient of variation CV of the astrovirus RNA positive control of certain concentration 25%, it was demonstrated that this method is used to have good repeatability when astrovirus quantitative detection.
Obviously, the above embodiment of the present invention be only to clearly illustrate example of the present invention, and not be pair The restriction of embodiments of the present invention.For those of ordinary skill in the art, may be used also on the basis of the above description To make other variations or changes in different ways.Here all embodiments can not be exhaustive.It is all to belong to this hair The obvious changes or variations that bright technical solution is extended out are still in the scope of protection of the present invention.

Claims (2)

1. a kind of kit of accurate quantification detection astrovirus, which is characterized in that the kit includes: starlike for detecting It is the primer and probe of viral RNA, astrovirus positive control RNA, one-step RT-ddPCR supermix, magnesium acetate, micro- Drop generates oil, droplet generates card, 96 orifice plates and aluminium foil heat-sealing film；
Wherein, the primer for detecting astrovirus RNA is nucleotide sequence shown in SEQ ID No.1 to SEQ ID No.2, Probe for detecting astrovirus RNA is nucleotide sequence shown in SEQ ID No.3, and probe 5 ' holds flag F AM, 3 ' ends Mark BHQ.
2. a kind of detection method of non-diagnostic purpose accurate quantification detection astrovirus, which is characterized in that this method comprises:
(1) sample to be tested RNA is extracted；
(2) RT-ddPCR reaction system is prepared, wherein the reaction system includes shown in SEQ ID No.1 to SEQ ID No.2 For detecting the primer of astrovirus RNA, for detecting the probe of astrovirus RNA, and probe shown in SEQ ID No.3 5 ' end flag F AM, 3 ' end label BHQ；
(3) the RT-ddPCR reaction system and droplet prepared step (2) generate oil and are added in droplet generation card, and it is raw to be placed in droplet Cheng Yizhong generates droplet；
(4) it carries out amplification reaction；
(5) result judgement calculates the copy number for obtaining astrovirus RNA according to Poisson distribution principle, and then passes through following formula Calculate the content for obtaining sample to be tested stellate virus:
Sample stellate viral level=RNA template body product,
Wherein, RNA template sample-adding amount is 2 μ L.
CN201610630403.3A 2016-08-04 2016-08-04 A kind of kit and detection method of accurate quantification detection astrovirus Active CN106119416B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610630403.3A CN106119416B (en) 2016-08-04 2016-08-04 A kind of kit and detection method of accurate quantification detection astrovirus

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610630403.3A CN106119416B (en) 2016-08-04 2016-08-04 A kind of kit and detection method of accurate quantification detection astrovirus

Publications (2)

Publication Number Publication Date
CN106119416A CN106119416A (en) 2016-11-16
CN106119416B true CN106119416B (en) 2019-11-26

Family

ID=57255526

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610630403.3A Active CN106119416B (en) 2016-08-04 2016-08-04 A kind of kit and detection method of accurate quantification detection astrovirus

Country Status (1)

Country Link
CN (1) CN106119416B (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107385111B (en) * 2017-08-18 2020-12-08 福建省农业科学院畜牧兽医研究所 Real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer of goose astrovirus and kit thereof
CN108085412A (en) * 2017-11-06 2018-05-29 北京出入境检验检疫局检验检疫技术中心 A kind of human astrovirus 1's type detection of nucleic acids standard substance and its preparation method and application
CN108103241A (en) * 2017-11-27 2018-06-01 西南大学 Citrus yellow vein virus droplet digital pcr immue quantitative detection reagent box and method

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102965453A (en) * 2012-09-25 2013-03-13 珠海国际旅行卫生保健中心 Astrovirus real-time isothermal amplification detection kit, its primers and probe
CN104017905A (en) * 2014-06-25 2014-09-03 长兴县人民医院 Probe, primer and kit for detecting human astrovirus and human noroviruses through real-time fluorescent RT-PCR and application of probe, primer and kit
WO2015013681A1 (en) * 2013-07-25 2015-01-29 Bio-Rad Laboratories, Inc. Genetic assays

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102965453A (en) * 2012-09-25 2013-03-13 珠海国际旅行卫生保健中心 Astrovirus real-time isothermal amplification detection kit, its primers and probe
WO2015013681A1 (en) * 2013-07-25 2015-01-29 Bio-Rad Laboratories, Inc. Genetic assays
CN105555972A (en) * 2013-07-25 2016-05-04 伯乐生命医学产品有限公司 Genetic assays
CN104017905A (en) * 2014-06-25 2014-09-03 长兴县人民医院 Probe, primer and kit for detecting human astrovirus and human noroviruses through real-time fluorescent RT-PCR and application of probe, primer and kit

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
星状病毒核酸检测标准物质的研制;徐蕾蕊;《食品科学》;20151015;第37卷(第6期);172-177 *

Also Published As

Publication number Publication date
CN106119416A (en) 2016-11-16

Similar Documents

Publication Publication Date Title
CN106222300B (en) A kind of kit and detection method of accurate quantification detection A rotavirus
Oberste et al. Comparative evaluation of Taqman real-time PCR and semi-nested VP1 PCR for detection of enteroviruses in clinical specimens
CN107513584B (en) A kind of five heavy fluorescence quantitative kits detecting enterovirus
WO2021175298A1 (en) Novel coronavirus detection reagent and detection method
CN106119417B (en) Kit and detection method for accurately and quantitatively detecting norovirus type I
CN106222301B (en) A kind of kit and detection method of accurate quantification detection II type of norovirus
CN106119416B (en) A kind of kit and detection method of accurate quantification detection astrovirus
CN107151711A (en) A kind of double fluorescent quantitative RT PCR kits for detecting dengue virus and zika virus
CN105936946A (en) One step method inverse transcription PCR kit for detecting and differentiating Zika viruses and detection method thereof
Conceição et al. A real-time PCR procedure for detection of dengue virus serotypes 1, 2, and 3, and their quantitation in clinical and laboratory samples
CN105624329A (en) Real-time fluorescence nucleic acid isothermal amplification detection kit for human herpesvirus 1
Chen et al. Development and evaluation of a real-time method of simultaneous amplification and testing of enterovirus 71 incorporating a RNA internal control system
CN103243179A (en) Shell-type PCR (polymerase chain reaction) amplification primer of porcine epidemic diarrhea virus and application thereof
CN113249522A (en) Method for detecting SARS-CoV-2variant strain nucleic acid and its application
CN108330211A (en) A group rotavirus/astrovirus/intestinal adenovirus nucleic acid multiple fluorescence PCR detection reagent box and application thereof
Chen et al. Development of a minor groove binder assay for real-time PCR detection of porcine Sapelovirus
CN106755572A (en) I types DHV and duck plague virus double fluorescent quantitative PCR method
CN103484568A (en) Method and kit for dual fluorescence quantitative PCR detection of grass carp reovirus types I and II
CN108531647A (en) A kind of zika virus one-step method fluorescence rt-PCR detection methods and kit
CN101948936A (en) Real-time fluorescence PCR method for detecting norovirus in aquatic product
CN102965453A (en) Astrovirus real-time isothermal amplification detection kit, its primers and probe
CN104073570B (en) For the identification of the primer pair of human adenovirus 55 type, primed probe composition and their application
CN102816865B (en) H1N1, PRRSV and CSFV quadruple detection kit
CN104894112B (en) A kind of Japanese B encephalitis virus real-time fluorescence isothermal amplification detection kit and its primer and probe
CN108130385A (en) A kind of human cytomegalovirus kit for detecting nucleic acid

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant