CN105907889A - Detection kit of human noroviruses and application of detection kit - Google Patents
Detection kit of human noroviruses and application of detection kit Download PDFInfo
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Abstract
The invention provides a detection kit of human noroviruses. The detection kit at least comprises the following reagents: a first primer pair used for detecting the human noroviruses in a GI group and a TaqMan probe matched with the first primer pair, a second primer pair used for detecting the human noroviruses in a GII group and a TaqMan probe matched with the second primer pair, and a one-step reverse transcription polymerase chain reaction reagent. The invention also provides application of the detection kit. The detection kit provided by the invention is good in specificity, high in sensitivity and simple and convenient to use, and is capable of efficiently detecting the human noroviruses.
Description
Technical field
The invention belongs to biology field, be specifically related to a kind of height based on virus-specific receptor capture
The original position Acquisition Detection test kit of effect detection by quantitative people source norovirus.
Background technology
Norovirus (noroviruses, NoVs) is also known as norwalk virus (Norwalk Virus), class promise
Watt gram virus (Norwalk-like Virus) or little round shape virus (Small Round Structured Virus,
SRSV), it is under the jurisdiction of Qian Bei section norovirus and belongs to member, for single strand RNA virus.Kapikian in 1972
Etc. utilize the method for immuno-electron microscope nineteen sixty-eight Ohio, USA Norwalk town vomit in acute winter of breaking out
Tell and disease sample is found that this virus first, and by its named Norwalk virus.Occur the most successively closing
In the report that this virus is broken out, cause this viral nomenclature chaotic.2002, ICTV (the
International Committee on Taxonomy of Viruses, ICTV) it is classified as embedding Caliciviridae,
And formally it is renamed as norovirus.
According to the difference of virus major structural protein nucleotide sequence, NoVs is divided into 7 groups (GI-GVII),
Wherein GI, GII and GIV group NoVs can infect the mankind, is referred to as people source norovirus (human
noroviruses,HuNoVs).Epidemic data shows, and: HuNoVs (GI and GII group is main) is
Cause the main arch-criminal of acute nonbacterial gastroenteritis, have that sickness rate is high, infective dose is low, to external world
The features such as resistance is strong.The epidemic monitoring result of this state is shown, between 2009-2010 by Disease Control and Prevention Center of the U.S.
Breaking out 1527 food origin disease events altogether, wherein the pathogen of 790 example epidemic situations is HuNoVs, accounts for total sudden and violent
The 42% of the event of sending out.In the end of the year 2012, break out different grades of in states such as Britain, Holland, Japan successively
HuNoVs popular event.HuNoVs present worldwide take place frequently and various places sickness rate occur increase year by year
High trend, with people to its recognize deeply, growing perfect, the sensitivity of detection method significantly improves
Relevant.
At present, the detection method of HuNoVs is set up at virion profile, specific nucleic acid section and main
On the basis of antigenic determinant, mainly there are electron microscopy, immunological method and molecular detecting method etc..China is specially
Profit CN201510487540.1, CN201510179138.7, CN201310553678.8,
CN201310271924.0 etc. contribute to the Molecular Detection of NoVs.But, these patents of invention are with at present
The defect of conventional HuNoVs detection method essentially consists in: 1) be required to the step by special extracting RNA
Suddenly obtain viral nucleic acid, waste time and energy；2) cannot be distinguished from expanding template ribonucleic acid and derive from infective NoVs
Viral RNA free in particle, or sample；3) environment and food samples usually contain some PCR
Inhibitor, easily causes false positive results.Therefore, research and development are a kind of highly sensitive, and accuracy rate is high, operation letter
Single, the shortest, and the detection kit of high flux HuNoVs has good using value.
Summary of the invention
Because the drawbacks described above of prior art, the invention provides the detection examination of a kind of new people source norovirus
Agent box, the detection kit specificity that this is provided is good, highly sensitive, easy to use, can efficient detection people
Source norovirus.
For solving the problems referred to above, the present invention adopts the technical scheme that: the detectable of a kind of people source norovirus
Box, described detection kit at least includes following reagent: for detecting the first of GI group people source norovirus
Primer to and the first primer to supporting TaqMan probe, for detecting the second of GII group people source norovirus
Primer to and the second primer to supporting TaqMan probe, a step inverse transcription polymerase chain reaction reagent；
Wherein, the sequence of the primer one of the first primer pair is as shown in SEQ ID NO:1, and the first primer is to drawing
The sequence of thing two is as shown in SEQ ID NO:2；First primer sequence to the probe three of supporting TaqMan probe
Arrange as shown in SEQ ID NO:3, the first primer sequence such as SEQ to the probe four of supporting TaqMan probe
Shown in ID NO:4；The sequence of the primer five of the second primer pair as shown in SEQ ID NO:5, the second primer pair
The sequence of primer six as shown in SEQ ID NO:6；Second primer probe seven to supporting TaqMan probe
Sequence as shown in SEQ ID NO:7.
Preferably, 5 ' ends of described TaqMan probe are marked with fluorescent reporter group FAM, and 3 ' ends are marked with
Fluorescent quenching group TAMRA.
Preferably, described detection kit also includes type III Intestinum Sus domestica gastric mucosa extract and bovine serum albumin/defat
Milk powder.
Preferably, described type III Intestinum Sus domestica gastric mucosa extract contains the specific receptor of people source norovirus.
Preferably, described detection kit also includes enzyme mark strip, enzyme mark frame, polyolefin film, absorbent filter, phosphorus
Phthalate buffer, sodium carbonate-bicarbonate buffer solution and DEPC water/ddH2O。
Present invention also offers the application of detection kit provided by the present invention, described application includes walking as follows
Rapid:
1) type III Intestinum Sus domestica gastric mucosa extract is coated in ELISA Plate；
2) sealase target, prevents ELISA Plate non-specific adsorption virion；
3) testing sample is joined ELISA Plate；
4) Pintsch process release RNA；
5) add the first primer to, the first primer to supporting TaqMan probe, the second primer to, second draw
Supporting TaqMan probe and a step inverse transcription polymerase chain reaction reagent are reacted by thing；
6) RT-qPCR detection is carried out.
Preferably, detection GI group people source norovirus and the cleaning of enzyme target of GII group people source norovirus and dilute
The buffer releasing virus is respectively pH=3.0 and pH=7.0.
Preferably, the temperature of Pintsch process release RNA is 95 DEG C.
Preferably, the reaction condition of RT-qPCR detection is: 42 DEG C of reverse transcription 10min of first stage, second
95 DEG C of denaturations 30s of stage；95 DEG C of degeneration 15s of phase III, 53 DEG C of annealing 15s, 60 DEG C extend 30s,
Expand 45 circulations；4 DEG C of insulations；And carry out FAM fluorescence signal acquisition in the phase III.
The rna polymerase region that the present invention relies on according to the sequence preservative area RNA of virus, uses Primer5.0
Software design specific primer, by using capture reverse transcription real time fluorescent quantitative poly chain type in situ anti-
Should the method for (RT-qPCR), the infection of people source norovirus is carried out auxiliary diagnosis, and this virus
Epidemiological study.
The present invention is characterized by: people source norovirus specific receptor is attached in ELISA Plate, sick in sample
Poison can be combined with its receptor-specific；Can retain with special with removal of contamination during detersive enzyme target
Property the virion that combines of receptor, detection object is carried out the screening of the first step；95 DEG C of releasing virus nucleic acid；
Designed primer and TaqMan probe have high degree of specificity, reaffirm detection object.This
Invention eliminates numerous and diverse step of purified virus RNA, not only greatly reduces reagent cost and cost of labor,
And also improve sensitivity and the specificity of detection, easy and simple to handle.
The invention have the benefit that 1) detection method is easy and simple to handle, and can the procedural operation of scale；2)
Detection method is highly sensitive, high specificity；3) testing result is reliable, directly perceived；4) testing cost is low.
Below with reference to accompanying drawing, the technique effect of design, concrete structure and the generation of the present invention is described further,
To be fully understood from the purpose of the present invention, feature and effect.
Accompanying drawing explanation
Fig. 1 be in the embodiment of the present invention 2 pH value on detection GI group and the impact of GII papova；
Fig. 2 is to carry out the comparison (GI group) of detection limit with tradition RT-qPCR method in the embodiment of the present invention 3；
Fig. 3 is to carry out the comparison (GII group) of detection limit with tradition RT-qPCR method in the embodiment of the present invention 3；
Fig. 4 is the amplification curve of the river detecting GI papova artificial contamination in the embodiment of the present invention 4；
Fig. 5 is the amplification curve of the river detecting GII papova artificial contamination in the embodiment of the present invention 4；
Fig. 6 is the amplification curve detecting disease prevention and control center's clinical sample in the embodiment of the present invention 5；
Fig. 7 is the amplification curve detecting artificial contamination's Oyster Tissue in the embodiment of the present invention 6；
Fig. 8 is the amplification curve that in the embodiment of the present invention 7, detection detects commercially available Oyster Tissue.
Detailed description of the invention
In the embodiment of the present invention, agents useful for same all can be by commercially available acquisition, institute's employment source norovirus clinical sample
From Beijing disease prevention and control center (Center for Disease Control and Prevention, CDC) and
Huzhou disease prevention and control center.
Embodiment 1 primer and TaqMan probe
Obtain primer and the method for TaqMan probe in the present invention: use Mega 5.0 software to
In GenBank, the gene order of existing people source norovirus carries out the bioinformatic analysis of sequence, by same
Source property compares, and obtains conservative region；Application ABI primer composite software (Primer Express version 2.0, Applied
Biosystems) primer and the TaqMan probe of amplification conserved region gene fragment are devised；Designed primer
It is respectively provided with the sequence being complementary to people source norovirus specific gene fragment with probe, applies NCBI/
Primer-BLAST (http://www.ncbi.nlm.nih.gov/BLAST) to the primer of designed synthesis and
TaqMan probe carries out specificity and availability detection, it is ensured that the most same with the nucleotide sequence of other pathogen
Source property；Containing the receptor of people source norovirus in PGM, during hatching, virus is tied with its receptor-specific
Close, and conjugate is washed, improve reliability and the accuracy of testing result.
Embodiment 2 detection kit and detection method
The reagent of detection kit includes: enzyme mark strip, enzyme mark frame, MicroAmp polyolefin film, absorbent filter,
Type III Intestinum Sus domestica gastric mucosa extract (porcine gastric mucin, PGM), bovine serum albumin (bovine
Serum albumin, BSA), the first primer to, the first primer to supporting TaqMan probe, the second primer
To, the second primer to supporting TaqMan probe, a step inverse transcription polymerase chain reaction (RT-qPCR)
Enzyme, phosphate buffer (phosphate buffer saline, PBS), sodium carbonate-bicarbonate buffer solution and
DEPC water.
Wherein, the sequence of the primer one of the first primer pair is as shown in SEQ ID NO:1, and the first primer is to drawing
The sequence of thing two is as shown in SEQ ID NO:2；First primer sequence to the probe three of supporting TaqMan probe
Arrange as shown in SEQ ID NO:3, the first primer sequence such as SEQ to the probe four of supporting TaqMan probe
Shown in ID NO:4；The sequence of the primer five of the second primer pair as shown in SEQ ID NO:5, the second primer pair
The sequence of primer six as shown in SEQ ID NO:6；Second primer probe seven to supporting TaqMan probe
Sequence as shown in SEQ ID NO:7.5 ' ends of TaqMan probe are marked with fluorescent reporter group FAM,
3 ' ends are marked with fluorescent quenching group TAMRA.Primer one, primer two, TaqMan probe three, TaqMan
Probe four, primer five, primer six, the concentration of TaqMan probe seven are all 10.0 μMs.Type III Intestinum Sus domestica stomach glues
In film extract, the specific receptor containing people source norovirus is (such as serum tissue antigen, Histo-blood
group antigens)。
Utilize above primer TaqMan probe can detect two groups of popular genotype (GI group and GII group) people source
Norovirus strain, the first primer to and the first primer to supporting TaqMan probe detection GI group people source promise such as
Virus, the second primer to and the second primer to supporting TaqMan probe detection GII group people source norovirus.
The detection method using this detection kit specifically includes following steps:
1) PGM is coated in ELISA Plate
PGM is diluted, extremely final concentration of 1.0mg/mL with sodium carbonate-bicarbonate buffer solution, with
PGM after dilution is added in enzyme mark hole by 100.0 μ L-200.0 μ L/ holes；4 DEG C of overnight incubation.
2) sealase target
BSA is dissolved, to final concentration of 1.0% (w/v) with PBS.Take out the ELISA Plate being coated overnight, PBS
Wash 3 times, with 120.0 μ L-230.0 μ L/ holes, the BSA of final concentration 1.0% added in enzyme mark hole,
Hatch 30min-60min for 37 DEG C；As the deformation of this specific embodiment, confining liquid also can use final concentration
It is about the defatted milk powder of 5.0%.
3) testing sample is added
Taking out ELISA Plate, PBS washs 3 times, with 100.0 μ L-200.0 μ L/ holes, testing sample joins enzyme mark
In plate hole, hatch 30min-60min for 37 DEG C.Wherein, detection GI papova and GII papova clean
The PBS of ELISA Plate and virus dilution be respectively pH=3.0 and pH=7.0 (pH value to detection GI group with
The impact of GII papova is as it is shown in figure 1, can improve virion and virus under the conditions of corresponding pH
The combination rate of specific receptor).
4) Pintsch process release RNA
Taking out ELISA Plate, discard liquid in ELISA Plate hole, PBS washs 3 times, adds DEPC with 8.5 μ L/ holes
Water, and cover with MicroAmp polyolefin film.Hatch 5min-10min for 95 DEG C, 4 DEG C of cooling 5min.
5) RT-qPCR reactant liquor preparation
Reactant liquor formula is as follows: 10.0 μMs of each 0.5 μ L of upstream and downstream primer, 10.0 μMs of TaqMan probe 1.0 μ L,
2 × reaction buffer 12.5 μ L, one-step method RT-qPCR reaction enzymes 2.0 μ L (include reverse transcriptase and DNA
Polymerase), add up to 16.5 μ L.With 16.5 μ L/ holes by above-mentioned reactant liquor addition enzyme mark hole, and mix homogeneously.
Enzyme mark strip is covered with new MicroAmp polyolefin film.
6) RT-qPCR detection
Enzyme mark strip is put in quantitative real time PCR Instrument, select FAM fluorescence channel.Reaction condition is as follows:
42 DEG C of reverse transcription 10min of first stage, 95 DEG C of denaturations 30s of second stage；Phase III 95 DEG C
Degeneration 15s, 53 DEG C of annealing 15s, 60 DEG C extend 30s, expand 45 circulations；4 DEG C of insulations；?
Phase III carries out FAM fluorescence signal acquisition.
Embodiment 3 compares with tradition RT-qPCR method
With PBS, 60.0 μ LGI group people source norovirus positive sample are carried out gradient dilution, extension rate from
101To 106, each dilution factor volume is 600.0 μ L, is equally divided into 2 parts；1 part provides by embodiment 2
Test kit and method detect, the repetition of three, each sample；Use viral RNA extraction agent for another 1 part
Box extracting RNA, RNA is dissolved in 30.0 μ LDPEC water, and taking 8.5 μ LRNA, to carry out RT-qPCR anti-
Should, the repetition of 3, each sample.Result reaches 10 as in figure 2 it is shown, work as extension rate5Time, embodiment 2
The test kit provided and method can detect GI papova, and tradition RT-qPCR method cannot detect,
Show the detection limit using the present invention can be effectively improved GI papova.
With PBS, 60.0 μ LGII group people source norovirus positive sample are carried out gradient dilution, extension rate from
101To 106, each dilution factor volume is 600.0 μ L, is equally divided into 2 parts, and 1 part provides by embodiment 2
Test kit and method detect, the repetition of 3, each sample；Use viral RNA extraction agent for another 1 part
Box extracting RNA, RNA is dissolved in 30 μ LDPEC water, takes 8.5 μ LRNA and carries out RT-qPCR reaction,
The repetition of 3, each sample.Result reaches 10 as it is shown on figure 3, work as extension rate5With 106Time, embodiment 2
The test kit provided and method all can detect that GII papova, and tradition RT-qPCR method cannot detect,
Show the detection limit using the present invention can be effectively improved GII papova.
Embodiment 4 detects the river of artificial contamination
River samples: the river choosing Huangpu River tributary carries out randomization.Use 50mL plastics from
Heart pipe is acquired, often pipe about 30mL, takes 3 parallel water samples altogether, respectively takes from 3 parallel water samples
5mL is mix homogeneously in new pipe, as experiment water sample, is stored in 4 DEG C of refrigerators stand-by.
Artificial contamination: by GI group and GII group people source norovirus positive sample, river is polluted respectively,
I.e. carry out viral dilution, totally 100 times and 1000 times of two dilution factors with river, and PBS dilution disease is set
Poison as negative control, uses test kit and the side of embodiment 2 offer as positive control, only detection river
Method detects.As shown in Figure 4 and Figure 5, that wherein A-E represents is A to result respectively: dilute with PBS
The virus-positive sample of 100 times；B: dilute the virus-positive sample of 1000 times with PBS；C: with river
Dilute the virus-positive sample of 100 times；D: dilute the virus-positive sample of 1000 times with river；E: river
Water.From result, the present invention can efficiently detect the HuNoVs in the river of artificial contamination, and result is steady
Fixed.
Embodiment 5 detects people source norovirus clinical sample
Choose totally 10 the people source norovirus clinical samples from Beijing and Huzhou CDC to detect, take
Employment source norovirus clinical sample 3.0 μ L, dilutes 100 times with PBS, and the repetition of three, each sample is adopted
The test kit and the method that there is provided by embodiment 2 detect.Testing result is stable (see Fig. 6), shows phase
Close primer and probe has good specificity.
Embodiment 6 detects the Concha Ostreae sample of artificial contamination
Simulation Concha Ostreae growing environment, selects 30 great-hearted Concha Ostreaes to be randomly divided into two after overnight pre-raising is chosen
Group, raises respectively in 15L sea water.By 106The people source norovirus positive clinical sample of copy is thrown respectively
Enter two groups of manual simulation Concha Ostreaes and raise water body.After raising Concha Ostreae 24h according to above-mentioned feeding manner, take out male
Oyster sled shell.Take Concha Ostreae different tissues (gill, stomach, Digestive diverticula and other) add physiology with the ratio of 1:4
Saline, homogenizing, after mixing with 50% glycerol 1:1, subpackage is in 2.0mL EP pipe, is stored in-80 DEG C.
Take 0.5mL tissue homogenate addition 1.0mL E.C. 3.4.21.64 (0.2mg/mL) fully to mix；37 DEG C are shaken
Bed hatches 60min；60 DEG C of water-bath 15min；12000 × g is centrifuged 15min, collects supernatant 1.0mL.With
100.0 μ L-200.0 μ L/ hole supernatant add in enzyme mark hole, use test kit and the method for embodiment 2 offer
Detect.Result as it is shown in fig. 7, GI group and GII papova the gill of Concha Ostreae, stomach, Digestive diverticula and
Its hetero-organization all presents positive test symbol.
Embodiment 7 detects commercially available Concha Ostreae sample
The Concha Ostreae sample that on market, stochastic buying is fresh, take Concha Ostreae different tissues (gill, stomach, Digestive diverticula and
Other) add normal saline with the ratio of 1:4, homogenizing, after mixing with 50% glycerol 1:1, subpackage is to 2.0mL
In EP pipe, it is stored in-80 DEG C.Take 0.5mL tissue homogenate and add 1.0mL E.C. 3.4.21.64 (0.2mg/mL)
Fully mixing；37 DEG C of shaking tables hatch 60min；60 DEG C of water-bath 15min；12000 × g is centrifuged 15min, receives
Collection supernatant 1.0mL.Add in enzyme mark hole with 100.0 μ L-200.0 μ L/ hole supernatant, use embodiment 2 to carry
Test kit and the method for confession detect.Result as shown in Figure 8, for GI papova, Concha Ostreae
The gill, stomach, Digestive diverticula all has positive amplification；For GII papova, there is no positive findings.
The preferred embodiment of the present invention described in detail above.Should be appreciated that those of ordinary skill in the art
Just many modifications and variations can be made according to the design of the present invention without creative work.Therefore, all technology neck
In territory, technical staff is the most on the basis of existing technology by logical analysis, reasoning or limited
Test available technical scheme, all should be in the protection domain being defined in the patent claims.
Claims (9)
1. the detection kit of a people source norovirus, it is characterised in that described detection kit is at least wrapped
Include following reagent: for detect the first primer of GI group people source norovirus to and the first primer to supporting
TaqMan probe, for detect the second primer of GII group people source norovirus to and the second primer to supporting
TaqMan probe, a step inverse transcription polymerase chain reaction reagent；
Wherein, the sequence of the primer one of the first primer pair is as shown in SEQ ID NO:1, and the first primer is to drawing
The sequence of thing two is as shown in SEQ ID NO:2；First primer sequence to the probe three of supporting TaqMan probe
Arrange as shown in SEQ ID NO:3, the first primer sequence such as SEQ to the probe four of supporting TaqMan probe
Shown in ID NO:4；The sequence of the primer five of the second primer pair as shown in SEQ ID NO:5, the second primer pair
The sequence of primer six as shown in SEQ ID NO:6；Second primer probe seven to supporting TaqMan probe
Sequence as shown in SEQ ID NO:7.
2. detection kit as claimed in claim 1, it is characterised in that the 5 ' of described TaqMan probe
End is marked with fluorescent reporter group FAM, and 3 ' ends are marked with fluorescent quenching group TAMRA.
3. detection kit as claimed in claim 1, it is characterised in that described detection kit also includes
Type III Intestinum Sus domestica gastric mucosa extract and bovine serum albumin/defatted milk powder.
4. detection kit as claimed in claim 3, it is characterised in that described type III Intestinum Sus domestica gastric mucosa carries
Take the specific receptor containing people source norovirus in thing.
5. detection kit as claimed in claim 3, it is characterised in that described detection kit also includes
Enzyme mark strip, enzyme mark frame, polyolefin film, absorbent filter, phosphate buffer, sodium carbonate-bicarbonate buffer
Solution and DEPC water/ddH2O。
6. the application of detection kit as claimed in claim 1, it is characterised in that described application include as
Lower step:
1) type III Intestinum Sus domestica gastric mucosa extract is coated in ELISA Plate；
2) sealase target, prevents ELISA Plate non-specific adsorption virion；
3) testing sample is joined ELISA Plate；
4) Pintsch process release RNA；
5) add the first primer to, the first primer to supporting TaqMan probe, the second primer to, second draw
Supporting TaqMan probe and a step inverse transcription polymerase chain reaction reagent are reacted by thing；
6) RT-qPCR detection is carried out.
Apply the most as claimed in claim 6, it is characterised in that in described step 3) in, detect GI group
The buffer of people source norovirus and the cleaning of enzyme target of GII group people source norovirus and virus dilution is respectively
PH=3.0 and pH=7.0.
Apply the most as claimed in claim 6, it is characterised in that in described step 4) in, Pintsch process is released
The temperature putting RNA is 95 DEG C.
Apply the most as claimed in claim 6, it is characterised in that in described step 6) in, RT-qPCR
The reaction condition of detection is: 42 DEG C of reverse transcription 10min of first stage, 95 DEG C of denaturations 30s of second stage；
95 DEG C of degeneration 15s of phase III, 53 DEG C of annealing 15s, 60 DEG C extend 30s, expand 45 circulations；4℃
Insulation；And carry out FAM fluorescence signal acquisition in the phase III.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108676920A (en) * | 2018-07-07 | 2018-10-19 | 广东省实验动物监测所 | It is a kind of quickly to detect mouse norovirus primer, kit and its RT-RPA methods |
| CN109207641A (en) * | 2018-11-02 | 2019-01-15 | 浙江省疾病预防控制中心 | A kind of multiple RT-PCR detection kit and application |
| CN110592284A (en) * | 2019-10-12 | 2019-12-20 | 福建谷科生物科技有限公司 | Kit for rapidly detecting norovirus |
| CN113186354A (en) * | 2021-05-28 | 2021-07-30 | 中国科学院微生物研究所 | Kit and special primer for detecting GI.1 type norovirus in clinical sample |
| CN113481328A (en) * | 2021-07-23 | 2021-10-08 | 中国科学院微生物研究所 | Kit and special primer for detecting GI.5 type norovirus in clinical sample |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101497928A (en) * | 2009-01-07 | 2009-08-05 | 珠海国际旅行卫生保健中心 | Method and special reagent kit for identifying GG I norovirus and GG II norovirus |
| CN105177185A (en) * | 2015-10-10 | 2015-12-23 | 山东出入境检验检疫局检验检疫技术中心 | Test and quality control kit and test method of Hepatitis A virus and Norovirus in vegetable |
-
2016
- 2016-05-05 CN CN201610293917.4A patent/CN105907889A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101497928A (en) * | 2009-01-07 | 2009-08-05 | 珠海国际旅行卫生保健中心 | Method and special reagent kit for identifying GG I norovirus and GG II norovirus |
| CN105177185A (en) * | 2015-10-10 | 2015-12-23 | 山东出入境检验检疫局检验检疫技术中心 | Test and quality control kit and test method of Hepatitis A virus and Norovirus in vegetable |
Non-Patent Citations (3)
| Title |
|---|
| DAPENG WANG ET AL: "Inactivation conditions for human norovirus measured by an in situ capture-qRT-PCR method", 《 INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY》 * |
| P. TIAN等: "Specificity and kinetics of norovirus binding to magnetic bead-conjugated histo-blood group antigens", 《JOURNAL OF APPLIED MICROBIOLOGY》 * |
| 钱明明等: "常规ＲＴ－ＰＣＲ与荧光定量ＲＴ－ＰＣＲ检测ＧⅠ、ＧⅡ型诺如病毒方法的建立及其应用", 《中国人兽共患病学报》 * |
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| CN113186354A (en) * | 2021-05-28 | 2021-07-30 | 中国科学院微生物研究所 | Kit and special primer for detecting GI.1 type norovirus in clinical sample |
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