CN102776293A - Method for auxiliary identification of rotavirus and special primers used therein - Google Patents
Method for auxiliary identification of rotavirus and special primers used therein Download PDFInfo
- Publication number
- CN102776293A CN102776293A CN2011101196896A CN201110119689A CN102776293A CN 102776293 A CN102776293 A CN 102776293A CN 2011101196896 A CN2011101196896 A CN 2011101196896A CN 201110119689 A CN201110119689 A CN 201110119689A CN 102776293 A CN102776293 A CN 102776293A
- Authority
- CN
- China
- Prior art keywords
- rotavirus
- sequence
- dna
- sequence table
- primer special
- 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.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for auxiliary identification of rotavirus and special primers used therein. The special primers provided by the invention comprise DNAs respectively represented by sequence 1, sequence 2, sequence 3 and sequence 4 in a sequence table. Rotavirus can be identified by using the group of the special primers through reverse transcription loop-mediated isothermal amplification, and therefore, the special primers can be applied to monitoring of rotavirus in a variety of water bodies (sewage, reclaimed water, surface water and drinking water) and excrement of patients (human beings and animals) with intestinal diseases.
Description
Technical field
The present invention relates to a kind of method and primer special thereof of assistant identification rotavirus.
Background technology
Infantile rotavirus enteritis is exactly a kind of virus infectious diarrhoea.According to investigations, this diarrhoea has 80%-90% to be caused by rotavirus.This virus is observed under electron microscope and is spherical shape, and the intermediary housing is outside radial arrangement as the spoke of wheel, the outer rim of the similar wheel of housing of outside, and the form utmost point is named as rotavirus (Rotavirus) as wheel so rise.Rotavirus all has distribution in the whole world, as far back as the thirties just once in the popular mistake of American-European countries, in the Asia, developing countries such as Africa, latin america, be to cause one of infant's main causes of death.China is from the fifties, also takes place popularly once more than 20 provinces and cities, and its popular scope almost spreads all over the whole nation.This virus also once caused in the Baby Room of maternity ward each other and infected, and caused several babies to suffer from diarrhoea, have in addition cause death.According to test, rotavirus is under 50 ℃ high temperature, and 1 hour still can be not dead; Under-20 ℃ severe cold condition, can survive 7 years; Can prolonged preservation in-70 ℃ environment.It also has stronger tolerance to soda acid, and general washing composition has no killing action to it, but irreproducible in external environment.Just because of these characteristics of this virus, just make it in hostile environment, can hide for a long time wait, in case have an opportunity to get into human body, just can breed pathogenic in a large number; Excrete with ight soil then, pollute outside atmosphere.Again infect others.Aunt Zhou Erfu like this, human so far also not finding can effectively be killed this viral active drug.
Ring mediated reverse transcription isothermal amplification technique (LAMP) is that the cyclic permutation amplification that nucleic acid chains is passed through in isothermal environment realizes the amplification to target sequence, and reaches testing goal.Because used two pairs of Auele Specific Primers in the reaction, amplified reaction just can carry out when having only two pairs of primers all to combine with special target sequence, therefore the specificity of this detection method is very high.5 ' end of wherein a pair of special primer contains an end and downstream amplified production complementary sequence; Therefore; This single stranded product to primer has just formed a similar dumbbell shaped palindrome, and this structure just in time provides the template that can be gone round and begun again and utilize for next step isothermal displacement amplified reaction, thus guaranteed amplification continue carry out; Finally, amplified production forms successive scalariform band on nucleic acid electrophoresis glue.Be not difficult to find out from principle and the characteristics of LAMP; This method should have incomparable advantage aspect traditional gene diagnosis in the context of detection of RNA viruses; Compare with some conventional gene test means, this technology has bigger advantage aspect operability and detection time and the equipment requirements.This is mainly reflected in the following aspects: at first, can shorten to its detection time in two hours, sensitivity can reach the viral nucleic acid molecule that detects about 10 copies; Except using some polysaccharases commonly used, hardware device there is not particular requirement basically, just can accomplish this testing at common biology laboratory; Second; This technology has been omitted the secondary amplification step of independent rt and nest-type PRC; Next step is accomplished at constant temperature for reverse transcription and amplification procedure in Experimental Establishment, do not have the change renaturation process of nucleic acid, has not only saved the opportunities for contamination that great amount of time has reduced RNA enzyme and amplification of nucleic acid again; The 3rd, amplified reaction only could carry out under the situation of six land couplings at four primers fully with in the target sequence of identification smoothly, thereby has improved the specificity of reaction greatly; The 4th, can form visual Pyrophosphate phosphohydrolase deposition in the amplified reaction of LAMP, so naked eyes can be observed the white casse thing in the positive reaction pipe; The 5th, also can in the LAMP reaction system, add the HNB dyestuff, can be through observing colour-change indication positive findings.
Summary of the invention
The method and the primer special thereof that the purpose of this invention is to provide a kind of assistant identification rotavirus.
The primer special of assistant identification rotavirus provided by the invention (primer sets compound), for (a) as follows or (b):
(a) primer special of forming by DNA shown in the sequence 4 of DNA shown in the sequence 3 of DNA shown in the sequence 2 of DNA shown in the sequence 1 of sequence table, sequence table, sequence table and sequence table (primer sets compound);
(b) primer special of forming by the reverse complemental DNA of DNA shown in the sequence 4 of the reverse complemental DNA of DNA shown in the sequence 3 of the reverse complemental DNA of DNA shown in the sequence 2 of the reverse complemental DNA of DNA shown in the sequence 1 of sequence table, sequence table, sequence table and sequence table (primer sets compound).
Said primer special can be used for preparing the test kit of assistant identification rotavirus.
The present invention also protects a kind of test kit of assistant identification rotavirus, comprises said primer special (primer sets compound).
Said test kit also can comprise positive plasmid; Said positive plasmid can be the plasmid of DNA shown in the sequence 5 that contains ordered list.Said positive plasmid specifically can be rotavirus vp7 gene partial sequence (Nucleotide as sequence like the sequence 5 of sequence table from shown in 5 ' the terminal 571-834 Nucleotide) is oppositely inserted the recombinant plasmid that the SmaI restriction enzyme site of pGH carrier obtains.
Said primer or said test kit can be used for the assistant identification rotavirus.
The present invention also protects a kind of method of assistant identification rotavirus, comprises the steps:
(1) RNA of extraction virus to be measured;
(2) RNA that step (1) is extracted adopts said primer special (primer sets compound) to encircle the mediated reverse transcription isothermal duplication;
(3) identify according to the amplified production of step (2) whether virus to be measured is rotavirus.
The response procedures of said ring mediated reverse transcription isothermal duplication specifically can be 64-65 ℃ of constant-temperature amplification 1-1.5h.
The amplified production that said step (3) specifically can be ring mediated reverse transcription isothermal duplication carries out agarose gel electrophoresis, identifies according to the amplified band of electrophoresis showed whether virus to be measured is rotavirus.
Said step (3) specifically also can be amplified production with ring mediated reverse transcription isothermal duplication carries out enzyme with restriction enzyme Fok I and cuts evaluation, cuts product according to enzyme and identifies whether virus to be measured is rotavirus.If obtain 280bp, 193bp and three dna fragmentations of 78bp after Fok I enzyme is cut, virus to be measured is candidate's rotavirus.
Said virus to be measured specifically can be human astrovirus virus, rotavirus or norovirus.
Said primer special (primer sets compound) or said test kit can be used for whether containing in the auxiliary detection sample to be tested rotavirus.
Among the present invention; Designed the one group of primer special (primer sets compound) that is used to identify rotavirus; Adopt and to identify rotavirus through ring mediated reverse transcription isothermal amplification technique by group primer special (primer sets compound), and then be applied to the monitoring of rotavirus in various water bodys (sewage, reuse water, surface water, tap water) and enteron aisle patient (people, animal) ight soil.
Use primer special of the present invention and identify rotavirus; Has accurate, special, sensitive, advantage efficiently; Be mainly reflected in the following aspects: at first; Amplified reaction only could carry out under the situation of six land couplings at four primers fully with in the target sequence of identification smoothly, and all these characteristics have reduced the background of amplified reaction to a great extent, thereby detection specificity is improved; Second; This technology has been omitted the secondary amplification step of independent rt and nest-type PRC; Amplified reaction carries out under 65 ℃ of isothermal conditions in addition; The change renaturation process that does not have nucleic acid has not only been saved great amount of time and has been reduced the opportunities for contamination of RNA enzyme and amplification of nucleic acid again, thereby has improved the susceptibility and the security that detect; The 3rd, all amplified reactions carry out under 65 ℃ of left and right sides isothermal conditions, need not the PCR appearance, have reduced the requirement to experiment hardware, help the popularization of basic unit's WQM and health and epidemic prevention etc.; The 4th, shortened the required time (can accomplish in 0.5-2 hour) of detecting than RT-PCR.
Description of drawings
Fig. 1 is the structural representation of rotavirus plasmid.
Fig. 2 is the electrophorogram that adopts the mg ion of different concns among the embodiment 2 in the reaction system.
Fig. 3 is the electrophorogram that adopts the trimethyl-glycine of different concns among the embodiment 2 in the reaction system.
Fig. 4 is the electrophorogram that adopts the differential responses temperature among the embodiment 2.
Fig. 5 is the electrophorogram that adopts the differential responses time among the embodiment 2.
Fig. 6 is the observations of positive control and blank in the step 2 of embodiment 3.
Fig. 7 is the observations of human astrovirus virus, rotavirus and norovirus in the step 2 of embodiment 3.
Fig. 8 is the observations of positive control and blank in (1) of step 3 of embodiment 3.
Fig. 9 is the observations of human astrovirus virus in (1) of step 3 of embodiment 3, rotavirus and norovirus.
Figure 10 is the observations of positive control and blank in (2) of step 3 of embodiment 3.
Figure 11 is the observations of positive control and blank among the embodiment 4.
Figure 12 is the observations of each diluent among the embodiment 5.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.The water that is used as negative control among the embodiment is the RNase-free ddH of day root company
2O.AMV ThermoScript II: Promega, the U.S..Bst-DNA polysaccharase and damping fluid thereof: NEB, Britain.10mM dNTP:Biodee, Beijing.Trimethyl-glycine: Sigma, the U.S..Maker D2000:TIANGEN, Beijing.Restriction enzyme Fok I:NEB, Britain.HNB:Sigma, the U.S..
The design of the primer special of embodiment 1, assistant identification rotavirus (primer sets compound)
One, the design of the primer special of assistant identification rotavirus
Through NCBI, Blast, Primerexplorer V4 program, obtain the Rotavirus gene order and design four primers, two outer primers (rF3, rB3) and two inner primers (rFIP, rBIP).Article four, the sequence of primer is following:
RF3:5 '-GCTACATTTTCTCTTGGACCTAA-3 ' (sequence 1 of sequence table);
RB3:5 '-ATGGGATCATCATGTACCG-3 ' (sequence 2 of sequence table);
RFIP:5 '-GGATGTCGTTGATGGGATAAATCAT-CAATTTCGAATAGTACATGTCGTA-3 ' (sequence 3 of sequence table);
RBIP:5 '-ATGAGTCTACGTTTGTTGTTTGACA-TGAAAGTGTGTCCGCTAA-3 ' (sequence 4 of sequence table).
Because the long-chain DNA that the LAMP amplified production connects for ring, different amplified fragments are difficult to make evaluation through electrophoretogram judgement size.Among the present invention,, after amplified reaction finishes, can adopt the method for digestion with restriction enzyme, realize the specificity length detection of amplified production preferably through in amplimer, introducing restriction enzyme site.
Two, the preparation of positive control (rotavirus plasmid)
The rotavirus plasmid is that rotavirus vp7 gene partial sequence (the vp7 gene shown in the sequence 5 of sequence table is from 5 ' terminal 571-834 Nucleotide) is oppositely inserted pGH carrier (Beijing AudioCodes biotechnology Ltd; Plasmid is numbered D9433-1) the recombinant plasmid that obtains of SmaI restriction enzyme site.The structural representation of rotavirus plasmid is seen Fig. 1 (said rotavirus vp7 gene partial sequence is a recombinant plasmid 997-1260 Nucleotide).
Three, the preparation of the test kit of assistant identification rotavirus
The test kit of assistant identification rotavirus is by four primers (rF3, rB3, rFIP and rBIP of step 1 design; Synthetic by Beijing AudioCodes biotechnology Ltd) with the rotavirus plasmid composition of step 2.
The optimization of embodiment 2, reaction conditions
Rotavirus plasmid with embodiment 1 preparation is a template, and the LAMP reaction conditions of four primers (rF3, rB3, rFIP and rBIP) of embodiment 1 design is optimized.
Adopt the electrophorogram of the mg ion of different concns to see Fig. 2 in the reaction system.Among Fig. 2, M represents Marker D2000, and KB represents blank (is template with water), and 0 to represent the magnesium ion concentration in the reaction system be 0mmolL
-1, 1 to represent the magnesium ion concentration in the reaction system be 1mmolL
-1, 2 to represent the magnesium ion concentration in the reaction system be 2mmolL
-1, 3 to represent the magnesium ion concentration in the reaction system be 3mmolL
-1, 4 to represent the magnesium ion concentration in the reaction system be 4mmolL
-1, 5 to represent the magnesium ion concentration in the reaction system be 5mmolL
-1, 6 to represent the magnesium ion concentration in the reaction system be 6mmolL
-1, 7 to represent the magnesium ion concentration in the reaction system be 7mmolL
-1Mg ion is very big to the influence of reaction, and low more its reaction efficiency of concentration is low more, and high more its reaction efficiency of concentration is high more, but atopic is poor more, and best magnesium ion concentration is 3mmol/L.
Adopt the electrophorogram of the trimethyl-glycine of different concns to see Fig. 3 in the reaction system.Among Fig. 3, M represents Marker D2000, and KB represents blank (is template with water), and 0 to represent the trimethyl-glycine concentration in the reaction system be 0mmolL
-1, 1 to represent the trimethyl-glycine concentration in the reaction system be 1mmolL
-1, 2 to represent the trimethyl-glycine concentration in the reaction system be 2mmolL
-1, 3 to represent the trimethyl-glycine concentration in the reaction system be 3mmolL
-1, 4 to represent the trimethyl-glycine concentration in the reaction system be 4mmolL
-1Under the situation of not adding trimethyl-glycine, the specific reaction band still occurs, but efficient is lower, when trimethyl-glycine concentration surpasses 2mmol/L, band thickens, and shows nonspecific reaction to have occurred, and best trimethyl-glycine concentration is 1mol/L.
Adopt the electrophorogram of different temperature of reaction to see Fig. 4.Among Fig. 4, M represents Marker D2000, and KB represents blank (is template with water); 60 ℃ temperature of reaction is adopted in 1 representative, and 61 ℃ temperature of reaction is adopted in 2 representatives, and 62 ℃ temperature of reaction is adopted in 3 representatives; 63 ℃ temperature of reaction is adopted in 4 representatives; 64 ℃ temperature of reaction is adopted in 5 representatives, and 65 ℃ temperature of reaction is adopted in 6 representatives, and 66 ℃ temperature of reaction is adopted in 7 representatives.Temperature is bigger to the temperature effect of reaction, but specific amplification is all arranged between 62-66 ℃, and 64 ℃, 65 ℃ of bands are more clear bright.
Adopt the electrophorogram in different reaction times to see Fig. 5.Among Fig. 5, M represents Marker D2000, and KB represents blank (is template with water); The reaction times that 30min is adopted in 1 representative, the reaction times that 45min is adopted in 2 representatives, the reaction times that 60min is adopted in 3 representatives; The reaction times that 75min is adopted in 4 representatives; The reaction times that 90min is adopted in 5 representatives, the reaction times that 105min is adopted in 6 representatives, the reaction times that 120min is adopted in 7 representatives.In theory the minimum reaction times of optimum reacting time for increasing and can accomplishing, to carry out in order reacting fully, to prevent that false positive from occurring, optimum reacting time is 90min.
The specificity of embodiment 3, test kit (primer special)
Experiment sample is human astrovirus virus, rotavirus and the norovirus that extracted the diarrhea patient from CDC (volunteer of the informed consent) ight soil in 2007, and the public can obtain from Ecological Environment Research Center, Chinese Academy of Sciences.Human astrovirus virus (astrovirus): reference: Liu C; Grillner L; Jonsson K et al.Identification of viral agents associated with diarrhea in young children during a winter season in Beijing; China.J Clin Virol.2006,35:69-72.Rotavirus (rotavirus): reference: He, X.Q.; Cheng, L.; Zhang, D.Y.; Li, W.; Xie, X.M.; Ma, M.; Wang; Z.J.First Molecular Detection of Group A Rotaviruses in Drinking Water Sources in Beijing; China.Bulletin of Environmental Contamination and Toxicology.2009,83:120-124.Norovirus (norovirus): reference: Liu C; Grillner L; Jonsson K et al.Identification of viral agentsassociated with diarrhea in young children during a winter season in Beijing; China.J Clin Virol.2006,35:69-72.
Adopt the test kit of embodiment 1 preparation respectively each experiment sample to be identified that step is following:
1, adopt day virus genome RNA of root company/DNA extraction test kit to extract the RNA of sample to be tested.
2, adopt four primers (rF3, rB3, rFIP and rBIP) to encircle mediated reverse transcription isothermal duplication (RT-LAMP) RNA that extracts.
RT-LAMP system (25 μ L) is as follows: rF3 and rB3 are 0.2 μ molL
-1, rBIP and rFIP are 1.6 μ molL
-1, dNTP 1mmol/L, Betaine (trimethyl-glycine) 1mol/L, MgSO
43mmol/L, 2.5 μ L, 10 * Bst-DNA Polymerase Buffer, 8U Bst-DNA polymerase, 10U AMV ThermoScript II, 2 μ L AMV5 * buffer, 5 μ L RNA samples, all the other are water.
RT-LAMP system mixing, 65 ℃ of constant-temperature amplification 1.5h.
Set up positive control and blank simultaneously: promptly use 5 μ L rotavirus plasmids (1.27 * 10
11Copy/5 μ L) replaces the RNA sample as positive control, replace the RNA sample as blank with 5 μ L water.
Positive control and positive sample can form visual Pyrophosphate phosphohydrolase deposition in amplified reaction, centrifugal back naked eyes can be observed the white casse thing in the reaction tubes.Blank and negatives can not obtain the white casse thing.The result of blank and positive control sees Fig. 6 (KB represents blank, and 1 represents positive control).The result of human astrovirus virus, rotavirus and norovirus sees Fig. 7 (KB represents blank, and 1 represents rotavirus, and 2 represent human Astrovirus, and 3 represent norovirus).Rotavirus is the same with positive control to have the white casse thing.Human astrovirus is viral, norovirus is the same with blank, does not obtain the white casse thing.
3, the amplified production of detection ring mediated reverse transcription isothermal duplication
In order further macroscopic judged result in the step 2 to be confirmed, operate as follows:
(1) after ring mediated reverse transcription isothermal duplication is accomplished, amplified production is carried out 1.5% agarose gel electrophoresis.
The result of blank and positive control sees Fig. 8 (M represents Marker D2000, and KB represents blank, and 1 represents positive control).The result of human astrovirus virus, rotavirus and norovirus sees Fig. 9 (M represents MarkerD2000, and KB represents blank, and 1 represents rotavirus, and 2 represent human Astrovirus, and 3 represent norovirus).Rotavirus is the same with positive control, and amplification obtains characteristic ring mediated isothermal amplification product (stepped band).Human astrovirus is viral, norovirus is the same with blank, does not obtain characteristic ring mediated isothermal amplification product (stepped band).
(2) after ring mediated reverse transcription isothermal duplication is accomplished, amplified production is carried out enzyme with restriction enzyme Fok I cut evaluation.20 μ L endonuclease reaction systems: 5 μ L amplified productions, 2 μ L Buffer, 4,1 μ L Fok I restriction endonucleases add sterilized water to 20 μ; 37 ℃ of reaction 3h, 65 ℃, the 20min termination reaction.Enzyme is cut product carry out 1.5% agarose gel electrophoresis, and reclaim special band and check order.
The electrophoresis result of blank and positive control is seen Figure 10 (M represents Marker D2000, and KB represents blank, and 1 represents positive control).Rotavirus is the same with positive control, shows below special banding pattern: 280bp, 193bp and three master tapes of 78bp and indivedual assorted band.Human astrovirus is viral, norovirus is the same with blank, does not show not obtain endonuclease bamhi.
The specificity of embodiment 4, test kit (primer special)
Experiment sample is with embodiment 3.
Adopt the test kit of embodiment 1 preparation respectively each experiment sample to be identified that step is following:
1, adopt day virus genome RNA of root company/DNA extraction test kit to extract the RNA of sample to be tested.
2, adopt four primers (rF3, rB3, rFIP and rBIP) to encircle mediated reverse transcription isothermal duplication (RT-LAMP) RNA that extracts.
RT-LAMP system (25 μ L) is as follows: rF3 and rB3 are 0.2 μ molL
-1, rBIP and rFIP are 1.6 μ molL
-1, dNTP lmmol/L, Betaine (trimethyl-glycine) 1mol/L, MgSO
43mmol/L, 2.5 μ L10 * Bst-DNA Polymerase Buffer, 8U Bst-DNA polymerase, 10U AMV ThermoScript II, 2 μ L AMV, 5 * buffer, 5 μ L RNA samples, 2 μ L (1.5mmol/L) HNB dyestuffs (hydroxynaphthol blue), all the other are water.
RT-LAMP system mixing, 65 ℃, water-bath 1.5h.
Set up positive control and blank simultaneously: promptly use 5 μ L rotavirus plasmids (1.27 * 10
11Copy/5 μ L) replaces the RNA sample as positive control, replace the RNA sample as blank with 5 μ L water.
The result of blank and positive control sees Figure 11 (KB represents blank, and 1 represents positive control).Rotavirus is the same with positive control, can be observed colour-change (sky blue when being become reaction and finished by the purple of reaction when initial).Human astrovirus is viral, norovirus is the same with blank, does not have colour-change (reaction of reaction initial sum is purple when finishing).
The sensitivity of embodiment 5, test kit (primer special)
Adopt the test kit of embodiment 1 preparation to carry out sensitivity determination, step is following:
1, water (the RNase-free ddH of day root company
2O) be each concentration in the table 1 with rotavirus plasmid gradient dilution.
The concentration of rotavirus plasmid in each diluent of table 1
2, the diluent with each numbering adopts four primers (rF3, rB3, rFIP and rBIP) to carry out ring mediated isothermal amplification (LAMP).
LAMP system (25 μ L) is as follows: rF3 and rB3 are 0.2 μ molL
-1, rBIP and rFIP are 1.6 μ molL
-1, dNTP 1mmol/L, Betaine (trimethyl-glycine) 1mol/L, MgSO
43mmol/L, 2.5 μ L, 10 * Bst-DNA Polymerase Buffer, 8U Bst-DNA polymerase, 5 μ L RNA samples, 2 μ L AMV5 * buffer, 2 μ L (1.5mmol/L) HNB dyestuffs (hydroxynaphthol blue), all the other are water.
LAMP system mixing, 65 ℃, water-bath 1.5h.
After ring mediated reverse transcription isothermal duplication is accomplished, amplified production is carried out 1.5% agarose gel electrophoresis.
Electrophorogram is seen Figure 12.Among Figure 12: M:Marker D2000; From left to right, the 1-11 numbering in the correspondence table 1 (first row) successively.Numbering 1 to 10 diluent all can increase obtain various different sizes amplified fragments.The RT-LAMP reaction can obviously detect the rotavirus gene that copy number concentration is 5.08copy/ μ L, explains that its detection sensitivity can reach more than 10 rotavirus copy numbers, is higher than general regular-PCR and detects.
Claims (10)
1. the primer special of assistant identification rotavirus, for (a) as follows or (b):
(a) primer special of forming by DNA shown in the sequence 4 of DNA shown in the sequence 3 of DNA shown in the sequence 2 of DNA shown in the sequence 1 of sequence table, sequence table, sequence table and sequence table;
(b) primer special of forming by the reverse complemental DNA of DNA shown in the sequence 4 of the reverse complemental DNA of DNA shown in the sequence 3 of the reverse complemental DNA of DNA shown in the sequence 2 of the reverse complemental DNA of DNA shown in the sequence 1 of sequence table, sequence table, sequence table and sequence table.
2. the application of the said primer special of claim 1 in the test kit of preparation assistant identification rotavirus.
3. the test kit of an assistant identification rotavirus comprises the said primer special of claim 1.
4. said primer special of claim 1 or the application of the said test kit of claim 3 in the assistant identification rotavirus.
5. the method for an assistant identification rotavirus comprises the steps:
(1) RNA of extraction virus to be measured;
(2) RNA that step (1) is extracted adopts the said primer special of claim 1 to encircle the mediated reverse transcription isothermal duplication;
(3) identify according to the amplified production of step (2) whether virus to be measured is rotavirus.
6. method as claimed in claim 5 is characterized in that: the response procedures of said ring mediated reverse transcription isothermal duplication is 64 ℃ of-65 ℃ of water-bath 1-1.5h.
7. like claim 5 or 6 described methods, it is characterized in that: said step (3) is carried out agarose gel electrophoresis for the amplified production that will encircle the mediated reverse transcription isothermal duplication, identifies according to the amplified band of electrophoresis showed whether virus to be measured is rotavirus.
8. like claim 5 or 6 described methods, it is characterized in that: said step (3) is cut evaluation for the amplified production that will encircle the mediated reverse transcription isothermal duplication carries out enzyme with restriction enzyme Fok I, cuts product according to enzyme and identifies whether virus to be measured is rotavirus.
9. like arbitrary described method in the claim 5 to 8, it is characterized in that: said virus to be measured is human Astrovirus, rotavirus or norovirus.
10. whether said primer special of claim 1 or the said test kit of claim 3 contain the application in the rotavirus in the auxiliary detection sample to be tested.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101196896A CN102776293A (en) | 2011-05-10 | 2011-05-10 | Method for auxiliary identification of rotavirus and special primers used therein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101196896A CN102776293A (en) | 2011-05-10 | 2011-05-10 | Method for auxiliary identification of rotavirus and special primers used therein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102776293A true CN102776293A (en) | 2012-11-14 |
Family
ID=47121395
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011101196896A Pending CN102776293A (en) | 2011-05-10 | 2011-05-10 | Method for auxiliary identification of rotavirus and special primers used therein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102776293A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101153342A (en) * | 2007-09-21 | 2008-04-02 | 珠海市疾病预防控制中心 | Primer, detection method and detection reagent kit for detection of group A type G3 rotavirus |
| CN101570798A (en) * | 2009-03-20 | 2009-11-04 | 陈颖 | Detection kit and detection method for 3 species of food-borne viruses in marine products |
| CN101671746A (en) * | 2009-10-21 | 2010-03-17 | 中华人民共和国北京出入境检验检疫局 | Kit and oligonucleotide sequences for detecting rotavirus A |
-
2011
- 2011-05-10 CN CN2011101196896A patent/CN102776293A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101153342A (en) * | 2007-09-21 | 2008-04-02 | 珠海市疾病预防控制中心 | Primer, detection method and detection reagent kit for detection of group A type G3 rotavirus |
| CN101570798A (en) * | 2009-03-20 | 2009-11-04 | 陈颖 | Detection kit and detection method for 3 species of food-borne viruses in marine products |
| CN101671746A (en) * | 2009-10-21 | 2010-03-17 | 中华人民共和国北京出入境检验检疫局 | Kit and oligonucleotide sequences for detecting rotavirus A |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | Development of a loop-mediated isothermal amplification assay for rapid detection of subgroup J avian leukosis virus | |
| Almasi et al. | Development of colorimetric loop-mediated isothermal amplification assay for rapid detection of the tomato yellow leaf curl virus | |
| Pallister et al. | Development of real‐time PCR assays for the detection and differentiation of Australian and European ranaviruses | |
| KR20180052317A (en) | Primer set for detection of MERS-coronavirus and uses thereof | |
| WO2022257663A1 (en) | Method and kit for detecting and screening n501y mutation in covid-19 | |
| CN106916906A (en) | A kind of Primer composition and its kit for detecting infectious diarrhea pathogen | |
| KR20150145771A (en) | Primer set for multiple detection lily viruses and method for detecting said viruses using the same | |
| CN110578019A (en) | Detection kit for distinguishing newcastle disease virulent strains and attenuated strains by double fluorescence LAMP (loop-mediated isothermal amplification) and primer group thereof | |
| CN102534050B (en) | Triple polymerase chain reaction (PCR) detecting primer group, reagent kit and method for diagnosing grass carp reovirus | |
| Lin et al. | The development and application of a duplex reverse transcription loop-mediated isothermal amplification assay combined with a lateral flow dipstick method for Macrobrachium rosenbergii nodavirus and extra small virus isolated in China | |
| CN104450961A (en) | Kit for detecting grass carp reovirus types I, II and III based on RT-LAMP visualization technology and method for detecting grass carp reovirus types I, II and III | |
| CN102296128B (en) | Reverse transcription-loop mediated isothermal amplification (RT-LAMP) quick detection method of citrus tristeza viruses (CTV) | |
| Khan et al. | Rapid detection of infectious bursal disease by loop-mediated isothermal amplification for field analysis | |
| CN106987657B (en) | Primer combination for identifying bovine virus diarrhea virus and bovine rotavirus and application thereof | |
| CN104099430A (en) | Ring mediate isothermal amplification kit for diagnosing H7N9 hypotype avian influenza virus and application method thereof | |
| CN101586168B (en) | Primer set for detecting J subgroup fowl leukemia virus gene, detection method and rapid detection kit | |
| CN106939357A (en) | H4 hypotypes, H6 hypotypes and the triple RT PCR primer combinations of H9 hypotypes AIV, kit and its application | |
| CN108060271B (en) | Loop-mediated isothermal amplification dengue virus detection method | |
| CN102776293A (en) | Method for auxiliary identification of rotavirus and special primers used therein | |
| CN116287441A (en) | Normal temperature monkey pox virus detection typing kit | |
| KR20180125141A (en) | Primer set for detection of MERS-coronavirus and uses thereof | |
| CN114032336A (en) | Method and kit for detecting cucumber mosaic virus | |
| CN102465184A (en) | Method for auxiliary identification of human astrovirus and special primers thereof | |
| Almasi et al. | Immunocapture loop mediated isothermal amplification for rapid detection of tomato yellow leaf curl virus (TYLCV) without DNA extraction | |
| Kou et al. | Development and application of a loop-mediated isothermal amplification assay on rapid and sensitive detection of rotavirus in fecal samples and artificially seeded oysters |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20121114 |