CN101153342B - Primer group and detection reagent kit for detection of group A type G3 rotavirus - Google Patents
Primer group and detection reagent kit for detection of group A type G3 rotavirus Download PDFInfo
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Abstract
The present invention relates to a fast analytical technique on foodborn pathogens based on loop-mediated isothermal amplification (LAMP), and provides a primer (or primers) which is (are) used for analysis on G3 of group A of rotavirus, and capable of amplifying the specific base sequence of the target gene, which is the VP7 gene (accession number of GenBank: D86272) of G3 of group A of rotavirus, and the primer(s) is (are) complemented with a partial sequence on the sites from 307 to 505 of the target gene, or with the complementary chain of the partial sequence. The present invention provides a group of primers or a primer with specificity on the specific gene segments of G3 of group A of rotavirus, with the utilization of the kit comprising the group of primers to analyze whether a specific gene segment of G3 of group A of rotavirus exists in the sample under analysis, thereby identifying whether G3 of group A of rotavirus exists in the sample.
Description
Technical field
The present invention relates to a kind of based on ring mediated isothermal amplification (loop-mediated isothermalamplification, LAMP) the food-borne causal agent Fast Detection Technique of technology.Be particularly related to group A type G 3 rotavirus specific gene fragment is had specific primer and primer sets; Also relate to and use the ring mediated isothermal amplification method to detect the detection method and the detection kit of group A type G 3 rotavirus described primer and primer sets.
Background technology
The food origin disease sickness rate is higher, by Salmonellas, Shigellae, streptococcus aureus, Bacillus proteus, vibrio cholerae, Vibrio parahemolyticus and E.coli O157:H7, the food poisoning that rotavirus and norovirus cause, its sickness rate accounts for very high ratio in China's food origin disease sickness rate, they be a serious public health problem, and the food poisoning that rotavirus causes is most for due to the G3 type.
Detection to food-borne causal agent at present mainly relies on pathogen isolation method, immunological method and various PCR method.Though pathogen isolation is a gold standard, loaded down with trivial details time-consuming, generally need 5 day time, the longlyest need one month, and the specificity of immunological method and susceptibility are all lower.
Along with the development of Protocols in Molecular Biology, people adopt round pcr to be applied to the diagnosis of bacterium.The for example patent application of PCR Chinese patent publication number CN1526825 has disclosed a kind of specificity of utilizing Vibrio parahemolyticus PR72H sequence, detects the method for Vibrio parahemolyticus by DNA extraction, pcr amplification, electrophoresis observation equimolecular biological means.Though this method sensitivity, accurately, fast, owing to need the plant and instrument of costliness, higher testing cost and make it not be suitable for field quick detection and basic unit's popularization and application the higher technical requirements of testing staff.
Ring mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) technology (International Patent Publication No. WO 00/28082) is that Notomi in 2000 etc. develop a kind of nucleic acid amplification new technology, promptly at 4 special primers of 6 zone design of target gene (can also be added with ring primer to) if needed, utilize a kind of strand displacement archaeal dna polymerase (Bst DNA polymerase) to be incubated about 60 minutes at 65 ℃ of left and right sides constant temperatures, can finish nucleic acid amplification reaction, amplification can directly be judged by naked eyes amplification by product magnesium pyrophosphate precipitation or its turbidity be detected, also available fluorescence dye SYBR Green I dyeing in conjunction with double-stranded DNA is judged by naked eyes.2 pairs of primers that are used for LAMP technology amplification are at 6 sections of gene, thereby have the specificity higher than PCR, and possessing does not simultaneously need in thermal cycling, its unit time amplification efficiency higher and do not need advantage such as specific apparatus yet.But detection method and the detection kit of not utilizing the ring mediated isothermal amplification method to detect rotavirus A group G3 type are arranged at present, and at the report of segmental LAMP primer of group A type G 3 rotavirus specific gene and primer sets.
Summary of the invention
One object of the present invention is, provides a kind of group A type G 3 rotavirus specific gene fragment is had specific primer sets.
Above-mentioned purpose is achieved by the following technical solution:
A kind of group A type G 3 rotavirus detects and uses primer sets, it is characterized in that described primer sets is made up of following primer:
Forward outer primer F3-G3 ACTGAAGCAGCAACAGAA
Reverse outer primer B3-G3 ACATGTCCAGTTGCAGTG
Forward inner primer FIP-G3
The reverse inner primer BIP-G3 of AGATCCTGTTGGCCATCCTTTAATAATTCATGGAAGGATACACTTTC
Can increase 307 of VP7 (D86272) gene order of group A type G 3 rotavirus of TATTGCCTCGTTTTCAGTTGATCCGCGTCGTATTTCATTAATACCAA---505
The part of nucleotide sequence or its complementary strand.
Another object of the present invention is, a kind of detection kit of utilizing above-mentioned primer or primer sets to detect group A type G 3 rotavirus based on the ring mediated isothermal amplification method is provided.
Above-mentioned purpose is achieved by the following technical solution:
A kind of group A type G 3 rotavirus detects and uses test kit, it is characterized in that described test kit comprises amplification reaction solution, the enzyme that contains above-mentioned primer or primer sets at least.
Comprise following reagent in the described amplification reaction solution:
Wherein 10 * Bst DNA Polymerase Buffer reaction buffer contains trihydroxy-methionine(Met) methane-hydrochloric acid (Tris-HCl), 100mM Repone K, 100mM ammonium sulfate, 20mM sal epsom and 1% triton x-100 of 200mM pH 8.8;
Described enzyme comprises that every microlitre contains the Bst archaeal dna polymerase of 8 activity units and the AMV ThermoScript II that every microlitre contains 20 activity units.
Described test kit also comprises feminine gender and positive control template, and described negative control template is a distilled water; Described positive control template is a group A type G 3 rotavirus genomic dna (1～100nM).
The present invention has specific primer or primer sets by providing a kind of to group A type G 3 rotavirus specific gene fragment, and detect in the sample whether have group A type G 3 rotavirus specific gene fragment, and then whether there is group A type G 3 rotavirus in definite sample with the test kit that includes above-mentioned primer sets.Detection reagent of the present invention and detection method have susceptibility height, high specificity, convenient and swift, do not need specific apparatus, advantage such as applied widely, can solve field quick detection and basic unit's popularization and application difficult problem of food-borne causal agent; Particularly on-the-spot detect and wartime field application.Simultaneously, compare with existing P CR method have high specificity, susceptibility than PCR high or quite, than PCR save time about 2 hours, do not need specific apparatus advantages such as (amplified reaction can be finished) in water-bath.
Description of drawings
Fig. 1 combination of primers of the present invention is carried out ring mediated isothermal amplification (LAMP) back by macroscopic result.Legend: 1, positive amplification; 2, negative amplification.
Fig. 2 combination of primers of the present invention is carried out ring mediated isothermal amplification (LAMP) back and is added the result that dyestuff is observed.Legend: 1, negative amplification; 2,3, positive amplification.
Fig. 3 combination of primers of the present invention is carried out ring mediated isothermal amplification (LAMP) result's electrophorogram;
Legend: 1:100bp marker; 2: the amplified production enzyme is cut evaluation; 3-4: group A type G 3 rotavirus (standard strain); 5-7: be respectively norovirus G II and detect sample, rotavirus A group G1 and rotavirus B group strain (standard strain) contrast, 8 negative contrasts for detecting virus.
Embodiment
The present invention will be described below by concrete group A type G 3 rotavirus testing process, but the present invention is not limited to these embodiment.
The amplification of a pair of known colyliform virus of A group G3VP7 gene of embodiment
One) design of primer sets
By consulting document and filtering out the 307---505bp nucleotide sequence of group A type G 3 rotavirus specific gene VP7 with the BLAST software analysis, design LAMP primer and synthetic at these segmental six sites (these six sites difference: 307-324bp, 332-356bp, 372-393bp, 417-440bp, 463-485bp, 488-505bp), obtain following primer; Design of primers is finished by LAMP primer special design software binding molecule biological analysis software Advance Vector NTI.
Sequence numbering 1
Forward outer primer F3G3 ACTGAAGCAGCAACAGAA
Sequence numbering 2
Reverse outer primer B3-G3 ACATGTCCAGTTGCAGTG
Forward inner primer FIP-G3
AGATCCTGTTGGCCATCCTTTAATAATTCATGGAAGGATACACTTTC
Sequence numbering 4
Reverse inner primer BIP-G3
The sequence in described 6 sites of TATTGCCTCGTTTTCAGTTGATCCGCGTCGTATTTCATTAATACCAA is respectively:
307-324bp：act?gaagcagcaacagaa
332-356bp ataattcatggaaggatacactttc
372-393bp taaaggatggccaacaggatct
417-440bp tattgcctcgttttcagttgatcc
463-485bp ttggtattaatgaaatacgacgc
488-505bp cactgcaactggacatgt
Wherein,
Described forward outer primer F3: amplification starts from 307-324bp (actgaagcagcaacagaa);
Described forward inner primer FIP: amplification starts from complementary sequence and the 332-356bp (ataattcatggaaggatacactttc) of 372-393bp (taaaggatggccaacaggatct);
Described reverse outer primer B3: amplification starts from the complementary sequence of 488-505bp (cac tgcaactgga catgt);
Reverse inner primer BIP: amplification starts from the complementary sequence of 417-440bp (tatt gcctcgtttt cagttgatcc) and 463-485bp (ttggtatt aatgaaatac gacgc).
The general character of each primer is in the above-mentioned primer sets: with the 307--505bp nucleic acid array complementation of group A type G 3 rotavirus specific gene VP7.
Two) strain of present embodiment experiment and the amplification such as the following table one of each strain
Table one (for the ease of understanding, the applicant with number among following sequence number and Fig. 1 be arranged to consistent)
| Sequence number | Molecular weight standard product and amplified production | The result |
| 1 | 100bp?marker | |
| 2 | Positive amplified production enzyme is cut evaluation | Meet |
| 3 | Group |
Sun |
| 4 | Group |
Sun |
| 5 | Norwalk virus G II type sample (through the RT-PCR checking) | Cloudy |
| 6 | Rotavirus A organizes G1 type (Wa type strain) | Cloudy |
| 7 | Rotavirus B organizes (CAL-34 type strain) | Cloudy |
| 8 | Negative control | Cloudy |
Above-mentioned partly is standard strain for the prelibation strain, available from China typical culture collection center; Part is preserved by the applicant for detecting strain sample (through the checking of Shenzhen Mrs's gene by fluorescence PCR test kit).
The no amplified reaction of " the moon " expression, " sun " expression has amplified reaction.
Three) extraction of above-mentioned each strain gene RNA
Get cell culture supernatant, ight soil, diarrhoea thing and dilute with an amount of physiological saline, the centrifuging and taking supernatant liquor is used for RNA extracting (QIAamp Viral RNAMini Kit).
Four) based on the gene amplification of LAMP method
Get amplification reaction solution 14.6 μ l, add 2.0 μ l template to be checked, add the ThermoScript II of Bst archaeal dna polymerase and the 0.5 μ l of 1 μ l, add 6.9 μ l ddH again
2O, forming as the following table cumulative volume is the reaction system of 25 μ l, is that 65 ℃ thermostat water bath is incubated about 90 minutes in temperature, places 80 ℃, 2 minutes inactivators then.
Reaction system is: (the reaction cumulative volume is 25 μ l)
Except that nucleic acid-templated, above-mentioned reaction system can be reduced to amplification reaction solution, enzyme and distilled water.
Amplification reaction solution: reverse outer primer B3-G3,1.4mM dNTP and the 1M trimethyl-glycine (betaine) of forward outer primer F3-G3,0.2 μ M that comprises 10 * Bst DNAPolymerase Buffer reaction buffer, 3.6mM sal epsom, 1.6 μ M forward inner primer FIP-G3, the reverse inner primer BIP-G3 of 1.6 μ M, the 0.2 μ M of 1/10 predetermined reaction volume;
Wherein 10 * Bst DNA Polymerase Buffer reaction buffer contains trihydroxy-methionine(Met) methane-hydrochloric acid (Tris-HCl), 100mM Repone K, 100mM ammonium sulfate, 20mM sal epsom and 1% triton x-100 of 200mM pH 8.8;
Enzyme: every microlitre contains the Bst archaeal dna polymerase of 8 activity units, final concentration 0.32U/ μ l and the AMV ThermoScript II that every microlitre contains 20 activity units, final concentration 0.4U/ μ l.
The adding principle of distilled water is after the amount of other reagent is determined, to add distilled water and make reaction volume arrive predetermined reaction volume.
Five) detection of amplified production
Along with the carrying out of amplified reaction, the magnesium ion that exists pyrophosphate ion of separating out from dNTP and the reaction solution will form the magnesium pyrophosphate precipitation.Therefore, only just white opacity can appear in the reaction solution that nucleic acid amplification takes place.Can judge in the following way like this.
A) naked eyes detect: show relatively that with the negative control pipe detector tube occurs obviously muddy positive, do not see muddy negative; (referring to Fig. 1) or,
B) detect after adding dyestuff: the reaction tubes of per 25 μ l systems adds 1000 * SYBR Green I (invitrogen) 1-2 μ l, 1-5 minute observations, and it is green positive that reaction solution turns, and keeps colourless or brown negative; (referring to Fig. 2) or,
C) electrophoresis detection: the 2-3% sepharose, the about 60-100 of 70V electrophoresis minute, the electrophoresis picture showed LAMP characteristic scalariform band, and minimal segment is about 160bp, and then the result is positive; As then the result is negative not have any band.(referring to Fig. 3)
Through detection to amplified production, show that above-mentioned primer sets only the LAMP amplified reaction occurs to the group A type G 3 rotavirus strain, amplified reaction does not appear in the contrast strain, has shown good specificity.
6) remolding sensitivity is:
, carrying RNA reverse transcription is become to do a series of dilutions behind the cDNA: 10 as detecting bacterium with group A type G 3 rotavirus
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, use LAMP method and PCR method (the upstream and downstream primer is respectively F3-G II, B3-G II) to detect respectively, detected result shows that the LAMP reaction sensibility reaches 10
-4The PCR reaction sensibility reaches 10
-3The result shows that the LAMP reaction sensibility is than responsive 10 times of PCR reaction.
Embodiment two
The difference of present embodiment and embodiment one is that in the present embodiment, the reaction system of using based on the gene amplification of LAMP method is:
Reaction system is: (the reaction cumulative volume is 25 μ l)
Except that nucleic acid-templated, above-mentioned reaction system can be reduced to amplification reaction solution, enzyme and distilled water.
Amplification reaction solution: reverse outer primer B3-G3,1.0mM dNTP and the 0.8M trimethyl-glycine (betaine) of forward outer primer F3-G3,0.15 μ M that comprises 10 * Bst DNA Polymerase Buffer reaction buffer, 2mM sal epsom, 1.0 μ M forward inner primer FIP-G3, the reverse inner primer BIP-G3 of 1.0 μ M, the 0.15 μ M of 1/10 predetermined reaction volume;
Wherein 10 * Bst DNA Polymerase Buffer reaction buffer contains trihydroxy-methionine(Met) methane-hydrochloric acid (Tris-HCl), 100mM Repone K, 100mM ammonium sulfate, 20mM sal epsom and 1% triton x-100 of 200mMpH 8.8;
Enzyme: every microlitre contains the Bst archaeal dna polymerase of 8 activity units and the AMV ThermoScript II that every microlitre contains 20 activity units.
The adding principle of distilled water is after the amount of other reagent is determined, to add distilled water and make reaction volume arrive predetermined reaction volume.
Reaction conditions: in temperature is that 60 ℃ thermostat water bath is incubated about 90 minutes, places 80 ℃, 2 minutes inactivators then.
Embodiment three
The difference of present embodiment and embodiment one is that in the present embodiment, the reaction system of using based on the gene amplification of LAMP method is:
Reaction system is: (the reaction cumulative volume is 25 μ l)
Except that nucleic acid-templated, above-mentioned reaction system can be reduced to amplification reaction solution, enzyme and distilled water.
Amplification reaction solution A: reverse outer primer B3-G3,1.6mM dNTP and the 1.5M trimethyl-glycine (betaine) of forward outer primer F3-G3,0.3 μ M that comprises 10 * Bst DNA PolymeraseBuffer reaction buffer, 6mM sal epsom, 2.0 μ M forward inner primer FIP-G3, the reverse inner primer BIP-G3 of 2.0 μ M, the 0.3 μ M of 1/10 predetermined reaction volume;
Wherein 10 * Bst DNA Polymerase Buffer reaction buffer contains trihydroxy-methionine(Met) methane-hydrochloric acid (Tris-HCl), 100mM Repone K, 100mM ammonium sulfate, 20mM sal epsom and 1% triton x-100 of 200mM pH 8.8;
Enzyme: every microlitre contains the Bst archaeal dna polymerase of 16 activity units and the AMV ThermoScript II that every microlitre contains 20 activity units.
The adding principle of distilled water is after the amount of other reagent is determined, to add distilled water and make reaction volume arrive predetermined reaction volume.
Reaction conditions: in temperature is that 65 ℃ thermostat water bath is incubated about 90 minutes, is warming up to 80 ℃, 2 minutes inactivators then.
Embodiment four
The difference of present embodiment and embodiment one is that in the present embodiment, the reaction system of using based on the gene amplification of LAMP method is:
Reaction system is: (the reaction cumulative volume is 100 μ l)
Embodiment five
The difference of present embodiment and embodiment one is that in the present embodiment, the reaction system of using based on the gene amplification of LAMP method is:
Reaction system is: (the reaction cumulative volume is 20 μ l)
The detection of embodiment six isolating strain from ight soil
The extraction of strain gene RNA and amplification, detection
1) sample preparation and template extraction, the sample scope is applicable to viral samples to be checked such as cell culture supernatant, ight soil, vomitus; Get ight soil, diarrhoea thing dilute with an amount of physiological saline, the centrifuging and taking supernatant liquor is used for RNA extracting (QIAamp Viral RNA Mini Kit);
The RNA of said extracted and the embodiment one same LAMP method of passing through is carried out the amplification of nucleic acid and the detection of amplified production.The result that the enzyme of amplification and positive amplified production is cut evaluation compares, as following table two.
| Sequence number | LAMP detects | The enzyme of amplified production is cut evaluation |
| Faecal samples 1 | Positive | Meet theoretical prediction |
| Faecal samples 2 | Positive | Meet theoretical prediction |
Enzyme based on positive amplified production is cut evaluation
The amplified production of LAMP test positive is carried out enzyme cut evaluation: utilize the restriction enzyme HaeIII on the target-gene sequence to be amplified, be positioned at 380-384bp, come enzyme to cut the positive amplified production of LAMP, the result shows disconnected 3 of enzyme section, be respectively 163bp, 128bp, 60bp size, conform to the theoretical prediction value in (referring to electrophorogram 3 the 2nd road), thereby determine that positive amplification is specific amplification.
As shown in Table 2, the enzyme of positive amplified production is cut authenticator and is rationally discussed predicted segment size and segments, has only the LAMP specific amplification that detects sample just to observe The above results.
Sequence table
＜110〉Zhuhai Disease Prevention And Control Center
＜120〉group A type G 3 rotavirus detects with primer sets, detection kit
<160>5
<210>1
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>1
ACTGAAGCAGCAACAGAA 18
<210>2
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>2
ACATGTCCAGTTGCAGTG 18
<210>3
<211>47
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>3
AGATCCTGTTGGCCATCCTTTAATAATTCATGGAAGGATACACTTTC 47
<210>4
<211>47
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>4
TATTGCCTCGTTTTCAGTTGATCCGCGTCGTATTTCATTAATACCAA 47
<210>5
<211>199
<212>RNA
＜213〉group A type G 3 rotavirus
<400>5
307?actgaagcagcaac?agaaataaat?gataattcat?ggaaggatac?actttctcag
361?ttatttttaa?ctaaaggatg?gccaacagga?tctgtttatt?ttaaagatta?tactgatatt
421?gcctcgtttt?cagttgatcc?acaactgtac?tgtgattata?atttggtatt?aatgaaatac
481?gacgctacac?tgcaactgga?catgt
Claims (6)
1. a group A type G 3 rotavirus detects and uses primer sets, it is characterized in that described primer sets
Form by following primer:
Forward outer primer F3-G3 ACTGAAGCAGCAACAGAA
Reverse outer primer B3-G3 ACATGTCCAGTTGCAGTG
Forward inner primer FIP-G3
The reverse inner primer BIP-G3 of AGATCCTGTTGGCCATCCTTTAATAATTCATGGAAGGATACACTTTC
TATTGCCTCGTTTTCAGTTGATCCGCGTCGTATTTCATTAATACCAA
The VP7---gene pool accession number of group A type G 3 rotavirus can increase: 307 of D86272 gene order--a part or its complementary strand of-505 nucleotide sequence.
2. a kind of group A type G 3 rotavirus according to claim 1 detects and uses primer sets, it is characterized in that described primer sets can the increase following fragment or the complementary strand of group A type G 3 rotavirus:
Described forward outer primer F3-G3: amplification starts from 307-324bp;
Described forward inner primer FIP-G3: amplification starts from complementary sequence and the 332-356bp of 372-393bp;
Described reverse outer primer B3-G3: amplification starts from the complementary sequence of 488-505bp;
Described reverse inner primer BIP-G3: amplification starts from the complementary sequence of 417-440bp and 463-485bp.
3. a group A type G 3 rotavirus detects and uses test kit, it is characterized in that described test kit comprises amplification reaction solution, the enzyme that contains any described primer sets of claim 1-2 at least.
4. a kind of group A type G 3 rotavirus according to claim 3 detects uses test kit, it is characterized in that, comprises following reagent in the described amplification reaction solution:
Wherein 10 * Bst DNA Polymerase Buffer reaction buffer contains Tris-HCl, 100mM Repone K, 100mM ammonium sulfate, 20mM sal epsom and 1% triton x-100 of 200mM pH 8.8;
Described enzyme comprises that the every microlitre of storage liquid concentration contains the Bst archaeal dna polymerase of 8 activity units, final concentration 0.16-0.64U/ μ l and the AMV ThermoScript II that the every microlitre of storage liquid concentration contains 20 activity units, final concentration 0.4U/ μ l.
5. a kind of group A type G 3 rotavirus according to claim 4 detects uses test kit, it is characterized in that described amplification reaction solution: comprise 1/10 predetermined reaction volume 10 * Bst DNAPolymerase Buffer reaction buffer, 3.6mM sal epsom, 1.6 μ M forward inner primer FIP-G3, the forward outer primer F3-G3 of the reverse inner primer BIP-G3 of 1.6 μ M, 0.2 μ M, reverse outer primer B3-G3,1.4mM dNTP and the 1M trimethyl-glycine of 0.2 μ M;
Described enzyme comprises that the every microlitre of storage liquid concentration contains the BstDNA polysaccharase of 8 activity units, final concentration 0.32U/ μ l and the AMV ThermoScript II that the every microlitre of storage liquid concentration contains 20 activity units, final concentration 0.4U/ μ l.
6. use test kit according to claim 3 or 4 or 5 described a kind of group A type G 3 rotavirus detections, it is characterized in that described test kit also comprises feminine gender and positive control template, described negative control template is a distilled water; Described positive control template is 1～100nM group A type G 3 rotavirus genomic dna.
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| CN101638685B (en) * | 2008-07-29 | 2013-01-09 | 杭州优思达生物技术有限公司 | Method for amplifying target nucleic acid sequence by using cross primer and kit for amplifying target nucleic acid sequence and application thereof |
| CN101413036B (en) * | 2008-12-02 | 2012-01-04 | 中山大学 | Primer for detecting porcine reproductive and respiratory syndrome virus, and use method thereof |
| CN101418351B (en) * | 2008-12-09 | 2011-08-31 | 苏州大学 | Shrimp white spot syndrome virus detection reagent kit and detecting method |
| CN101671746B (en) * | 2009-10-21 | 2012-04-25 | 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 |
| CN102776293A (en) * | 2011-05-10 | 2012-11-14 | 北京林业大学 | Method for auxiliary identification of rotavirus and special primers used therein |
| CN102260752B (en) * | 2011-08-25 | 2013-04-10 | 成都新基因格生物科技有限公司 | Composition, kit and method for detecting group A rotavirus |
| CN103725755A (en) * | 2012-10-10 | 2014-04-16 | 苏州四同医药科技有限公司 | Method for highly-specific highly-sensitive detection of salmonella |
| CN103014178A (en) * | 2012-12-18 | 2013-04-03 | 广东省微生物研究所 | Loop-mediated isothermal amplification detection primer group for rotavirus and detection method thereof |
| CN103014177A (en) * | 2012-12-18 | 2013-04-03 | 广东省微生物研究所 | Loop-mediated isothermal amplification detection kit for rotavirus |
| CN103224998A (en) * | 2013-04-24 | 2013-07-31 | 深圳市生科源技术有限公司 | Rotavirus PCR detection kit and detection method thereof |
| CN114854734B (en) * | 2022-04-13 | 2023-11-21 | 福建省农业科学院畜牧兽医研究所 | Pigeon rotavirus A-type loop-mediated isothermal amplification detection primer set and kit thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1687454A (en) * | 2005-03-23 | 2005-10-26 | 上海市血液中心 | Method for sceening viral nucleic acid of blood through isothermal amplification based on loop mediated technique |
-
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1687454A (en) * | 2005-03-23 | 2005-10-26 | 上海市血液中心 | Method for sceening viral nucleic acid of blood through isothermal amplification based on loop mediated technique |
Non-Patent Citations (2)
| Title |
|---|
| 蔡哲钧等.核酸环介导等温扩增技术.国际检验医学杂志27 12.2006,27(12),1092-1093. |
| 蔡哲钧等.核酸环介导等温扩增技术.国际检验医学杂志27 12.2006,27(12),1092-1093. * |
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