CN103451315A - Loop-mediated isothermal amplification detection primer set and kit for rotaviruses as well as detection method - Google Patents
Loop-mediated isothermal amplification detection primer set and kit for rotaviruses as well as detection method Download PDFInfo
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Abstract
The invention discloses a loop-mediated isothermal amplification (LAMP) detection primer set and kit for rotaviruses as well as a detection method. The primer set comprises an upstream outer primer F3, a downstream outer primer B3, an upstream inner primer FIP and a downstream inner primer BIP which are represented by SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively. An LAMP detection method adopts an LAMP reaction solution formed by taking four LAMP specific primers and a BstDNA (Bst Deoxyribonucleic Acid) polymerase and the like as main components, so as to establish a loop-mediated isothermal amplification rapid detection method for pig rotaviruses; the speed and the accuracy of clinical diagnosis can be improved, the operation is simple and the cost is low; the detection method is applicable to a base layer and can be used for effectively diagnosing pig rotavirus infection caused by the etiology; after the disease is controlled, the slaughtering rate of live pigs is improved obviously, the death rate of the live pigs is reduced and the economic benefits of a live pig industry are improved.
Description
Technical field
The present invention relates to biological technical field, the ring mediated isothermal amplification that is specifically related to a kind of rotavirus detects primer sets and test kit and detection method.
Background technology
Rotavirus (Rotavirus) belongs to Reoviridae (Reoviridae), and rotavirus (Rotaviras) is one of infecting both domestic animals and human important pathogen, can infect many animals.The rotavirus of pig (porcine rotavirus, RV) infects and is most commonly in piglet, and 1-4 piglet sickness rate in age in week surpasses 80%, mortality ratio 7%-20%.The affected pig main manifestations is severe diarrhea, and draining sample ight soil is yellow to white, and containing floss in various degree, some cases is because of serious dehydration, acid base imbalance, secondary infection death.
The detection method of rotavirus cause of disease has electron microscopy, viral separation and Culture technology, immunological technique and Protocols in Molecular Biology at present, but first three above-mentioned kind technology often exists, and sensitivity is lower, complex operation is consuming time, the shortcomings such as plant and instrument of the large-scale costliness of needs, when application, its limitation is often arranged, especially can not be applicable to the practical applications such as large-scale cause of disease examination and detection.Protocols in Molecular Biology, wherein especially with the RT-PCR detection technique because it is quick, sensitive, be applicable to processing the characteristics such as sample of large flux, demonstrate its superiority and more and more applied in virus detects, but the method still needs multiple instrumentation from the interpretation that result detected, operate comparatively loaded down with trivial details, required time still, more than 5 hours, often has the defects such as non-specific appearance.
In recent years, a kind of novel nucleic acids amplification technique one ring mediated isothermal amplification (LAMP) technology, due to its characteristics such as quick, simple, special, more and more disease diagnosis that apply to.The LAMP technology is T.Notomi in a kind of novel nucleic acids amplification technique of invention in 2000, and this technology mainly utilizes 4 kinds of different Auele Specific Primers identification 6 specific regions of target gene and a kind of archaeal dna polymerase with strand displacement characteristic carries out efficiently under isothermal condition, quick, high amplified target gene specifically.This technology can be used for the rapid detection of microorganism, has been widely used in the fields such as food, agricultural, health.In recent years, the LAMP technology develops rapidly aspect the animal epidemic diagnosis, has developed the LAMP detection methods such as Pestivirus suis, transmissible gastro-enteritis virus, Porcine epidemic diarrhea virus, but has not yet to see the report that detects porcine rotavirus LAMP detection technique.
Summary of the invention
The ring mediated isothermal amplification that the purpose of this invention is to provide a kind of rotavirus detects primer sets and detects the detection kit of primer sets based on this ring mediated isothermal amplification, utilizes this detection kit can specific detection rotavirus (rotavirus).
The ring mediated isothermal amplification of rotavirus of the present invention detects primer sets, and it is comprised of following primer;
Upstream primer F3:5 '-ACCATTCCTTAACGCACAA-3 ' (SEQ ID NO.1);
Downstream primer B3:5 '-GGCAACATCAGCATAGTCTT-3 ' (SEQ ID NO.2);
Upstream inner primer FIP:5 '-
CAAGCACAAAGTCGAAGTTAGAAATAAATCTTCCAATTACTGGGTC-3′（SEQ?ID?NO.3）；
Downstream inner primer BIP:5 '-CTGAAGCTGCAACAGAAATAAACGATCCTGTTGGCCATCCT-3 ' (SEQ ID NO.4).
It is two specificity inner primers (upstream primer and downstream primer) and two the specificity outer primers (upstream inner primer and downstream inner primer) according to six sequence fragment designs comparatively conservative in porcine rotavirus VP4 gene that the ring mediated isothermal amplification of rotavirus of the present invention detects primer sets, this conservative gene sequence is that porcine rotavirus is common, detects the reliability of the porcine rotavirus of different sources with the detection kit that guarantees the ring mediated isothermal amplification detection primer sets based on this rotavirus.
The ring mediated isothermal amplification of the rotavirus based on above-mentioned detects the detection kit of primer sets, it comprises that RNA extracts reagent, RNA reverse transcription reagent and loop-mediated isothermal amplification reaction reagent and colouring reagents, described loop-mediated isothermal amplification reaction reagent comprises loop-mediated isothermal amplification liquid and Bst archaeal dna polymerase, described loop-mediated isothermal amplification liquid is comprised of following component: the upstream primer F3 of 10 μ mol/L, the downstream primer B3 of 10 μ mol/L, the upstream inner primer FIP of 10 μ mol/L, the downstream inner primer BIP of 10 μ mol/L, 5mol/L Betaine, 100mmol/L MgS0
4, 1.6mmol/L dNTPs, 10 * Thermo pol reaction buffer.
The detection method of the loop-mediated isothermal amplification detection kit of described rotavirus, it comprises the following steps:
1) extraction of sample viral RNA;
2) viral RNA reverse transcription step 1 extracted becomes cDNA;
3) first set up the loop-mediated isothermal amplification system, then under 60-65 ℃ of isothermal condition, insulation 30-60min completes the cDNA amplified reaction;
Wherein, the loop-mediated isothermal amplification system is totally 25 μ L, specifically comprises: 10 * Bst Thermo pol Buffer2.5 μ L, 10 μ mol/L upstream primer F30.5 μ L, 10 μ mol/L downstream primer B30.5 μ L, 10 μ mol/L upstream inner primer FIP4 μ L, 10 μ mol/L downstream inner primer BIP4 μ L, 1.6mmol/L dNTPs2 μ L, 5mol/L Betaine5 μ L, 100mmol/LMgS0
40.9 μ L, 8U/ μ L Bst archaeal dna polymerase 1 μ L, cDNA1 μ L, all the other are by ddH
2o is supplemented to 25 μ L;
4) be confirmed whether to exist amplified production by developer or agarose gel electrophoresis.
The present invention is directed to the technological operation of traditional detection porcine rotavirus loaded down with trivial details, need expensive instrument, the problems such as susceptibility is poor, adopt loop-mediated isothermal amplification technique, set up the LAMP detection kit and the detection method that detect porcine rotavirus, the method use instrument is simple, cost is low, detection is quick, high specificity, susceptibility is high, but the naked eyes judged result, be suitable for basic unit's Site Detection, thereby can solve its problem of difficult diagnosis clinically.In the above-mentioned detection method of the present invention, the concrete operations of described step 1 are:
1) every 50～100mgRNA sample carries out cracking with 1mL Trizol liquid reagent to tissue;
2) the Trizol liquid of above-mentioned RNA sample is proceeded in the EP pipe, place 5 minutes under 15-30 ℃;
3) in above-mentioned EP pipe, the amount that adds the 0.2mL chloroform according to every 1mL Trizol liquid adds chloroform, covers EP pipe lid, firmly shake 15 seconds, and place 2～3 minutes under 15 ℃～30 ℃ after, under 2 ℃～8 ℃ with the centrifugal force of 12000g centrifugal 15 minutes; Get the upper strata water and be placed in another root EP pipe, the amount that adds the 0.5mL Virahol according to every 1mL Trizol liquid adds Virahol, places then under 2 ℃～8 ℃ with the centrifugal force of 12000g centrifugal 10 minutes under 15 ℃～30 ℃ 10 minutes; Abandon supernatant liquor, add 1mL75% ethanol according to every ImL Trizol liquid and washed, vortex mixed, under 2 ℃～8 ℃ with the centrifugal force of 7500g centrifugal 5 minutes, abandon supernatant liquor; Allow the precipitation at room temperature seasoning of RNA; Then dissolve the RNA precipitation with Rnase-free water.
The RNA sample OD that described step 1 is extracted
260/ OD
280between 1.7-2.0, concentration is 10-100g/ μ L.
In the above-mentioned detection method of the present invention, the concrete operations of described step 2 are: be formulated as follows reaction solution in the 1.5mL centrifuge tube: by RNA solution 10 μ L, random primer 1 μ L, DEPC water 1, μ L is centrifugal mixes; Then hatch 5min at-70 ℃, set to 0 immediately ℃ cooling, then in centrifuge tube, add 5 * M-MLV buffer4 μ L, RNA enzyme to suppress 1 μ L, dNTP mixture l μ L, the dark 1 μ L of M-MLV reverse transcription, DEPC water 5 μ L, above-mentioned reaction solution is put to 42 ℃ of incubation 60min, after reverse transcription completes, 95 deactivation M-MLV, obtain the cDNA product.
The LAMP reaction solution that the present invention adopts the main components such as 4 LAMP Auele Specific Primers and Bst archaeal dna polymerase are to form, adopt loop-mediated isothermal amplification technique, sets up the LAMP detection kit and the detection method that detect porcine rotavirus.Because loop-mediated isothermal amplification technique has sensitivity, special, quick, reliable, easy characteristics, make it compare with other diagnostic techniques, be more suitable for diagnosing in clinical labororatory.The porcine rotavirus loop-mediated isothermal amplification fast detection method that the present invention sets up, can improve speed and the accuracy of clinical diagnosis, and simple to operate, with low cost, be applicable to basic unit and use, the porcine rotavirus that this cause of disease of energy efficient diagnosis causes infects, after this disease is controlled, to significantly improve slaughter rate of hogs, reduce the live pig mortality ratio, improve the economic benefit of live pig industry.
The accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation:
The check electrophorogram that Fig. 1 is embodiment 3 sensitivity experiments;
The check electrophorogram that Fig. 2 is the experiment of embodiment specificity.
Embodiment
Below in conjunction with embodiment, the present invention is further detailed explanation:
The ring mediated isothermal amplification of rotavirus of the present invention detects primer sets, and it is comprised of following primer;
Upstream primer F3:5 '-ACCATTCCTTAACGCACAA-3 ';
Downstream primer B3:5 '-GGCAACATCAGCATAGTCTT-3 ';
Upstream inner primer FIP:5 '-
CAAGCACAAAGTCGAAGTTAGAAATAAATCTTCCAATTACTGGGTC-3′；
Downstream inner primer BIP:5 '-CTGAAGCTGCAACAGAAATAAACGATCCTGTTGGCCATCCT-3 '.
The ring mediated isothermal amplification of the rotavirus based on above-mentioned detects the detection kit of primer sets, it comprises that RNA extracts reagent, RNA reverse transcription reagent and loop-mediated isothermal amplification reaction reagent and colouring reagents, described loop-mediated isothermal amplification reaction reagent comprises loop-mediated isothermal amplification liquid and Bst archaeal dna polymerase, described loop-mediated isothermal amplification liquid is comprised of following component: the upstream primer F3(SEQ ID NO.1 of 10 μ mol/L), the downstream primer B3(SEQ ID NO.2 of 10 μ mol/L), the upstream inner primer FIP(SEQ ID NO.3 of 10 μ mol/L), the downstream inner primer BIP(SEQ ID NO.4 of 10 μ mol/L), 5mol/L Betaine, 100mmol/L MgS0
4, 1.6mmol/L dNTPs, 10 * Thermo pol reaction buffer.
The detection method of the loop-mediated isothermal amplification detection kit of described rotavirus, it comprises the following steps:
1) extraction of sample viral RNA;
2) viral RNA reverse transcription step 1 extracted becomes cDNA;
3) first set up the loop-mediated isothermal amplification system, then under 60-65 ℃ of isothermal condition, insulation 30-60min completes the cDNA amplified reaction;
Wherein, the loop-mediated isothermal amplification system is totally 25 μ L, specifically comprises: 10 * Bst Thermo pol Buffer2.5 μ L, 10 μ mol/L upstream primer F30.5 μ L, 10 μ mol/L downstream primer B30.5 μ L, 10 μ mol/L upstream inner primer FIP4 μ L, 10 μ mol/L downstream inner primer BIP4 μ L, 1.6mmol/L dNTPs2 μ L, 5mol/L Betaine5 μ L, 100mmol/LMgS0
40.9 μ L, 8U/ μ L Bst archaeal dna polymerase 1 μ L, cDNA1 μ L, all the other are by ddH
2o is supplemented to 25 μ L;
4) be confirmed whether to exist amplified production by developer or agarose gel electrophoresis.
The doubtful RV of 1 pair of a certain example of specific embodiment infects sample and carries out the LAMP detection
(1) extraction of sample RNA;
1) tissue sample, every 50～100mgRNA sample carries out cracking with 1mL Trizol liquid reagent to tissue;
2) the Trizol liquid of above-mentioned RNA sample is proceeded in the EP pipe, place 5 minutes under 15-30 ℃;
3) in above-mentioned EP pipe, the amount that adds the 0.2mL chloroform according to every 1mL Trizol liquid adds chloroform, covers EP pipe lid, firmly shake 15 seconds, and place 2～3 minutes under 15 ℃～30 ℃ after, under 2 ℃～8 ℃ with the centrifugal force of 12000g centrifugal 15 minutes; Get the upper strata water and be placed in another root EP pipe, the amount that adds the 0.5mL Virahol according to every 1mL Trizol liquid adds Virahol, places then under 2 ℃～8 ℃ with the centrifugal force of 12000g centrifugal 10 minutes under 15 ℃～30 ℃ 10 minutes; Abandon supernatant liquor, add 1mL75% ethanol according to every ImL Trizol liquid and washed, vortex mixed, under 2 ℃～8 ℃ with the centrifugal force of 7500g centrifugal 5 minutes, abandon supernatant liquor; Allow the precipitation at room temperature seasoning of RNA; And then dissolve the RNA precipitation with Rnase-free water.
(2) viral RNA reverse transcription step 1 extracted becomes cDNA;
Be formulated as follows reaction solution in the 1.5mL centrifuge tube: by RNA solution 10 μ L, random primer 1 μ L, DEPC water 1, μ L is centrifugal mixes; Then hatch 5min at-70 ℃, set to 0 immediately ℃ cooling, then in centrifuge tube, add 5 * M-MLV buffer4 μ L, RNA enzyme to suppress 1 μ L, dNTP mixture l μ L, the dark 1 μ L of M-MLV reverse transcription, DEPC water 5 μ L, above-mentioned reaction solution is put to 42 ℃ of incubation 60min, after reverse transcription completes, 95 deactivation M-MLV, obtain the cDNA product.
(3) first set up the loop-mediated isothermal amplification system, then under 60-65 ℃ of isothermal condition, insulation 30-60min completes the cDNA amplified reaction; Wherein, the loop-mediated isothermal amplification system is totally 25 μ L, specifically comprises: 10 * Bst Thermo pol Buffer2.5 μ L, 10 μ mol/L upstream primer F30.5 μ L, 10 μ mol/L downstream primer B30.5 μ L, 10 μ mol/L upstream inner primer FIP4 μ L, 10 μ mol/L downstream inner primer BIP4 μ L, 1.6mmol/L dNTPs2 μ L, 5mol/L Betaine5 μ L, 100mmol/L MgS0
40.9 μ L, 8U/ μ L Bst archaeal dna polymerase 1 μ L, cDNA1 μ L, all the other are by ddH
2o is supplemented to 25 μ L;
(4) be confirmed whether to exist amplified production by developer.
Add 1 μ L SYBR Green I dyestuff in each reaction tubes to be checked, the visual inspection colour-change, green if positive control is, it is orange that negative control is, and illustrates that reaction is effectively.If detected example reaction liquid color becomes green, illustrate that sample to be checked is for containing porcine rotavirus.
Its result is: negative electrode contrast (template is Porcine epidemic diarrhea virus nucleic acid) yellow, do not contain porcine rotavirus, and the doubtful RV that No. 2 samples are the present embodiment infects sample, and its color be green, contains porcine rotavirus.
(1) extraction of sample RNA;
1) every 50～100mgRNA sample carries out cracking with 1mL Trizol liquid reagent to tissue;
2) the Trizol liquid of above-mentioned RNA sample is proceeded in the EP pipe, place 5 minutes under 15-30 ℃;
3) in above-mentioned EP pipe, the amount that adds the 0.2mL chloroform according to every 1mL Trizol liquid adds chloroform, covers EP pipe lid, firmly shake 15 seconds, and place 2～3 minutes under 15 ℃～30 ℃ after, under 2 ℃～8 ℃ with the centrifugal force of 12000g centrifugal 15 minutes; Get the upper strata water and be placed in another root EP pipe, the amount that adds the 0.5mL Virahol according to every 1mL Trizol liquid adds Virahol, places then under 2 ℃～8 ℃ with the centrifugal force of 12000g centrifugal 10 minutes under 15 ℃～30 ℃ 10 minutes; Abandon supernatant liquor, add 1mL75% ethanol according to every ImL Trizol liquid and washed, vortex mixed, under 2 ℃～8 ℃ with the centrifugal force of 7500g centrifugal 5 minutes, abandon supernatant liquor; Allow the precipitation at room temperature seasoning of RNA; Dissolve the RNA precipitation with Rnase-free water.
(2) viral RNA reverse transcription step 1 extracted becomes cDNA;
Be formulated as follows reaction solution in the 1.5mL centrifuge tube: by RNA solution 10 μ L, random primer 1 μ L, DEPC water 1, μ L is centrifugal mixes; Then hatch 5min at-70 ℃, set to 0 immediately ℃ cooling, then in centrifuge tube, add 5 * M-MLV buffer4 μ L, RNA enzyme to suppress 1 μ L, dNTP mixture l μ L, the dark 1 μ L of M-MLV reverse transcription, DEPC water 5 μ L, above-mentioned reaction solution is put to 42 ℃ of incubation 60min, after reverse transcription completes, 95 deactivation M-MLV, obtain the cDNA product.
(3) first set up the loop-mediated isothermal amplification system, then under 60-65 ℃ of isothermal condition, insulation 30-60min completes the cDNA amplified reaction; Wherein, the loop-mediated isothermal amplification system is totally 25 μ L, specifically comprises: 10 * Bst Thermo pol Buffer2.5 μ L, 10 μ mol/L upstream primer F30.5 μ L, 10 μ mol/L downstream primer B30.5 μ L, 10 μ mol/L upstream inner primer FIP4 μ L, 10 μ mol/L downstream inner primer BIP4 μ L, 1.6mmol/L dNTPs2 μ L, 5mol/L Betaine5 μ L, 100mmol/L MgS0
40.9 μ L, 8U/ μ L Bst archaeal dna polymerase 1 μ L, cDNA1 μ L, all the other are by ddH
2o is supplemented to 25 μ L;
(4) be confirmed whether to exist amplified production by agarose gel electrophoresis.
After the LAMP reaction finishes, get 1 μ L reaction product and carry out the agarose gel electrophoresis analysis, the stepped specific amplified of positive control, and negative control without amplification explanation reaction effectively, on this basis, if the stepped specific amplified of detected sample is the RV positive, if be the RV feminine gender without amplification.
Its result is: negative electrode contrast (template is Porcine epidemic diarrhea virus nucleic acid), without stepped specific amplified, does not contain porcine rotavirus, the doubtful RV infection sample that No. 2 samples are the present embodiment, and its stepped specific amplified, contain porcine rotavirus.
By the RNA of embodiment 2 samples from 10
0～10
-7carry out 10 times of gradient dilutions, then according to the method for the step 2 to 4 in embodiment 2, carry out the check of reverse transcription, ring mediated isothermal amplification and amplified production, verify sensitivity of the present invention.As shown in Figure 1, adopt the agarose gel electrophoresis discovery of testing, the 1-5 swimming lane is respectively 10
-3, 10
-4, 10
-5, 10
-6, 10
-7dilution cDNA template, wherein, 10
-3, 10
-4, 10
-5, 10
-6dilution cDNA template is stepped specific amplified all.Test-results shows, LAMP method detection sensitivity of the present invention can reach 10
-6extent of dilution (content is 10pg).
Transmissible gastro-enteritis virus, Porcine epidemic diarrhea virus, Pestivirus suis and embodiment 2 are determined to the sample that contains the porcine rotavirus positive, again detect according to the LAMP method of embodiment 2 steps 2 to 4.As shown in Figure 2, the 1-3 swimming lane is respectively transmissible gastro-enteritis virus, Porcine epidemic diarrhea virus, Pestivirus suis to detected result, without stepped specific amplified, negative; The sample of these three kinds of known negative still presents feminine gender in the LAMP detected result; 4 swimming lanes are porcine rotavirus, stepped specific amplified, and this sample still presents the positive in the LAMP detected result.Show that LAMP detected result and PCR detected result match, and have verified the specificity of institute's establishment method of the present invention and test kit.
Sequence table
<110 > Longyan School's Life Science College
<120 > ring mediated isothermal amplification of rotavirus detects primer sets and test kit and detection method
<160>4
<210>1
<211>19
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<400>1
ACCATTCCTT?AACGCACAA19
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GGCAACATCA?GCATAGTCTT20
<210>3
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CAAGCACAAA?GTCGAAGTTA?GAAATAAATC?TTCCAATTAC?TGGGTC46
<210>4
<211>41
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<213 > artificial sequence
<400>4
CTGAAGCTGC?AACAGAAATA?AACGATCCTG?TTGGCCATCC?T41
<110 > Longyan School's Life Science College
<120 > ring mediated isothermal amplification of rotavirus detects primer sets and test kit and detection method
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<210>?1
<211>?19
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ACCATTCCTT?AACGCACAA?19
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GGCAACATCA?GCATAGTCTT?20
<210>?3
<211>?46
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CAAGCACAAA?GTCGAAGTTA?GAAATAAATC?TTCCAATTAC?TGGGTC?46
<210>?4
<211>?41
<212>?DNA
<213 > artificial sequence
<400>?4
CTGAAGCTGC?AACAGAAATA?AACGATCCTG?TTGGCCATCC?T?41
Claims (6)
1. the ring mediated isothermal amplification of rotavirus detects primer sets, and it is characterized in that: it is comprised of following primer;
Upstream primer F3:5 '-ACCATTCCTTAACGCACAA-3 ';
Downstream primer B3:5 '-GGCAACATCAGCATAGTCTT-3 ';
Upstream inner primer FIP:5 '-
CAAGCACAAAGTCGAAGTTAGAAATAAATCTTCCAATTACTGGGTC-3′；
Downstream inner primer BIP:5 '-CTGAAGCTGCAACAGAAATAAACGATCCTGTTGGCCATCCT-3 '.
2. the ring mediated isothermal amplification based on rotavirus claimed in claim 1 detects the detection kit of primer sets, it is characterized in that: it comprises that RNA extracts reagent, RNA reverse transcription reagent and loop-mediated isothermal amplification reaction reagent and colouring reagents, described loop-mediated isothermal amplification reaction reagent comprises loop-mediated isothermal amplification liquid and Bst archaeal dna polymerase, described loop-mediated isothermal amplification liquid is comprised of following component: the upstream primer F3 of 10 μ mol/L, the downstream primer B3 of 10 μ mol/L, the upstream inner primer FIP of 10 μ mol/L, the downstream inner primer BIP of 10 μ mol/L, 5mol/L Betaine, 100mmol/LMgS0
4, 1.6mmol/L dNTPs, 10 * Thermo pol reaction buffer.
3. the detection method of the loop-mediated isothermal amplification detection kit of rotavirus according to claim 2, it is characterized in that: it comprises the following steps:
1) extraction of sample viral RNA;
2) viral RNA reverse transcription step 1 extracted becomes cDNA;
3) first set up the loop-mediated isothermal amplification system, then under 60-65 ℃ of isothermal condition, insulation 30-60min completes the cDNA amplified reaction;
Wherein, the loop-mediated isothermal amplification system is totally 25 μ L, specifically comprises: 10 * Bst Thermo pol Buffer2.5 μ L, 10 μ mol/L upstream primer F30.5 μ L, 10 μ mol/L downstream primer B30.5 μ L, 10 μ mol/L upstream inner primer FIP4 μ L, 10 μ mol/L downstream inner primer BIP4 μ L, 1.6mmol/L dNTPs2 μ L, 5mol/L Betaine5 μ L, 100mmol/LMgS0
40.9 μ L, 8U/ μ L Bst archaeal dna polymerase 1 μ L, cDNA1 μ L, all the other are by ddH
2o is supplemented to 25 μ L;
4) be confirmed whether to exist amplified production by developer or agarose gel electrophoresis.
4. the detection method of the loop-mediated isothermal amplification detection kit of rotavirus according to claim 3, it is characterized in that: the concrete operations of described step 1 are:
1) every 50～100mgRNA sample carries out cracking with 1mL Trizol liquid reagent to tissue;
2) the Trizol liquid of above-mentioned RNA sample is proceeded in the EP pipe, place 5 minutes under 15-30 ℃;
3) in above-mentioned EP pipe, the amount that adds the 0.2mL chloroform according to every 1mL Trizol liquid adds chloroform, covers EP pipe lid, firmly shake 15 seconds, and place 2～3 minutes under 15 ℃～30 ℃ after, under 2 ℃～8 ℃ with the centrifugal force of 12000g centrifugal 15 minutes; Get the upper strata water and be placed in another root EP pipe, the amount that adds the 0.5mL Virahol according to every 1mL Trizol liquid adds Virahol, places then under 2 ℃～8 ℃ with the centrifugal force of 12000g centrifugal 10 minutes under 15 ℃～30 ℃ 10 minutes; Abandon supernatant liquor, add 1mL75% ethanol according to every ImL Trizol liquid and washed, vortex mixed, under 2 ℃～8 ℃ with the centrifugal force of 7500g centrifugal 5 minutes, abandon supernatant liquor; Allow the precipitation at room temperature seasoning of RNA; Then dissolve the RNA precipitation with Rnase-free water.
5. the detection method of the loop-mediated isothermal amplification detection kit of rotavirus according to claim 4, is characterized in that: the RNA sample OD that described step 1 is extracted
260/ OD
280between 1.7-2.0, concentration is 10-100g/ μ L.
6. the detection method of the loop-mediated isothermal amplification detection kit of rotavirus according to claim 3, it is characterized in that: the concrete operations of described step 2 are:
Be formulated as follows reaction solution in the 1.5mL centrifuge tube: by RNA solution 10 μ L, random primer 1 μ L, DEPC water 1, μ L is centrifugal mixes; Then hatch 5min at-70 ℃, set to 0 immediately ℃ cooling, then in centrifuge tube, add 5 * M-MLV buffer4 μ L, RNA enzyme to suppress 1 μ L, dNTP mixture l μ L, the dark 1 μ L of M-MLV reverse transcription, DEPC water 5 μ L, above-mentioned reaction solution is put to 42 ℃ of incubation 60min, after reverse transcription completes, 95 deactivation M-MLV, obtain the cDNA product.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104611464A (en) * | 2015-02-04 | 2015-05-13 | 四川农业大学 | LAMP detection kit for detecting porcine rotavirus |
| CN104694670A (en) * | 2015-03-20 | 2015-06-10 | 东北农业大学 | LAMP (Loop-Mediated Isothermal Amplification) detection method of PRV (Porcine Rotavirus) inverse transcription and application |
| CN105734175A (en) * | 2016-04-25 | 2016-07-06 | 广西壮族自治区兽医研究所 | Pig rotavirus reverse transcription loop-mediated isothermal amplification reagent kit and application thereof |
| CN113981148A (en) * | 2021-11-18 | 2022-01-28 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Method for identifying RV infection and virus genotype and application thereof |
| CN114854734A (en) * | 2022-04-13 | 2022-08-05 | 福建省农业科学院畜牧兽医研究所 | Primer group for pigeon rotavirus type A loop-mediated isothermal amplification detection and kit thereof |
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2013
- 2013-06-26 CN CN201310258549.6A patent/CN103451315B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
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| MANABU NEMOTO: "Detection of Equine Rotavirus by Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP)", 《J. VET. MED. SCI》 * |
| 张维谊等: "RT-PCR方法检测上海地区猪轮状病毒", 《畜牧与兽医》 * |
| 杨丽: "环介导等温扩增技术（LAMP）检测三种典型肠病毒方法的建立及其在水环境中的应用", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611464A (en) * | 2015-02-04 | 2015-05-13 | 四川农业大学 | LAMP detection kit for detecting porcine rotavirus |
| CN104694670A (en) * | 2015-03-20 | 2015-06-10 | 东北农业大学 | LAMP (Loop-Mediated Isothermal Amplification) detection method of PRV (Porcine Rotavirus) inverse transcription and application |
| CN105734175A (en) * | 2016-04-25 | 2016-07-06 | 广西壮族自治区兽医研究所 | Pig rotavirus reverse transcription loop-mediated isothermal amplification reagent kit and application thereof |
| CN113981148A (en) * | 2021-11-18 | 2022-01-28 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Method for identifying RV infection and virus genotype and application thereof |
| CN114854734A (en) * | 2022-04-13 | 2022-08-05 | 福建省农业科学院畜牧兽医研究所 | Primer group for pigeon rotavirus type A loop-mediated isothermal amplification detection and kit 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 |
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