CN103014178A - Loop-mediated isothermal amplification detection primer group for rotavirus and detection method thereof - Google Patents
Loop-mediated isothermal amplification detection primer group for rotavirus and detection method thereof Download PDFInfo
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- CN103014178A CN103014178A CN2012105536726A CN201210553672A CN103014178A CN 103014178 A CN103014178 A CN 103014178A CN 2012105536726 A CN2012105536726 A CN 2012105536726A CN 201210553672 A CN201210553672 A CN 201210553672A CN 103014178 A CN103014178 A CN 103014178A
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Abstract
The invention discloses a loop-mediated isothermal amplification detection primer group for rotavirus and a detection method thereof. The detection primer group comprises an upstream outer primer F3, a downstream outer primer B3, an upstream inner primer FIP and a downstream inner primer BIP, wherein the upstream outer primer F3, the downstream outer primer B3, the upstream inner primer FIP and the downstream inner primer BIP are expressed by SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively. The invention builds the LAMP detection method for the rotavirus; the detection method designs two specific inner primers and two specific outer primers according to gene conserved sequences of the rotavirus; and the gene conserved sequences are shared by the rotavirus. The primer group adopts the LAMP technology, so that the specificity is strong; the primer group has higher sensitivity compared with the PCR detection method, but the expensive PCR amplifier is not needed, and all that is needed is the general water bath tank; and the result is not required to be observed by the gel electrophoresis method, but is observed by fluorescent dye, so that the operation is easy and fast; and the primer group can be used for detecting the rotavirus, and is particularly suitable for basic level field application.
Description
Technical field:
The invention belongs to biological technical field, the ring mediated isothermal amplification that is specifically related to a kind of rotavirus detects primer sets and detection method.
Background technology:
Rotavirus (rotavirus) is the Important cause of disease of world wide inner virus gastro-enteritis.The gastro-enteritis treatment that at present rotavirus infection is caused there is no specific medicament.No matter in developed country or developing country, the diarrhoea that is caused by rotavirus all has higher sickness rate, is the main cause of disease that the infant falls ill and causes death.
Rotavirus is very strong in the in vitro viability, and various physical and chemical factors are had stronger resistibility, and ether-resistant and weak acid all can not make its inactivation with chloroform, multigelation, ultrasonication, and the sequence variations of this viroid is larger, has various serotype to exist.In recent years, the popular trend that continuous rising is worldwide arranged of the gastro-enteritis that rotavirus causes, and Epidemic Scope is wide, and infectivity is strong, and the secondary attack rate is high, often causes acute serious vomiting and diarrhoea.Although the symptom weight that different virus infects differs, general patient has the symptom of the serious dehydration sample gastro-enteritis such as low fever, vomiting, diarrhoea, headache.The crowd is to the general susceptible of these several viruses, and children and old man are the most serious, is the population at risk of marcy agent.Although rotavirus gastroenteritis popular has certain seasonality, all can occur the whole year, can be outbreak of epidemic, also can be to distribute popularly, therefore causes and can not protect timely and effectively it.
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, sensitivity is lower, complex operation is consuming time, the shortcomings such as plant and instrument of the large-scale costliness of needs but first three above-mentioned kind technology often exists, when using, 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 RT-PCR detection technique characteristics such as sample owing to the large flux of its quick, sensitive, suitable processing, demonstrating its superiority in virus detects is also more and more used, but the method still needs multiple instrumentation from the interpretation that detects the result, operate comparatively loaded down with trivial details, required time still more than 5 hours, often has the defectives such as non-specific appearance.Compare with above-mentioned several laboratory diagnostic methods, newly-established ring mediated isothermal amplification (LAMP) technology accomplishes easily that clinically detection time is also shorter because it is less demanding to plant and instrument in recent years, be no more than 3 hours, explore a kind of novel method for fast, accurately detecting rotavirus.
Summary of the invention:
The first purpose of the present invention provides the detection primer sets that a kind of application ring mediated isothermal amplification detects rotavirus (rotavirus), utilizes this detection primer sets can specific detection rotavirus (rotavirus).
Application ring mediated isothermal amplification of the present invention detects the detection primer sets of rotavirus (rotavirus), it is characterized in that, is comprised of following primer:
Upstream outer primer F3:5 '-ACCACTCCTTAATGCACAA-3 '; (shown in SEQ ID NO.1);
Downstream outer primer B3:5 '-GCCATCCTTTGATTAAAAATAGCT-3 '; (shown in SEQ ID NO.2);
Upstream inner primer FIP:5 '-CCTCTCGCGTTGAGTTCGTAT-GGAATAAATCTTCCGATTACTGG-3 '; (shown in SEQ ID NO.3)
Downstream inner primer BIP:5 '-ATGTTTGTATTACCCAACTGAAGCA-AGAAAGTGTATCCTTCCATGAA-3 ' (shown in SEQ ID NO.4).
Second purpose of the present invention provides the detection method of the rotavirus of a kind of diagnosis of non-disease and therapeutic purpose, it is characterized in that, sample is carried out viral RNA to be extracted, reverse transcription becomes cDNA again, then with above-mentioned detection primer sets cDNA is carried out selective amplification by the method for ring mediated isothermal amplification, be confirmed whether to have amplified production.
Described sample can be the diarrhoea sample of hospital, and water, food etc.
Described method by ring mediated isothermal amplification is carried out selective amplification with above-mentioned detection primer sets to cDNA and is specially: the ring mediated isothermal amplification system is system cumulative volume 25 μ L, comprises 2.5 μ L, 10 * Thermopol reaction buffer, 0.4 μ L10mmol/LdNTPs, 0.5 μ L, 10 μ mol/L upstream outer primer F3,0.5 μ L, 10 μ mol/L downstream outer primer B3,1 μ L40 μ mol/L upstream inner primer FIP, 1 μ L, 40 μ mol/L downstream inner primer BIP, 0.6 μ L100mmol/L MgSO
4, 12.5 μ L2mol/L trimethyl-glycines, 4 μ L sterilization distilled water ddH
2O, 1 μ L8U/ μ L Bst archaeal dna polymerase and 1 μ L cDNA, reaction conditions is hatched 60min for being 65 ℃ in temperature.
Describedly be confirmed whether to have amplified production and can utilize electrophoresis detection, fluorescence developing to detect or whether turbidity detection ring mediated isothermal amplification product has amplified production, preferably detect with fluorescence developing, specifically in the reaction tubes of termination reaction, add 1 μ L, 10% SYBR Green I developer, observations behind the 10min, if color is yellow, then the sample rotavirus is negative, without rotavirus, if color is green, then the sample rotavirus is positive, contains rotavirus.
Described 10 * Thermopol reaction buffer is material of the prior art, can buy from reagent company, it contains trihydroxy methyl aminomethane-hydrochloric acid (Tris-HCl), 100mmol/L Repone K (KCl), the 100mmol/L ammonium sulfate (NH of 200mmol/L pH8.8
4)
2SO
4, 20mmol/L sal epsom (MgSO
4) and 1% triton x-100 (TtitonX-100), surplus is water.
The present invention has set up the LAMP detection method of rotavirus, and this detection method has designed two specificity inner primers and two specificity outer primers according to the gene conserved sequence of rotavirus, and this conservative gene sequence is that rotavirus is common.The present invention adopts the LAMP technology, high specificity, and than PCR detection method higher sensitivity is arranged, but do not need expensive PCR instrument, only need common water bath to get final product, and the result needn't observe with gel electrophoresis method, observe with fluorescence dye and to get final product, simple and quick, can be used for the detection of rotavirus, be particularly suitable for basic unit's rig-site utilization.
Ring mediated isothermal amplification (Loop-mediated Isothermal Amplification, LAMP) technology has high specificity, highly sensitive, quick and low cost and other advantages, be characterized in the 4 kinds of primers of 6 position designs to target gene, utilize strand replacement reaction under constant temperature, target gene efficiently to be increased.Because its reaction is that multiple primer starts jointly, so that reaction result is more special than PCR, in addition, because the isothermal duplication of carrying out, reaction just can be finished in constant water bath box, not only saves instrument cost, and working method is simpler, is applicable to field quick detection.
The invention solves long, lower, the high in cost of production defective of sensitivity of required cycle of the method that detects rotavirus in the prior art, can be widely used in department and the fields such as hospital, food service industry, inspection and quarantining for import/export and quality surveillance.
Embodiment:
Following examples are to further specify of the present invention, rather than limitation of the present invention.
Embodiment 1: utilize rotavirus LAMP detection kit to detect rotavirus
Sample to be tested preparation: take clinical diarrhoea sample as example
Present embodiment has been collected 2 parts of clinical diarrhoea samples that hospital collects, and detects in accordance with the following methods respectively.
(1) sample process
Get the clinical diarrhoea sample that hospital collects, be diluted to 10% suspension with the PBS of pH7.4,4 ℃, 10000g centrifuging and taking supernatant are used for the extraction viral RNA.
(2) viral RNA extracts
(a) get the supernatant of 200 μ L steps (1), add 800 μ L Trizol reagent, with pipettor repeatedly mixing leave standstill afterwards several times 5min;
(b) add 200 μ L chloroforms, firmly jolting 15S leaves standstill 2～3min again;
(c) 4 ℃, 12000g, centrifugal 15min discards supernatant liquid;
(d) add 500 μ L Virahols, abundant mixing, room temperature is placed 10min;
(e) 4 ℃, 12000g, centrifugal 10min, again supernatant discarded;
(f) ethanol of adding 75 ﹪, jolting, fully washing precipitation;
(g) 4 ℃, 7500g, centrifugal 5min carefully discards ethanol;
(h) after the abundant drying, the RNA precipitation is suspended in an amount of water without the RNA enzyme, obtains thus the RNA sample.
(3) viral RNA reverse transcription
Get 2 μ L RNA samples, 1 μ L dNTP, 0.5 μ L RNasin, 1 μ L MLV, the outer primer F3(5 ' of each 1 μ L-ACCACTCCTTAATGCACAA-3 ') and outer primer B3(5 '-GCCATCCTTTGATTAAAAATAGCT-3 '), 2.5 μ L, 10 * RT buffer, 1.5 μ LMgCl
2, adding RNase free water is 25 μ L to final volume, the reverse transcription reaction condition is: and 42 ℃, 30min, then 95 ℃, 2min obtains cDNA thus.
(4) ring mediated isothermal amplification
The ring mediated isothermal amplification system is system cumulative volume 25 μ L, comprises that 2.5 μ L, 10 * Thermopol reaction buffer, 0.4 μ L10mmol/L dNTPs, 0.5 μ L, 10 μ mol/L upstream outer primer F3(are shown in SEQ ID NO.1), 0.5 μ L, 10 μ mol/L downstream outer primer B3(are shown in SEQ ID NO.2), 1 μ L, 40 μ mol/L upstream inner primer FIP(are shown in SEQ ID NO.3), 1 μ L, 40 μ mol/L downstream inner primer BIP(are shown in SEQ ID NO.4), 0.6 μ L100mmol/L MgSO
4, 12.5 μ L2mol/L trimethyl-glycines, 4 μ L sterilization distilled water ddH
2O, the cDNA of 1 μ L Bst archaeal dna polymerase (8U/ μ L) and 1 μ L step (3), reaction conditions is hatched 60min for being 65 ℃ in temperature.
The detection primer sets is:
Upstream outer primer F3:5 '-ACCACTCCTTAATGCACAA-3 ';
Downstream outer primer B3:5 '-GCCATCCTTTGATTAAAAATAGCT-3 ';
Upstream inner primer FIP:5 '-CCTCTCGCGTTGAGTTCGTAT-GGAATAAATCTTCCGATTACTGG-3 ';
Downstream inner primer BIP:5 '-ATGTTTGTATTACCCAACTGAAGCA-AGAAAGTGTATCCTTCCATGAA-3 '.
(5) color developing detection of amplified production
Add 1 μ L, 10% SYBR Green I developer in above-mentioned reaction tubes, room temperature is placed 10min, and the colour-change that detects by an unaided eye becomes green such as color, then contains rotavirus in the interpret sample; If color is yellow, illustrate that sample to be checked does not contain rotavirus.
Its result is: negative control (template is water) yellow, do not contain rotavirus, No. 2 samples are the clinical diarrhoea sample of present embodiment, its color all is yellow, do not contain rotavirus, the clinical diarrhoea sample of other portion that No. 3 samples are present embodiments, its color all are green, contain rotavirus.This and coming to the same thing of obtaining by the detection of conventional RT-PCR detection method.
Embodiment 2: specific test
Sample with 1 known rotavirus feminine gender and 2 known colyliform virus-positives, RT-LAMP method according to embodiment 1 detects again, the result shows, the sample of known negative, still present feminine gender in the LAMP detected result, the sample of the known positive 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.
Embodiment 3: sensitivity test
With the rotavirus RNA gradient dilution, carry out the color developing detection of reverse transcription, ring mediated isothermal amplification and amplified production according to the method for step 3-5 among the embodiment 1, verify sensitivity of the present invention, detected result is: negative control (template is water) is for yellow, do not contain rotavirus, positive control (rotavirus that RT-PCR is positive and the order-checking conclusive evidence is positive) is green, contain rotavirus, and its color of rotavirus RNA sample of 100pg, 10pg, 1pg, 0.1pg template amount all is green, all positive, determine that its detection sensitivity is 0.1pg.
Claims (5)
1. use the detection primer sets that ring mediated isothermal amplification detects rotavirus (rotavirus) for one kind, it is characterized in that, formed by following primer:
Upstream outer primer F3:5 '-ACCACTCCTTAATGCACAA-3 ';
Downstream outer primer B3:5 '-GCCATCCTTTGATTAAAAATAGCT-3 ';
Upstream inner primer FIP:5 '-CCTCTCGCGTTGAGTTCGTAT-GGAATAAATCTTCCGATTACTGG-3 ';
Downstream inner primer BIP:5 '-ATGTTTGTATTACCCAACTGAAGCA-AGAAAGTGTATCCTTCCATGAA-3 '.
2. the detection method of the rotavirus of the diagnosis of a non-disease and therapeutic purpose, it is characterized in that, sample is carried out viral RNA to be extracted, reverse transcription becomes cDNA again, then with detection primer sets claimed in claim 1 cDNA is carried out selective amplification by the method for ring mediated isothermal amplification, be confirmed whether to have amplified production.
3. detection method according to claim 2 is characterized in that, described sample is diarrhoea sample, water or the food of hospital.
4. detection method according to claim 2, it is characterized in that, described method by ring mediated isothermal amplification is carried out selective amplification with detection primer sets claimed in claim 1 to cDNA and is specially: the ring mediated isothermal amplification system is system cumulative volume 25 μ L, comprises 2.5 μ L, 10 * Thermopol reaction buffer, 0.4 μ L 10mmol/LdNTPs, 0.5 μ L10 μ mol/L upstream outer primer F3,0.5 μ L 10 μ mol/L downstream outer primer B3,1 μ L, 40 μ mol/L upstream inner primer FIP, 1 μ L, 40 μ mol/L downstream inner primer BIP, 0.6 μ L100mmol/L MgSO
4, 12.5 μ L 2mol/L trimethyl-glycines, 4 μ L sterilization distilled water ddH
2O, 1 μ L 8U/ μ L Bst archaeal dna polymerase and 1 μ L cDNA, reaction conditions is hatched 60min for being 65 ℃ in temperature.
5. according to claim 2 or 3 or 4 described detection methods, it is characterized in that, described to be confirmed whether to have amplified production be to detect with fluorescence developing, specifically adds 1 μ L, 10% SYBR Green I developer, observations behind the 10min in the reaction tubes of termination reaction, if color is yellow, then the sample rotavirus is negative, without rotavirus, if color is green, then the sample rotavirus is positive, contains rotavirus.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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| CN101245350A (en) * | 2007-02-17 | 2008-08-20 | 王健伟 | Encoding nucleotide sequence of codons optimizing rotavirus protein, recombinant and uses thereof |
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| 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 |
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