CN1814790A - Rotavirus RT PCR detecting kit and its detecting method - Google Patents
Rotavirus RT PCR detecting kit and its detecting method Download PDFInfo
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- CN1814790A CN1814790A CN 200510033159 CN200510033159A CN1814790A CN 1814790 A CN1814790 A CN 1814790A CN 200510033159 CN200510033159 CN 200510033159 CN 200510033159 A CN200510033159 A CN 200510033159A CN 1814790 A CN1814790 A CN 1814790A
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Abstract
Thisa invention relates to a semi-set RT-PCR test reagent box for testing and verifying wheel-like virus including a MLV inverse transcriptase, Rnasin, DNA poly-enzyme, primers, dNTP, MgCl<SUB>2</SUB> and 10 x buffer solution, in which, the upstream primer P1 is 5'-GTA TGG TAT TGA ATA TAC CAC-3', the downstream primer P2 is 5'-GAT CCT GTT GGC CAT CC-3. The RT-PCR test method includes: picking up a due test sample template, adding MLV inverse transcriptases, Rnasin, DNA poly-enzymes, primers, dNTP, MgCl<SUB>2</SUB>, 10 x buffer solution, the due test sample template and DECP water to be mixed uniformly to be enlarged on a PRC instrument, then the product is electrophoresis-enlarged in a device to get a result, which is recorded, analyzed and judged.
Description
[affiliated technical field]
The present invention relates to a kind of RT-PCR detection kit and using method thereof that detects and identify rotavirus, belong to biological technical field.
[background technology]
The human rotavirus belongs to arc reovirus virus section rotavirus.The rotavirus that can cause the people to suffer from diarrhoea has A, B, three groups of C.Wherein A group is the infantile diarrhea rotavirus, and B, C group are for becoming the human rotavirus.No matter in developed country or developing country, the diarrhoea that the A rotavirus causes all has higher sickness rate, is that the infant falls ill and the lethal main cause of disease, in China, situation is more serious, and the diarrhoea that infantile diarrhea rotavirus and adult diarrhea rotavirus cause is all very popular.According to the WHO1997 statistics, there are 1,400,000,000 rotavirus infection persons in the whole world every year, in developing country 870,000 people so and dead is arranged.In the U.S. 3,500,000 rotavirus infection persons there is every year, loses nearly 2,500 ten thousand dollars.The rotavirus infection onset is anxious, and arrive tens of times and do not wait the every day ten of suffering from diarrhoea, and normal companion gently or moderate is dewatered or metabolic is poisoned, and some cases often has upper respiratory tract infection symptoms before symptom of digestive tract occurring.The course of disease is about a general week, but the minority infant still has the disaccharide malabsorption in a short time, suffers from diarrhoea sustainable several weeks, is the several months individually.
Rotavirus mainly infects by fecal-oral route, and the water source of pollution and food are important contagium.Wherein shellfish is because its life water body is easy to be subjected to the pollution of human feces, and virus is had inrichment, becomes rotavirus and eats the high-risk food that the source sexuality is dyed.In addition, the infective dose of rotavirus is very low, is estimated as 10-100 virus particle.Stronger to the physical and chemical factor resistibility, ether-resistant and weak acid all can not make its inactivation with chloroform, multigelation, ultrasonication.Heating just can make its inactivation in 1 hour under 56 degrees centigrade, but prolonged preservation under-20 degrees centigrade.
In recent years, the outburst of many food origin diseases all causes by virus, food-borne virus be meant can be in food stable existence, and be carrier and the virus of disease mediated generation with it.Along with improving constantly of standard of living, the security of food is more and more paid close attention to, but for a long time, there is very big careless omission in detection viral in the food always, even approaches blank.Existing food hygiene standard can't describe the pollution situation of virus in the food, and irreproducible in food though virus is strict cytozoon, the virus of trace will cause disease in the food.Show according to foreign data, food-borne virus and the disease that causes thereof have suitable number and kind, it is wide wherein to be no lack of popular scope, infectivity is strong, easily outburst is easily popular, endangers serious disease, and this is not only the risk factor in the food sanitation field, to the significant threat of people's public health and nutrient health level, and foodstuffs industry and national economy brought more serious loss.
Rotavirus is a kind of important food-borne virus, the detection method of virus has electron microscopic observation, cell cultures, nucleic acid hybridization, enzyme linked immunological and polymerase chain reaction in the conventional food, but the sensitivity of electron microscopic observation, nucleic acid hybridization and enzyme-linked immune detection method is relatively all very low, can't be applied to separately detect; The operation of cell culture method is loaded down with trivial details, take longer, generally need just can observe in a week cell pathology reaction, therefore, develop molecular Biological Detection method quick, highly sensitive, easy and simple to handle and test kit thereof for the detection of eating source property rotavirus with prevent significant.
[summary of the invention]
The objective of the invention is to overcome the weak point in the existing traditional detection technology, provide a kind of be used to detect rotavirus highly sensitive, speed is fast, easy, accurate, PCR detection kit that sense cycle is short.
The PCR detection method of rotavirus is designed the very strong primer of specificity by using the pcr amplification principle according to the known viruse gene order, is that template amplification goes out special gene fragment with the viral RNA of extremely low concentration, thereby reaches the purpose that detects virus.The present invention is after setting up the RT-PCR detection method of rotavirus, with the used reagent of experiment and medicine make up with the optimization experiment condition after, but but made up the PCR detection kit that a kind of rapid detection rotavirus and industrialization are produced.The combination of components that the PCR detection method need be used during use, only needs to extract testing sample together, adds this test kit reagent, just can carry out quick, the easy detection of sensitivity through comparatively simple operation program.And adopt this test kit to detect the employed experimental installation of rotavirus, and the detection of other virus in food, clinical sample, the environmental sample is had good practicability, also lay a good foundation for tracing to the source fast of burst disease.Experiment shows, use PCR detection kit provided by the present invention to detect rotavirus, a sample generally only needs 3-4h can finish detection, the traditional method of comparing, sense cycle shortens greatly, and sensitivity also is greatly improved, can detect the sample nucleic acid about 10pg, it is long to have overcome conventional sense method sense cycle, and sensitivity is low, the shortcomings such as needs that incompatibility market is detected.
Purpose of the present invention can realize by following scheme:
The PCR detection kit of rotavirus of the present invention comprises following reagent: MLV ThermoScript II, Rnasin, archaeal dna polymerase, primer, dNTP, MgCl
2, 10 * damping fluid, DEPC water.Wherein upstream primer P1 is: 5 '-GTA TGG TAT TGA ATA TAC CAC-3 ', downstream primer P2 is: 5 '-GAT CCT GTT GGC CATCC-3 ', these two primers are the general primers of each serotype according to the high conserved region section design of rotavirus vp7 gene, pairing base position is respectively 51-71bp, 376-392bp.
The invention provides a kind of composition of rotavirus P CR detection kit: rotavirus positive control 20ul, rotavirus negative control 20ul, MLV ThermoScript II 10ul, Rnasin10ul, archaeal dna polymerase 15ul, primer 100ul, 2mmol/LdNTP50ul, 25mmol/LMgCl
250ul, 10 * damping fluid 100ul, DEPC water.The dosage that is added during concrete the detection is: rotavirus positive control 1ul, rotavirus negative control 1ul, MLV ThermoScript II 1ul, Rnasin0.5ul, archaeal dna polymerase 1.5ul, primer 4ul, 2mmol/LdNTP2.5ul, 25mmol/LMgCl
22.5ul, 10 * damping fluid 5ul, DEPC water 6ul.Used archaeal dna polymerase is TaqE.
The present invention also provides the using method of this rotavirus detection kit:
(1) extraction of rotavirus nucleic acid
A. get the sample of 200-300ul, add 900ulTrizol reagent, with pipettor repeatedly mixing leave standstill 5min several times;
B. add the 200ul chloroform, firmly jolting 15S leaves standstill 2-3min again;
C.4 ℃, 12000g, centrifugal 15min discards supernatant liquid;
D. add the 500ul Virahol, abundant mixing, room temperature is placed 10min;
E.4 ℃, 12000g, centrifugal 10min, supernatant discarded once more;
F. add 75 ethanol, jolting, thorough washing precipitation;
G.4 ℃, 7500g, centrifugal 5min carefully discards ethanol;
H. after the thorough drying, the RNA precipitation is suspended in the water of an amount of no RNA enzyme.
(2) pcr amplification
Concrete process is as follows: the first step adds the reaction solution of RT-PCR in the PCR thin-walled tube, comprises MLV ThermoScript II, Rnasin, archaeal dna polymerase, P1, P2 primer, dNTP, MgCl
2, 10 * damping fluid, testing sample template and DEPC water, totally be 25ul, increase on the rearmounted PCR instrument of mixing.
RT-PCR amplification comprises that the first chain cDNA of 42 ℃ of following 30min is synthetic, 94 ℃ of sex change 5min and then, and at 94 ℃ of 40S, 60 ℃ of-50 ℃ of 50S, 72 ℃ of 40S circulations 30 times, 72 ℃ are extended 7min, fully extend to guarantee amplified production.
(3) electrophoresis, PCR product sequencing
Get amplified production 5ul twice, the sepharose of preparation 1.4% directly carries out electrophoresis, observations in the ultraviolet imagery system, and with the contrast of 100bp standard molecular weight, the PCR positive findings is 342bp.
Using the purpose of positive or negative contrast is the electrophoresis result that is used for the comparison amplified production, whether contains rotavirus so that judge testing sample.If contain rotavirus, then can observe the band identical with the positive findings control site from electrophoresis result; If do not contain rotavirus, then the same with negative control do not have this band.
[description of drawings]
Fig. 1 is among the embodiment one: rotavirus detected result in the ight soil.M:Marker; The 1-7:RT-PCR detected result has a band at the 342bp place, shows the rotavirus detected result positive.
Fig. 2 is among the embodiment two: shellfish sample detection result.M:Marker; The 1-7:RT-PCR detected result has a band at the 342bp place, shows the rotavirus detected result positive.
[embodiment]
Embodiment one: the detection of rotavirus in the ight soil
(1) extract sample nucleic acid with the Trizol test kit: get the 200-300ul sample, add 900ulTrizol reagent, with pipettor repeatedly mixing leave standstill 5min several times; Add the 200ul chloroform then, firmly jolting 15S leaves standstill 2-3min again; 4 ℃ of centrifugal 15min of 12000g discard and add the 500ul Virahol behind the supernatant liquid, abundant mixing, and room temperature is placed 10min; 4 ℃, 12000g, centrifugal 10min, supernatant discarded once more; The ethanol of adding 75, jolting, thorough washing precipitation; 4 ℃, 7500g, centrifugal 5min carefully discards ethanol; After the thorough drying, the RNA precipitation is suspended in the water of an amount of no RNA enzyme.
(2) draw MLV ThermoScript II 1ul, archaeal dna polymerase 1ul, Rnasin0.5ul, primer 4ul, 2mmol/LdNTP2.5ul, 10 * damping fluid 2.5ul, 25mmol/LMgCl in the PCR test kit respectively with microsyringe
24Ul, the testing sample nucleic acid of adding 1ul is supplied volume to 25ul with DEPC water then, fully mixing.
(3) set up the rotavirus positive and negative control simultaneously, institute's dosage is the same, just changes sample nucleic acid into provide in the test kit positive control and negative control.
(4), on the PCR instrument, increase: 42 ℃ of following 30min, and then 94 ℃ of sex change 5min according to following temperature and time with after the mixture high speed centrifugation several seconds, at 94 ℃ of 40S, 60 ℃ of-50 ℃ of 50S, 72 ℃ of 40S landing circulate 30 times, 72 ℃ are extended 7min, fully extend to guarantee amplified production.
(5) get the 5ul amplified production, sepharose electrophoresis in electrophoresis equipment of preparation 1.4%.
(6) on the gel imaging instrument, observe and write down experimental result.
(7) carrying out the result according to following result judges: the rotavirus positive findings should have a band at the 342bp place.
(8) electrophoresis result as shown in Figure 1.
Embodiment two: the detection of rotavirus in the shellfish
(1) Bing Du biomagnification
Buy shellfish from the aquatic products market of this locality, it is put in a suitable place to breed in a container that fills seawater, throw in the rotavirus sample then in container, the environment of simulating nature water body makes the enrichment of shellfish nature.Simultaneously, set up negative control with same condition (not poisoning).Change water and poisoning again once every 8h, the whole biomagnification time is 24h.
(2) activation of virus and concentrated in the shellfish
With top shellfish through biomagnification, five shellfishes are a sample, peel off, get its stomach and digestive tube 1.5g, change over to then in the Falcon pipe of 50ml, add the cold 0.05mol/L glycine-0.14mol/L sodium-chlor (pH7.5) of 15ml, use Waring blender then at high-speed homogenization on ice through sterilization.In order to prevent crossed contamination, the alcohol of application 70% is sterilized to Waring blender, and heats 1min with Bursen beuner after each sample homogenization.Concrete process is as follows:
(a): 4 ℃, 5000g, centrifugal 20min collects supernatant, deposits for 4 ℃;
(b): precipitation is suspended in 15ml again, in the sodium-chlor of Threonine-0.14M of the 0.5M of PH7.5, vortex 60s;
(c): 4 ℃, 5000g, centrifugal 20in.Twice supernatant liquor mixed, place another Falcon pipe, discard precipitation;
(d): in supernatant liquor, add 15mlPEG6000 (12%wt/vol, i.e. PEG6000, the sodium-chlor of 0.3M), place 2h down at 4 ℃;
(e): 8000g, 4 ℃, centrifugal 30min abandons supernatant.To precipitate among the PBS that is suspended in 15ml again (PH7.5), add the chloroform of 15ml again, vortex 60s;
(f): 4 ℃, the centrifugal 30min of 2000g gets supernatant;
(g): add the PEG of 7.5ml12% again, placed two hours down for 4 ℃;
(h): 4 ℃, 14000g, centrifugal 15min gets supernatant;
(i): PEG precipitation bead is used 1ml again, the guanidinium isothiocyanate solution resuspending of 6M, and room temperature is placed 10min;
(j): 4 ℃, the centrifugal 10min of 12000g gets supernatant, is used for the RNA extracting.
(3) RNA extracting
With the Trizol test kit that U.S. Life Technologies, Inc. produces, the viral RNA in the extracting shellfish enriched material.Get the 200-300ul sample, add 900ulTrizol reagent, with pipettor repeatedly mixing leave standstill 5min several times; Add the 200ul chloroform then, firmly jolting 15S leaves standstill 2-3min again; 4 ℃ of centrifugal 15min of 12000g discard and add the 500ul Virahol behind the supernatant liquid, abundant mixing, and room temperature is placed 10min; 4 ℃, 12000g, centrifugal 10min, supernatant discarded once more; The ethanol of adding 75, jolting, thorough washing precipitation; 4 ℃, 7500g, centrifugal 5min carefully discards ethanol; After the thorough drying, the RNA precipitation is suspended in the water of an amount of no RNA enzyme.On spectrophotometer, measure RNA concentration and the purity extracted.
(4) test kit detects
(a) draw MLV ThermoScript II 1ul, archaeal dna polymerase 1ul, Rnasin0.5ul, primer 4ul, 2mmol/LdNTP2.5ul, 10 * damping fluid 2.5ul, 25mmol/LMgCl in the PCR test kit respectively with microsyringe
24ul adds the testing sample nucleic acid of 1ul, supplies volume to 25ul with DEPC water then, fully mixing.
(b) set up the rotavirus positive and negative control simultaneously, institute's dosage is the same, just changes sample nucleic acid into provide in the test kit positive control and negative control.
(c), on the PCR instrument, increase: 42 ℃ of following 30min, and then 94 ℃ of sex change 5min according to following temperature and time with after the mixture high speed centrifugation several seconds, at 94 ℃ of 40S, 60 ℃ of-50 ℃ of 50S, 72 ℃ of 40S landing circulate 30 times, 72 ℃ are extended 7min, fully extend to guarantee amplified production.
(d) get the 5ul amplified production, sepharose electrophoresis in electrophoresis equipment of preparation 1.4%.
(e) on the gel imaging instrument, observe and write down experimental result.
(f) carrying out the result according to following result judges: the rotavirus positive findings should have a band at the 342bp place.
(g) electrophoresis result as shown in Figure 2.
Claims (10)
1. the RT-PCR detection kit of rotavirus, comprise MLV ThermoScript II, Rnasin, archaeal dna polymerase, primer, dNTP, 10 * damping fluid, it is characterized in that, upstream primer wherein is: 5 '-GTA TGG TATTGA ATA TAC CAC-3 ', downstream primer is: 5 '-GAT CCT GTT GGC CAT CC-3 '.
2. the RT-PCR detection kit of the described rotavirus of claim 1 comprises rotavirus positive control 20ul, rotavirus negative control 20ul, MLV ThermoScript II 10ul, Rnasin10ul, archaeal dna polymerase 15ul, primer 100ul, 2mmol/LdNTP50ul, 25mmol/LMgCl
250ul, 10 * damping fluid 100ul, DEPC water 1ml.
3. the RT-PCR detection kit of the described rotavirus of claim 1, employed amount is when it is characterized in that one-time detection: MLV ThermoScript II 1ul, Rnasin0.5ul, archaeal dna polymerase 1.5ul, primer 4ul, 2mmol/LdNTP2.5ul, 25mmol/LMgCl
22.5ul, 10 * damping fluid 5ul, remaining volume is supplied 25ul with DEPC water after the amount of removing the 1ul test sample.
4. the RT-PCR detection kit of the described rotavirus of claim 1-3, used archaeal dna polymerase is TaqE.
5. the RT-PCR detection method of rotavirus, it detects step and comprises:
(1) extracts the sample rotavirus nucleic acid with the Trizol test kit
(2) draw MLV ThermoScript II, archaeal dna polymerase, Rnasin, primer, dNTP, 10 * damping fluid, MgCl in the PCR test kit respectively with microsyringe
2, testing sample nucleic acid, DEPC water in the PCR thin-walled tube, abundant mixing.
(3) set up the rotavirus positive and negative control simultaneously, it is the same that institute adds reagent, just changes sample nucleic acid into provide in the test kit positive control and negative control.
(4) mixture is increased on the PCR instrument.
(5) in electrophoresis equipment, carry out electrophoresis, observe The results in electrophoresis with the gel imaging instrument then.
(6) analyze and carry out the result and judge.
6. the RT-PCR detection method of the described rotavirus of claim 5 is characterized in that, the pcr amplification parameter is: 42 ℃ of following 30min, and 94 ℃ of sex change 5min and then, at 94 ℃ of 40S, 60 ℃ of-55 ℃ of 50S, 72 ℃ of 40S landing circulations 30 times, 72 ℃ are extended 7min.
7. the RT-PCR detection method of the described rotavirus of claim 5, it is characterized in that the amount that detects used reagent is: rotavirus positive control 1ul, rotavirus negative control 1ul, MLV ThermoScript II 1ul, Rnasin0.5ul, archaeal dna polymerase 1.5ul, primer 4ul, 2mmol/LdNTP2.5ul, 25mmol/LMgCl
22.5ul, 10 * damping fluid 5ul, remaining volume is supplied 25ul with DEPC water after the amount of removing the 1ul test sample.
8. the RT-PCR detection method of the described rotavirus of claim 5 is characterized in that adopting the Trizol test kit to extract sample nucleic acid: get quantity of sample, add 900ulTrizol reagent, with pipettor repeatedly mixing leave standstill 5min several times; Add the 200ul chloroform then, firmly jolting 15S leaves standstill 2-3min again; 4 ℃ of centrifugal 15min of 12000g discard and add the 500ul Virahol behind the supernatant liquid, abundant mixing, and room temperature is placed 10min; 4 ℃, 12000g, centrifugal 10min, supernatant discarded once more; The ethanol of adding 75, jolting, thorough washing precipitation; 4 ℃, 7500g, centrifugal 5min carefully discards ethanol; After the thorough drying, the RNA precipitation is suspended in the water of an amount of no RNA enzyme.
9. the RT-PCR detection method of the described rotavirus of claim 5 is characterized in that, at the electrophoresis equipment amplified production, record result's concrete steps are:
(1) gets the 5-10ul amplified production, prepare sepharose electrophoresis in electrophoresis equipment of 1.4%, select the Marker of 1000bp for use.
(2) on the gel imaging instrument, observe and write down experimental result.
10. according to the RT-PCR detection method of claim 5 or the described rotavirus of claim 9, it is characterized in that the rotavirus positive findings should have a band at the 342bp place.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100434535C (en) * | 2007-05-24 | 2008-11-19 | 清华大学 | Method of quantificationally detecting rota virus and special-purpose standard substance for the same |
| CN102260752A (en) * | 2011-08-25 | 2011-11-30 | 成都新基因格生物科技有限公司 | Composition, kit and method for detecting group A rotavirus |
| CN101660001B (en) * | 2008-08-27 | 2011-12-14 | 中山大学达安基因股份有限公司 | Reagent kit for detecting rotavirus nucleic acid |
| CN102382907A (en) * | 2011-11-10 | 2012-03-21 | 中华人民共和国大榭出入境检验检疫局 | Degenerate reverse transcription-polymerase chain reaction (RT-PCR) detection reagent and kit for hantavirus group |
| CN102382906A (en) * | 2011-11-10 | 2012-03-21 | 中华人民共和国大榭出入境检验检疫局 | Degenerate RT-PCR detection method of Hantavirus group |
| 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 |
| 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 |
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- 2005-02-06 CN CN 200510033159 patent/CN1814790A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100434535C (en) * | 2007-05-24 | 2008-11-19 | 清华大学 | Method of quantificationally detecting rota virus and special-purpose standard substance for the same |
| CN101660001B (en) * | 2008-08-27 | 2011-12-14 | 中山大学达安基因股份有限公司 | Reagent kit for detecting rotavirus nucleic acid |
| 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 |
| CN102260752A (en) * | 2011-08-25 | 2011-11-30 | 成都新基因格生物科技有限公司 | Composition, kit and method for detecting group A rotavirus |
| 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 |
| CN102382907A (en) * | 2011-11-10 | 2012-03-21 | 中华人民共和国大榭出入境检验检疫局 | Degenerate reverse transcription-polymerase chain reaction (RT-PCR) detection reagent and kit for hantavirus group |
| CN102382906A (en) * | 2011-11-10 | 2012-03-21 | 中华人民共和国大榭出入境检验检疫局 | Degenerate RT-PCR detection method of Hantavirus group |
| 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 |
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