CN101570798B - Detection kit and detection method for 3 species of food-borne viruses in marine products - Google Patents
Detection kit and detection method for 3 species of food-borne viruses in marine products Download PDFInfo
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- CN101570798B CN101570798B CN2009100108049A CN200910010804A CN101570798B CN 101570798 B CN101570798 B CN 101570798B CN 2009100108049 A CN2009100108049 A CN 2009100108049A CN 200910010804 A CN200910010804 A CN 200910010804A CN 101570798 B CN101570798 B CN 101570798B
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Abstract
The invention discloses a detection kit and a detection method for 3 species of food-borne viruses in marine products. The kit comprises reverse transcription Tag DNA polymerase with a concentration of 5U/mu L, inverse transcriptase with a concentration of 5U/muL and an RT-PCR reaction solution, wherein the RT-PCR reaction solution contains 10 millimols of Tris.HCl, 50 millimols of KCl, 25 millimols of MgCl2, 10 millimols of dNTP, a ribonuclease inhibitor with a concentration of 40 U/mu L, and 20 mu mols of downstream primer pair and 20 mu mols of upstream primer pairs of the food-borne viruses. The kit can synchronously detect hepatitis A viruses, rotaviruses and norwalk viruses. The kit has extremely high sensitivity, can detect 1*10<-7> nano gram of viral nuclei and is superior to the prior reported detection method for viruses in the marine products. In addition, the method is short in detection time, simple and quick, and suitable for quick detection.
Description
Technical field
The present invention relates to be used for the test kit that three kinds of food-borne virus of fishery products detect, relate in particular to the test kit that utilizes multiple virus in multiplex PCR and sex change high performance liquid chromatography (DHPLC) the technology for detection fishery products.The method that also relates to detection.
Background technology
Hepatitis A virus (HAV), rotavirus, Norwalk virus are common food-borne virus in the fishery products.They very easily contaminate environment, food, water source, sea-food (as blood clam etc.), tableware, process for processing with utensil etc. and healthy people's hand.And then cause the susceptible person to infect outburst ill or the initiation area pandemic.Since the globalization of foodstuff production development, the application of new food production process, and the various factorss such as change of people's food habits make the sickness rate of food-borne viral disease more and more strong to a certain extent.According to authoritative document announcement, the food origin disease that virus causes is one of principal element that influences food safety.In recent years, worldwide pernicious, the unexpected incidents of food safety aspect take place repeatedly.Show that according to The World Health Organization's statistical report the actual sickness rate of food origin disease is Duoed 300～500 times than the cases reported number, global trouble patient is several hundred million to reach, and annual statistical value all remains high, and has consumed lot of manpower and material resources.Yet, detect differently with the virus of routine, the contained pathogenic virus in the food is generally all multiple and a small amount of, and these viruses often are difficult to enrichment and vitro culture, and the detection of this decision food-borne virus is difficult point and the blank spot in the microorganism detection always.All there are some problems in existing detection technique such as microorganism culturing and biochemical identification, immunological technique, round pcr etc.: conventional biochemical identification method complicated operation, and length consuming time, often the one-time detection experiment can only be identified a kind of or a few bacterium; Though immunological technique susceptibility height easily pollutes, and the false positive phenomenon often occurs; Round pcr generally will with the gel electrophoresis technology coupling, also only can reach virus is detected, do not possess high-throughout characteristics.At present,, still do not have complete cause of disease to detect and identification of means, especially be applicable to the detection technique that quick, many bacterial classifications detect simultaneously, therefore suddenly wait to develop for food-borne virus disease in the fishery products.
The present invention promptly is intended to utilize mPCR and DHPCL technology, and foundation can be fast, the Fast Detection Technique of 3 kinds of common food-borne virus in sensitive, the high well-illuminated detection fishery products.
Summary of the invention
The object of the present invention is to provide a kind of 3 kinds of common food-borne virus of sensitive rapid detection fishery products, i.e. mPCR-DHPLC detection kit of hepatitis A virus, rotavirus and Norwalk virus of being used for.
3 kinds of food-borne virus detection kit comprise that concentration is that reverse transcription-Tag archaeal dna polymerase, the concentration of 5U/ μ L is ThermoScript II and the RT-PCR reaction solution of 5U/ μ L in the fishery products of the present invention; Contain 10mM TrisHCl, 50mM KCl, 25mM MgCl in the RT-PCR reaction solution wherein
2, each 10mM of dNTP (dATP, dGTP, dCTP and dTTP), RNA enzyme inhibitors 40U/ μ L and 3 kinds of food-borne virus the upstream and downstream primer to each 20 μ M;
Described primer sequence is as follows:
Test kit preservation condition :-20 ℃ of preservations.
The present invention also provides the method for utilizing the mentioned reagent box to detect above-mentioned 3 kinds of food-borne virus in the fishery products, comprises the steps:
1. get 2 μ l testing sample RNA solution, add PCR reaction solution, 1.0 μ L reverse transcription-Tag archaeal dna polymerases and 1.0 μ L ThermoScript II in the 22 μ l test kits, sterilization ultrapure water 24 μ L, cumulative volume 50 μ L; The centrifugal 10s of 5000r/min, carry out the multiple RT-PCR amplification according to following condition then:
Reverse transcription: RT reacts 42 ℃ of 30min, 94 ℃ of 2min of RTase inactivation;
Cyclic amplification: 94 ℃ of sex change 60s, 48 ℃ of annealing 2min, 72 ℃ are extended 3min, 35 circulations;
Stop extending: 72 ℃ of 10min;
2. the RT-PCR product is carried out DHPLC and analyze, the DHPLC analysis condition is as follows:
Chromatographic column: PS-DVB ﹠amp; C18 DNASep chromatographic column, 4.6mm * 50mm, granularity 3 μ m;
Column temperature: 50 ℃;
Moving phase (volume percent): 0.0min, 55.0% buffered soln A, 45.0% buffered soln B,
0.5min, 50.2% buffered soln A, 49.8% buffered soln B,
3.0min, 42.9% buffered soln A, 57.1% buffered soln B,
5.5min, 39.4% buffered soln A, 60.6% buffered soln B,
8.0min, 37.3% buffered soln A, 62.7% buffered soln B,
10.5min, 36.0% buffered soln A, 64.0% buffered soln B,
Wherein, buffered soln A is that 50ml TEAA and 250 μ l acetonitriles mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution; Buffered soln B is that 50ml TEAA and 250ml acetonitrile mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution;
Flow velocity: 0.9mL/min;
Detector: fluorimetric detector, light source: 150W Xenon lamp; Excitation spectrum bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR product 10 μ L.
Utilize test kit of the present invention and detection method can detect hepatitis A virus in the fishery products, rotavirus and 3 kinds of viruses of Norwalk virus simultaneously.The detection sensitivity height can detect 1 * 10
-7The ng viral nucleic acid promptly is equivalent to detect 30 virus particle, method for detecting virus in the fishery products that its sensitivity is better than being reported at present.Use this test kit can realize multinomial order food-borne virus high throughput testing in the based food, solve the problem of the multiple food-borne virus of rapid detection simultaneously.
Description of drawings
Accompanying drawing 5 width of cloth of the present invention, wherein:
Fig. 1 is the specificity test-results spectrogram of embodiment 2;
Fig. 2 is the sensitivity test spectrogram as a result of embodiment 3;
Fig. 3 is the Precision test result spectrogram of embodiment 4;
Fig. 4 is the test-results spectrogram that is used for hepatitis A virus (HAV) and Norwalk virus artificial contamination sample detection in the test of embodiment 5 simulating pollution sample detection;
Fig. 5 is the test-results spectrogram that is used for rotavirus and Norwalk virus artificial contamination sample detection in the test of embodiment 5 simulating pollution sample detection;
In the above-mentioned accompanying drawing, X-coordinate is retention time, and (unit: minute min), ordinate zou is represented absorption peak strength of signal (unit: millivolt mV).
Embodiment
Be specific embodiments of the invention below, its foundation and application thereof to present method is further described, but does not limit content of the present invention in any form.
If no specified otherwise, the employed main agents in this part, instrument and merchandise resources thereof are: reagent such as RNA/DNA a small amount of purification kit (Takara MiniBEST Viral RNA/DNA Extraction Kit), RT-PCR reaction solution, AMV-Optimized Tag polysaccharase, AMV RTase XL are all available from precious biotechnology (Dalian) company limited; Triethylamine acetyl salt (TEAA, chromatographically pure) is available from Transgenomic company; Acetonitrile (chromatographically pure) is available from Fisher company; Regular-PCR instrument PE24000 (PerkinElmer company, the U.S.); Sex change high performance liquid chromatograph NAV-99-4500 (Transgenomic company, the U.S.); Supercentrifuge centrifuge 5804 (Eppendorf company, Germany).
Test related virus strain source in this part is respectively: HAV virus is separated from the hepatitis A patient specimen of having made a definite diagnosis during 2006～2007 years for The Central Hospital of Dalian City, specimen inoculation monkey embryonic kidney passage cell (FRHK4) cultivation of going down to posterity, behind propagation 1～3d, cell dissociation is disperseed to make the antigen sheet, check intracellular antigen with indirect immunofluorescence again, detect the hepatitis A virus infection titer.
Rotavirus is that Dalian Medical Univ separated from the patient specimen of the rotavirus infection of having made a definite diagnosis during 2006～2007 years, the specimen inoculation rhesus monkey embryonic kidney cells MA104 strain cultivation of going down to posterity, behind propagation 1～3d, cell dissociation is disperseed to make the antigen sheet, check intracellular antigen with indirect immunofluorescence again, detect the rotavirus infection titre;
The specific gene fragment of hepatitis A virus, Norwalk virus and the rotavirus that DHPLC is analyzed is carried out fragment and is reclaimed, the DNA purifying, and the clone makes up the cloned plasmids pMD18-T that contains hepatitis A virus, Norwalk virus and rotavirus target sequence.
Norwalk virus, poliovirus (I, II, III type, vaccine strain), Coxsackie virus (1-6 type, vaccine strain), pseudorabies virus and hepatitis B virus RNA or DNA are presented by Dalian City Clinical Examination Center.
Fishery products samples such as the employed shellfish fish shrimp crab in this part are during 2007～2008 years, from Dalian, Dandong etc. is coastal and respectively distinguish each big supermarket and gather the market of farm produce.Gather in the 2h of back and put into-20 ℃ of refrigerators.Testing location is delivered in refrigeration.
(1) foundation of test kit
The primer that present embodiment is identified for detecting is as shown in the table:
On this basis, be designed for 3 kinds of food-borne virus detection kit in the fishery products of pcr amplification:
This test kit comprises that concentration is that reverse transcription-Tag archaeal dna polymerase, the concentration of 5U/ μ L is ThermoScript II and the RT-PCR reaction solution of 5U/ μ L; Contain 10mM TrisHCl, 50mM KCl, 25mM MgCl in the RT-PCR reaction solution wherein
2, each 10mM of dNTP (dATP, dGTP, dCTP and dTTP), RNA enzyme inhibitors 40U/ μ L and above-mentioned 3 kinds of food-borne virus the upstream and downstream primer to each 20 μ M;
This test kit preservation condition :-20 ℃ of preservations.
The mPCR-DHPLC method of using this test kit to detect 3 kinds of food-borne virus in the fishery products sample comprises the steps:
1. the preparation of sample to be checked: adopt the test kit extraction method to prepare testing sample rna gene group:
A. the enriching method of hepatitis A virus (HAV): polyethylene glycol 6000 settling process
The cheek and the Digestive tract of water intaking product, 0.05mol/L glycine-NaCl with pH9.5 makes suspension, after-20 ℃ of freeze thawing, with 10 000r/min, in supernatant liquor, add 8% polyoxyethylene glycol 6 000 behind 4 ℃ of centrifugal 0.5h, 0.4mol/L NaCl 4 ℃ of joltings repeatedly, are abandoned supernatant and precipitation is dissolved in is made suspension among the PBS and promptly get sample at 4 ℃ of centrifugal half an hour of 10 000r/min again.
B. the enrichment of Norwalk virus
The mid intestinal gland tissue of getting the 5g shellfish adds 35mL glycine buffer (pH9.5).Homogenizer high-speed homogenization 3min, with the homogenate 50mL centrifuge tube of packing into, the 30min that vibrates under 37 ℃ of incubation 30min or the room temperature, 4 ℃, the centrifugal 30min of 10 000g.
Pipette supernatant and newly manage to 50mL, add isopyknic PEG 8 000 solution, put upside down mixing 5 times.Place 1h at least on ice, 4 ℃, the centrifugal 5min of 10000g, supernatant discarded keeps precipitation.
C. the enrichment of rotavirus
Get and add abundant broken mixing 2min in 70ml glycine buffer (example weight is 1: 7 with the ratio of the damping fluid volume) agitator in the 10g shellfish digestive tube tissue.Take out 30ml, hatch 30min for 37 ℃.15000g, 4 ℃ of centrifugal 20min.Ask liquid in the collection, add isopyknic PEG8000 solution, the sedimentation virus (the PEG final concentration is 8%) of spending the night on ice, 10000g, 4 ℃, centrifugal 5min.
More than three kinds of viral enriched substance adopt Takara MiniBEST Viral RNA/DNA Extraction Kit, extract the RNA of virus.Directly standby as RT-PCR template or-20 ℃ of preservations.
2. pcr amplification:
Get 2 μ l testing sample RNA solution, add PCR reaction solution, 1.0 μ L reverse transcription-Tag archaeal dna polymerases and 1.0 μ L ThermoScript II in the 22 μ l test kits, sterilization ultrapure water 24 μ L, cumulative volume 50 μ L; The centrifugal 10s of 5000r/min, carry out the multiple RT-PCR amplification according to following condition then:
Reverse transcription: RT reacts 42 ℃ of 30min, 94 ℃ of 2min of RTase inactivation;
Cyclic amplification: 94 ℃ of sex change 60s, 48 ℃ of annealing 2min, 72 ℃ are extended 3min, 35 circulations;
Stop extending: 72 ℃ of 10min;
3. DHPLC analyzes, and the DHPLC analysis condition is as follows:
Chromatographic column: PS-DVB ﹠amp; C18 DNASep chromatographic column, 4.6mm * 50mm, granularity 3 μ m;
Column temperature: 50 ℃;
Moving phase (volume percent): 0.0min, 55.0% buffered soln A, 45.0% buffered soln B,
0.5min, 50.2% buffered soln A, 49.8% buffered soln B,
3.0min, 42.9% buffered soln A, 57.1% buffered soln B,
5.5min, 39.4% buffered soln A, 60.6% buffered soln B,
8.0min, 37.3% buffered soln A, 62.7% buffered soln B,
10.5min, 36.0% buffered soln A, 64.0% buffered soln B,
Wherein, buffered soln A is that 50ml TEAA and 250 μ l acetonitriles mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution; Buffered soln B is that 50ml TEAA and 250ml acetonitrile mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution;
Flow velocity: 0.9mL/min;
Detector: fluorimetric detector, light source: 150W Xenon lamp; Excitation spectrum bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR product 10 μ L.
With numerous food virus reference material, the operation steps of simulation routine check is extracted nucleic acid according to the method that embodiment 1 is set up, and carries out mPCR-DHPLC and detects.
Extract the RNA of hepatitis A virus, Norwalk virus and rotavirus respectively, use poliovirus (I, II, the III type, vaccine strain) DNA and Coxsackie virus (1-6 type, vaccine strain) DNA, Pseudorabies Virus DNA and hepatitis B virus DNA are blank with the aqua sterilisa simultaneously.With the Auele Specific Primer of three viruses, the method for being set up according to embodiment 1 increases to extracting template, carries out the test of mPCR-DHPLC specificity.Detected result as shown in Figure 1.
The specificity test-results shows, detection kit that embodiment set up and detection method can detect three kinds of target virals specifically, have only hepatitis A virus, Norwalk virus and rotavirus to detect special sample peak, the detected result of other contrast virus is all negative, does not have false positive and false negative result.
Related virus strain source, this part is respectively: HAV virus is separated from the hepatitis A patient specimen of having made a definite diagnosis during 2006～2007 years for The Central Hospital of Dalian City, specimen inoculation monkey embryonic kidney passage cell (FRHK4) cultivation of going down to posterity, behind propagation 1～3d, cell dissociation is disperseed to make the antigen sheet, check intracellular antigen with indirect immunofluorescence again, detect the hepatitis A virus infection titer.
Rotavirus is that Dalian Medical Univ separated from the patient specimen of the rotavirus infection of having made a definite diagnosis during 2006～2007 years, the specimen inoculation rhesus monkey embryonic kidney cells MA104 strain cultivation of going down to posterity, behind propagation 1～3d, cell dissociation is disperseed to make the antigen sheet, check intracellular antigen with indirect immunofluorescence again, detect the rotavirus infection titre;
The specific gene fragment of hepatitis A virus, Norwalk virus and the rotavirus that DHPLC is analyzed is carried out fragment and is reclaimed, the DNA purifying, and the clone makes up the cloned plasmids pMD18-T that contains hepatitis A virus, Norwalk virus and rotavirus target sequence.
Norwalk virus, poliovirus (I, II, III type, vaccine strain), Coxsackie virus (1-6 type, vaccine strain), pseudorabies virus and hepatitis B virus RNA or DNA are presented by Dalian City Clinical Examination Center.
With the food virus reference material of known viruse amount, adopt embodiment 1 test kit of setting up and detection method to make template and mPCR-DHPLC detection, determine the sensitivity that detects.Table 1 be 3 kinds of viruses when the different vaccination amount, the genomic nucleic acids fragment of extraction, spectrophotometric instrumentation OD value is calculated the gained nucleic acid content.
For verifying the detection limit of this detection method, measure the positive plasmid pMD18-T concentration of (containing hepatitis A virus, Norwalk virus, rotavirus target sequence respectively) respectively with spectrophotometer, be diluted to 1 * 10 respectively then
-1Ng, 1 * 10
-2Ng, 1 * 10
-3Ng, 1 * 10
-4Ng, 1 * 10
-5Ng, 1 * 10
-6Ng, 1 * 10
-7Ng, 1 * 10
-8Ng extracts plasmid DNA according to the method for embodiment 1, carries out mPCR-DHPLC and detects.Following table (table 1) is the virus concentration of gradient dilution:
Table 1
Detected result as shown in Figure 2, the absorption of sample peak is followed successively by from high to low: 1 * 10
-4Ng positive plasmid, 1 * 10
-5Ng positive plasmid, 1 * 10
-6Ng positive plasmid, 1 * 10
-7The ng positive plasmid shows that the minimum detection limit of this multiplex PCR-DHPLC detection method is 1 * 10-7ng positive plasmid.Be multiplex PCR-DHPLC method that present embodiment is set up, can detect 1 * 10
-7The ng viral DNA is equivalent to detect 30 virus particle.Method for detecting virus in the fishery products that its sensitivity is better than being reported at present.
The precision test of embodiment 4, multiplex PCR-DHPLC
Between the viral RNA that different batches extracts in embodiment 3, get 1 * 10
-5The viral RNA of ng carries out PCR-DHPLC as template and detects, and determines to detect precision.Precision test result as shown in Figure 3.By accompanying drawing 3 as seen, for 1 * 10 of different batches
-5The viral RNA of ng detectable level detects through PCR-DHPLC, repeats all can present good stability and circulation ratio for 10 times.
The various viruses of concentration known are added in the fishery products, or, extract genomic nucleic acids, carry out mPCR-DHPLC and detect according to the method that embodiment 1 sets up with positive plasmid simulating pollution fish tissue.
1, quantitative hepatitis A virus (HAV) and Norwalk virus positive plasmid and the fishery products Digestive tract suspension of milling oppose doubly mixed, abundant mixing, after the freeze thawing 3 times, 4 ℃ of centrifugal 30min of 6000r/min get supernatant.Extract the nucleic acid of microorganism according to the detection method that embodiment 1 is set up, mPCR-DHPLC detects.Detected result as shown in Figure 4.
2, quantitative rotavirus and Norwalk virus virus-positive plasmid, with the fishery products Digestive tract of milling (lefteye flounder blood, the gill, liver, spleen, stomach and intestines suspension) oppose doubly mixed, behind the abundant mixing, multigelation 3 times, 4 ℃ of centrifugal 30min of 6000r/min get supernatant.Extract the nucleic acid of microorganism according to the detection method that embodiment 1 is set up, mPCR-DHPLC detects.Detected result as shown in Figure 5.
Effect was very desirable when from detected result as can be seen, test kit of the present invention and method application simulation contaminated samples detected.
The application in actual detected of embodiment 6, present method
During 7 months of year June in December, 2007 to 2008, the check that test kit that embodiment 1 is set up and detection method are applied to actual sample detects 1341 kinds of samples of 5 big classes altogether.Test sample does not have the amplification absorption peak and occurs, and the decidable sample result is negative, and directly report does not detect corresponding pathogenic bacterium; Typical PCR product absorption peak appears in test sample, and absorption peak is during greater than 3mV, and this sample result of decidable is a probable positive; Typical PCR product absorption peak appears in test sample, but absorption peak is during less than 3mV, and the suggestion sample is reformed.The results peaks of reforming absorption value is still then negative less than 3mV, otherwise is probable positive.
Adopt standard GB/T 22287-2008 (hepatitis A virus detection method in the shellfish simultaneously, common RT-PCR method and real-time fluorescence RT-PCR method), inspection and quarantine industry standard SN/T 1635-2005 (Norwalk virus detection method in the shellfish, common RT-PCR method and real-time fluorescence RT-PCR method) verifies comparison.The result shows that the test kit and the detection method that adopt embodiment 1 to be set up are screened 3 parts of positive sample that detect, and wherein 2 duplicate samples detect hepatitis A virus (HAV), and 1 duplicate samples detects Norwalk virus.Result consistent with the national standard method detected result (GB/T 22287-2008 and SN/T 1635-2005).Concrete detected result is shown in following table (table 2):
Table 2
SEQUENCE?LISTING
＜110〉old, grain husk
＜120〉3 kinds of food-borne virus detection kit and detection method in the fishery products
<130>N/A
<160>6
<170>PatentIn?version?3.3
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ggaaatgtct?caggtacttt?ctttg 25
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ataccactat?gatgcagatt?a 21
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Claims (2)
1. 3 kinds of food-borne virus detection kit in the fishery products is characterized in that this test kit comprises that concentration is that reverse transcription-Taq archaeal dna polymerase, the concentration of 5U/ μ L is ThermoScript II and the RT-PCR reaction solution of 5U/ μ L; Contain 10mM TrisHCl, 50mM KCl, 25mM MgCl in the RT-PCR reaction solution wherein
2, each 10mM of dNTP, RNA enzyme inhibitors 40U/ μ L and 3 kinds of food-borne virus the upstream and downstream primer to each 20 μ M;
Described primer sequence is as follows:
Test kit preservation condition :-20 ℃ of preservations.
2. the detection method of 3 kinds of food-borne virus in the fishery products is characterized in that using the described test kit of claim 1, comprises the steps:
1. get 2 μ L testing sample RNA solution, add RT-PCR reaction solution, 1.0 μ L reverse transcription-Taq archaeal dna polymerases and 1.0 μ L ThermoScript II in the 22 μ L test kits, sterilization ultrapure water 24 μ L, cumulative volume 50 μ L; The centrifugal 10s of 5000r/min, carry out the multiple RT-PCR amplification according to following condition then:
Reverse transcription: RT reacts 42 ℃ of 30min, 94 ℃ of 2min of RTase inactivation;
Cyclic amplification: 94 ℃ of sex change 60s, 48 ℃ of annealing 2min, 72 ℃ are extended 3min, 35 circulations;
Stop extending: 72 ℃ of 10min;
2. the RT-PCR product is carried out DHPLC and analyze, the DHPLC analysis condition is as follows:
Chromatographic column: PS-DVB ﹠amp; C18 DNASep chromatographic column, 4.6mm * 50mm, granularity 3 μ m;
Column temperature: 50 ℃;
Moving phase (volume percent): 0.0min, 55.0% buffered soln A, 45.0% buffered soln B,
0.5min, 50.2% buffered soln A, 49.8% buffered soln B,
3.0min, 42.9% buffered soln A, 57.1% buffered soln B,
5.5min, 39.4% buffered soln A, 60.6% buffered soln B,
8.0min, 37.3% buffered soln A, 62.7% buffered soln B,
10.5min, 36.0% buffered soln A, 64.0% buffered soln B,
Wherein, buffered soln A is that 50mL triethylamine acetyl salt and 250 μ L acetonitriles mix, and adds the sterilization ultrapure water and is settled to 1000mL gained solution; Buffered soln B is that 50mL triethylamine acetyl salt and 250mL acetonitrile mix, and adds the sterilization ultrapure water and is settled to 1000mL gained solution;
Flow velocity: 0.9mL/min;
Detector: fluorimetric detector, light source: 150W Xenon lamp; Excitation spectrum bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR product 10 μ L.
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| 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 |
| CN102719564B (en) * | 2012-06-25 | 2013-09-25 | 广西壮族自治区兽医研究所 | Triple polymerase chain reaction (PCR) kit for duck hepatitis virus type I, duck circoviruses and Muscovy duckling parvovirosis and application of triple PCR kit |
| CN103088151B (en) * | 2012-08-15 | 2014-07-23 | 浙江大学 | Kit for hepatitis B virus four-color fluorescence quantitative PCR (polymerase chain reaction) assay and application |
| CN103255226A (en) * | 2013-05-27 | 2013-08-21 | 郑秋月 | Trichina PCR-DHPLC (Polymerase Chain Reaction-Denaturing High Performance Liquid Chromatography) detection primer as well as kit and detection method |
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| CN1763196A (en) * | 2004-09-13 | 2006-04-26 | 首都医科大学宣武医院 | Gene mutation type and gene sequencing method |
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