CN100434535C - Method of quantificationally detecting rota virus and special-purpose standard substance for the same - Google Patents
Method of quantificationally detecting rota virus and special-purpose standard substance for the same Download PDFInfo
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- CN100434535C CN100434535C CNB2007100995842A CN200710099584A CN100434535C CN 100434535 C CN100434535 C CN 100434535C CN B2007100995842 A CNB2007100995842 A CN B2007100995842A CN 200710099584 A CN200710099584 A CN 200710099584A CN 100434535 C CN100434535 C CN 100434535C
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Abstract
The invention discloses a quantitative detecting rotavirus method and the specific standard product. The quantitative detecting rotavirus standard product contains a nucleic acid sequence recombinant carrier of sequence 1 in the sequence table or transgenosis host cell of the recombinant carrier. The quantitative detecting rotavirus method is characterized by the following: regarding detecting sample and the genome DNA of standard product as the model or regarding detecting sample and cDNA by total RNA reverse transcription of the standard product as the model; proceeding PCR with nucleic acid sequence( sequence 2 nucleic acid fragment in the sequence table) and nucleic acid sequence( the primer comprising sequence 3 nucleic acid fragment in the sequence table); proceeding rotavirus definite quantity of the detecting sample. The standard product is provided with the association characteristic of broad spectrum identification and infectious nucleic specific, which provides good sensibility, simple manufacturing method, can be used for various sample and quick quantitative determination of rotavirus.
Description
Technical field
The present invention relates to a kind of method and specialized standard product thereof of detection by quantitative rotavirus.
Background technology
Rotavirus is one of main pathogenic microorganism of humans and animals acute diarrhea in the worldwide.Wherein the A group rotavirus is to cause the serious most important reason of children acute gastro-enteritis in the world wide, reaches 600 every year, 000-800,000 lethality rate (Umesh, D.P., Joseph, S.B., Jon, R.G., Roger, I.G., 1998.Rotavirus.Emerg.Infect.Dis.4 (4), 561-570); In recent years research data shows, the infantile diarrhea of China autumn and winter season 50%～60% causes (Chang Ruxu by rotavirus, He Cuijuan. the rotavirus detection method compares [J] in the diarrhoea infant stool sample. Chinese paediatrics magazine, 2000,38 (11): 703～704).Rotavirus is a kind of diplornavirus, is made up of 11 genomic fragments, and its core is made up of the double capsid of 70nm diameter.This viral inner casing has four structural protein (VP1, VP2, VP3 and VP6), and shell has two structural protein (VP4 and VP7).Wherein VP4 and VP7 are two main structural protein, they have determined the serotype and the (Estes of virus neutralization (neutralization) of rotavirus, M.K., Palmer, E.L., Obijeski, J.F., 1983.Rotaviruses.Curr.Top.Microbiol.Immunol.105,123-184).Rotavirus mainly is to propagate by fecal oral route, and rotavirus exists very commonly in sanitary sewage and contaminated surface water, and it mainly is to propagate (parashar, U.D by being grown in the Crustacean in the polluted water and the tap water of pollution; R.C Holman; M.J.Clarke; J.S.Bresee; And R.I.Glass.Hospitalizations associated with rotavirus diarrhea in the United States, 1993 through 1995:surveillance based on the new ICD-9-CM rotavirus-specificdiagnostic code.1998.J.Infect.Dis.177:13-17; Le Guyader, F; L.Haugarrean; L.Miossec; E.Dubois; And M.Pommepuy.Three-year study to assess human entericviruses in shellfish.Appli.Environ.Microbiol.2000.66:3241-3248).
Main both at home and abroad at present the utilization substitutes the content (D.Hot. that index is estimated virus in the water body, O.Legeay, J.Jacquesa, C.Gantzerc, Y.Caudrelierb, K.Guyardb, M.Langeb, L.Andreolettia.Detection of somatic phages, infectious enteroviruses andenterovirus genomes as indicators of human enteric viral pollution in surfacewater, Water Research 2003 37:4703-4710).Alternative index is mainly utilized the phage of specific type in bacterium movement indicator (colibacillus of excrement and suis) and the human feces, as have a liking for the meat coliphage, F-specific RNA phage and bacteriophage fragilis phage are as quality standard (A.Arrajl, J.Bohatier, H.Laveran and O.Traore.Comparison ofbacteriophage and enteric virus removal in pilot scale activated sludgeplants.Journal of Applied Microbiology 2005.98:516-524).But, studies show that virus still exists under the situation that intestinal bacteria and coliphage do not exceed standard, and coliphage and viral dependency are undesirable in the water body, can not react the infectivity of virus.In recent years, a large amount of research work have been launched at the rapid detection of rotavirus in the water surrounding in countries in the world, and wherein the detection method based on real-time quantitative PCR (real-time PCR) has presented good prospects for application.
Real-time quantitative PCR (real-time PCR) is by the accumulation of fluorescent signal in the real-time monitoring PCR reaction process, and sample is detected and quantitative analysis.The whole process of Real-time PCR is carried out fragment amplification and product analysis under totally enclosed state, reduced so effectively and polluted the harm that reaches human body, can carry out detection by quantitative fast in large quantities simultaneously.The Real-time round pcr is fast and convenient quantitative measurement technology, and it can adopt absolute quantitation and 2 kinds of modes of relative quantification to realize detection by quantitative.Wherein absolute quantitation be adopt at present maximum a kind of, but need to adopt the quantitative preparation of outer standard substance to realize absolute quantitation (Lan.M.Mackay et al.Real-time PCRin Virology.Nucleic Acids Research.2006 30:1292-1305).In Real-time PCR detection by quantitative, can the preparation of these outer standard substance becomes it accurate quantitative key.
Bibliographical information molecular biology RT-PCR technology has susceptibility height, high specificity, advantage such as quick, can detect each geneome RNA of the enteron aisle Causative virus in the polluted water body; The wherein genomic detection of enterovirus, there is (the D.Hot. that has great importance in virus in the prediction water body, O.Legeay, J.Jacquesa, C.Gantzerc, Y.Caudrelierb, K.Guyardb, M.Langeb, L.Andreolettia.Detectionof somatic phages, infectious enteroviruses and enterovirus genomes asindicators of human enteric viral pollution in surface water, Water Research2003 37:4703-4710).It is the method (D.Hot. of virus pollution index in a kind of suitable detection water surrounding that propositions such as while L.Andreoletti utilize real-time quantitative RT-PCR detection by quantitative enterovirus genome, O.Legeay, J.Jacquesa, C.Gantzerc, Y.Caudrelierb, K.Guyardb, M.Langeb, L.Andreolettia.Detection of somatic phages, infectious enteroviruses andenterovirus genomes as indicators of human enteric viral pollution in surfacewater, Water Research 2003 37:4703-4710).He and Jiang etc. utilizes human adenovirus and the enterococcin in the real-timePCR detection by quantitative environmental sample, proves that this method has higher susceptibility than viral plaque analysis or method for cultivation of bacteria.Jim Gray etc. compares enzyme immunoassay (EIA) and real-time RT-PCR both techniques subsequently, the result shows more fast, economical (GagandeepKang, Miren Iturriza-Gomara, Jeremy G.Wheeler of real-time RT-PCR, Premila Crystal, BindhuMonica, Sasirekha Ramani, Beryl Primrose, Prabhakar D.Moses, Chris I.Gallimore, David W.Brown, and Jim Gray.Quantitation of Group A Rotavirusby Real-Time Reverse-Transcription-Polymerase Chain Reaction：CorrelationWith Clinical Severity in Children in South India。Journal of Medical Virology.2004.73：118-122)。Report such as Charlotte C.Yu Ip application real-time PCR, nested-PCR and these three kinds of technology for detection of electron microscope (electron microsopy) are from rotavirus in diarrhoea children's the stool sample, it is fast simple that the result shows that real-time RT-PCR analysis has, advantages such as susceptibility height and minimizing exogenous pollution; And definite real-time RT-PCR can detect multiple situation and comprises rotavirus (Xiaoli L.Pang in diarrhoea, eruption and prevalence, the environmental sample etc. at random, Bonita Lee, Nasim Boroumand, BarbaraLeblanc, Jutta K.Preiksaitis, and Charlotte C.Yu Ip.Increased Detectionof Rotavirus Using a Real Time Reverse transcription-Polymerase ChainReaction(RT-PCR)Assay in Stool Specimens From Children With Diarrhea。Journal of Medical Virology.2004.72：496-501)。Pang etc. utilize real-time quantitative RT-PCR to detect the rotavirus infection of 123 routine children's diarrhae samples, and RT-PCR and nest-type PRC that detection sensitivity is more traditional have improved 10
2-10
4Doubly, consuming time then is half of the latter, and prove that this method can also monitor virus pollution situation (the Pang XL in the water surrounding sample, Lee B, Boroumand N, et al.Increased detect ion ofrotavirus using a real time reserve transcription-polymerase chain reaction (RT-PCR) assay in stool specimens from children with diarrhea.J Med Virol.2004.72 (3): 496-501).In sum, some investigators have adopted the rotavirus of real-time round pcr detection from the diarrhoea sample in recent years, but the rotavirus in the water surrounding sample and infective detection thereof are also in heuristic process.These investigators consider that at sample the key of real-time round pcr is quantitatively in regard to process, so they adopt the structure of TaqMan probe technique or outer standard substance to realize absolute quantitation.The gordian technique that is configured to research of wherein different outer standard substance.
With regard to the water quality control criterion, USEPA takes much count of water Jie pathogenic micro-organism to health effects, microbial standard is 8 in its water quality standard, comprising viral index, and the virus of in the water quality standard of China water Jie being propagated is not also clearly stipulated, only stipulates must not detect (CJ/T206-2005 Urban water supply water quality standard) in the every 100mL water sample of heat-resisting coliform.And in revision water quality standard process, the foundation of corresponding detecting method is particularly important.At present, still do not have the standard method that detects rotavirus in the water surrounding both at home and abroad, Real-time PCR is considered to the quantitative detection method of the most promising quick sensitivity.Therefore in Real-time PCR detection by quantitative, the preparation of outer standard substance is its quantitative gordian techniquies.
Summary of the invention
The method and the specialized standard product thereof that the purpose of this invention is to provide a kind of detection by quantitative rotavirus.
Quantitative detecting rotavirus standard product provided by the present invention is the recombinant vectors that contains the nucleotide sequence of sequence 1 in the ordered list, or contains the genetically modified host cell of described recombinant vectors.
The sequence of all VP7 of monkey rotavirus strain SA-11 has the homology more than 99% among sequence 1 and the GenBank, has homology more than 75% with the VP7 sequence of human rotavirus's strain among the GenBank.Utilize the prepared standard substance of sequence 1 outside can be used as the standard substance that detect the monkey rotavirus strain, also can be used as the outer standard substance that detect other rotavirus strains, these standard substance have broad spectrum.
Described standard substance can be used in the quantitative PCR detection.
Described quantitative PCR can be conventional PCR or real-time quantitative PCR.
Anyly can bring the exogenous dna fragment of carrying into recipient cell, and the dna molecular that is maintained therein all can be used for making up described recombinant vectors, as plasmid, phage vector, cement grain carrier engineering carriers such as (cosmid).
Described recombinant vectors can be according to ordinary method, is that the multiple clone site that the nucleotide fragments of sequence 1 in the sequence table inserts carrier obtains with nucleotide sequence.
Described genetically modified host cell can be bacterium, fungi, plant or zooblast.
The method of detection by quantitative rotavirus provided by the present invention, be to be template with the genomic dna of detected sample and above-mentioned any standard substance respectively, or be template with the cDNA that obtains by total RNA reverse transcription of described detected sample and above-mentioned any standard substance respectively, utilization is the primer formed of the nucleotide fragments of sequence 2 in the sequence table and nucleotide fragments that nucleotide sequence is sequence 3 in the sequence table to carrying out PCR by nucleotide sequence, and it is quantitative that detected sample is carried out rotavirus.
In the sequence table nucleotide sequence of sequence 2 be 5 '-CCTCACTTATACACTTTGCC-3 '; In the sequence table nucleotide sequence of sequence 3 be 5 '-TTCGCTTCGTCAGTTTGCT-3 '.
In one embodiment of the present of invention, provide a quantitative detecting rotavirus standard product, these standard substance are to be that the nucleotide fragments of sequence 1 in the sequence table is connected the recombinant vectors pGEM-T Easy-VP7 that obtains with pGEM-T Easy carrier with nucleotide sequence.It prepares according to following method:
1) separation and cultivation monkey rotavirus type strain from the MA-104 cell;
2) utilize the Trizol cracking process to carry out the extraction of the total RNA of cell, obtain total RNA;
3) adopt the SuperscriptIII reversed transcriptive enzyme to carry out reverse transcription and obtain cDNA, utilize Auele Specific Primer (sequence 2 and sequence 3), the pcr amplification sequence is the nucleotide fragments of sequence 1 in the sequence table; Adopt the T-A clone technology, this nucleotide fragments is connected into pGEM-T Easy carrier, obtain recombinant vectors pGEM-T Easy-VP7.
Among the preparation method of above-mentioned outer standard substance pGEM-T Easy-VP7, rotavirus RNA cultivates preparation from the MA-104 cell, be that infective rotavirus is typically represented therefore.It is specific to belong to infective rotavirus based on these outer standard substance that obtain, and the real-time PCR method of Jian Liing is except that having the advantage aspect accuracy, susceptibility on this basis, has bigger advantage aspect virus infectious estimating simultaneously; The primer of being made up of sequence in the sequence table 2 and sequence 3 has comprised the part with specific part and structural protein, and therefore the method based on this foundation can detect widely, have infective rotavirus, has improved sensing range and precision.
Quantitative detecting rotavirus standard product of the present invention has wide spectrum identification and the infectious special characteristics that combine, the susceptibility height, the preparation method is easy, but prolonged preservation, purity is good, detect linear a wider range, the fast quantification that can generally be used for rotavirus in the various samples, particularly water surrounding detects.
Description of drawings
Figure 1A is that the enzyme of recombinant vectors is cut the evaluation collection of illustrative plates
Figure 1B is the part sequencing result of pGEM-T Easy-VP7 recombinant plasmid
Fig. 2 is the conventional PCR of the pGEM-T Easy-VP7 of different dilution titers
Fig. 3 A is the typical curve of real-time quantitative PCR
Fig. 3 B is the melting curve of real-time quantitative PCR
Fig. 4 detects the cDNA sample result of the Caco-2 cell that has infected monkey rotavirus type strain SA-11 for conventional PCR
Fig. 5 detects the amplification curve of plasmid standard pGEM-T Easy-VP7 for real-time quantitative PCR
Fig. 6 A is the typical curve of the real-time quantitative PCR of detection unknown sample (the cDNA sample of Caco-2 cell)
Fig. 6 B is the amplification curve that real-time quantitative PCR detects plasmid standard pGEM-T Easy-VP7 and unknown sample (the cDNA sample of Caco-2 cell)
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Be example only below with the recombinant plasmid that contains the nucleotide sequence of sequence 1 in the ordered list, illustrate the effect of quantitative detecting rotavirus standard product detection by quantitative rotavirus, other contains the recombinant vectors of the nucleotide sequence of sequence 1 in the ordered list or contains the effect of genetically modified host cell detection by quantitative rotavirus of this recombinant vectors all identical with these standard substance.
The preparation of embodiment 1, quantitative detecting rotavirus standard product pGEM-T Easy-VP7
A. the cultivation of rotavirus: utilize the DMEM+10% foetal calf serum, at 5%CO
2(ATCC number is HTB-37 to 37 ℃ of cultivation rhesus monkey clone MA-104 cells in the incubator
TM), when this clone at 25cm
2Culturing bottle in when being grown to individual layer, utilize 1 * PBS to clean cell twice, to remove dead cell; (ATCC number is VR-1565 to inoculation 500mL monkey rotavirus type strain SA-11
TM) in the culturing bottle of cultivating the MA-104 cell, 5%CO
2Hatch 1-2h for 37 ℃ in the incubator, add the DMEM virus that contains the 5mL2% foetal calf serum and keep liquid, cultivate after 5 days, the observation of cell state, when obvious viral plaque unit forms, collecting cell, and the collecting cell supernatant liquor, obtain amplification monkey rotavirus type strain SA-11.
B. the extraction of rotavirus RNA: adopt the Trizol cracking process that the MA-104 cell of collecting infected monkey rotavirus type strain SA-11 is carried out the extraction of cell total rna.Concrete steps are as follows:
1) at 25cm
2Culturing bottle about 3 * 10
5Add 1mL Trizol in the individual cell immediately, evenly shake 5min under the room temperature on the shaking table, utilize the 1mL suction pipe to blow and beat cell gently, make lysis;
2) add the 0.2mL chloroform, the vibration mixing, room temperature is placed 2min;
3) 4 ℃ of centrifugal 15min of 2000g;
4) get supernatant, add 0.5mL Virahol mixing, place 10min under the room temperature;
5) 4 ℃ of centrifugal 10min of 12000g;
6) abandon supernatant, in the RNA precipitation, add the ethanol mixing of 1mL 75%;
7) 4 ℃ of centrifugal 5min of 7500g;
8) abandon supernatant, room temperature or 55 ℃ are drying precipitated, and are water-soluble with 20 μ L DEPC, put the 5min dissolving in 55 ℃-65 ℃.
9) use ultraviolet spectrophotometer evaluation RNA concentration and purity to use or place afterwards-80 ℃ of preservations immediately,
In the entire operation process, note wearing mouth mask, gloves will often be relieved a garrison and be ended the degraded of RNA.
The design of the synthetic and Auele Specific Primer of C.cDNA:
CDNA's is synthetic: the total RNA that gets 4ug uses reversed transcriptive enzyme SuperScriptIII to carry out the synthetic of cDNA by reverse transcription, and operating process is as follows: the synthetic system of the cDNA of 20uL comprises 1uLOligo (dT)
12-18(200-500ng), the mRNA of 5uL (0.8ug/ul), the 10mMdNTP of 1uL, the no RNA enzyme distilled water of 6uL; This system is at 65 ℃ of water-bath 5min, hatch 2min on ice, carry out of short duration centrifugal after, add following several reagent immediately in this pipe: 5 * article one chain reaction damping fluid of 4uL, the 0.1M DTT of 1uL, the RNA enzyme inhibitors (40u/uL) of 1uL, the SuperScriptIII RT (200u/ul) of 1uL; The soft mixing, cultivate 60min 50 ℃ of water-baths, in 70 ℃ of water-bath 15min deactivation.-20 ℃ of preservations, standby.
The design of Auele Specific Primer:
In GenBank, download sequence and the human rotavirus's strain part of V P7 sequence (seeing Table 1) of all VP7 of monkey rotavirus type strain SA-11.With carrying out its homology of multisequencing comparative analysis in these sequence inputs Clastla W software, carry out design of primers and synthetic, the GC content of primer is between 40-60%, and distance is greater than 30bp between the primer.Primer sequence is as follows: SA-11-Sense:5 '-CCTCACTTATACACTTTGCC-3 ' (sequence 2); SA-11-Antisense:5 '-TTCGCTTCGTCAGTTTGCT-3 ' (sequence 3).With aseptic double-distilled water primer is mixed with the storage liquid of 200umol/uL, packing ,-20 degree are preserved.
The sequence information of monkey rotavirus type strain SA-11 VP7 and human rotavirus VP 7 among all GenBank of table 1.
| Title | The number of presenting | Molecule type | Length (bp) |
| Simian rotavirus SA-11 |
X66158.1 GI：312109 | DNA | 1063bp |
| Simian 11 |
V01190.1 GI：61682 | DNA | 1062bp |
| Simian 11 |
V01546.1 GI：61870 | | 1062bp |
| Sequence | |||
| 2 from Patent EP 0251467 | I05272.1 GI：591173 | DNA | 1063bp |
| Simian 11 rotavirus major outer capsid protein VP7(seg 9) | K02028.1 GI：333791 | cDNA | 1062bp |
| |
M21843.1 GI：333862 | RNA | 1059bp |
| Rotavirus A strain Dhaka16-03 VP7 gene | DQ492674.1 GI：94982715 | RNA | 1062bp |
| Human |
K02033.1 GI：333800 | ds-RNA | 1062bp |
D. the preparation of plasmid standard:
Concrete steps are: 1) segmental preparation of purpose and purifying: get the template of 8ul cDNA as the PCR reaction, the PCR reaction system is 100uL: 2uL 20uM upstream primer (sequence 2), 2uL 20uM downstream primer (sequence 3), 2uL 10mM dNTP, 1.2uL TaqDNA polysaccharase (5u/uL), 6.4uL 25mM MgCl
2, 10uL10 * PCR damping fluid, 76.4ul aseptic double-distilled water.The PCR response procedures is 94 ℃ of 4min of elder generation; 94 ℃ of 1min then, 59 ℃ 40 seconds, 72 ℃ of 1min30 seconds, carry out 35 circulations; 72 ℃ are extended 10min again.Get capable 2% agarose gel electrophoresis of 10uL PCR product, the result shows that the amplified production size for 318bp, proves that amplified production is the purpose fragment.Get capable 2% agarose of 90ul PCR product and reclaim glue, utilize glue to reclaim test kit and carry out the segmental recovery purifying of purpose.Get 2ul behind the recovery purifying and carry out 2% agarose gel electrophoresis, the result shows that the effect that reclaims purifying is better, can be used for T-A and other double digestions clone.
2) T-A clone technology: a. ligation: the purpose fragment that will reclaim purifying is connected with pGEM-T Easy carrier, detailed process is as follows: according to the brightness of carrying out 2% agarose gel electrophoresis finding band after reclaiming, regulating carrier and the segmental amount of purpose carried out 1: 1 respectively, the ligation of 1: 2 and 1: 3, the connection ratio that finally is defined as 1: 3 is better, and its linked system is 10uL: 3uL purpose fragment, 1uL pGEM-T Easy carrier, 5uL 2 * ligase enzyme damping fluid, 1uL T
4Dna ligase, 4 ℃ connect 12h.
B. the preparation of competence bacterium: utilize CaCl
2Legal system is equipped with DH5 α competence bacterium, will connect product and be transformed in this competent cell, and detailed process is as follows:
I. picking list bacterium colony from 37 ℃ of plates of cultivating 12～16h, transfer to and contain 3mL LB in vitro, 37 ℃ are spent the night, take out next day in the 500mL bottle that 1mL is seeded to 100mL LB, 37 ℃ of violent shakes are cultivated 2～3h (200～300r/min), treat that the OD600 value reaches at 0.3～0.4 o'clock, flask is taken out put ice bath 10～15min immediately.
II. bacterium liquid is transferred in the 50mL centrifuge tube of an ice-cold sterilization, and 4 ℃ of centrifugal 10min of 4000g reclaim bacterium.
III. abandon nutrient solution, ice-cold 10mL 0.1M CaCl
2Resuspended thalline is put ice bath 30min.4 ℃ of centrifugal 10min of 4000g, supernatant discarded.
IV. add the 0.1M CaCl of 2mL again with the ice bath precooling
2Resuspended thalline (wanting light).Put 4 ℃ of refrigerator 12～24h, promptly can be used for transforming.
V. by every part of 100uL packing cell, transform at once.
C. the conversion of bacterium
The competence bacteria of prepared fresh, every 100uL competence bacteria add 10uL and connect product, ice bath 30h.42 ℃ of heat-shocked 90s insert in the ice immediately, place 2min.Add 500uL LB, 37 ℃, 225rpm cultivates 1h.The 8000rpm room temperature is centrifugal, removes the part supernatant, and get behind the mixing and be coated with the LB culture dish that contains 100ug/mLAmp+ in right amount, 37 ℃ of overnight incubation (12～16h), can not surpass 16h.
3) evaluation of positive colony: the DNA that utilizes improved alkaline lysis method of extracting plasmid.Detailed process is as follows:
Take out being coated with the culture dish that bacterium colony appearred in dish after transforming, choose 12 single bacterium colonies in containing 5mL LB (Amp+) nutrient solution, cultivate 10～12h at 37 ℃; Get 1.5mL bacterium liquid in 1.5mL Eppendorf pipe, room temperature, the centrifugal 5min of 12000rpm; Discard supernatant liquid, add solution I (50mM glucose, the Tris-HCl of 25mM pH8.0, the EDTA of 10mM pH8.0 and the 10ug/mL RNaseA) thermal agitation that 120uL contains the RNA enzyme, room temperature is placed 10-15min; The solution II (1%SDS, 200mM NaOH) that adds the new configuration of 240uL, mixing gently, room temperature is no more than 5min; Add 180uL solution III (60mL 5M potassium acetate, the 11.5mL Glacial acetic acid adds water to 100mL), put upside down mixing, room temperature 1-5min; The centrifugal 10min of room temperature 12000rpm carefully moves into supernatant liquor in another 1.5mL centrifuge tube; Add the saturated phenol of equal-volume Tris that mixes in advance: chloroform (1: 1) mixing, the centrifugal 5min of room temperature 12000rpm; Suct layer water in another centrifuge tube, add the Virahol of 0.6-0.8 times of volume, mixing, the centrifugal 15-20min of room temperature 12000rpm abandons supernatant; Add 70% ethanol 1mL, the centrifugal 5min of room temperature 12000rpm; Abandon supernatant, 55 ℃ of dryings add 30uL TE dissolving.-20 ℃ of preservations are equipped with enzyme and cut evaluation usefulness.
Utilize restriction enzyme mapping in the Cutter2.0 software on-line analysis purpose fragment, determine not have the EcoRI restriction enzyme site, adopting EcoRI that institute's upgrading grain is carried out enzyme cuts, enzyme is cut product carry out 2% agarose gel electrophoresis, select the Marker of 100bp for use, observe on the gel imaging instrument and the record experimental result, the result shows a few strain positive colonies of preliminary evaluation.Enzyme is cut the bacterium liquid that proves positive colony send order-checking, after sequencing result carries out the blast comparison, the result proves positive recombinant chou, and the sequence of downloading all VP7 of monkey rotavirus strain SA-11 among this recombinant chou and the GenBank has the homology more than 99%, the nucleotide sequence of the rotavirus DNA that is carried in this recombinant chou is the sequence 1 in the sequence table, and will contain nucleotide sequence is the recombinant vectors called after pGEM-T Easy-VP7 of the nucleotide fragments of the sequence 1 in the sequence table.Plasmid standard pGEM-T Easy-VP7 successfully constructs, and carries out a large amount of extractions and the purifying of pGEM-T Easy-VP7.The enzyme of recombinant vectors is cut qualification result shown in Figure 1A, shows the recombinant vectors that has obtained to contain the 318bp exogenous dna fragment.Among Figure 1A, M:DNA Marker; Negative: as not have the enzyme of carrier to cut system; 1-12: enzyme is cut the numbering of recombinant vectors.The part order-checking collection of illustrative plates of pGEM-T Easy-VP7 is shown in Figure 1B.
E. the detection of recombinant plasmid standard substance pGEM-T Easy-VP7:
Step is: the 1) calculating of the mensuration of recombinant plasmid concentration and copy number: the plasmid standard 1ul that gets purifying utilizes the OD value of UV spectrophotometry 260nm in the TE of 200ul, determine that the purity of plasmid standard is better.The concentration of utilizing the formula of DNA mass concentration=A260 * nucleic acid extension rate * 50/1000 to calculate plasmid standard is 3.22ug/uL.The copy number that needs to calculate plasmid standard after the concentration of acquisition plasmid standard, the wherein copy number of DNA=(molar mass of quality/DNA of DNA) * 6.02 * 10
231bp=649Da in the double-stranded DNA wherein; The bp of molar mass=649Da of DNA * (3000+318); Bringing into and calculating acquisition in the formula is 9 * 10
11Copy/uL.
2) determining of recombinant plasmid pGEM-T Easy-VP7 stability: getting 5 batches of concentration is 9 * 10
8-4The plasmid extract of copy number/uL, carry out frozen in batches in-20 the degree, the frozen time is respectively 1,20,60 days.Get 5 batches of concentration of these 3 time points and carry out the real-time quantitative PCR reaction, fluorescent agent used in this real-time quantitative PCR is SYBRgreen I, is a kind of double-stranded DNA combination dye that is incorporated in the ditch.Carry out the parallel hole repeated experiments.The real-time quantitative PCR reaction system is as follows through optimizing: 20uM upstream primer (sequence 2), and 20uM downstream primer (sequence 3) and 10mM dNTP are respectively 0.5ul; The Taq archaeal dna polymerase is 0.3uL; 10 * PCR reaction buffer is 2uL; 25mM MgCl
2Be 1.6uL; 20 * SYBR green I dyestuff is 1.2uL, and 1uL 9 * 10
8-4Copy number/uL is a template, supplies volume to 20uL with the distilled water of 12.4ul.The real-time quantitative PCR reaction process is through being optimized for 94 ℃ of pre-sex change of 2min earlier, then according to 94 ℃ 20 seconds, 59 ℃ 30 seconds, 72 ℃ 20 seconds, carry out 50 circulations.The process of melt curve analysis is: 95 ℃ of 1min, and 55 ℃ of 1min began to carry out 81 circulations in 30 seconds to 55 ℃, and range of temperature is 0.5 ℃, and end temp is 95 ℃.Analysis through melt curve analysis determines that the Tm value of specific reaction is 81 ℃.The result shows poor＜0.5 between the Ct value of parallel sample, and repeatability better in the explanation group; Three independent experiment results' CV value＜0.9% shows that this plasmid standard has good repeatability and stable.
Table 2. plasmid standard Detection of Stability
4) real-time quantitative PCR detects recombinant plasmid standard substance pGEM-T Easy-VP7 linearity range: recombinant plasmid pGEM-T Easy-VP7 is carried out 10 times doubling dilution, and dilution titer is respectively 9 * 10
9, 9 * 10
89 * 10
0Copy number/uL carries out real-time quantitative PCR and detects, and it is 9 * 10 that the same step 3) of reaction parameter and process, experimental result determine to utilize the real-time quantitative PCR reaction parameter of above-mentioned optimization and linearity range that program can detect plasmid standard
0-9 * 10
11Copy number/uL.The result as shown in Figure 5, the linearity range that real-time quantitative PCR detects plasmid standard is 9 * 10
0-9 * 10
11Copy number/uL.
5) conventional PCR detects the scope of recombinant plasmid standard substance pGEM-T Easy-VP7
Rotavirus plasmid standard pGEM-T Easy-VP7 is carried out 10 times doubling dilution, and dilution titer is respectively 9 * 10
11, 9 * 10
10, 9 * 10
9, 9 * 10
89 * 10
-2Copy number/uL, carry out conventional PCR and detect: 20uM upstream primer (sequence 2), 20uM downstream primer (sequence 3) and 10mM dNTP are respectively 0.5ul, and the Taq archaeal dna polymerase is 0.3uL, and 10 * PCR reaction buffer is 2uL, 25mM MgCl
2Be 1.6uL, the plasmid standard of 10 times of doubling dilutions of 1uL is a template, supplies volume to 20uL with the distilled water of 13.6ul.Increase by following condition: 94 ℃ of pre-sex change of 4min, then according to 94 ℃ of 1min, 59 ℃ 40 seconds, 72 ℃ of 1min 30 seconds carry out 35 circulations, last 72 ℃ are extended 10min.The PCR product is carried out 2% agarose electrophoresis, the result as shown in Figure 2, the sensing range that shows conventional PCR is 9 * 10
3～9 * 10
11Copy number/uL, this result are three reproducible results.Among Fig. 2, M:DNA Marker (100bp DNA ladder); 1～15: represent with 9 * 10 respectively
11, 9 * 10
10, 9 * 10
9, 9 * 10
8, 9 * 10
-2Copy number/uL and negative control (distilled water) carry out conventional PCR for template and detect the purpose fragment that is amplified, and the segmental size of purpose is 318bp, is shown as the base pair size between 300bp and the 400bp in the drawings; The band at 100bp place is the primer dimer that forms in the amplification procedure.
F. the preparation of real-time quantitative PCR examination criteria curve:
The recombinant plasmid standard substance pGEM-T Easy-VP7 that gets known 5 doubling dilutions is respectively 9 * 10
8, 9 * 10
7, 9 * 10
6, 9 * 10
5, 9 * 10
4Copy number/uL carries out the drafting of the typical curve of real-time quantitative PCR detection.Each extent of dilution is made 3 parallel samples simultaneously, carries out the real-time quantitative PCR reaction.All reaction parameters and process are with 3 of step E).The result as shown in Figure 3A, X-coordinate is represented the Log of the initial dilution titer of plasmid standard
10Value, ordinate zou Threshold Cycle represents the critical cycle number of the plasmid standard of serial dilution titer.The rotavirus plasmid standard is from 9 * 10
4～9 * 10
7R in the scope of/uL copy number
2=0.9995, illustrate that this system linear dependence is better; Slope is that-3.207 these system amplification efficiencies of explanation are higher.The result is the result of three independent repeated experiments among the figure.
The melting curve of G, real-time quantitative PCR examination criteria curve
The measuring method of pGEM-T Easy-VP7 melt curve analysis is with 3 of step E), the result shows that the Tm value of specific reaction is 81 ± 0.5 ℃ (Fig. 3 B).
Utilization contains the DMEM substratum of 15% foetal calf serum, at 37 ℃ of 5%CO
2(ATCC number is CRL-2378.1 to cultivator colon carcinoma cell line Coco-2 cell in the incubator
TM); When this cell line growth is individual layer, inoculate four kinds of titres (10 respectively
3.5TCID
50, 10
1.5TCID
50, 10
-0.5TCID
50, 10
-1.5TCID
50) monkey rotavirus type strain SA-11 (ATCC number is VR-1565
TM), each concentration is established the cell contrast of three parallel samples and not infection simultaneously.Infect after 120 hours, collecting cell utilizes the Trizol cracking process, carries out the extraction of cell total rna.Utilize the method for C among the embodiment 1 to carry out the synthetic of cDNA, obtain the cDNA of three infection titers of Coco-2 cell respectively, as testing sample.CDNA and pGEM-T Easy-VP7 (outer standard substance) with three infection titers of Coco-2 cell is template respectively, carries out the amplification of conventional PCR and real-time quantitative PCR.The reaction system of conventional PCR and real-time quantitative PCR amplification and temperature of reaction condition respectively with embodiment 1 in identical.Used primer is all identical except that β-actin.
The primer of monkey rotavirus SA-11:
SA-11-Sense:5 '-20 bases of CCTCACTTATACACTTTGCC-3 ' (sequence 2)
SA-11-Antisense:5 '-19 bases of TTCGCTTCGTCAGTTTGCT-3 ' (sequence 3)
House-keeping gene β in the cell-actin Auele Specific Primer:
β-actin-sense:5 '-GGAGAAAATCTGGCACCACAC-3 '; 21 bases
β-actin-antisense:5 '-CGTACAGGTCTTTGCGGATGT-3 '; 21 bases
Conventional PCR detected result as shown in Figure 4, the Caco-2 cell detected rotavirus at 120 hours infection titer is 10
3.5TCID
50Wherein: A is the infection titer of conventional PCR at Caco-2 cell detection rotavirus, and the primer is a monkey rotavirus strain Auele Specific Primer.M is DNA Marker (DL 2000); C is the cDNA of the Caco-2 cell that do not infect rotavirus; N is the negative control (distilled water) of PCR reaction system.Lane1～3 are for having infected 10
-1.5TCID
50The cDNA of the Caco-2 cell of rotavirus; Lane4～6 are for having infected 10
-0.5TCID
50The cDNA of the Caco-2 cell of rotavirus; Lane7～9 are for having infected 10
1.5TCID
50The cDNA of the Caco-2 cell of rotavirus; Lane10～12 are for having infected 10
3.5TCID
50The cDNA of the Caco-2 cell of rotavirus; B is the infection titer of conventional PCR at Caco-2 cell detection rotavirus, and the primer is house-keeping gene β in the cell-actin Auele Specific Primer.M is DNA Marker (DL 2000); C is the cDNA of the Caco-2 cell that do not infect rotavirus; N is the negative control (distilled water) of PCR reaction system.Lane1～3 are for having infected 10
-1.5TCID
50The cDNA of the Caco-2 cell of rotavirus; Lane4～6 are for having infected 10
-0.5TCID
50The cDNA of the caco-2 cell of rotavirus; Lane7～9 are for having infected 10
1.5TCID
50The cDNA of the Caco-2 cell of rotavirus; Lane10～12 are for having infected 10
3.5TCID
50The cDNA of the Caco-2 cell of rotavirus.
Utilize pGEM-T Easy-VP7 to be summarized as follows in the fluorescence quantitative PCR detection result that the caco-2 cell carries out infective rotavirus as outer standard substance:
As the prepared typical curve of Fig. 6 A, calculate 10 according to this experiment
3.5TCID
50In the cDNA sample of the Caco-2 cell of rotavirus infection, the copy number of rotavirus is 4.467 * 10
6Individual/uL, 10
1.5TCID
50In the cDNA sample of the Caco-2 cell of rotavirus infection, the copy number of rotavirus is 1.354 * 10
5Individual/uL.By Fig. 6 B as can be known in real time amplification curve be typical serpentine, have good amplification plateau; By the amplification of melting curve analysis revealed is that rotavirus is specific.Among Fig. 6 B, curve 1 is negative control (distilled water), and curve 2 is to have infected 10
-0.5TCID
50The cDNA of the caco-2 cell of rotavirus, curve 3 is to have infected 10
1.5TCID
50The cDNA of the caco-2 cell of rotavirus, curve 4 is to have infected 10
3.5TCID
50The cDNA of the caco-2 cell of rotavirus, curve 5-8 are respectively 9 * 10 of pGEM-T Easy-VP7
4, 9 * 10
5, 9 * 10
6, 9 * 10
7Copy number/uL.These results show that the result of detection of real-time quantitative PCR is more reliable, and the detection sensitivity of the more conventional PCR of susceptibility is high 100 times; These results show that also plasmid standard prepared among the present invention has better linearity relation and sensing range simultaneously.
Sequence table
<160>3
<210>1
<211>318
<212>DNA
＜213〉monkey rotavirus SA-11 (Simian rotavirus)
<400>1
cctcacttct acactttgcc tatattatcc gactgaggct gcgactgaaa taaacgataa 60
ttcatggaaa gacacactgt cacaactatt tcttacgaaa gagtggccaa ctggatccgt 120
atattttaaa gaatatacta acattgcatc gttttctgtt gatccgcagt tgtattgtga 180
ttataacgta gtactaatga aatatgacgc gacgttgcaa ttggatatgt cagaacttgc 240
ggatctaata ttaaacgaat ggttgtgtaa tccaatggat attactctgt attattatca 300
gcaaactgac gaagcgaa 318
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
<210>3
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
ttcgcttcgt cagtttgct 19
Claims (10)
1, quantitative detecting rotavirus standard product is the recombinant vectors that contains the nucleotide sequence of sequence 1 in the ordered list, or contains the genetically modified host cell of described recombinant vectors.
2, standard substance according to claim 1 is characterized in that: detection by quantitative is a quantitative PCR detection.
3, standard substance according to claim 2 is characterized in that: described quantitative PCR is conventional PCR or real-time quantitative PCR.
4, according to claim 1,2 or 3 described standard substance, it is characterized in that: described recombinant vectors is plasmid, phage vector or cement grain carrier.
5, according to claim 1,2 or 3 described standard substance, it is characterized in that: described genetically modified host cell is bacterium, fungi, plant or zooblast.
6, according to claim 1,2 or 3 described standard substance, it is characterized in that: described standard substance are to be that the nucleotide fragments of sequence 1 in the sequence table is connected the recombinant vectors that obtains with pGEM-T Easy carrier with nucleotide sequence.
7, the application of the described standard substance of arbitrary claim in the quantitative PCR detection rotavirus in the claim 1 to 5.
8, application according to claim 7 is characterized in that: described quantitative PCR is conventional PCR or real-time quantitative PCR.
9, a kind of method of detection by quantitative rotavirus, be to be template with the genomic dna of the described standard substance of arbitrary claim in detected sample and the claim 1 to 6 respectively, or be template with the cDNA that obtains by total RNA reverse transcription of the described standard substance of arbitrary claim in described detected sample and the claim 1 to 6 respectively, utilization is the primer formed of the nucleotide fragments of sequence 2 in the sequence table and nucleotide fragments that nucleotide sequence is sequence 3 in the sequence table to carrying out PCR by nucleotide sequence, and it is quantitative that detected sample is carried out rotavirus.
10, the test kit that contains the described standard substance of arbitrary claim in the claim 1 to 6.
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| CN104726609A (en) * | 2015-04-15 | 2015-06-24 | 北京凯思百奥科技发展有限公司 | Rotavirus detection kit and application thereof |
| CN105969766A (en) * | 2016-05-19 | 2016-09-28 | 山东出入境检验检疫局检验检疫技术中心 | Pear blister canker viroid (PBCVd) molecular standard sample and preparation method thereof |
| CN106769928A (en) * | 2016-12-14 | 2017-05-31 | 中国医学科学院医学生物学研究所 | Wheel virus antigen ELISA quantitative detecting methods |
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