CN102965452A - Norovirus real-time isothermal amplification detection kit, its primers and probe - Google Patents
Norovirus real-time isothermal amplification detection kit, its primers and probe Download PDFInfo
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Abstract
The invention relates to an enteropathogen rapid detection technology based on real-time nucleic acid sequence-based amplification (NASBA). Specifically, the invention provides a Norovirus real-time isothermal amplification detection kit, and a pair of primers and a molecular beacon probe thereof. The kit includes: a 2*real-time NASBA reaction solution containing the primers and the probe, a 5*enzyme mixed solution and a positive control template, a negative control and a blank control. The sequences of the primers and the probe are the sequences numbered as SEQIDNO:1-3, and the primers and the probe can specifically amplify and detect a Norovirus ORFs1 gene fragment. The kit provided in the invention has the characteristics of fastness, high efficiency, sensitivity and specificity, and real-time detection analysis, etc., and can be used in the fields of conventional detection and disease control and prevention in clinical practice and ports.
Description
Technical field
The present invention relates to a kind of enteropathogen Fast Detection Technique based on real-time nucleic acid sequence dependent amplification (Nucleic acid sequence-based amplification, NASBA) technology.Be specifically related to promise such as real-time NASBA isothermal amplification fast detecting reagent kit; Also relate to use in the described test kit for the norovirus genome have specific primer to and molecular beacon probe.
Background technology
Norovirus (Norovirus, NV) claim again norwalk group viruses, take norwalk virus (Norwalk virus) as representative, be a kind of distribution very widely, can cause being grown up and the virus of children acute gastro-enteritis, be the topmost virus causing disease that except rotavirus, causes diarrhoea, break out closely related with the acute gastroenteritis that the pollution at food, water source etc. causes.The norovirus genome is the sub-thread positive chain RNA, and total length 7642nt comprises 3 open reading frame (open reading frames, ORFs) ORFs1, ORFs2 and ORFs3.The conservative Nonstructural Protein with RNA polymerase activity of ORFs1 coding; ORFs2 coding molecule amount is about the capsid protein of 56Kda; ORFs3 a kind of molecular weight of encoding is about the strong basicity micro-structure albumen of 22.5Kda, and subregion and capsid protein generation interaction form capsid jointly.Although the acute gastroenteritis symptom that norovirus causes (vomiting, diarrhoea and abdominal cramps etc.) is self limiting, if treat the untimely death that still can cause, so it is all significant for disease control and clinical diagnosis to detect this Viral infection situation.Therefore, a cover is easy fast, the norovirus detection method of high specificity, can provide strong technical support for prevention and control and the clinical diagnosis of dysentery.
At present, for the detection of norovirus, classical method has electron microscopic observation, nucleic acid hybridization, enzyme linked immunological etc.Electron microscopic observation not only low but also expensive, the equipment of susceptibility and technical qualification requires very highly, can't be applied to routine clinical detection and large-scale epidemiology survey; Nucleic acid hybridization and enzyme linked immunosorbent detection are because of the restriction of technology itself, and its sensitivity is all lower, and loss is high.
Along with the fast-developing and combination of molecular biology and information biology, rapid based on the technical development of nucleic acid amplification, make rapid progress.Easy, the routine techniques accurately of a kind of RNA of detection viroid such as RT-polymerase chain reaction (RT-PCR).But the technology of PCR-based is the end-point method analysis, needs repeatedly the cyclic amplification of heating and cooling and the gel electrophoresis analysis result behind the PCR, and is consuming time longer, and sensitivity still remains to be improved.Afterwards, the real-time fluorescence RT-PCR that need not electrophoretic analysis becomes more efficiently a kind of nucleic acid amplification and RNA viruses detection technique.This technology utilizes the fluorescent probe realization to the specific detection of target nucleic acid, thereby utilizes the accumulation of fluorescent signal to realize that the whole process of Real-Time Monitoring pcr amplification reaction can the real-time analysis detected result.Because real-time fluorescence RT-PCR still needs to have reverse transcription step and three temperature spots such as sex change, annealing and extension to carry out tens heating and cooling circulation, each temperature spot and time also need accurately to set, and whole reaction still needs consuming time more than 2～2.5 hours.
In recent years, a kind of novel nucleic acid amplification technologies-isothermal amplification technique is implemented in the detection under the constant temperature.Such as dna circle mediated isothermal amplification technology (Loop-mediated Isothermal Amplification of DNA, that LAMP) uses is more extensive, related application is also arranged, but the LAMP Technology Need uses 4 primers, identify altogether 6 different zones of target DNA, although improved specificity, very difficult in design for the viral nucleic acid molecule that variation is large.
Nucleotide sequence dependent amplification (Nucleic acid sequence-based amplification, NASBA) technology is another kind of rapid isothermal amplification technique, it has overcome the in the past deficiency of nucleic acid amplification, and more simple and effective is particularly useful for the detection of RNA viruses.Different from PCR, NASBA is under the common cooperation of three kinds of enzymes such as AMV ThermoScript II, RNA enzyme H, T7 RNA polymerase, at 41～42 ℃, and the external specific nucleotide sequence isothermal duplication enzymic process of the continuous homogeneous that is instructed by pair of primers.Wherein its 5 ' end of primer has the promoter sequence of being identified by the T7 RNA polymerase.Binding molecule beacon on the NASBA basis (molecular beacon) probe can be set up real-time NASBA(Real-time NASBA) isothermal amplification technique.This technology has the following advantages: (1) isothermal duplication: reaction is carried out under same constant temperature system condition; (2) rapidly and efficiently, highly sensitive: in real time NASBA reaction is a kind of continuous rapid amplifying that need not heating and cooling, do not need the transcriptive process,reversed of 15～30 minutes fs in the RT-PCR reaction yet, the whole reaction times is short, the accumulation of single stranded RNA product is rapid, can in 60～90 minutes, finish detection, and make template ribonucleic acid amplification 10
9Doubly, sensitivity even can reach and detect copy number and be a template ribonucleic acid of position.The NASBA/ECL detection kit of for example researching and developing for all hypotypes of A type avian influenza virus at present, the experimental data that it is formally delivered shows, the NASBA technology is than 10～1000 times of present commercial immunity detection reagent sensitivities, and is also responsive more many than conventional PCR method; (3) high, the in real time detection of specificity: reaction does not need high-temperature denatured, can not be amplified even there is double-stranded DNA to pollute yet, and further improve specificity and sensitivity by the detection of molecular beacon probe, has realized real-time detection and interpretation of result yet; (4) simplicity of design: its only needs 1 pair of primer of design and 1 probe to identify 3 different zones of target RNA, and for detecting hypervariable viral nucleic acid, relative LAMP technology is more easy in the design.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, provides a kind of based on real-time NASBA(Real-time NASBA) technology the norovirus genome is had the primer of specific detection effect; It is a kind of based on real-time NASBA(Real-time NASBA that the present invention also provides) technology the norovirus genome is had the molecular beacon probe of specific detection effect; In addition the present invention also provide a kind of utilize above-mentioned primer to the detection kit of molecular beacon probe based on real-time nucleic acid sequence dependent amplification (Real-time NASBA) technology for detection norovirus.
A kind of norovirus of the present invention detects the technical scheme that adopts with primer, the present invention includes forward primer F1 and reverse primer R1, and the base sequence of described forward primer F1 and described reverse primer R1 is expressed as follows:
Forward primer F1:5 '-TGGAAYTCCATYGCCCACTG-3 ';
Reverse primer R1:
5’-AATTCTAATACGACTCACTATAGGGGTRCTNACAATYTCATCATCACC-3’。
25 bases of the 5 ' end of described reverse primer R1 are the T7 bacteriophage promoter sequences that increases, and 23 bases of 3 ' end are and the specific sequence of norovirus ORFs1 complementation, and the viral nucleic acid variant sites of the 3 ' end of described forward primer F1 and described reverse primer R1 is used the degeneracy base.
It is that the base sequence of described molecular beacon probe is expressed as follows that a kind of norovirus of the present invention detects the technical scheme that adopts with molecular beacon probe:
Molecular beacon probe P1:
5’-ROX-CCGAGCTGTGCVCTBTCYGARRTBACGCTCGG-DABCYL-3’，
Described molecular beacon probe P1 is in 5 ' end mark fluorescent reporter group ROX of its oligonucleotide DNA molecule, 3 ' end mark fluorescent quenching group DABCYL, viral nucleic acid variant sites use degeneracy base.
The technical scheme that the real-time isothermal amplification detection kit of a kind of norovirus of the present invention adopts is, described test kit comprises 2 * in real time NASBA reaction solution and 5 * enzyme mixations at least, and described 2 * real-time NASBA reaction solution includes aforesaid forward primer F1 and reverse primer R1 and molecular beacon probe P1.
Described 2 * real-time NASBA reaction solution comprises component as shown in table 1:
Table 12 * real-time NASBA reaction solution
Table 25 * enzyme mixation
Described 5 * enzyme mixation comprises following component:
Described test kit also comprises positive control, negative control and blank.Described positive control is the geneome RNA fragment of norovirus in-vitro transcription.Described negative control is when nucleic acid extraction and sample while parallel extraction stroke-physiological saline solution.Described blank is the ultrapure water of nuclease free.
The invention has the beneficial effects as follows: the present invention has specific pair of primers and a molecular beacon probe by providing a kind of to norovirus ORFs1, and comprise above-mentioned primer to the test kit of molecular beacon probe, detect whether there is norovirus ORFs1 specific gene fragment in the isolated preparation, and then whether have norovirus in definite isolated preparation.Detection kit of the present invention has the following advantages: 1. isothermal duplication: all inconvenience of having avoided regular-PCR that the particular requirement of temperature cycle is brought; 2. high specificity: by well-designed and in conjunction with specificity forward primer, reverse primer and molecular beacon probe that clinical sample examination optimized choice goes out, identify synchronously 3 different zones of norovirus, guaranteed the specificity of amplification; 3. sensitive efficient: because the amplification characteristics of NASBA self, so that detection sensitivity further improves, copy number is that 100 template can be detected; 4. amplification efficiency is high: can finish detection in 60～90 minutes, and make template ribonucleic acid increase about 10
9Doubly; 5. detect in real time: can obtain detected result by analytical data after the NASBA amplification finishes at once.
Description of drawings
Fig. 1 is among the embodiment four, and norovirus adopts the real-time NASBA detection kit of the present invention to carry out result's demonstration of sensitivity analysis, wherein, and 1:1 * 10
8Copy; 2:1 * 10
7Copy; 3:1 * 10
6Copy; 4:1 * 10
5Copy; 5:1 * 10
4Copy; 6:1 * 10
3Copy; 7:1 * 10
2Copy; 8:1 * 10
1Copy; 9:1 * 10
0Copy; The 10:NTC(negative control);
Fig. 2 is among the embodiment four, and norovirus employing real-time RT-PCR detection reagent case carries out result's demonstration of sensitivity analysis, wherein, and 1:1 * 10
8Copy; 2:1 * 10
7Copy; 3:1 * 10
6Copy; 4:1 * 10
5Copy; 5:1 * 10
4Copy; 6:1 * 10
3Copy; 7:1 * 10
2Copy; 8:1 * 10
1Copy; 9:1 * 10
0Copy; The 10:NTC(negative control);
Fig. 3 is among the embodiment four, and norovirus adopts the Conventional RT-PCR technology to carry out result's demonstration of sensitivity analysis, wherein, and 1:1 * 10
8Copy; 2:1 * 10
7Copy; 3:1 * 10
6Copy; 4:1 * 10
5Copy; 5:1 * 10
4Copy; 6:1 * 10
3Copy; 7:1 * 10
2Copy; 8:1 * 10
1Copy; 9:1 * 10
0Copy; The 10:NTC(negative control); M:100bp DNA Marker;
Fig. 4 is specificity analyses result's demonstration that real-time NASBA detection kit detects norovirus, wherein, and 1:GII type norovirus; The 2:A group rotavirus; The 3:B group rotavirus; 4: Astrovirus; 5: prick as virus; 6: coxsackie virus A 16-type; 7: negative control; 8: blank (H
2O).
Embodiment
The present invention will be described below by concrete norovirus testing process.
Embodiment one: the in real time design of NASBA primer and probe
According to the genomic RdRp of NVs and capsid protein nucleotide sequence difference, NV can be divided into 5 heredity groups (Genogroup), wherein heredity group I (Genogroup I, GGI) and heredity group II (Genogroup II, GGII) and tentative heredity group IV (Genogroup IV, GGIV) infect human.Heredity group GI and GII can be divided into several gene hypotypes again.Epidemic data shows that GGII/4 type NV is the modal genotype of world wide, and the NV of China's patient infection also is as main take genome GGII/4 type.At first download 100 GII type norovirus complete genome DNA sequences from the gene pool of U.S. NCBI, then utilize molecular biology software DNAMAN 6.0(Lynnon Corporation, Quebec, Canada) sequence of downloading is carried out the homology compare of analysis, seek the conserved sequence of height homology as the candidate regions of primer and probe design, simultaneously in conjunction with software Oligo 7.56(Molecular Biology Insights, Inc. USA) carry out the design of primer and probe.Design philosophy is: consider the principle of universality that specificity that norovirus is detected and versatility (processing with the degeneracy base in the viral nucleic acid variant sites), primer and probe design should follow (such as Tm value, 3 ' end free energy, GC content, avoid occurring internal structure and formation dimer etc.), and reverse primer adds the impact after the T7 bacteriophage promoter sequences etc.Carry out screening verification by RT-PCR experiment and the NASBA experiment of 50 parts of clinical diarrhoea samples of norovirus infant again after synthesizing to return for the primer of designing and probe, at last the present invention optimizes following primer and probe:
Forward primer F1:
5 '-TGGAAYTCCATYGCCCACTG-3 ', sequence numbering are SEQ ID NO:1;
Reverse primer R1:
5 '-AATTCTAATACGACTCACTATAGGGGTRCTNACAATYTCATCATCACC-3 ', sequence numbering are SEQ ID NO:2;
Molecular beacon probe P1:
5 '-ROX-CCGAGCTGTGCVCTBTCYGARRTBACGCTCGG-DABCYL-3 ', sequence numbering are SEQ ID NO:3.
25 bases of the 5 ' end of above-mentioned reverse primer R1 are the T7 bacteriophage promoter sequences that increases, and 23 bases of 3 ' end are and the specific sequence of norovirus ORFs1 complementation, and the viral nucleic acid variant sites of primer 3 ' end is used the degeneracy base;
Above-mentioned molecular beacon probe P1 is at 5 ' end mark fluorescent reporter group ROX of its oligonucleotide DNA molecule, 3 ' end mark fluorescent quenching group DABCYL; 6 bases of 5 ' end and 3 ' end are the reverse complementary sequences that forms probe characteristic hairpin structure stem petiolarea, 20 bases in the middle of the probe molecule are and the specific sequence of norovirus ORFs1 antisense strand complementation that wherein the viral nucleic acid variant sites is used the degeneracy base.
Know together 3 different zones of other norovirus ORFs1 of the molecular beacon probe of above-mentioned pair of primers and amplification region inside, thus the specificity of amplification fully guaranteed, and the viral nucleic acid variant sites that probe relates to then adopts the degeneracy base.For avoiding the long amplification efficiency that affects of reverse primer, the present invention has only used the T7 bacteriophage promoter sequences of 25 bases, experimental result show after the T7 RNA polymerase is to its identification to transcribe efficient still very high.For guaranteeing the quality of primer and probe, entrust Dalian precious biotechnology company limited to synthesize with the purity rubric of HPLC.
Embodiment two: the Establishment and optimization of the real-time NASBA detection system of norovirus
Carry out the optimization of condition for some important influence factors of real-time NASBA detection system.
1. method
(1) K
+Concentration: prepare 2 * in real time NASBA reaction solutions according to the reaction system of table 1, fix other parameter, regulate respectively K
+Final concentration to 40mM, 60mM, 80mM, 100mM, 120mM, 240mM, carry out real-time NASBA isothermal duplication take norovirus ORFs1 portion gene fragment in-vitro transcription RNA as template.After finishing, experiment compares different K
+Concentration is on the impact of amplification efficiency and fluorescence curve.
(2) concentration combination of dNTP/NTP: prepare 2 * in real time NASBA reaction solutions according to the reaction system of table 1, fix other parameter, regulate respectively the final concentration of dNTP/NTP to 0.1mM/1.6mM, 0.2mM/1.6mM, 0.4mM/1.6mM, 0.2mM/0.4mM, 0.2mM/0.8mM, 0.2mM/2.0mM, carry out real-time NASBA isothermal duplication take norovirus ORFs1 portion gene fragment in-vitro transcription RNA as template.The concentration combination of more different dNTP/NTP was on the impact of amplification efficiency and fluorescence curve after experiment finished.
(3) trehalose concentration: prepare 2 * in real time NASBA reaction solutions according to the reaction system of table 1, fix other parameter, regulate respectively the final concentration of trehalose to 0.05mM, 0.10mM, 0.15mM, 0.20mM, 0.25mM, 0.30mM, carry out real-time NASBA isothermal duplication take norovirus ORFs1 portion gene fragment in-vitro transcription RNA as template.More different trehalose concentration were on the impact of amplification efficiency and fluorescence curve after experiment finished.
(4) trimethyl-glycine concentration: prepare 2 * in real time NASBA reaction solutions according to the reaction system of table 1, fix other parameter, regulate respectively the final concentration of trimethyl-glycine to 0.05mM, 0.10mM, 0.15mM, 0.20mM, 0.30mM, 0.40mM, carry out real-time NASBA isothermal duplication take norovirus ORFs1 portion gene fragment in-vitro transcription RNA as template.More different trimethyl-glycine concentration were on the impact of amplification efficiency and fluorescence curve after experiment finished.
(5) concentration ratio of primer and probe: prepare 2 * in real time NASBA reaction solutions according to the reaction system of table 1, fix other parameter, the concentration ratio of regulating respectively primer and probe is 1: 1,2: 1,3: 1,4: 1, carries out real-time NASBA isothermal duplication take norovirus ORFs1 portion gene fragment in-vitro transcription RNA as template.After finishing, experiment compares the concentration ratio of different primers and probe to the impact of amplification efficiency and fluorescence curve.
2. result: for each Dynamics Optimization experiment, the fluorescence growth curve situation that the real-time NASBA reaction of systematic comparison obtains found that and works as K
+It is 2: 1 o'clock that concentration selects the concentration combination of 120mM, dNTP/NTP to select 0.2mM/1.6mM, trehalose concentration to select 0.15mM, trimethyl-glycine concentration to select the concentration ratio of 0.20mM, primer and probe, best by the expanding effect of 90 minutes NASBA isothermal duplication gained.Therefore select above-mentioned optimal conditions to be defined as the composition of each component of test kit.
Embodiment three: the composition and the detection method that detect the real-time NASBA test kit of norovirus
1. the composition of test kit (being stored in-20 ℃)
(1) 2 * in real time NASBA reaction solution: its component is: 120mM Tris-HCl (pH8.0), 240mM KCl, 20mM MgCl2,24mM DTT, 4mM spermidine, 0.4mM dNTP, 3.2mM NTP, 0.3mM trehalose, 0.4mM trimethyl-glycine, 30% DMSO, 0.8 μ M forward primer F1,0.8 μ M reverse primer R1,0.4 μ M molecular beacon probe P1; Wherein forward primer F1 is that sequence numbering is the nucleotide sequence shown in the SEQ ID NO:1, reverse primer R1 is that sequence numbering is the nucleotide sequence shown in the SEQ ID NO:2, and molecular beacon probe P1 is that sequence numbering is the nucleotide sequence shown in the SEQ ID NO:3;
(2) 5 * enzyme mixations: its component is: 1.5 U/ μ L AMV ThermoScript II, 6 U/ μ L T7 RNA transcriptases, 0.0625 U/ μ L RNAase H enzyme, 5 U/ μ L Ribonuclease Inhibitor, 20 mg/mL BSA, 8 mM sorbyl alcohols;
(3) positive control: be the ORFs1 portion gene fragment of norovirus in-vitro transcription;
(4) negative control: be stroke-physiological saline solution, when nucleic acid extraction and sample simultaneously parallel extraction to use as negative control;
(5) DEPC water: the ultrapure water of the nuclease free of processing for DEPC, use as blank.
2. the preparation of positive reference substance
Select at random the clinical infantile diarrhea sample of a norovirus, extract viral RNA.Design ORFs1 portion gene fragment amplification primer Noro_T7F1 and Noro_R1 utilize the amplification of RT-PCR technology to obtain total length and are the norovirus ORFs1 portion gene fragment of 852bp.Entrust the English Weihe River prompt base (Shanghai) trade Co., Ltd to carry out T-A clone construction recombination plasmid pMD18-NoroV the RT-PCR product that obtains, and carry out sequencing.10 norovirus genome sequences selecting at random among order-checking row result and the GenBank carry out the homology compare of analysis, guarantee that its homology reaches more than 95%.The positive colony bacterial strain extracts plasmid DNA after increasing bacterium, take its as template recycles above-mentioned primer Noro_T7F1 and Noro_R1, obtain the norovirus ORFs1 gene fragment of 852bp by the round pcr amplification, then utilize the T7 RNA polymerase to carry out in-vitro transcription and obtain single stranded RNA, transcribe and remove wherein dna molecular with DNase I digestion afterwards, then utilize the test kit RNeasy MiniElute Cleanup kit of German QIAGEN company to carry out column purification.Utilize micro-ultraviolet spectrophotometer to measure the concentration of RNA behind the purifying, calculate copy number by its molecular weight.After the packing in-80 ℃ of preservations, as the positive control of test kit.
The sequence of above-mentioned primer Noro_T7F1 is:
5’-AATTCTAATACGACTCACTATAGGGAGAATACCACTATGATGCAGATTA-3’；
The sequence of primer Noro_R1 is:
5’-CTTCGACGCCATCTTCATTCACA-3’。
3. the detection method of test kit is as follows:
(1) RNA of extraction sample, be template ribonucleic acid: test kit of the present invention does not provide RNA sample extraction reagent, the user can select suitable commercial kit to extract viral nucleic acid according to sample type, such as the test kit QIAamp MinElute Virus Spin Kit(article No. of German QIAGEN company: 57704);
(2) prepare in accordance with the following methods reaction solution: reaction volume is 20 μ L; Get 10 μ L, 2 * NASBA reaction solution and add 0.2ml optics PCR reaction tubes, adding 6 μ L sample rnas (suitably adjusts according to concentration again, the volume insufficient section can replace with the sterilization ultrapure water, making the template cumulative volume is 6 μ L) or positive control, negative control and blank, get again the inner face that 4 μ L, 5 * enzyme mixation is added in PCR pipe pipe lid, then will manage gently and build, and avoid pipe to cover containing the drop of enzyme to slip in the pipe;
(3) reaction pre-treatment: above-mentioned reaction tubes is placed on constant temperature bath or the regular-PCR instrument, and after elder generation respectively heated 3 minutes by 65 ℃ and 42 ℃, reaction tubes is of short duration centrifugal, and the enzyme mixation that allows pipe cover enters in the pipe, flicks mixing, and was again of short duration centrifugal;
(4) upper machine testing: pretreated reaction tubes is put into nucleic acid amplification detect in real time analytical system (Nuclisens EasyQ Analyzer (BioM é rieux, Boxtel, The Netherlands)) or the fluorescent PCR instrument carry out isothermal duplication and detect in real time.Reaction conditions is: 41～42 ℃ were reacted 90 minutes; Increase if be chosen on the fluorescent PCR instrument, then fluorescence detection channel is selected ROX, arrange accordingly according to different instruments, if 7500 type fluorescent PCR instrument of American AB I company, can arrange 35 seconds at 42 ℃ and 41 ℃～60 seconds circulation, each cycle detection first order fluorescence signal, cycle number is made by oneself, makes the isothermal duplication cumulative time reach 90 minutes and gets final product;
(5) result judges: according to fluorescent signal value (WT Signal) judged result that detects sample.With 1.2 times of threshold values as positive fluorescent signal (threshold) of negative control reaction end fluorescent signal value, the time point that positive fluorescence curve passes threshold value is defined as positive time point.At first be that quality control system judges that namely positive control WT Signal must be greater than threshold value, blank is less than threshold value, otherwise system's detection is invalid.When system detects when being effective, judge sample results to be checked, when the WT of sample to be checked Signal 〉=threshold value, be judged as positive sample, when the WT of sample to be checked Signal＜threshold value, be judged as negative sample;
(6) precaution: the experiment whole process all should be used powder-free gloves.For fear of the experiment in crossed contamination, in adding the process of template, should at first add blank and negative control, next adds sample to be checked, adds at last positive control.
Embodiment four: the sensitivity analysis of the real-time NASBA detection kit of norovirus
1. method:
(1) sample process: the RNA with the norovirus ORFs1 gene fragment of above-mentioned in-vitro transcription uses the RNA template as detecting, utilize micro-ultraviolet spectrophotometer to measure the concentration of RNA behind the purifying, the copy number that calculates Initial R NA template by molecular weight is 1.67 * 10
11Then/μ L by the mode of 6 μ L samples+54 μ L water, takes turns doing 10 times of gradient dilutions with it, chooses 9 gradients of lower concentration and tests, and each concentration gradient is respectively got 6 μ L and is used for amplification as template, and the copy number of 9 concentration gradients is: 1 * 10
8To 1 * 10
0
(2) three groups of parallel control experiments: select respectively real-time NASBA detection kit of the present invention, real-time RT-PCR detection reagent case (available from the Shenzhen City Yirui Bioisystech Co., Ltd, lot number: 111205) and Conventional RT-PCR technology (basic reagent is available from precious biotechnology (Dalian) company limited, article No. DRR055A) above-mentioned 9 gradient dilution samples and 1 negative control are carried out augmentation detection.
NASBA detects in real time: adopt embodiment three described detection methods to carry out the reagent preparation, each gradient dilution template 6 μ L, then reaction tubes is placed regular-PCR instrument (U.S. C1000 of BIORAD company type PCR instrument) upper successively in 65 ℃ and 42 ℃ respectively heat 3 minutes after, reaction tubes is of short duration centrifugal, the enzyme mixation that allows pipe cover enters in the pipe, flick mixing, again of short duration centrifugal, then reaction tubes is placed and carry out isothermal duplication and in real time detection on the fluorescent PCR instrument (American AB I company 7500 type fluorescent PCR instrument), reaction conditions is set is: (42 ℃ of 25sec, 41 ℃ of 35 sec) * 90 circulations, fluorescence detection channel is selected ROX, when circulating in 41 ℃ of 35 sec, each gathers the first order fluorescence signal, about 90 minutes of working time of whole isothermal duplication.Observation fluorescence curve collection of illustrative plates after reaction is finished arranges the fluorescent signal thresholding, analyzes template copy number minimum and corresponding proliferation time that test kit of the present invention can detect.
Real-time RT-PCR detects: carry out the reagent preparation according to the test kit specification sheets, and each gradient dilution template 6 μ L, the reaction conditions of real-time fluorescence RT-PCR is: 42 ℃ of 15min; 95 ℃ of 1min; FAM is selected in (95 ℃ of 15sec, 56 ℃ of 35sec, 72 ℃ of 30sec) * 45 circulations, fluorescence detection channel, gathers fluorescent signal when each circulates in 56 ℃ of annealing, about 100 minutes of working time of whole fluorescence RT-PCR.Observation fluorescence curve collection of illustrative plates after reaction is finished, the Ct value (Cycle threshold, circulation thresholding) of analysis template copy number lowest detectable limit.
Conventional RT-PCR detects: contain 2 * 1 Step RT-PCR Buffer, 10 μ L in the 20 μ L RT-PCR reaction systems, each 0.8 μ L of 20 μ M forward primers and reverse primer, PrimeScript 1 Step Enzyme Mix 0.8 μ L, each gradient dilution template 6 μ L.The reaction conditions of RT-PCR is: 50 ℃ of 30min; 94 ℃ of 3min; (94 ℃ of 30sec, 56 ℃ of 30sec, 72 ℃ of 30sec) * 40 circulations, 72 ℃ of 7min.The amplified fragments size is 138bp.About 150 minutes of working time of the upper machine of whole RT-PCR.RT-PCR reaction is got 5 μ L products and carry out electrophoresis in 2.0% sepharose after finishing, and electrophoresis time is 40 minutes, after electrophoresis finishes with the gel imaging system observations and take pictures.
2. result: the detected result of three groups of parallel control experiments shows that it is hundred (1 * 10 that real-time NASBA test kit of the present invention can detect copy number
2) transcribe rna, and the fluorescence growth curve just reached the thresholding fluorescent signal and risen the 29th circulation and entered the index amplification phase when (about 29 minutes of isothermal duplication time), the results are shown in Figure 1; The detection of real-time fluorescence RT-PCR reaction is minimum to be limited to 1 * 10
3, its Ct value is 30, is about 85 minutes and PCR runs to the 30th circulation time required time, the results are shown in Figure 2; The detection of Conventional RT-PCR reaction is minimum to be limited to 1 * 10
3, the results are shown in Figure 3.
Can find that from the sensitivity analysis of present embodiment the sensitivity of real-time NASBA detection kit of the present invention is the highest, can detect hundred figure places, i.e. 100 copies/reaction, and real-time fluorescence RT-PCR and Conventional RT-PCR all can only detect thousands digit (1000 copies/reaction).
Simultaneously, present embodiment can find that also the amplification efficiency of real-time NASBA detection kit of the present invention is very high.(the amplification pattern that PCR is desirable is: Y=X * 2 because NASBA reacts the amplification pattern that is different from PCR
n, wherein Y represents the amplified production amount, and X represents the primary template number in the PCR reactant, n is the amplification number of times), it is to carry out the amplification of template in the mode of per 1 minimum transcribed 90 RNA molecule of cDNA molecule.Therefore, from detecting the required time analysis, real-time NASBA detection kit of the present invention just can be observed copy number at isothermal duplication after 29 minutes be that the positive fluorescence growth curve of 100 transcribe rna sample reaches the thresholding fluorescent signal, be that positive time point is 29 minutes, speed of response outclass real-time fluorescence RT-PCR and Conventional RT-PCR.As seen, real-time NASBA detection kit of the present invention fast, efficiently, and is highly sensitive, can play a significant role in the detection of Emergent detection and trace sample fast.
Embodiment five: the specificity analyses of the real-time NASBA detection kit of norovirus
1. sample: select the clinical positive RNA sample of one group of diarrhoea correlated virus, number consecutively is respectively 1:GII type norovirus, the 2:A group rotavirus, the 3:B group rotavirus, 4, Astrovirus, 5, prick as virus, 6, coxsackie virus A 16-type, 7, negative control, 8, blank (H
2O), being this laboratory preserves.
2. method: use real-time NASBA detection kit of the present invention, adopt embodiment three described methods that above 8 increments are originally detected, observe this test kit and whether nonspecific detected result can occur.
3. result: according to the fluorescence pattern analysis of real-time NASBA amplification, test kit of the present invention is only to the test positive of No. 1 sample-GII type norovirus, the detection of other virus and negative control, blank is all negative, proves that the method has preferably specificity.The results are shown in Figure 4.
Embodiment six: the real-time NASBA detection kit of norovirus is to the detection of clinical sample
1. sample: from 300 parts of infantile diarrhea stool samples that preserve in this laboratory, select at random 20 parts to detect.Originally to be year April in October, 2009 to 2010 collect and be stored in-80 ℃ in Zhuhai City clinical laboratory of healthcare hospital for women ﹠ children this lot sample.
2. method: 20 parts of samples that adopt double-blind method to select are at random upset numbering, extract the viral RNA of sample again, adopt respectively real-time NASBA detection kit of the present invention and fluorescent RT-PCR technology to detect, last comparison and detection result's coincidence rate.
3. detecting step:
(1) viral RNA extracts: the stool sample of picking soya bean size is in the 800 μ L mixing that vibrates in the physiological saline, centrifugal 5 minutes of 8000rpm, get 200 μ L supernatants and be used for the RNA extraction, adopt the test kit QIAamp MinElute Virus Spin Kit(article No. of German QIAGEN company: 57704), operate by the test kit specification sheets;
(2) NASBA detects in real time: the method by embodiment three operates;
(3) fluorescence RT-PCR detects: the test kit (lot number: 111205) operate that adopts the Shenzhen City Yirui Bioisystech Co., Ltd.
4. detected result: utilize real-time NASBA detection kit provided by the invention to detect 7 parts of norovirus positive sample, recall rate is 35%, and is in full accord with the detected result of real-time RT-PCR, and the coincidence rate of two kinds of methods is 100%, and detected result sees Table 3.
20 parts of clinical diarrhoea pattern detection results of table 3
The present invention can be applicable to clinical and conventional sense and control and prevention of disease the field port.
It should be noted that, above-mentionedly only describe the present invention with preferred embodiment, can not limit to interest field of the present invention at this point, therefore in the situation that does not break away from inventive concept, the equivalence that the content of all utilizations specification sheets of the present invention and accompanying drawing part is carried out changes, and all reason is with being included in the claim scope of the present invention.
SEQUENCE LISTING
＜110〉Zhuhai International Travel Health Care Centre
＜120〉the real-time isothermal amplification detection kit of a kind of norovirus and primer and probe
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＜223〉forward primer F1
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tggaaytcca tygcccactg 20
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＜223〉reverse primer R1
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aattctaata cgactcacta taggggtrct nacaatytca tcatcacc 48
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＜223〉molecular beacon probe P1
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ccgagctgtg cvctbtcyga rrtbacgctc gg 32
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Claims (10)
1. a norovirus detects and uses primer, it is characterized in that comprise forward primer F1 and reverse primer R1, the base sequence of described forward primer F1 and described reverse primer R1 is expressed as follows:
Forward primer F1:5 '-TGGAAYTCCATYGCCCACTG-3 ';
Reverse primer R1:
5’-AATTCTAATACGACTCACTATAGGGGTRCTNACAATYTCATCATCACC-3’。
2. a kind of norovirus according to claim 1 detects and uses primer, it is characterized in that: 25 bases of the 5 ' end of described reverse primer R1 are the T7 bacteriophage promoter sequences that increases, and 23 bases of 3 ' end are and the specific sequence of norovirus ORFs1 complementation, and the viral nucleic acid variant sites of the 3 ' end of described forward primer F1 and described reverse primer R1 is used the degeneracy base.
3. a norovirus detects and uses molecular beacon probe, it is characterized in that the base sequence of described molecular beacon probe is expressed as follows:
Molecular beacon probe P1:
5’-ROX-CCGAGCTGTGCVCTBTCYGARRTBACGCTCGG-DABCYL-3’，
Described molecular beacon probe P1 is at 5 ' end mark fluorescent reporter group ROX of its oligonucleotide DNA molecule, 3 ' end mark fluorescent quenching group DABCYL.
4. a norovirus according to claim 3 detects and uses molecular beacon probe, it is characterized in that: the 5 ' end of described molecular beacon probe P1 and 6 bases of 3 ' end are the reverse complementary sequences that forms probe characteristic hairpin structure stem petiolarea, 20 bases in the middle of the probe molecule are and the specific sequence of norovirus ORFs1 antisense strand complementation that wherein the viral nucleic acid variant sites is used the degeneracy base.
5. real-time isothermal amplification detection kit of norovirus, it is characterized in that: described test kit comprises 2 * in real time NASBA reaction solution and 5 * enzyme mixations at least, and described 2 * real-time NASBA reaction solution includes forward primer F1 as claimed in claim 1 and reverse primer R1 and molecular beacon probe P1 as claimed in claim 3.
6. the real-time isothermal amplification detection kit of a kind of norovirus according to claim 5 is characterized in that, described 2 * real-time NASBA reaction solution comprises following component:
7. the real-time isothermal amplification detection kit of a kind of norovirus according to claim 5 is characterized in that described 5 * enzyme mixation comprises following component:
8. the real-time isothermal amplification detection kit of a kind of norovirus according to claim 5, it is characterized in that: described test kit also comprises positive control, negative control and blank.
9. the real-time isothermal amplification detection kit of a kind of norovirus according to claim 8, it is characterized in that: described positive control is the ORFs1 gene fragment of norovirus in-vitro transcription.
10. the real-time isothermal amplification detection kit of a kind of norovirus according to claim 8 is characterized in that: described negative control for when nucleic acid extraction with sample while parallel extraction stroke-physiological saline solution, described blank is the ultrapure water of nuclease free.
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| CN103397105A (en) * | 2013-07-01 | 2013-11-20 | 上海海洋大学 | Kit for detecting GII type norovirus and applications thereof |
| CN103525947A (en) * | 2013-09-09 | 2014-01-22 | 中国水产科学研究院黄海水产研究所 | NASBA (Nucleic Acid Sequence Based Amplification) detection primer, probe and detection method for GII type norovirus in sea foods |
| CN103757130A (en) * | 2013-11-28 | 2014-04-30 | 中国科学院广州生物医药与健康研究院 | Hand-foot-mouth disease virus isothermal amplification detection kit |
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| CN112760423A (en) * | 2021-03-03 | 2021-05-07 | 大连民族大学 | Primer group, kit and method for detecting GI (gastrointestinal tract) type and GII (gastrointestinal tract infection) type isothermal amplification of norovirus |
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| CN103397105A (en) * | 2013-07-01 | 2013-11-20 | 上海海洋大学 | Kit for detecting GII type norovirus and applications thereof |
| CN103397105B (en) * | 2013-07-01 | 2015-07-01 | 上海海洋大学 | Kit for detecting GII type norovirus and applications thereof |
| CN103525947A (en) * | 2013-09-09 | 2014-01-22 | 中国水产科学研究院黄海水产研究所 | NASBA (Nucleic Acid Sequence Based Amplification) detection primer, probe and detection method for GII type norovirus in sea foods |
| CN103757130A (en) * | 2013-11-28 | 2014-04-30 | 中国科学院广州生物医药与健康研究院 | Hand-foot-mouth disease virus isothermal amplification detection kit |
| CN103757130B (en) * | 2013-11-28 | 2017-01-18 | 中国科学院广州生物医药与健康研究院 | Hand-foot-mouth disease virus isothermal amplification detection kit |
| CN104894112A (en) * | 2015-05-18 | 2015-09-09 | 湖北省农业科学院畜牧兽医研究所 | Kit, primer, and probe for detecting Japanese encephalitis virus via real-time fluorescence isothermal amplification |
| CN104894112B (en) * | 2015-05-18 | 2018-04-13 | 湖北省农业科学院畜牧兽医研究所 | A kind of Japanese B encephalitis virus real-time fluorescence isothermal amplification detection kit and its primer and probe |
| CN106521032A (en) * | 2016-11-30 | 2017-03-22 | 博奥生物集团有限公司 | Nucleic acid amplification technology and application thereof in detection of mosquito-borne viruses |
| CN106521032B (en) * | 2016-11-30 | 2019-09-06 | 博奥生物集团有限公司 | A kind of nucleic acid amplification technologies and its application in carapuru virus detection |
| CN112760423A (en) * | 2021-03-03 | 2021-05-07 | 大连民族大学 | Primer group, kit and method for detecting GI (gastrointestinal tract) type and GII (gastrointestinal tract infection) type isothermal amplification of norovirus |
| CN112760423B (en) * | 2021-03-03 | 2022-06-14 | 大连民族大学 | Primer group, kit and method for detecting GI (gastrointestinal tract) type and GII (gastrointestinal tract infection) type isothermal amplification of norovirus |
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