CN105039591A - Method for quickly identifying and detecting N1, N2, N6 and N8 subtype influenza viruses - Google Patents
Method for quickly identifying and detecting N1, N2, N6 and N8 subtype influenza viruses Download PDFInfo
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- CN105039591A CN105039591A CN201510325784.XA CN201510325784A CN105039591A CN 105039591 A CN105039591 A CN 105039591A CN 201510325784 A CN201510325784 A CN 201510325784A CN 105039591 A CN105039591 A CN 105039591A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention provides a method for quickly identifying and detecting N1, N2, N6 and N8 subtype influenza viruses. A one-step fluorescent quantitation RT-PCR detection method is adopted. The method comprises the following steps of premixing Buffer, dNTP, Enhance and solution into 2X one-step buffer; and mixing various enzymes in a matching manner. Reaction liquid of experimental operation is easy and convenient to prepare, operation time is shortened, operation pollution is avoided, and dozens of copy numbers and even several copy numbers can be quantified. The method is easy and convenient to operate, is high in sensitivity, is convenient and speedy, is suitable for developing N1, N2, N6 and N8 identifying and detecting fluorescent quantitation RT-PCR kits for detection, is used for clinical identifying and diagnosing and epidemiological investigation of N1, N2, N6 and N8 subtypes, and has important significance on prevention and control of influenza of China.
Description
Technical field
The present invention relates to a kind of method differentiating influenza virus different subtype, be specifically related to a kind of method utilizing multiple fluorescence quantitative to differentiate N1, N2, N6, N8 subtype influenza virus fast.
Background technology
Influenza virus (InfluenzaVirus) belongs to orthomyxoviridae family, is RNA viruses.According to the difference of virus membrane antigen (M) and nucleoprotein (NP), be divided into by influenza virus first (A), second (B), third (C) three large-scale.Wherein the fashionable colors of A type influenza is the longest, endangers maximum, and popular host range is also the widest.According to virus surface hemagglutinin (HA) and the structure of neuraminidase (NA) and the difference of genetic characteristics thereof, different subtype can be divided into again.The hemagglutinin (HA) that current influenza A has found has 16 hypotypes, uses H1, H2 respectively ... H16 represents; Neuraminidase has 9 hypotypes, uses NI, N2 respectively ... N9 represents.HA with NA between each hypotype intersects and forms different hypotype strains, as H5N1, H5N2, H9N2, H5N6, H5N8, H6N6, H10N8, H4N6, H1N1 etc.The gene order of each hypotype is different, and virus characteristic and antigenicity etc. are also different.Wherein with regard to NA gene, NA1, NA2, NA6, NA8 are the most often separated in aviculture in recent years, and the loss caused by these hypotypes is also maximum.Detection for single hypotype can not show result fast timely, and expense is also expensive.Therefore the multiple fluorescence quantitative RT-PCR method for N1, N2, N6, N8 tetra-subtype influenza virus is set up, for clinical prevention treatment poultry diease is provided fundamental basis.
Influenza virus easily makes a variation, and variant form has the variation of the aspects such as the variation of antigenic variation, temperature sensitivity, host range, but most importantly antigenic variation.Antigenic variation mainly surface antigen HA and NA easily makes a variation.The size of influenza virus surface antigens variation amplitude, directly has influence on the ability of virus disseminating.If variation amplitude is little, belong to quantitative change, be called antigenic drift.If antigenic variation amplitude is large, belongs to qualitative change, be called antigenic shift, form new hypotype, harm is general comparatively large, often causes larger popular, even worldwide popular.Easily there is antigenic shift in HA, NA gene as influenza A virus, causes the part of HA and NA or Most amino-acids to change, and occurs the diverse new subtype of antigenicity.
The detection of influenza virus mainly relies on laboratory definitely to diagnose, and conventional diagnostic method mainly comprises isolation identification two aspect of Serologic detection and cause of disease.The separation of virus is one of method diagnosing this disease the most frequently used, usually adopts egg inoculation separated flow Influenza Virus, and the virus after cultivating suppresses (HI) to test through blood clotting (HA) and blood clotting, preliminary evaluation can go out influenza virus.But virus purification is egg inoculation or cellular segregation all exists excessive cycle, accurately can not distinguish the problem of virus subtype, accuracy and susceptibility difference.
Along with molecular biological develop rapidly, Protocols in Molecular Biology has been widely used in the mensuration of influenza virus.Especially in recent years fast-developing fluorescent quantitative RT-PCR method.Quantitative fluorescent PCR by detecting in real time each circulation products fluorescent signal in pcr amplification reaction, thus realizes quantitatively and qualitatively analyzing starting template.Reverse transcription and pcr amplification complete again in same pipe, contribute to reducing operational pollution, and the time is shorter, sensitivity is higher.In real-time fluorescence quantitative PCR reaction, introduce a kind of fluorescent chemical, along with the carrying out of PCR reaction, PCR reaction product constantly adds up, and fluorescence signal intensity also equal proportion increases.Often through a circulation, collect a fluorescence intensity signals, we just can monitor the change of product amount by the change of fluorescence intensity like this, thus obtain an amplified fluorescence graphic representation.The most frequently used probe is TaqMan probe, and this probe is a kind of oligonucleotide probe, and its fluorescence is relevant to the amplification of aim sequence.Its sequence and the sequence height between target sequence upstream primer and downstream primer match.Wherein, fluorophor is connected to 5 ' end of probe, and quenching group is then at 3 ' end.Conventional fluorophor has FAM, TET, VIC, HEX etc.When complete probe and target sequence match, the quenching group held of fluorogene and 3 ' launched of fluorophor is close and be quenched.But when carrying out extension, probe is carried out enzyme and cuts by 5 ' 5 prime excision enzyme activity of polysaccharase, makes fluorophor be separated with quenching group.Like this along with the continuous increase of amplification cycles number, the fluorophor discharged also in continuous accumulation, the therefore proportional relation of the quantity of fluorescence intensity and amplified production.
The quenching group of MGB probe adopts non-fluorescence quenching group (Non-FluorescentQuencher), and itself does not produce fluorescence, greatly can reduce the intensity of background signal.Probe is also connected with MGB (MinorGrooveBinder) modification group simultaneously, can by Tm value raising 10 ° of about C of probe.Therefore in order to obtain same Tm value, MGB probe can obtain shorter than general T aqMan probe design, both reduced synthesis cost, also makes the success ratio of probe design greatly improve.
At present, fluorescence quantitative PCR detection is widely used in the pathogenic microorganism examination, genopathy diagnosis, transmissible disease detection and lesion detection etc.
The AIVs of N1, N2, N6 and N8 hypotype is infected poultry and the common NA hypotype that can cause a disease.Mostly the method for current detection AIV is the detection for its HA hypotype, and as hypotypes such as H1, H3, H4, H5, H7, H9, the rare report of the detection technique for NA hypotype, especially for the differential diagnosis of multiple NA hypotype.Therefore, this research establishes a kind of quick, responsive, special multiple fluorescence quantitative RT-PCR method, can detect the influenza virus of N1, N2, N6, N8 hypotype simultaneously.
Summary of the invention
In order to solve above technical problem, the invention provides a kind of method that quick discriminating detects N1, N2, N6, N8 subtype influenza virus.
Concrete scheme of the present invention is as follows:
The reagent getting following concentration and volume is placed in reaction tubes and mixes:
2Xone-stepRT-PCRBuffer12.5μL；
Mixed enzyme RNaseinhibitor5U, M-MLV40U and TaqHS3U;
N1-fwd20μM0.25μL；
N1-rev20μM0.25μL；
N2-fwd20μM0.25μL；
N2-rev20μM0.25μL；
N6-fwd20μM0.25μL；
N6-rev20μM0.25μL；
N8-fwd20μM0.25μL；
N8-rev20μM0.25μL；
N1probe10μM0.25μL；
N2probe10μM0.25μL；
N6probe10μM0.25μL；
N8probe10μM0.25μL；
N1RNA template 10ng-1 μ g;
N2RNA template 10ng-1 μ g;
N6RNA template 10ng-1 μ g;
N8RNA template 10ng-1 μ g;
RNase-freeddH
2o mends to 25 μ L;
Start fluorescent PCR instrument, reaction tubes is put into PCR instrument device, RT-PCR reaction conditions: the first step 20min42 DEG C, second step 94 DEG C of 1min, the 3rd step 95 DEG C 15s, 60 DEG C of 30s, carry out 45 circulations, 4 DEG C of preservations, note: 60 DEG C gather fluorescence, record amplification curve;
The fluorescent reporter group of N1probe5 ' end mark is FAM, and the quenching group of 3 ' end mark is BQ1; The fluorescent reporter group of N2probe5 ' end mark is VIC, and the quenching group of 3 ' end mark is BQ2; The fluorescent reporter group of N6probe5 ' end mark is FAM, and the quenching group of 3 ' end mark is MGB; The fluorescent reporter group of N8probe5 ' end mark is VIC, and the quenching group of 3 ' end mark is MGB;
The preparation method of described N1, N2, N6, N8RNA template is as follows:
Get the influenza virus containing getting N1, N2, N6, N8 hypotype respectively, illustrate according to Trizol reagent and extract viral RNA: 1.5mL without the EP pipe of RNA enzyme in add the Trizol of 200 μ L allantoic fluids and 1mL, 5 minutes are placed in room temperature, with thorough dissociate nucleoprotein complex body after abundant mixing; Add the chloroform of 0.2mL, add and build rear concuss 15 seconds, place 2-3min in room temperature, then centrifugal 12,000 × g, 15min, 4 ° of C; Be divided into three layers in centrifugal rear pipe, redness is below phenol-chloroform phase, a middle layer, and one side is colourless aqueous phase; RNA precipitation careful by upper water phase transition to another without in the EP pipe of RNA enzyme, add the pre-cold isopropanol of equivalent volumes, room temperature leaves standstill 10min, centrifugal 12,000 × g, 15min, 4 ° of C; Can see that cotton-shaped glue sample precipitates at the sidewall of pipe and bottom after centrifugal; Supernatant is removed in RNA washing, adds the washing with alcohol RNA precipitation of 1mL75% precooling, mix gently, centrifugal 12,000 × g, 5min, 4 ° of C in precipitation; RNA dissolves and removes supernatant, and in super clean bench, air-dry RNA precipitation, adds 20 μ LDEPC water and fully dissolve RNA.
The influenza virus of discriminating detection N1, N2, N6 and N8 hypotype that the present invention is passable.
Accompanying drawing explanation
Fig. 1 is the amplification curve of N1 gene;
Fig. 2 is the amplification curve of N2 gene;
Fig. 3 is the amplification curve of N3 gene;
Fig. 4 is the amplification curve of N4 gene;
Fig. 5 is the common amplification curve of four kinds of genes.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
embodiment 1
materials and methods
one, material
viral template:
N1 is the RNA that two strain H5N1 balanced mix are extracted, and N2 is the total serum IgE that H5N2 and H9N2 sample equivalent carries out mixed extraction, and N6 is the total serum IgE that H5N6 and H4N6 balanced mix is extracted, and N8 is the total serum IgE that two strain H5N8 balanced mix are extracted.
the preparation of template:illustrate according to Trizol reagent and extract viral RNA: 1.5mL without the EP pipe of RNA enzyme in add the Trizol of 200 μ L allantoic fluids and 1mL, place 5 minutes in room temperature, with thorough dissociate nucleoprotein complex body after fully mixing.Add the chloroform of 0.2mL, add and build rear concuss 15 seconds, place 2-3min in room temperature, then centrifugal 12,000 × g, 15min, 4 ° of C.Be divided into three layers in centrifugal rear pipe, redness is below phenol-chloroform phase, a middle layer, and one side is colourless aqueous phase.RNA precipitation careful by upper water phase transition to another without in the EP pipe of RNA enzyme, add the pre-cold isopropanol of equivalent volumes, room temperature leaves standstill 10min, centrifugal 12,000 × g, 15min, 4 ° of C.Can see that cotton-shaped glue sample precipitates at the sidewall of pipe and bottom after centrifugal.Supernatant is removed in RNA washing, adds the washing with alcohol RNA precipitation of 1mL75% precooling, mix gently, centrifugal 12,000 × g, 5min, 4 ° of C in precipitation.RNA dissolves and removes supernatant, and in super clean bench, air-dry RNA precipitation, adds 20 μ LDEPC water and fully dissolve RNA.
main agents:one-stepReal-timeRT-PCRkit is purchased from TaKaRa company.
the Design and synthesis of primer and probe:choose the high conservative region of above strain N6 and N8 gene, carry out the design of primer and probe with PrimerExpress.The fluorescent reporter group of N1 probe 5 ' end mark is FAM, and the quenching group of 3 ' end mark is BQ1; The fluorescent reporter group of N2 probe 5 ' end mark is VIC, and the quenching group of 3 ' end mark is BQ2; The fluorescent reporter group of N6 probe 5 ' end mark is FAM, and the quenching group of 3 ' end mark is MGB; The fluorescent reporter group of N8 probe 5 ' end mark is VIC, and the quenching group of 3 ' end mark is MGB.Primer and probe are by the synthesis of Life company, and wherein primer is PAGE level purifying, and probe is HPLC level purifying.The detailed sequence of primer and probe and the unknown, in table 1.
key instrument:quantitative real time PCR Instrument is CFX384touchReal-timePCRdetectionsystems, purchased from American BIO-RAD company.
Table 1
。
two, amplification method
single primer increases separately
One step RT-PCR premix system
2Xone-stepRT-PCRBuffer12.5μL
Mixed enzyme RNaseinhibitor5U, M-MLV40U and TaqHS3U
The each 0.5 μ L of N1, N2, N6 or N8 upstream and downstream primer (20 μMs)
Corresponding probe (10 μMs) 0.5 μ L
Corresponding templates 10ng-1 μ g
RNase-freeddH
2o mends to 25 μ L.
multi-fluorescence RT-PCR
Add equivalent many covers probe primer, specific as follows:
2Xone-stepRT-PCRBuffer12.5μL
Mixed enzyme RNaseinhibitor5U, M-MLV40U and TaqHS3U
The each 0.25 μ L of N1 upstream and downstream primer (20 μMs)
The each 0.25 μ L of N2 upstream and downstream primer (20 μMs)
The each 0.25 μ L of N6 upstream and downstream primer (20 μMs)
The each 0.25 μ L of N8 upstream and downstream primer (20 μMs)
N1 probe (10 μMs) 0.25 μ L
N2 probe (10 μMs) 0.25 μ L
N6 probe (10 μMs) 0.25 μ L
N8 probe (10 μMs) 0.25 μ L
N1, N2, N6 and N8RNA template each 10ng-1 μ g
RNase-freeddH
2o mends to 25 μ L.
Start fluorescent PCR instrument, reaction tubes is put into PCR instrument device.RT-PCR reaction conditions: the first step 20min42 DEG C, second step 94 DEG C of 1min, the 3rd step 95 DEG C 15s, 60 DEG C of 30s, carry out 45 circulations, 4 DEG C of preservations.Note: 60 DEG C gather fluorescence.Record amplification curve.
three, result
the amplification curve of N1 gene
In Fig. 1, N1 gene obtains specific amplification (shown in wavy line), and N2 gene does not obtain amplification (shown in dotted line).Solid black lines amplification is empty template contrast.
N2
the amplification curve of gene
In Fig. 2, N2 gene obtains specific amplification (dotted line), and N1 gene does not obtain amplification (black).
N6
the amplification curve of gene
In Fig. 3, N6 gene obtains specific amplification (solid line), and NA8 gene (dotted line) and contrast (wavy line) do not obtain amplification.
N8
the amplification curve of gene
In Fig. 4, N8 gene obtains specific amplification (black amplification curve), and NA6 gene and contrast do not obtain amplification (black horizontal line).
N1
, N2, N6 and N8 specific detection-quadruple fluorescence quantitative RT-RCR
Four templates all obtain the specific amplification of expection, and four curves are followed successively by from top to bottom: N6, N1, N2, N8.
comprehensive analysis:
Above results proved that specificity and the accuracy of this multiple discriminating detection method.Conventional virus isolation procedure needs the time of 3-5 days, and quantitative fluorescent PCR only needs a few hours, and it has good specificity and susceptibility.And the single stage method fluorescence quantitative RT-PCR detecting method that the inventive method adopts, Buffer, dNTP, Enhancesolution are blended together 2Xone-stepbuffer in advance, multiple enzyme proportioning is mixed, reaction solution is prepared more simple and convenient, shorten the operating time, avoid operational pollution simultaneously.Therefore present method establishes the method for quick for N1, N2, N6 and N8 subtype influenza virus, can quantitative tens copy numbers, even several copy. the method is easy and simple to handle, susceptibility good, convenient and swift, be applicable to N1, N2, N6 and N8 discriminating detection fluorescence quantitative RT-PCR kit that exploitation detects, for Clinical differential diagnosis and the epidemiology survey of N1, N2, N6 and N8 hypotype, significant to the prevention and control of China's influenza.
Claims (1)
1. differentiate the method detecting N1, N2, N6, N8 subtype influenza virus fast, it is characterized in that, comprise the steps:
The reagent getting following concentration and volume is placed in reaction tubes and mixes:
2Xone-stepRT-PCRBuffer12.5μL；
Mixed enzyme RNaseinhibitor5U, M-MLV40U and TaqHS3U;
N1-fwd20μM0.25μL；
N1-rev20μM0.25μL；
N2-fwd20μM0.25μL；
N2-rev20μM0.25μL；
N6-fwd20μM0.25μL；
N6-rev20μM0.25μL；
N8-fwd20μM0.25μL；
N8-rev20μM0.25μL；
N1probe10μM0.25μL；
N2probe10μM0.25μL；
N6probe10μM0.25μL；
N8probe10μM0.25μL；
N1RNA template 10ng-1 μ g;
N2RNA template 10ng-1 μ g;
N6RNA template 10ng-1 μ g;
N8RNA template 10ng-1 μ g;
RNase-freeddH
2o mends to 25 μ L;
Start fluorescent PCR instrument, reaction tubes is put into PCR instrument device, RT-PCR reaction conditions: the first step 20min42 DEG C, second step 94 DEG C of 1min, the 3rd step 95 DEG C 15s, 60 DEG C of 30s, carry out 45 circulations, 4 DEG C of preservations, note: 60 DEG C gather fluorescence, record amplification curve;
The base sequence of described N1-fwd is as SEQINNO:1;
The base sequence of N1-rev is as SEQINNO:2;
The base sequence of N1probe is as SEQINNO:3;
The base sequence of N2-fwd is as SEQINNO:4;
The base sequence of N2-rev is as SEQINNO:5;
The base sequence of N2probe is as SEQINNO:6;
The base sequence of N6-fwd is as SEQINNO:7;
The base sequence of N6-rev is as SEQINNO:8;
The base sequence of N6probe is as SEQINNO:9;
The base sequence of N8-fwd is as SEQINNO:10;
The base sequence of N8-rev is as SEQINNO:11;
The base sequence of N8probe is as SEQINNO:12;
The fluorescent reporter group of N1probe5 ' end mark is FAM, and the quenching group of 3 ' end mark is BQ1; The fluorescent reporter group of N2probe5 ' end mark is VIC, and the quenching group of 3 ' end mark is BQ2; The fluorescent reporter group of N6probe5 ' end mark is FAM, and the quenching group of 3 ' end mark is MGB; The fluorescent reporter group of N8probe5 ' end mark is VIC, and the quenching group of 3 ' end mark is MGB;
The preparation method of described N1, N2, N6, N8RNA template is as follows:
Get the influenza virus containing getting N1, N2, N6, N8 hypotype respectively, illustrate according to Trizol reagent and extract viral RNA: 1.5mL without the EP pipe of RNA enzyme in add the Trizol of 200 μ L allantoic fluids and 1mL, 5 minutes are placed in room temperature, with thorough dissociate nucleoprotein complex body after abundant mixing; Add the chloroform of 0.2mL, add and build rear concuss 15 seconds, place 2-3min in room temperature, then centrifugal 12,000 × g, 15min, 4 ° of C; Be divided into three layers in centrifugal rear pipe, redness is below phenol-chloroform phase, a middle layer, and one side is colourless aqueous phase; RNA precipitation careful by upper water phase transition to another without in the EP pipe of RNA enzyme, add the pre-cold isopropanol of equivalent volumes, room temperature leaves standstill 10min, centrifugal 12,000 × g, 15min, 4 ° of C; Can see that cotton-shaped glue sample precipitates at the sidewall of pipe and bottom after centrifugal; Supernatant is removed in RNA washing, adds the washing with alcohol RNA precipitation of 1mL75% precooling, mix gently, centrifugal 12,000 × g, 5min, 4 ° of C in precipitation; RNA dissolves and removes supernatant, and in super clean bench, air-dry RNA precipitation, adds 20 μ LDEPC water and fully dissolve RNA.
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