CN105039591B - A kind of method of quick discriminating detection N1, N2, N6, N8 subtype influenza virus - Google Patents

A kind of method of quick discriminating detection N1, N2, N6, N8 subtype influenza virus Download PDF

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CN105039591B
CN105039591B CN201510325784.XA CN201510325784A CN105039591B CN 105039591 B CN105039591 B CN 105039591B CN 201510325784 A CN201510325784 A CN 201510325784A CN 105039591 B CN105039591 B CN 105039591B
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董以雷
张玉霞
袁小远
徐怀英
宋敏训
艾武
亓丽红
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Poultry Research Institute Shandong Academy of Agricultural Sciences
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Abstract

The invention provides a kind of detection and identification N1,N2,The method of N6 and N8 subtype influenza virus,Specifically employ an one-step fluorescence quantitative RT PCR detection methods,By Buffer,dNTP,Enhance solution blend together 2X one step buffer in advance,A variety of enzymes proportioning is mixed,Make the reaction solution preparation of experimental implementation more simple and convenient,Shorten the operating time,Avoid operational pollution simultaneously,Tens copy numbers can be quantified,Even several copies,This method is easy to operate,Sensitiveness is good,It is convenient and swift,It is adapted to the N1 of exploitation detection,N2,N6 and N8 differentiates detection fluorescent quantitation RT PCR kits,For N1,N2,The Clinical differential diagnosis of N6 and N8 hypotypes and epidemiology survey,Prevention and control to China's influenza are significant.

Description

A kind of method of quick discriminating detection N1, N2, N6, N8 subtype influenza virus
Technical field
The present invention relates to a kind of method for differentiating influenza virus different subtype, and in particular to one kind utilizes multiple fluorescence quantitative The method of quick discriminating N1, N2, N6, N8 subtype influenza virus.
Background technology
Influenza virus（Influenza Virus）Belong to orthomyxoviridae family, be RNA virus.According to virus membrane antigen（M）And core Albumen（NP）Difference, it is three large-scale that influenza virus is divided into first (A), second (B), third (C).The wherein fashionable colors of A types influenza Most long, harm is maximum, and popular host range is also most wide.According to the structure of virus surface hemagglutinin (HA) and neuraminidase (NA) And its difference of genetic characteristics, different subtype can be divided into again.The hemagglutinin that influenza A has found at present（HA）There are 16 Asias Type, represented respectively with H1, H2 ... H16；Neuraminidase has 9 hypotypes, is represented respectively with NI, N2 ... N9.Each hypotype Between HA and NA intersect and form different hypotype strain, 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 genes Speech, NA1, NA2, NA6, NA8 are most often separated in aviculture in recent years, are lost as caused by these hypotypes also maximum.For The detection of single hypotype quickly can not timely show result, and expense is also expensive.Therefore set up for N1, N2, N6, N8 tetra- The multiple fluorescence quantitative RT-PCR method of subtype influenza virus, treat poultry diease for clinical prevention and provide fundamental basis.
Influenza virus easily makes a variation, and variant form has antigenic variation, temperature sensitivity variation, host range etc. Variation, but most importantly antigenic variation.Antigenic variation is mainly that surface antigen HA and NA easily make a variation.Influenza surface The size of antigenic variation amplitude, directly influence the ability of viral transmission.If variation amplitude is small, belong to quantitative change, referred to as antigen floats Move.If antigenic variation amplitude is big, belong to qualitative change, referred to as antigenic shift, form new hypotype, harm is generally large, often Cause larger prevalence, or even worldwide prevalence.Antigenic shift easily occurs for HA, NA gene such as influenza A virus, causes HA and NA partially or mostly amino acid changes, and the entirely different new subtype of antigenicity occurs.
The detection of influenza virus relies primarily on laboratory and definitely diagnosed, and conventional diagnostic method mainly includes serology Detection and the aspect of the separation of cause of disease identification two.The separation of virus is to diagnose one of the most frequently used method of the disease, generally use chicken embryo Inoculation separation influenza virus, virus after culture suppress (HI) experiment by blood clotting (HA) and blood clotting, can Preliminary Identification go out stream Influenza Virus.But all there is excessive cycle, can not accurately distinguish virus in virus purification either egg inoculation or cell separation The problem of hypotype, accuracy and sensitiveness difference.
With the rapid development of molecular biology, Protocols in Molecular Biology is widely used to the measure of influenza virus.Especially It is fluorescent quantitative RT-PCR method fast-developing in recent years.Quantitative fluorescent PCR to each in pcr amplification reaction by following Ring product fluorescence signal is detected in real time, so as to realize quantitative to starting template and qualitatively analyze.Reverse transcription and PCR amplifications Completed again in same pipe, help to reduce operational pollution, the time is shorter, sensitivity is higher.It is anti-in real-time fluorescence quantitative PCR A kind of fluorescent chemical is introduced in answering, with the progress that PCR reacts, PCR reaction products are constantly accumulative, fluorescence signal intensity Also equal proportion increase.Often by a circulation, a fluorescence intensity signals are collected, so our cans pass through fluorescence intensity Change to monitor the change of product amount, so as to obtain a fluorescent amplification curve figure.The most frequently used probe is Taq Man probes, The probe is a kind of oligonucleotide probe, and its fluorescence is related to the amplification of aim sequence.Its sequence is drawn with target sequence upstream Sequence between thing and anti-sense primer is highly matched.Wherein, fluorophor is connected to 5 ' ends of probe, and quenching group is then In 3 ' ends.Conventional fluorophor has FAM, TET, VIC, HEX etc..When complete probe and target sequence match, fluorescence The fluorogene of group transmitting and the quenching group at 3 ' ends are approached and are quenched.But when carrying out extension, polymerase Probe is carried out digestion by 5 ' 5 prime excision enzyme activities so that fluorophor separates with quenching group.So with amplification cycles number not Disconnected increase, the fluorophor discharged is also constantly accumulating, therefore fluorescence intensity and the proportional relation of the quantity of amplified production.
The quenching group of MGB probes uses non-fluorescence quenching group (Non-Fluorescent Quencher), and itself is not Fluorescence is produced, the intensity of background signal can be substantially reduced.Simultaneously MGB (Minor Groove are also associated with probe Binder) modification group, the Tm values of probe can be improved 10 °C or so.Therefore in order to obtain same Tm values, MGB probes can It is shorter to be designed to than general T aq Man probes, synthesis cost had both been reduced, has also caused the success rate of probe design greatly to carry It is high.
At present, fluorescence quantitative PCR detection is widely used in the pathogenic microorganism examination, genopathy diagnosis, infectious disease detection With lesion detection etc..
The AIVs of N1, N2, N6 and N8 hypotype is the common NA hypotypes that infected poultry can simultaneously cause a disease,.Detection AIV at present Method be detection for its HA hypotype mostly, such as H1, H3, H4, H5, H7, H9 hypotype is few for the detection technique of NA hypotypes Have been reported that, in particular for the antidiastole of multiple NA hypotypes.Therefore, this research establishes a kind of quick, sensitive, special more Weight fluorescent quantitative RT-PCR method, the influenza virus of N1, N2, N6, N8 hypotype can be detected simultaneously.
The content of the invention
In order to solve the above technical problems, the invention provides a kind of quick discriminating to detect N1, N2, N6, N8 subtype influenza The method of virus.
The concrete scheme of the present invention is as follows：
Take the reagent of following concentration and volume to be placed in reaction tube to mix：
2X one-step RT-PCR Buffer 12.5μL；
Mixed enzyme RNase inhibitor 5U, M-MLV 40U and Taq HS 3U；
N1-fwd 20μM 0.25μL；
N1-rev 20μM 0.25μL；
N2-fwd 20μM 0.25μL；
N2-rev 20μM 0.25μL；
N6-fwd 20μM 0.25μL；
N6-rev 20μM 0.25μL；
N8-fwd 20μM 0.25μL；
N8-rev 20μM 0.25μL；
N1probe 10μM 0.25μL；
N2 probe 10μM 0.25μL；
N6 probe 10μM 0.25μL；
N8probe 10μM 0.25μL；
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-free ddH2O is mended to 25 μ L；
Start fluorescent PCR instrument, reaction tube is put into PCR instrument device, RT-PCR reaction conditions：The min 42 of the first step 20 DEG C, second step 94 DEG C of 1 min, the 3rd 95 DEG C of step 15s, 60 DEG C of 30s, carry out 45 circulations, 4 DEG C of preservations, note：60 DEG C of collections Fluorescence, record amplification curve；
The fluorescent reporter group of the ends of N1 probe 5 ' mark is FAM, and the quenching group of 3 ' end marks is BQ1；N2 probe The fluorescent reporter group of 5 ' end marks is VIC, and the quenching group of 3 ' end marks is BQ2；The fluorescence report of the ends of N6 probe 5 ' mark It is FAM to accuse group, and the quenching group of 3 ' end marks is MGB；The fluorescent reporter group of the ends of N8 probe 5 ' mark is VIC, and 3 ' hold The quenching group of mark is MGB；
The N1, the preparation method of N2, N6, N8 RNA template are as follows：
Take respectively containing the influenza virus for taking N1, N2, N6, N8 hypotype, illustrate extraction viral RNA according to Trizol reagents： 200 μ L allantoic fluids and 1mL Trizol are added in the 1.5mL EP pipes without RNase, are placed 5 minutes in room temperature after fully mixing, To be completely separated nucleoprotein complex；0.2 mL chloroform is added, good rear acutely concussion 15 seconds is capped, 2- is placed in room temperature 3min, is then centrifuged for the min of 12,000 × g, 15,4 °C；It is divided into three layers after centrifugation in pipe, red below is phenol-chloroform phase, One intermediate layer, it is simultaneously colourless aqueous phase；Upper strata aqueous phase is carefully transferred in the EP pipes of another no RNase by RNA precipitate, The pre- cold isopropanol of equivalent volumes is added, is stored at room temperature 10 min, centrifugation 12,000 × g, 15min, 4 °C；Can be with after centrifugation See cotton-shaped glue sample precipitation in the side wall of pipe and bottom；Supernatant is removed in RNA washings, and the second of the precoolings of 1mL 75% is added into precipitation Alcohol washs RNA precipitate, gently mixes, centrifugation 12,000 × g, 5min, 4 °C；Supernatant is removed in RNA dissolvings, is air-dried in super-clean bench RNA precipitate, add 20 μ L DEPC water and fully dissolve RNA.
The present invention can with discriminating detect N1, N2, N6 and N8 hypotype influenza virus.
Brief description of the drawings
Fig. 1 is the amplification curve of N1 genes；
Fig. 2 is the amplification curve of N2 genes；
Fig. 3 is the amplification curve of N3 genes；
Fig. 4 is the amplification curve of N4 genes；
Fig. 5 is the common amplification curve of four kinds of genes.
Embodiment
With reference to embodiment, the present invention is further illustrated.
Embodiment 1
Materials and methods
First, material
Viral template：
N1 is the RNA of two plants of H5N1 mixed in equal amounts extractions, and N2 is that H5N2 and H9N2 samples equivalent carries out mixing the total of extraction RNA, N6 are the total serum IgE of H5N6 and H4N6 mixed in equal amounts extraction, and N8 is the total serum IgE of two plants of H5N8 mixed in equal amounts extractions.
The preparation of template：Illustrate extraction viral RNA according to Trizol reagents：Added in the 1.5mL EP pipes without RNase The Trizol of 200 μ L allantoic fluids and 1mL, placed 5 minutes in room temperature after fully mixing, to be completely separated nucleoprotein complex.Add Enter 0.2 mL chloroform, be capped good rear acutely concussion 15 seconds, place 2-3min in room temperature, be then centrifuged for 12,000 × g, 15 Min, 4 °C.It is divided into three layers after centrifugation in pipe, red below is phenol-chloroform phase, an intermediate layer, is simultaneously colourless aqueous phase. Upper strata aqueous phase is carefully transferred in the EP pipes of another no RNase by RNA precipitate, adds the pre- cold isopropanol of equivalent volumes, room Temperature 10 min of standing, centrifugation 12,000 × g, 15min, 4 °C.It can see cotton-shaped glue sample in the side wall of pipe and bottom after centrifugation Precipitation.Supernatant is removed in RNA washings, and the ethanol washing RNA precipitate of the precoolings of 1mL 75% is added into precipitation, gently mixes, centrifuges 12,000 × g, 5min, 4 °C.Supernatant is removed in RNA dissolvings, air-dries RNA precipitate in super-clean bench, it is abundant to add 20 μ L DEPC water Dissolve RNA.
Main agents：One-step Real-time RT-PCR kit are purchased from TaKaRa companies.
The design and synthesis of primer and probe：The high conservative region of above strain N6 and N8 gene is chosen, uses Primer Express carries out the design of primer and probe.The fluorescent reporter group of the end of N1 probes 5 ' mark is FAM, and 3 ' end marks are quenched Group is BQ1；The fluorescent reporter group of the end of N2 probes 5 ' mark is VIC, and the quenching group of 3 ' end marks is BQ2；N6 probes 5 ' The fluorescent reporter group of end mark is FAM, and the quenching group of 3 ' end marks is MGB；The fluorescence report base of the end of N8 probes 5 ' mark Group is VIC, and the quenching group of 3 ' end marks is MGB.Primer and probe synthesizes by Life companies, and wherein primer is that PAGE levels are pure Change, probe purifies for HPLC levels.The detailed sequence of primer and probe and unknown, is shown in Table 1.
Key instrument：Quantitative real time PCR Instrument is the touch Real-time PCR detection of CFX 384 Systems, purchased from BIO-RAD companies of the U.S..
Table 1
。
2nd, amplification method
Single primer is individually expanded
One step RT-PCR premixes system
2X one-step RT-PCR Buffer 12.5μL
Mixed enzyme RNase inhibitor 5U, M-MLV 40U and Taq HS 3U
Each 0.5 μ L of N1, N2, N6 or N8 upstream and downstream primer (20 μM)
Corresponding probe (10 μM) 0.5 μ L
Corresponding templates 10ng-1 μ g
RNase-free ddH2O is mended to 25 μ L.
Multi-fluorescence RT-PCR
Add equivalent to cover probe primer more, it is specific as follows：
2X one-step RT-PCR Buffer 12.5μL
Mixed enzyme RNase inhibitor 5U, M-MLV 40U and Taq HS 3U
Each 0.25 μ L of N1 upstream and downstream primers (20 μM)
Each 0.25 μ L of N2 upstream and downstream primers (20 μM)
Each 0.25 μ L of N6 upstream and downstream primers (20 μM)
Each 0.25 μ L of N8 upstream and downstream primers (20 μM)
The μ L of N1 probes (10 μM) 0.25
The μ L of N2 probes (10 μM) 0.25
The μ L of N6 probes (10 μM) 0.25
The μ L of N8 probes (10 μM) 0.25
Each 10ng-1 μ g of N1, N2, N6 and N8RNA template
RNase-free ddH2O is mended to 25 μ L.
Start fluorescent PCR instrument, reaction tube is put into PCR instrument device.RT-PCR reaction conditions：The min 42 of the first step 20 DEG C, second step 94 DEG C of 1 min, the 3rd 95 DEG C of step 15s, 60 DEG C of 30s, carry out 45 circulations, 4 DEG C of preservations.Note：60 DEG C of collections Fluorescence.Record amplification curve.
3rd, result
The amplification curve of N1 genes
N1 genes obtain specific amplification in Fig. 1（Shown in wave）, and N2 genes do not obtain amplification（Shown in dotted line）.It is black The amplification of color solid line compares for empty template.
The amplification curve of N2 genes
N2 genes obtain specific amplification in Fig. 2（Dotted line）, and N1 genes do not obtain amplification（Black）.
The amplification curve of N6 genes
N6 genes obtain specific amplification in Fig. 3（Solid line）, and NA8 genes（Dotted line）And control（Wave）Expansion is not obtained Increase.
The amplification curve of N8 genes
N8 genes obtain specific amplification in Fig. 4（Black amplification curve）, and NA6 genes and control do not obtain amplification（It is black Color horizontal line）.
N1, N2, N6 and N8 specific detection-quadruple fluorescence quantitative RT-RCR
Four templates obtain expected specific amplification, and four curves are followed successively by from top to bottom：N6、N1、N2、 N8。
Comprehensive analysis：
Above results proved that the multiple specificity and accuracy for differentiating detection method.Conventional virus isolation procedure needs The time of 3-5 days is taken, and quantitative fluorescent PCR only needs a few hours, and also it has good specificity and sensitiveness.It is and of the invention The one-step method fluorescence quantitative RT-PCR detecting method that method uses, blendes together 2X in advance by Buffer, dNTP, Enhance solution One-step buffer, a variety of enzymes proportioning is mixed, prepare reaction solution more simple and convenient, shorten the operating time, together When avoid operational pollution.Therefore this method establishes the quick determination method for N1, N2, N6 and N8 subtype influenza virus, Tens copy numbers can be quantified, or even several copy this method are easy to operate, sensitiveness is good, convenient and swift, are adapted to exploitation detection N1, N2, N6 and N8 differentiate detection fluorescence quantitative RT-PCR kit, and clinical differentiate for N1, N2, N6 and N8 hypotype is examined Disconnected and epidemiology survey, the prevention and control to China's influenza are significant.

Claims (1)

  1. A kind of 1. method of quick discriminating detection N1, N2, N6, N8 subtype influenza virus, it is characterised in that comprise the following steps：
    Take the reagent of following concentration and volume to be placed in reaction tube to mix：
    2X one-step RT-PCR Buffer 12.5μL；
    Mixed enzyme RNase inhibitor 5U, M-MLV 40U and Taq HS 3U；
    N1-fwd 20μM 0.25μL；
    N1-rev 20μM 0.25μL；
    N2-fwd 20μM 0.25μL；
    N2-rev 20μM 0.25μL；
    N6-fwd 20μM 0.25μL；
    N6-rev 20μM 0.25μL；
    N8-fwd 20μM 0.25μL；
    N8-rev 20μM 0.25μL；
    N1probe 10μM 0.25μL；
    N2 probe 10μM 0.25μL；
    N6 probe 10μM 0.25μL；
    N8probe 10μM 0.25μL；
    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-free ddH2O is mended to 25 μ L；
    Start fluorescent PCR instrument, reaction tube is put into PCR instrument device, RT-PCR reaction conditions：42 DEG C of 20 min of the first step, Second step 94 DEG C of 1 min, the 3rd 95 DEG C of step 15s, 60 DEG C of 30s, carry out 45 circulations, 4 DEG C of preservations, note：60 DEG C of collections are glimmering Light, record amplification curve；
    It is described
    N1-fwd base sequence such as SEQ IN NO:1；
    N1-rev base sequence such as SEQ IN NO:2；
    N1probe base sequence such as SEQ IN NO:3；
    N2-fwd base sequence such as SEQ IN NO:4；
    N2-rev base sequence such as SEQ IN NO:5；
    N2 probe base sequence such as SEQ IN NO:6；
    N6-fwd base sequence such as SEQ IN NO:7；
    N6-rev base sequence such as SEQ IN NO:8；
    N6 probe base sequence such as SEQ IN NO:9；
    N8-fwd base sequence such as SEQ IN NO:10；
    N8-rev base sequence such as SEQ IN NO:11；
    N8probe base sequence such as SEQ IN NO:12；
    The fluorescent reporter group of the ends of N1 probe 5 ' mark is FAM, and the quenching group of 3 ' end marks is BQ1；N2 probe 5’ The fluorescent reporter group of end mark is VIC, and the quenching group of 3 ' end marks is BQ2；The fluorescence report of the ends of N6 probe 5 ' mark Group is FAM, and the quenching group of 3 ' end marks is MGB；The fluorescent reporter group of the ends of N8 probe 5 ' mark is VIC, and 3 ' ends are marked The quenching group of note is MGB；
    The N1, the preparation method of N2, N6, N8 RNA template are as follows：
    Take respectively containing the influenza virus for taking N1, N2, N6, N8 hypotype, illustrate extraction viral RNA according to Trizol reagents：In 1.5mL The EP pipes without RNase in add 200 μ L allantoic fluids and 1mL Trizol, fully mix after room temperature place 5 minutes, with thorough Bottom separates nucleoprotein complex；0.2 mL chloroform is added, good rear acutely concussion 15 seconds is capped, places 2-3min in room temperature, so After centrifuge the min of 12,000 × g, 15,4 °C；It is divided into three layers after centrifugation in pipe, following red is phenol-chloroform phase, in one Interbed, it is simultaneously colourless aqueous phase；Upper strata aqueous phase is carefully transferred in the EP pipes of another no RNase by RNA precipitate, is added The pre- cold isopropanol of equivalent volumes, is stored at room temperature 10 min, centrifugation 12,000 × g, 15min, 4 °C；Can be in pipe after centrifugation Side wall and bottom see cotton-shaped glue sample precipitation；Supernatant is removed in RNA washings, and the ethanol that the precoolings of 1mL 75% are added into precipitation is washed RNA precipitate is washed, is gently mixed, centrifugation 12,000 × g, 5min, 4 °C；Supernatant is removed in RNA dissolvings, and air-drying RNA in super-clean bench sinks Form sediment, add 20 μ L DEPC water and fully dissolve RNA.
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