CN101712999B - Dual real-time fluorescent RT-PC detection kit for subtypes of flu viruses N1 and N2 - Google Patents
Dual real-time fluorescent RT-PC detection kit for subtypes of flu viruses N1 and N2 Download PDFInfo
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Abstract
The invention relates to belongs to the field of flu virus detection, in particular to a dual real-time fluorescent RT-PC detection kit for the subtypes of flu viruses N1 and N2. The kit comprises a specific amplification flu virus N1 gene sequence primer with base sequences shown as SEQ ID NO I and SEQ ID NO:2, a Taqman probe which is used for detecting a flu virus N1 gene and has a base sequence shown as SEQ UD NO:3, a specific amplification flu virus N2 gene sequence primer with base sequences shown as SEQ ID NO:4 and SEQ IDNO:5 and a Taqman probe which is used for detecting the flu virus N2 gene and has a base sequence shown as SEQ IDNO:6; and all the Taqman probe sequences can be replaced by base complementation sequences thereof. The kit is used for detecting the subtypes of the fluviruses N1 and N2 and has the advantages of rapidness, simpleness, low cost, stable result, and the like.
Description
Technical field
The present invention relates to the influenza virus detection range, relate in particular to a kind of influenza virus N1, N2 hypotype dual real-time fluorescent RT detection kit.
Background technology
Influenza virus belongs to orthomyxoviridae family (Orthomyxoviridae), and for the minus-stranded rna virus of cyst membrane is arranged, the member comprises first, second, the third three type influenza viruses, and infected person mainly is first, Influenza B virus at present.First, Influenza B virus genome contains 8 sections, is the RNA of strand, minus strand, respectively the albumen of coding more than at least 10 kinds.According to the virus surface hemagglutinin (Hemaglutinin, HA) and neuraminidase (influenza A virus can be divided into 1-16 kind HA hypotype and 1-9 kind NA hypotype again for Neuraminidase, the NA) difference of antigenic property.The influenza virus that causes the human infection at present mainly is H1 (N1), H3 (N2) hypotype and Type B influenza virus.Since 1997, the people infects H5N1 subtype highly pathogenic influenza virus has also the time case case to take place.
Influenza virus is the cause of disease of birds, the mankind and mammiferous serious disease such as low.As far back as the B.C. record that similar influenza was just arranged in 412 years.19 th Century German uncommon and execute once tabulate write up since 1173 similar influenza be very popular have 30 approximately surplus time, the definite flu outbreak first time occurs in the Britain in 1510 years.1742 to 1743, there is 90% bohunk to get influenza.Swept across " the Russian influenza " in West Europe in 1889 to 1894, number of the infected is many, and mortality ratio is high.20th century, people's parainfluenza whole world that 3 pestilence property once took place was very popular.1918 to 1919, sweep away " spanish influenza " in the world with virulence, 3 epidemic peak appear in a few years, people's morbidity of 40% is arranged, cause various pneumonia complication thus, cause the about 2500-5000 ten thousand people death in the whole world; 1957-1958 " Asia influenza " causes 2,800,000 people dead; 1968-1969 " Mao flu " outburst involves the U.S., and 1,030,000 people are in peril of one's life, and wherein three or four ten thousand people die.
And bird flu was found so far in Italy from 1878 first and since migratory bird migrate frequent day by day with international bird trade, make this disease be global distribution, cause enormous economic loss to aviculture.Particularly 1997 " Hong Kong bird flu " incidents, since 2003 in South East Asia, China's Mainland and the world other countries human and bird fluenza epidemic situation and the flu/human avian influenza epidemic situation on the horizon great outburst that take place, caused of the very big concern of International Technology circle more with the World Health Organization and national governments.By the end of on January 3rd, 2008, the global people of World Health Organization's statistics infected the bird flu case and has reached 367 examples, and wherein dead 222 examples, mortality ratio are 64%; Since two thousand three, the people is taken place and is infected bird flu case 27 examples in middle Kuomintang-Communist, and wherein dead 17 examples, mortality ratio are 62.96%.
To sum up analyze, influenza, human and bird fluenza cause serious economic impact to whole international community, and especially highly pathogenic human and bird fluenza epidemic situation has become the great public health event that whole international community and the World Health Organization show great attention to after SARS.The World Health Organization (WHO) is in order to tackle the flu outbreak and effective control human and bird fluenza epidemic situation that is about to outburst; A plurality of preventions and control scheme have been formulated continuously; And spell out and lay special stress on: actively effectively perform the influenza virus monitoring; In the very first time, accomplishing the influenza virus diagnosis of (comprising human and bird fluenza virus H5N1), is the key job of great outburst of reply influenza and the highly pathogenic human and bird fluenza epidemic situation of control.Therefore; Must set up fast and accurately molecule and screen technical system, for the quick diagnosis of accurately the tracing to the source in real time of the real-time monitoring of influenza virus sub-strain, flu outbreak epidemic situation, human and bird fluenza virus H 5 N 1 subtype provide science accurately rapid molecular screen technology platform.
Influenza virus detects and comprises traditional method and molecular biology method two big classes.Traditional method comprise viral separation and Culture (chicken embryo culture and cell (MDCK) cultivate), blood clotting experiment (HA), blood clotting suppress experiment (HAI), (trace) neutralization experiment, golden mark method, directly (indirectly) immunofluorescence experiment, single radioimmunodiffusion haemolysis technological (Single radial hemolysis technique, SRH) etc.Traditional influenza virus detection method exists many defectives and deficiency; For example the operational cycle is long, sensitivity is low, poor specificity, cost an arm and a leg etc.; What is more important; According to the Biosafety relevant regulations of the Ministry of Health, have only limited laboratory can adopt traditional detection method that highly pathogenic human and bird fluenza virus H5N1 is diagnosed.Fast development along with Protocols in Molecular Biology; Increasing respective detection technique means is applied in the influenza virus molecule examination field; Particularly after the SARS; Successively there is multiple molecular diagnostic techniques to obtain using widely, specifically can comprises PCR, RT-PCR, Multiplex RT-PCR, MDD, Real-time RT-PCR, Duplex real time PCR.In recent years, with other molecular Biological Detection compared with techniques, the real-time fluorescence PCR technology is with fastest developing speed in the influenza virus molecular diagnosis field.
(polymerase chain reaction, PCR) since the reaction, round pcr becomes the hot spot technology of scientific research, clinical diagnosis very soon since nineteen eighty-three Mullis invention polymerase chain.But traditional P CR technology one is can not be accurately quantitative in application, the 2nd, and easy crossed contamination produces false positive.Up to FRET in recent years (fluorescence resonance energy transfer, FRET) technology be used for PCR quantitatively after, the problems referred to above are just solved preferably.(real-time fluorescence quantitative PCR FQ-PCR) is the nucleic acid quantification technology that on the qualitative technical foundation of PCR, grows up to real-time fluorescence quantitative PCR.It is a kind ofly in the PCR reaction system, to add fluorophor, utilizes the fluorescent signal accumulation whole PCR process of monitoring in real time, the method for through typical curve unknown template being carried out quantitative analysis at last.This technology has not only realized the qualitative, quantitative to the DNA/RNA template; And have characteristics highly sensitive, high specificity; Can realize multiple reaction; Advantage with level of automation height, nonstaining property, real-time and accuracy has been widely used in fields such as molecular biology research and medical research at present, is the development trend of rapid molecular diagnosis.
With regard to the influenza virus molecular diagnosis, all carried out a series of correlative study in recent years both at home and abroad, obtain certain achievement and breakthrough, but also had very big defective and deficiency.Domestic the development utilizes the Taqman-MGB fluorescent quantitation to intend transcriptase polymerase chain reaction rapid detection influenza A virus, fluorescence quantitative RT-RCR rapid detection A type influenza virus, fluorescence quantitative RT-RCR rapid detection Type B influenza virus, fluorescence quantitative RT-RCR rapid detection A, Type B influenza virus.These detection methods can only detect the type of influenza virus, can not carry out the rapid molecular diagnosis of influenza virus sub-strain.Country has set up influenza virus by RT-PCR (comprising one-step, nested RT-PCR, multiplex RT-PCR, real-timeRT-PCR) molecular diagnosis method in the influenza center; But one-step RT-PCR, nested RT-PCR, real-time RT-PCR can not accomplish the molecule of influenza virus type and hypotype simultaneously and screen only to the gene target spot on the single type of the influenza virus basis in these methods; And multiplex RT-PCR need carry out gel electrophoresis; Though can accomplish the molecule of influenza virus type and hypotype simultaneously screens; But this method required time is long; And this method system ratio is easier to crossed contamination, and specificity and sensitivity are relatively poor, shall not be applied to the rapid molecular diagnosis of influenza virus gradually.In addition; Country influenza center utilized the Tem-PCR technology in 2007; On Luminex xMAP (Multiple Analytes Profiling) platform, develop MDD (MolecularDifferential Diagnosis) molecule that carries out influenza virus type and hypotype and other Respiroviruses simultaneously and screened technology platform, and be successfully applied to the molecule examination of common Respirovirus (comprising SARS and H5N1 virus).The greatest drawback of this technology platform is to need special Luminex xMAP platforms, and is patented technology (develop cooperatively with the holder of the patent right at national influenza center) to the Tem-PCR technology of Respirovirus, so this technology platform is not effectively used.
Many molecular diagnostic techniques have also been developed to influenza virus abroad.Jurgen A.Richt in 2004 etc. utilize real-time RT-PCR technology to carry out the molecular diagnosis of type of American pig influenza virus (A type) and hypotype (H1, H3, N1, N2); But this system is not multiple real-time RT-PCR, can only carry out the molecular diagnosis of a type or hypotype at every turn.Freymnth F. in 2006 etc. utilize the multiplex round pcr that the acute respiratory symptom children that are in hospital are carried out the molecular diagnosis of Respirovirus, and compare with ordinary method (DFA), and discovery multiplex PCR is higher 2 times than DFA sensitivity.Yoo SJ in 2007 etc. utilize 2 cover multiplex PCR methods that 12 kinds of Respiroviruses have been carried out molecular diagnosis.These two kinds of multiplex round pcr systems are all carried out interpretation of result by gel electrophoresis, and itself there is the disadvantage of poor sensitivity, easy crossed contamination in this technical system.Ellis JS in 2007 etc. utilize the real-time PCR method to carry out the molecular diagnosis of human and bird fluenza virus H5N1HA-5 and NA-1 hypotype simultaneously; Brittain-long R in 2008 etc. adopt the method for multiple real-time PCR to common Respirovirus; For example: IFA, IFB, PIV1-3, MPV, RSV, RV, EV, ADV etc. have carried out molecular diagnosis, and the multiple real-time PCR molecule that does not carry out influenza virus sub-strain (HA and NA) is screened the foundation of system.
Summary of the invention
The object of the present invention is to provide a kind of influenza virus N1, N2 hypotype dual real-time fluorescent RT detection kit, this test kit can be accomplished the rapid molecular of influenza virus NA gene N1 and N2 hypotype and identify in 4 hours.
For achieving the above object, the present invention adopts following technical scheme:
A kind of influenza virus N1, N2 hypotype dual real-time fluorescence PCR detection kit is characterized in that said test kit comprises:
The primer of specific amplification influenza virus N1 gene order, its base sequence is shown in SEQ ID NO:1 and SEQ ID NO:2;
Be used to detect the Taqman probe of influenza virus N1 gene, its base sequence is shown in SEQ ID NO:3;
The primer of specific amplification influenza virus N2 gene order, its base sequence is shown in SEQ ID NO:4 and SEQ ID NO:5;
Be used to detect the Taqman probe of influenza virus N2 gene, its base sequence is shown in SEQ ID NO:6;
Above-mentioned all probe sequences can be by its complementary base sequences thereof replacement.
The said Taqman probe that is used to detect influenza virus N1 gene, its 5 ' end mark fluorescent group HEX, its 3 ' end mark cancellation fluorophor BHQ1.
The said Taqman probe that is used to detect influenza virus N2 gene, its 5 ' end mark fluorescent radicals R OX, its 3 ' end mark cancellation fluorophor BHQ2.
The present invention has designed 2 pairs of PCR primers specific amplification influenza virus N1, N2 gene order respectively; 2 pairs of PCR primers can increase in same PCR reaction tubes simultaneously; Realize double check, after optimizing, in four hours, accomplish the rapid molecular of influenza virus NA gene N1 and N2 hypotype and identify.
Primer that test kit according to the invention is included and probe are through up to a hundred different areas of MEGA comparison, the N1 of different times, N2 gene order; Select conservative region; Design with Primer Express, and influenza virus NAsequence all among designed primer probe and the genebank and virus database are carried out blast, when utilizing Primer Express to carry out the primer probe design mark is the highest; Be in the high conserved region of sequence; Do not have other sequence pairing situation when carrying out BLAST, and the preliminary experiment amplification curve is good, finally selects optimum primer and probe.Invent said primer and probe, in the design phase, the high conservative of assurance primer and probe and special, and guarantee the no complementary pairing or the situation appearance of increasing that intersects between two pairs of primers and the probe.The wavelength of fluorescence of two fluorescence groups that the present invention selects for use differs bigger, and both fluorescence signal intensities are close, to guarantee no phase mutual interference between two kinds of fluorescent signals.
Adopt test kit according to the invention that influenza virus N1, N2 hypotype are detected, have advantages such as simple fast, low-cost, that the result is stable.
Description of drawings
Fig. 1 is sample fluorescence quantitative PCR detection figure as a result.
Fig. 2 is N1 type Influenza Virus RNA standard substance amplification curve diagrams.
Fig. 3 is a N1 type Influenza Virus RNA canonical plotting.
Fig. 4 is N2 type Influenza Virus RNA standard substance amplification curve diagrams.
Fig. 5 is a N2 type Influenza Virus RNA canonical plotting.
Embodiment
A kind of influenza virus N1, N2 hypotype dual real-time fluorescence PCR detection kit comprise:
The primer P1 and the P2 of specific amplification influenza virus N1 gene order, P1 base sequence are shown in SEQ ID NO:1, and the P2 base sequence is shown in SEQ ID NO:2;
Be used to detect the Taqman probe T1 of influenza virus N1 gene, its base sequence shown in SEQ ID NO:3, its 5 ' end mark fluorescent group HEX, its 3 ' end mark cancellation fluorophor BHQ1;
The primer P3 and the P4 of specific amplification influenza virus N2 gene order, P3 base sequence are shown in SEQ IDNO:4, and the P4 base sequence is shown in SEQ ID NO:5;
Be used to detect the Taqman probe T2 of influenza virus N2 gene, its base sequence shown in SEQ ID NO:6, its 5 ' end mark fluorescent radicals R OX, its 3 ' end mark cancellation fluorophor BHQ2.
Embodiment 2 usefulness embodiment 1 said test kit detects sample
One, preparation detects and uses control sample
With High Pure Viral RNA Kit (Roche Diagnostics GmbH; Mannheim; Germany) extract the RNA of H1N1 standard strain (A1/ Guangdong Luohu/216/06) and the RNA of H3N2 standard strain (East Lake, A3/ Jiangxi/312/06), target gene fragment is increased afterwards and pGEM-T Easy vector (Promega, Madison with the primer of specific amplification influenza virus N1 gene order and the primer of specific amplification influenza virus N2 gene order; WI; Buy) connect, transformed into escherichia coli DH5 α extracts transformed bacteria mRNA as the positive RNA of system construction; Positive bacteria liquid is the positive reference substance that provides in the detection architecture, and the negative control article transform the bacterium liquid that DH5 α obtains for the pGEM-T carrier.
Two, detection method
Utilize takara One Step PrimeScript TM RT-PCR Kit (Perfect Real Time) on ABI7500 fluorescent PCR appearance, to make up double fluorescent RT-PCR system; Through each component concentrations and reaction conditions in the adjustment system; Make amplification curve best, N1, N2 fluorescent value are the most approaching.
The final concentration of each composition is in the RT-PCR amplification system: 1 * buffer
Primer P1 0.6uM
Primer P2 0.6uM
Primer P3 0.6uM
Primer P4 0.6uM
Probe T1 0.4uM
Probe T2 0.4uM
RNA template 5ul.
The RT-PCR amplification condition is: 42 ℃ of 5min;
95℃ 10s；
95℃ 5s，58℃40s，40cycles；
Fluorescent signal is selected HEX and ROX, in the time of 58 ℃, collects fluorescence.
Detected result is seen Fig. 1; Fig. 1 is the result who utilizes test kit according to the invention that N1, N2 positive colony body mRNA are detected, and top curve is that fluorescence RT-PCR amplification curve, the lower curve of N1 positive colony body is the fluorescence RT-PCR amplification curve of N2 positive colony body.
Three, sensitivity and amplification efficiency are measured
Extract the transformed bacteria plasmid, make N1, the linearizing of N2 recombinant plasmid, utilize RiboMaxT7 Express In Vitro Transcription System (WI buys for Promega, Madison) that N1, N2 goal gene are carried out in-vitro transcription with the SalI restriction endonuclease.Back RNA is transcribed in recovery, and (Eppendorf, Hamburg Germany) measure N1, N2 RNA concentration under the 260nm wavelength, calculate the RNA copy number through formula with BioPhotometer.N1, N2 RNA10 doubly are diluted to 10
7-10
0Copies does 6 repetitions to each extent of dilution RNA by the system that makes up, and does typical curve and calculates amplification efficiency and the variation coefficient, sees table one.
The RNA copy number calculates: Copies=(RNA ng number * 10
-9* 6.02 * 10
23)/(RNA base number * 345)
The final concentration of result: N1, N2 amplification in vitro RNA is respectively 1698ng/ul and 848ng/ul,
Calculate its copy number of back and be respectively 1.665 * 10
13With 6.49 * 10
12
Table one each concentration mean CT-number and variation coefficient
The variance and the variation coefficient by table 1 can find out, this reaction system repeated fine, and its variation coefficient majority is between 1-2%, and is little to the CT value variation of same concentration.
See also Fig. 2 and Fig. 3, its fluorescent signal does not reach threshold line when N1 RNA concentration reaches 100, so this system is 16.65copies/ul to the minimum sensitivity of N1.With the typical curve of the logarithmic value and the CT value of copy number, its relation conefficient reaches 99.6%, and amplification efficiency is 109.9%.
See also Fig. 4 and Fig. 5, when N2RNA concentration reaches 100, detect, so this system is 64.9copies/ul to the sensitivity of N2 less than fluorescent signal.With the typical curve of the logarithmic value and the CT value of copy number, its relation conefficient reaches 99.8%, and amplification efficiency is 106%.
Sequence table
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Claims (3)
1. an influenza virus N1, N2 hypotype dual real-time fluorescence PCR detection kit is characterized in that said test kit comprises:
The primer of specific amplification influenza virus N1 gene order, its base sequence is shown in SEQ ID NO:1 and SEQ ID NO:2;
Be used to detect the Taqman probe of influenza virus N1 gene, its base sequence is shown in SEQ ID NO:3;
The primer of specific amplification influenza virus N2 gene order, its base sequence is shown in SEQ ID NO:4 and SEQ ID NO:5;
Be used to detect the Taqman probe of influenza virus N2 gene, its base sequence is shown in SEQ ID NO:6;
Above-mentioned all Taqman probe sequences can be by its complementary base sequences thereof replacement.
2. influenza virus N1 according to claim 1, N2 hypotype dual real-time fluorescence PCR detection kit; It is characterized in that: the said Taqman probe that is used to detect influenza virus N1 gene; Its 5 ' end mark fluorescent group HEX, its 3 ' end mark cancellation fluorophor BHQ1.
3. influenza virus N1 according to claim 1, N2 hypotype dual real-time fluorescence PCR detection kit; It is characterized in that: the said Taqman probe that is used to detect influenza virus N2 gene; Its 5 ' end mark fluorescent radicals R OX, its 3 ' end mark cancellation fluorophor BHQ2.
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| WO2023077490A1 (en) * | 2021-11-06 | 2023-05-11 | 江汉大学 | Combination of mnp markers of influenza a, b and c viruses, primer pair combination, kit, and uses of combination, primer pair combination and kit |
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| CN101070557A (en) * | 2007-04-05 | 2007-11-14 | 广州市疾病预防控制中心 | Bird-flu-virus PCR increasing and hybridization detection and type-classifying method and reagent box |
| CN101363063A (en) * | 2007-08-10 | 2009-02-11 | 程小雯 | Primer, probe, kit and method for detecting A, B and H5 subtype influenza virus by triple fluorescent quantitative RT-PCR |
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| CN101070557A (en) * | 2007-04-05 | 2007-11-14 | 广州市疾病预防控制中心 | Bird-flu-virus PCR increasing and hybridization detection and type-classifying method and reagent box |
| CN101363063A (en) * | 2007-08-10 | 2009-02-11 | 程小雯 | Primer, probe, kit and method for detecting A, B and H5 subtype influenza virus by triple fluorescent quantitative RT-PCR |
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| 徐昌平.双重荧光定量RT-PCR快速检测A、B型流感病毒的研究.《中国卫生检验杂志》.2007,第17卷(第9期), * |
| 方浩.禽流感H5和H9亚型基因探针的制备.《湖北农业科学》.2007,第46卷(第2期), * |
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Effective date of registration: 20141218 Address after: 518000 West Block 3, 28 Lang Shan Road, Shenzhen, Guangdong, 202, Nanshan District, China Patentee after: SHENZHEN ZIJIAN BIOTECH CO., LTD. Address before: 518020 No. 1, No. 21, Tian Bei Road, Shenzhen, Guangdong, Luohu District Patentee before: Center of Diseases Prevention & Control, Shenzhen City |