CN101736089B - Kit for real-time fluorescence RT-PCR detection of avian influenza virus - Google Patents
Kit for real-time fluorescence RT-PCR detection of avian influenza virus Download PDFInfo
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Abstract
The invention relates to a kit for real-time fluorescence RT-PCR detection, in particular to a kit for early and rapid diagnosis of the infection of an avian influenza virus by using the one-step real-time fluorescence reverse transcription-polymerase chain reaction (RT-PCR) technology. Since the kit has high sensitivity and specificity, the rapid and early detection and quantitative analysis of the avian influenza virus in samples, such as the throat swab, the cloacae swab, the muscle or the viscera sample, the sera or the plasma and the like, can be realized by the kit.
Description
Technical field
The present invention relates to a kind of test kit of real-time fluorescence RT-PCR detection of avian influenza virus, particularly relate to test kit early stage with one-step method real-time fluorescent reverse-transcription polymerase chain reaction (RT-PCR) technology, the quick diagnosis avian influenza.This test kit has very high sensitivity and specificity, realizes the bird flu virus in brush,throat, cloaca swab, muscle or the samples such as internal organs sample, serum or blood plasma is carried out quick early detection and quantitative analysis through test kit of the present invention.
Background technology
(Avian Influenza AI) is the abbreviation of avian influenza, claims fowl plague or European checken pest again, is a kind of acute infectious disease from respiratory tract disease to multiple symptoms such as seriousness body tissue hemorrhagic pathologies in bird flu.1878, Perroncito found bird flu first in gondola chicken crowd, and report is all arranged all over the world thereafter; Break out at every turn and all cause enormous economic loss; As, Pennsylvania, America broke out bird flu in 1993, government 1,700 ten thousand chickens of furnishing funds for 6,000 ten thousand dollars to be used to catch and kill; Nineteen ninety-five, because the bird flu reason, about 5,000 ten thousand chickens are catched and killed, blocked in Mexico, and more than one hundred million chickens are carried out urgent vaccination, directly causes 1,000,000,000 dollars of financial losses.China found since 1994 bird flu popular in the chicken crowd (gold dollar is prosperous, Li Jingpeng, Zhang Long etc. the bird flu virus Progress on Molecular Biology. animal medicine progress, 2003,24 (1): 12-15), a lot of incidents of breaking out have taken place.Bird flu brings about great losses for world's aviculture, is the crushing transmissible disease of internationally recognized a kind of bird, and World Organization for Animal Health's epizootic disease tissue (IOE) regulation should disease be the category-A deadly infectious disease, and China also classifies it as type of Quarantine Objects.
Causing the pathogenic arch-criminal of bird flu---(Avian Influenza Virus AIV) belongs to orthomyxoviridae family to bird flu virus, A type (or first type) influenza virus; Be a kind of minus-stranded rna virus, according to its membranin hemagglutinin (Hemagglutinin, HA) antigenicity can be divided into different hypotypes; Found altogether that up to now (H1～H16), wherein exhausted big number AIV is inapparent infection to 16 kinds of HA hypotypes, does not show any clinical symptom; Have only minority AIV to have the characteristic of bamboo telegraph and high lethality; Be referred to as highly pathogenic avian influenza virus, as, AIV-H5, AIV-H7, AIV-H9.The same with other RNA viruses, because RNA polymerase lacks correct functioning, morph easily; More particularly, the genome of influenza virus all is made up of 8 discrete, segmented RNA fragments, reset easily, so AIV very easily morphs, and virulence is more and more stronger.In recent years, high pathogenic avian influenza is broken out into ascendant trend, not only serious harm bird all over the world; Also threaten Mammals, especially can infect human and can cause death, the particularly bird flu that breaks out of Hong Kong in 1997; Not only lose 100,000,000 Hongkong dollars and catched and killed 1,500,000 chickens; And human health has been caused very big threat, up till now, hundreds of people have been taken place and have been infected the lethal report of bird flu in China and other countries.This shows that the research bird flu has important public hygienics meaning and economics meaning! Current emphasis is the propagation of prevention and control AIV, and it is most important therefore to set up viral fast and accurately detection means.
The detection method commonly used of AIV comprises: virus culture method, immunological method and nucleic acid detection method.The virus culture method is one of laboratory detection method of widespread use; Highly sensitive, but the operational cycle long (7～10 days), and to also higher (the Kaiser L of the requirement of laboratory condition; Briones MS; Hayden FG.Performance of virus isolation andDirectigen Flu A to detect influenza A virus in experimental human infection.J ClinVirol, 1999,14:191-197).By comparison, immunological method is fast and convenient relatively, but sensitivity and poor specificity (Boivin G; Hardy I; Kress A.Evaluation of a rapid optical immunoassay for influenzaviruses (FLU OIA test) in comparison with cell culture and reverse transcription-PCR.J Clin Microbiol, 2001,39:730-732); And the result of antibody test only explains that bird to be detected or animal once infected bird flu virus; But can not accurately reflect currently whether infect or carry virus, therefore, have certain limitation in application facet.Particularly present poultry by the vaccinate immunity, therefore can not use this method to carry out examination mostly.In recent years, the nucleic acid molecule biological method of develop rapidly is like reverse-transcription polymerase chain reaction (reverse-transcriptionPCR; RT-PCR) technology is applied to the detection of AIV; Through the reversed transcriptive enzyme effect, become cDNA to AIV RNA rt, and then utilize the Taq enzyme that cDNA is carried out pcr amplification earlier; Highly sensitive and can quick and precisely detect and whether carry virus; Can combine other detection method further to judge (Ellis JS, Fleming DM, Zambon MC.Multiplex reversetranscription-PCR for surveillance of influenza A and B viruses in England and Walesin 199 5and 1996.J Clin Microbiol in addition; 1997,35:2076-2082; Magnard C; Valette M, AymardM, et al.Comparison of two nested PCR; Cell culture; And antigen detection for thediagnosis of upper respiratory tract infections due to influenza viruses.J Med Virol, 1999,59:215-220.)。
The real-time fluorescence PCR technology is a kind of rapidly nucleic acid detection technique of development in recent years, uses a kind of pcr amplification appearance that has nuclear power coupling devices (CCD), reflects each round-robin level of amplification of PCR in real time through the dynamic change that detects fluorescent signal.CCD can periodically send the exciting light of specific wavelength according to certain procedure, collect to detect fluorescent signal, and is aggregated into workstation through software analysis and obtains amplification curve.One-step method real-time fluorescent RT-PCR technology is a kind of (Martell, M., the J.Gomez of real-time fluorescence PCR; J.Esteban; S.Sauleda, J.Quer, B.Cabot; R.Esteban, and J.Guardia.1999.High-throughput real-time reverse transcription-PCR quantitation of hepatitis Cvirus RNA.J.Clin.Microbiol.37:327-332; Lee CW, Suarez DL.2004.Application ofreal-time RT-PCR for the quantitation and competitive replication study of H5 andH7subtype avian influenza virus.J Virol Methods.119 (2): 151-8.), it is the method for a kind of direct rapid detection RNA, compares with the real-time fluorescence PCR that detects DNA, difference is that the former has increased reversed transcriptive enzyme in reaction system, many simultaneously reverse transcription reaction steps; Something in common is that both all have the probe of a two ends difference mark fluorescent reporter group and quenching group in reaction system, and when probe structure was complete, the energy that the fluorescence report group sends fluorescence shifted to quenching group, presents quenching effect.If the existence of target sequence is arranged in the amplification procedure, the process middle probe molecule of amplification is hydrolyzed cut-out gradually, and fluorescence report group and quenching group dissociate each other, have blocked the two FRET effect, and the fluorescence report group sends fluorescent signal.Along with the carrying out of amplification, fluorescent signal presents linear the enhancing along with the segmental amplification of purpose.
Traditional RT-PCR is accomplished by two independent response procedures or step, and the first step is through the reversed transcriptive enzyme effect, and the RNA rt is become cDNA; Second step was under the effect of Taq enzyme, was that template is carried out pcr amplification with the cDNA that forms in the first step.Compare with traditional RT-PCR; One-step method real-time fluorescent RT-PCR technology has following advantage: two response procedures that 1, rt and two of pcr amplifications separately carried out in the differential responses pipe are merged into a response procedures; Stopped pipe is accomplished testing process fully in a PCR reaction tubes, has saved the reaction times greatly; 2, through analysis, abandon the regular-PCR method and receive multiple factor interferential end point analysis method, can carry out accurate quantitative analysis to test sample, thereby effectively monitor the effect of pharmacological agent the cycle threshold (Ct) of amplification curve and increased logarithmic phase; 3, be one with RNA rt, DNA cloning and detection three process fusion, can be in real time, the whole process of dynamic monitoring RNA amplification, saved PCR product postprocessing process, shortened analysis time as a result greatly, make that this method is more quick and easy; 4, owing to take a kind of detecting pattern of sealing, pollute and the false positive that causes thus thereby reduced aerosol; 5, since on common RT-PCR basis, increased by one can with the fluorescent probe of template complementary pairing, further improved the specificity that detects target polynucleotide.Therefore, this technology replaces traditional RT-PCR method gradually in the detection and quantitative analysis of RNA sample, obtains very using widely.
China FDA (SFDA) has ratified production and the clinical application such as the one-step method real-time fluorescent RT-PCR detection kit of pathogenic agent such as HIV-1, HCV.Therefore, the test kit of developing a kind of AIV of detection through one-step method real-time fluorescent RT-PCR method is feasible technically, and the needs of AIV rapid detection and monitoring will be greatly satisfied in its application.
As everyone knows; In using known one-step method real-time fluorescent RT-PCR technology for detection and quantitative analysis sample in the practice of certain particular target nucleic acid; In order to reduce and avoid the false negative or the false positive of detected result; Improve the accuracy of quantitative analysis, critical basic fundamental link is how based on known target polynucleotide sequences Design and prepare suitable primer and oligonucleotide probe.The inventor the has utilized one-step method real-time fluorescent RT-PCR technological invention on this basis test kit of a kind of AIV of detection is applied to detection and the quantitative analysis of AIV.
Summary of the invention
The present invention relates to a kind of test kit of real-time fluorescence RT-PCR detection of avian influenza virus, particularly relate to test kit early stage with one-step method real-time fluorescent RT-PCR reaction technology, the quick diagnosis avian influenza.This test kit can detect the AIV in brush,throat, cloaca swab, muscle or the samples such as internal organs sample, serum or blood plasma.
The purpose of this invention is to provide a kind of test kit that uses one-step method real-time fluorescent RT-PCR technology to come AIV in qualitative and the detection by quantitative sample, particularly relate to the application of early infection in laboratory diagnosis of AIV.Its ultimate principle is to utilize the Auele Specific Primer of a pair of oligonucleotide and the specific probe of an oligonucleotide, at reversed transcriptive enzyme (RT enzyme), hot resistant DNA polymerase (Taq enzyme), RNA enzyme inhibitors (RNasin), high-quality deoxyribonucleoside triphosphate (dNTPs) and Mg
2+In the RT-PCR reaction buffer, realize the cyclic amplification of target polynucleotide through the fluorescent PCR amplification appearance that DA7600, ABI7500 etc. are commercially available, thereby reach purpose quick, real-time, the detection by quantitative target polynucleotide.
The test kit of AIV comprises in the test sample provided by the present invention: (1) is equipped with RNA extracting solution (being TRIZOL nucleic acid extraction agent), RT-PCR amplification reaction solution, negative quality control product, positive quality control product and quantitative packing box with reference to article and a plurality of reagent bottles that seal or pipe and (2) separation and concentrated these reagent bottles of packing or pipe respectively.It is characterized in that being used in the RT-PCR amplification reaction solution forward primer AIV-F of target polynucleotide amplification and the sequence of reverse primer AIV-R is respectively: 5 '-CAAAAGCAGGTAGATGTTTAAAGATGA-3 ' (SEQ ID NO:1) and 5 '-GATTGGTCTTGTCTTTAGCCATTCCAT-3 ' (SEQ ID NO:2).
According to a preferred embodiment of the invention, the sequence that is used for the oligonucleotide probe AIV-P of target polynucleotide amplification and monitoring system in the RT-PCR amplification reaction solution is 5 '-GTTTTCTCTATCGTTCCATCAGGCCCCCTCA-3 ' (SEQ ID NO:3).
According to another preferred embodiment of the present invention; Wherein the RT-PCR amplification reaction solution by (a) hot resistant DNA polymerase (Taq enzyme), (b) reversed transcriptive enzyme (RT enzyme), (c) RNA enzyme inhibitors (RNasin), (d) deoxyribonucleoside triphosphate (dNTPs), (e) can with the forward primer of article one chain combination of double-stranded target polynucleotide; (f) can with the reverse primer of the second chain combination of double-stranded target polynucleotide, (g) with can combine with target polynucleotide and two ends are combined with the oligonucleotide probe composition of fluorescence report group and fluorescent quenching group respectively.
According to another preferred embodiment of the present invention, the best primer concentration that wherein is used for the RT-PCR amplification reaction solution is that 0.44 μ mol/L, concentration and probe concentration are 0.22 μ mol/L;
According to another preferred embodiment of the present invention, the mg ion optimum concn that wherein is used for the RT-PCR amplification reaction solution is that 2.0mmol/L, Taq enzyme optimum amount are that 5U/ reaction, RT enzyme optimum amount are that 100U/ reaction, RNasin optimum amount are that 20U/ reaction, deoxyribonucleoside triphosphate (dNTPs) optimum concn are 0.24mmol/L.
According to another preferred embodiment of the present invention, the optimal reaction temperature and the time that wherein are used for the RT-PCR amplification are: 40 ℃ of rt 25min; 94 ℃ of preparatory sex change 3min then; Last 93 ℃ of 15s, 55 ℃ of 45s, 40 circulations.
According to another preferred embodiment of the present invention, wherein negative quality control product is a saline water.
According to another preferred embodiment of the present invention, wherein positive quality control product is the in-vitro transcription RNA that contains the target fragment that increases, and concentration is 1.0 * 10
2Individual EID50.
According to another preferred embodiment of the present invention, be the in-vitro transcription RNA that contains the target fragment that increases quantitatively wherein with reference to article, comprise 4 concentration gradients: 1.0 * 10
6Individual EID50,1.0 * 10
5Individual EID50,1.0 * 10
4Individual EID50,1.0 * 10
3Individual EID50.
The minimum concentration that one-step method real-time fluorescent RT provided by the invention-PCR detection kit can detect AIV is 1.0 * 10
1Individual EID50 explains that this test kit has extraordinary sensitivity.
The present invention is directed to AIV genome conservative gene fragment design special primer and probe; Can detect AIV; But can not detect non-AIV pathogenic agent; Like NDV, infectious bursa of Fabricius virus, avian infectious bronchitis virus, DHV, duck plague virus, gosling plague virus etc., explain that this test kit has excellent specificity.
Real-time fluorescent RT-PCR detection reagent box provided by the invention can detect the AIV in brush,throat, cloaca swab, muscle or the samples such as internal organs sample, serum or blood plasma; Can be sensitivity, fast early diagnosis AIV infects that reliable experimental evidence is provided; Simultaneously owing to can be accurately quantitative, so can effectively monitor to curative effect.
Description of drawings
Fig. 1 shows linearity and the detected result figure of sensitivity with reference to article.To the template number is 1.0 * 10
1～1.0 * 10
7The linearity of individual EID50 and sensitivity are carried out the real-time fluorescence RT-PCR check and analysis with reference to article, and detected result shows, when virus quantity is 1.0 * 10
1During individual EID50, the Ct value of test sample is about 35, and promptly detection sensitivity can arrive 1.0 * 10
1Individual EID50.
Fig. 2 shows quantitative canonical plotting with reference to the article amplification.To the template number is 1.0 * 10
3～1.0 * 10
6The quantitative of individual EID50 carries out the real-time fluorescence RT-PCR check and analysis with reference to article, and drawing the slope of standard curve (Slope) that obtains is-4.6563, is 50.1330 at Y y-intercept (Intercept), square (R of relation conefficient
2)=0.9986.
Fig. 3 shows 10 positive detected result figure with reference to article.10 positive amplified fluorescence curves with reference to article have the obvious Exponential growth phase; And one intersection point is arranged with the preset threshold line; Draw the Ct value by the intersection point position and be respectively 16.95,19.60,20.08,21.51,22.46,25.37,26.40,26.75,27.09,28.14, can clearly be judged to be the positive.10 positive reference article are respectively AIV-H1, AIV-H3, AIV-H4, AIV-H5, AIV-H7, AIV-H9, AIV-H11, AIV-H13, AIV-H14, AIV-H15.
Fig. 4 shows 10 negative detected result figure with reference to article.Straight or oblique time of 10 negative amplification curves with reference to article and do not have with threshold line intersect, and do not have the Ct value, can clearly be judged to be feminine gender.10 negative being respectively with the AIV infection with reference to article have 6 kinds of viral sample (NDV, infectious bursa of Fabricius virus, avian infectious bronchitis virus, DHV, duck plague virus, gosling plague virus) of similar symptom and brush,throat and the cloaca swab sample of 4 parts of normal birds.
Fig. 5 shows the amplification curve of 6 clinical positive samples.The Ct value of 6 samples is respectively 20.20,21.23,22.44,28.75,29.40,31.85, in conjunction with amplification curve is arranged the obvious Exponential growth phase, all can be judged to be the positive.
Embodiment
The following example is intended to illustrate rather than limit the present invention.
Embodiment 1: the development of bird flu virus one-step method real-time fluorescent RT-PCR detection reagent
1, the design of primer and probe: through to the existing AIV nucleotide sequence of Genbank DB and both at home and abroad delivered the nucleotide sequence of reporting in the document and carried out the sequence alignment analysis; Conservative fragments with the genomic stromatin encoding sox of AIV (M gene) is the amplified target site; Select the section of no secondary structure and high conservative; According to the fundamental principle of primer probe design, utilize software, artificial design is many to primer and probe.
2, choice of sample: show according to domestic and international pertinent literature report, can select brush,throat, cloaca swab, muscle or samples such as internal organs sample, serum or blood plasma.
3, the foundation of reaction system and optimization
The preparation of sample: be accredited as 10 parts of AIV samples of male as positive reference article with virus culture, be respectively AIV-H1, AIV-H3, AIV-H4, AIV-H5, AIV-H7, AIV-H9, AIV-H11, AIV-H13, AIV-H14, AIV-H15; Be accredited as 10 parts of negative non-AIV samples as negative reference article with virus culture, be respectively brush,throat and the cloaca swab sample of 6 kinds of viral sample (NDV, infectious bursa of Fabricius virus, avian infectious bronchitis virus, DHV, duck plague virus, gosling plague virus) and 4 parts of normal birds.Extract above-mentioned positive in article and negative RNA, for use respectively with reference to article with TRIZOL.
The screening of primer probe: the many groups primer probe with design in above-mentioned 1 detects the above-mentioned positive respectively with reference to the RNA of article with negative reference article; Through TE, filter out the best primer probe combinations (like SEQ ID NO:1 in the sequence table～SEQ ID NO:3) of specificity, sensitivity and good reproducibility.
The optimization of primer concentration and probe concentration: in reaction system under the constant situation of other components; Use respectively from the primer of 0.20 μ mol/L to 0.5 μ mol/L concentration gradient with from the probe of 0.10 μ mol/L to 0.5 μ mol/L concentration gradient and carry out the PCR reaction; Warp is revision test repeatedly, and final definite best primer concentration is that 0.44 μ mol/L, concentration and probe concentration are 0.22 μ mol/L.
The optimization of Taq enzyme dosage: in 25 μ L reaction systems under the constant situation of other components; Use enzyme dosage/reaction to carry out the RT-PCR reaction respectively from 1U (unit of enzyme) to the 8U concentration gradient; Warp is revision test repeatedly, and final definite best Taq enzyme dosage is the 5U/ reaction.
The optimization of RT enzyme dosage: in 25 μ L reaction systems under the constant situation of other components; Use enzyme dosage/reaction to carry out the PCR reaction respectively from 50U (unit of enzyme) to the 400U concentration gradient; Warp is revision test repeatedly, and final definite best RT enzyme dosage is the 100U/ reaction.
The optimization of RNasin consumption: in 25 μ L reaction systems under the constant situation of other components; Use enzyme dosage/reaction to carry out the RT-PCR reaction respectively from 5U (unit of enzyme) to the 40U concentration gradient; Warp is revision test repeatedly, and final definite best RNasin consumption is the 20U/ reaction.
The optimization of dNTPs concentration: in reaction system under the constant situation of other components; Use respectively from the dNTPs of 0.1mmol/L to 0.25mmol/L concentration gradient and carry out the RT-PCR reaction; Warp is revision test repeatedly, and final definite best dNTPs concentration is 0.24mmol/L.
The optimization of temperature of reaction: according to the activity of enzyme and the length of few polynucleotide, mainly annealing temperature and extension time are optimized, warp is revision test repeatedly, and final definite best temperature of reaction and time are: 40 ℃ of rt 25min; 94 ℃ of preparatory sex change 3min then; Last 93 ℃ of 15s, 55 ℃ of 45s, 40 circulations.
4, sensitivity experiment: with concentration is 1.0 * 10
8The deactivation AIV-H5 culture supernatant of individual EID50,10 times of gradient dilutions become 1.0 * 10
7Individual EID50,1.0 * 10
6Individual EID50,1.0 * 10
5Individual EID50,1.0 * 10
4Individual EID50,1.0 * 10
3Individual EID50,1.0 * 10
2Individual EID50,1.0 * 10
1Individual EID50 with reference to article, carries out the real-time fluorescence RT-PCR check and analysis, when virus quantity is 1.0 * 10 as linear and sensitivity
1During individual EID50, the Ct value of test sample is about 35, and promptly detection sensitivity can arrive 1.0 * 10
1Individual EID50 (shown in accompanying drawing 1).
5, sample detects: with brush,throat, cloaca swab, muscle or internal organs sample, serum or blood plasma etc. as sample to be checked; After extracting the RNA of sample with TRIZOL respectively; The nucleic acid amplification system of setting up through above-mentioned optimization detects, and the result shows: this test kit very sensitive detects the AIV in the clinical samples.
Embodiment 2: bird flu virus one-step method real-time fluorescent RT-PCR detection kit and use thereof
1, preparation comprises the test kit of following moity: RNA extracting solution (50ml/ pipe) 1 is managed, RT-PCR amplification reaction solution (20 μ l/ pipe) 24 is managed, negative quality control product (100 μ l/ pipe) 1 is managed, positive quality control product (100 μ l/ pipe) 1 is managed, quantitatively reference article (50 μ l/ pipe) 4 are managed.
2, collection of specimens, transport and preserve
2.1 be suitable for sample type: brush,throat, cloaca swab, muscle or internal organs sample, serum or blood plasma etc.
2.2 sample collecting and pre-treatment (attention aseptic technique)
2.2.1 live-bird sample---get brush,throat, cloaca swab or fresh excreta swab, concrete acquisition method is following:
1) brush,throat: will swab goed deep into larynx and goes up cleft palate and scrape back and forth 3～5 times when taking, get the throat juice;
2) cloaca swab: swab is goed deep into cloaca turn around and pick ight soil; Little rare bird for being prone to cause damage directly picks proper amount of fresh ight soil with swab;
3) brush,throat and cloaca swab are put into the centrifuge tube that fills 1.0ml PBS together, number subsequent use.
2.2.2 internal organ or muscle samples---cut gripping sample 2.0g to be checked with aseptic tweezer and in mortar, fully grind; Add the 10mlPBS mixing again, or place tissue homogenizer, add 10ml PBS homogenate; Change in the aseptic centrifuge tube tissue suspension over to 3 then; The centrifugal 10min of 000rpm gets supernatant and changes in the centrifuge tube, numbers subsequent use.
2.2.3 serum or blood plasma---directly draw in (being no less than 400 μ l) to the aseptic centrifuge tube with asepsis injector, number subsequent use.
2.3 preserve and transport: the sample of gathering or handling is preserved under 2～8 ℃ of conditions and should be no more than 24h; If need prolonged preservation, must place-70 ℃ of refrigerators, but should avoid multigelation (freeze thawing is 3 times at most).After the sample sealing of gathering, adopt thermo jug or insulated tank sealing on the rocks, be transported to the laboratory as early as possible.
3, detect step
(1) RNA extracts
A. get the 1.5ml centrifuge tube of N (N=1 manages negative quality control product+number of awaiting test sample) sterilization, perform mark.
B. every pipe adds 600 μ l Trizol reagent respectively, adds negative quality control product or the testing sample supernatant (fully drawing behind the mixing) of 200 μ l then respectively, the mixing 15s that fully vibrates, and room temperature left standstill 3～5 minutes;
C. every pipe adds 200 μ l chloroforms, turns upside down mixing after 10 seconds, and 12, centrifugal 4 minutes of 000rpm;
D. carefully draw colourless supernatant liquid (noting untouchable middle batt layer), be transferred to the 1.5ml centrifuge tube of Amoxcillin, add 10 μ l RNA extracting solution A (fully drawing behind the mixing) then; Fully inhale and beat mixing; 8,000rpm carefully discards all liquid after centrifugal 1 minute;
E. add solution C (confirming to have added absolute ethyl alcohol) 800 μ l, fully inhale and beat mixing, 8, centrifugal 1 minute of 000rpm removes clean (avoiding touching deposit seeds) with liquid as far as possible;
F. centrifuge tube is opened wide lid, pendulum is air-dry 15 minutes (also can use open well heater in 60 ℃ of dryings 5 minutes, need prevent sample cross contamination) in stink cupboard, adds 30 μ l DEPC H then
2O inhales to beat in the mixing pipe and precipitates, and obtains the suspension liquid of white, can directly be used for detection, also can be stored in-70 ℃ for use.
(2) RT-PCR reaction and interpretation of result
Get negative quality control product, sample, positive quality control product, quantitative respectively, add and carry out the RT-PCR amplification in the PCR reaction tubes with reference to each 5 μ l of article.The RT-PCR cycling condition is: 40 ℃ of rt 25min; 94 ℃ of preparatory sex change 3min then; Last 93 ℃ of 15s, 55 ℃ of 45s, 40 circulations.
Reaction finishes the back and preserves the detection data file.Regulate analytical parameters according to the resulting graphic representation of RT-PCR amplification, making quantitative reference article typical curve typical curve (Std curve) window under reach the best (is square (R of relation conefficient
2)>0.97) (shown in accompanying drawing 2).Can find out by accompanying drawing 3; 10 positive amplified fluorescence curves with reference to article have the obvious Exponential growth phase; And one intersection point is arranged with the preset threshold line, draw the Ct value by the intersection point position and be respectively 16.95,19.60,20.08,21.51,22.46,25.37,26.40,26.75,27.09,28.14; In accompanying drawing 4, because 10 negative amplification curves with reference to article are straight or obliquely descend and do not have with threshold line to intersect, there is not the Ct value, can clearly be judged to be feminine gender.At last calculate the not mensuration numerical value of key sample by the instrument automatic analyser.
Embodiment 3: use bird flu virus one-step method real-time fluorescent RT-PCR detection kit detection by quantitative clinical sample
All experimentations are the bird flu reference laboratory completion of veterinary institute country in Harbin, and clinical sample is provided by Harbin veterinary institute country bird flu reference laboratory, comprises sample types such as brush,throat, cloaca swab, muscle or internal organs sample; Sample RNA extracts, the RT-PCR reaction is carried out with reference to embodiment 2 with interpretation of result.
After RT-PCR reaction finishes, regulate analytical parameters earlier according to amplification curve, making quantitative reference article typical curve typical curve (Std curve) window under reach the best (is square (R of relation conefficient
2)>0.97), analyze clinical sample then.The detected result of clinical sample is shown in accompanying drawing 5: the Ct value of 6 clinical positive sample amplification curves is respectively 20.20,21.23,22.44,28.75,29.40,31.85, in conjunction with amplification curve is arranged the obvious Exponential growth phase, all can be judged to be the positive; With reference in once testing, quantitatively judging that with reference to the canonical plotting (shown in accompanying drawing 2) of article the AIV concentration of 6 clinical positive samples is respectively: 6.08 * 10
6Individual EID50,3.58 * 10
6Individual EID50,1.92 * 10
6Individual EID50,7.38 * 10
4Individual EID50,5.30 * 10
4Individual EID50,1.50 * 10
4Individual EID50.
Sequence table
< 110>Da
< 120>a kind of test kit of real-time fluorescence RT-PCR detection of avian influenza virus
<140>
<141>
<160>3
<210>1
<211>27
<212>DNA
< 213>artificial sequence
<220>
< 223>according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>1
<210>2
<211>27
<212>DNA
< 213>artificial sequence
<220>
< 223>according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>2
<210>3
<211>31
<212>DNA
< 213>artificial sequence
<220>
< 223>according to the specific nucleotide sequence design, with probe as pcr amplification.
<400>3
Claims (8)
1. the test kit that bird flu virus exists in the test sample; This test kit comprises: a plurality of reagent bottles or pipe that (1) is equipped with RNA extracting solution, RT-PCR amplification reaction solution, negative quality control product, positive quality control product and quantitative reference article respectively and is sealed; (2) packing box of separation and concentrated these reagent bottles of packing or pipe; Wherein the RT-PCR amplification reaction solution comprises hot resistant DNA polymerase, reversed transcriptive enzyme, RNA enzyme inhibitors, deoxyribonucleoside triphosphate, forward primer, reverse primer; Two ends are combined with the oligonucleotide probe of fluorescence report group and fluorescent quenching group respectively, it is characterized in that being used in the RT-PCR amplification reaction solution forward of target polynucleotide amplification and the sequence of reverse primer and are respectively:
Forward primer AIV-F:5 '-CAAAAGCAGGTAGATGTTTAAAGATGA-3 ';
Reverse primer AIV-R:5 '-GATTGGTCTTGTCTTTAGCCATTCCAT-3 '; The sequence of employed oligonucleotide probe AIV-P is in the RT-PCR amplification reaction solution: 5 '-GTTTTCTCTATCGTTCCATCAGGCCCCCTCA-3 '.
2. according to the test kit of claim 1, its characteristic is that also forward primer concentration is that 0.44 μ mol/L, reverse primer concentration are that 0.44 μ mol/L, oligonucleotide probe concentration are 0.22 μ mol/L in the RT-PCR amplification reaction solution.
3. according to the test kit of claim 1, its characteristic is that also the concentration of hot resistant DNA polymerase in the RT-PCR amplification reaction solution is the 5U/ reaction.
4. according to the test kit of claim 1, its characteristic is that also the concentration of reversed transcriptive enzyme in the RT-PCR amplification reaction solution is the 100U/ reaction.
5. according to the test kit of claim 1, its characteristic is that also the concentration of RNA enzyme inhibitors in the RT-PCR amplification reaction solution is the 20U/ reaction.
6. according to the test kit of claim 1, its characteristic is that also the concentration of deoxyribonucleoside triphosphate in the RT-PCR amplification reaction solution (dNTPs) is 0.24mmol/L.
7. according to the test kit of claim 1, its characteristic is that also the temperature of reaction and the time that are used for the RT-PCR amplification is: 40 ℃ of rt 25min; 94 ℃ of preparatory sex change 3min then; Last 93 ℃ of 15s, 55 ℃ of 45s, 40 circulations.
8. according to the test kit of claim 1, its characteristic is that also the sample that is detected is selected from brush,throat, cloaca swab, muscle or internal organs sample, serum or blood plasma.
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| CN102251062A (en) * | 2011-08-05 | 2011-11-23 | 江苏硕世生物科技有限公司 | Dual quantitative fluorescence PCR (Polymerase Chain Reaction) assay kit for nucleic acid of influenza A1/A3 virus |
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| CN1690220A (en) * | 2004-04-19 | 2005-11-02 | 中国人民解放军军需大学军事兽医研究所 | Method and kit for testing chain reaction of reverse transcription polymerase of avian influenza virus |
| CN1814805A (en) * | 2005-12-01 | 2006-08-09 | 上海交通大学 | H5, H7, H9 subtype auian flu virus real-time fluo rescent quantixative PCR detecting method |
| CN1828299A (en) * | 2005-02-28 | 2006-09-06 | 中华人民共和国北京出入境检验检疫局 | Nucleotide sequence, reagent kit and checking method for detecting fowl influenza virus |
| 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 |
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| CN1690220A (en) * | 2004-04-19 | 2005-11-02 | 中国人民解放军军需大学军事兽医研究所 | Method and kit for testing chain reaction of reverse transcription polymerase of avian influenza virus |
| CN1828299A (en) * | 2005-02-28 | 2006-09-06 | 中华人民共和国北京出入境检验检疫局 | Nucleotide sequence, reagent kit and checking method for detecting fowl influenza virus |
| CN1814805A (en) * | 2005-12-01 | 2006-08-09 | 上海交通大学 | H5, H7, H9 subtype auian flu virus real-time fluo rescent quantixative PCR detecting method |
| 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 |
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