CN103320543B - Avian pneumovirus SYBR Green I real-time fluorescent quantitative RT-PCR detection kit - Google Patents
Avian pneumovirus SYBR Green I real-time fluorescent quantitative RT-PCR detection kit Download PDFInfo
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Abstract
The invention discloses an avian pneumovirus SYBR Green I real-time fluorescent quantitative RT-PCR detection kit and an application thereof. The kit contains a primer pair for detecting an avian pneumovirus, the primer pair is composed of a primer 1 and a primer 2, the primer 1 is single-stranded DNA represented by sequence 1 in a sequence table, and the primer 2 is single-stranded DNA represented by sequence 2 in the sequence table. Experiments prove that the kit has the advantages of short detection time, strong specificity, high sensitivity, simple operation and low price when the kit is used for the SYBR Green I fluorescent quantitative RT-PCR detection of the avian pneumovirus.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of avian pneumovirus SYBR Green I real-time fluorescence quantitative RT-PCR detection reagent kit and application thereof.
Background technology
Avian pneumovirus (avian pneumovirus, APV) is the member that the inclined to one side pneumonitis virus of fowl belongs to, and belongs to avian paramyxovirus section, this virus main harm turkey, but found in recent years that this virus also encroached on the chicken of different varieties, comprised kind of a chicken, commercial meat bird and egg, caused these chickens morbidities.The age in days that this virus causes chicken morbidity generally, in 4-7 age in week, the most easily occurs during age in week at 5-6.Mainly cause the symptom of the upper respiratory tract, with sneeze, eye conjunctiva flush, lachrymal gland is swollen, and significantly symptom is that subcutaneous dropsy appears in head, occurs head enlargement; Laying hen infects this virus and mainly causes egg drop reduction, and fall is between 2%-40%, and hatching of breeding eggs rate reduces; Along with the development of the state of an illness, can go out nervous symptoms; The sickness rate that this disease causes, between 1%-90%, causes that chicken death rate is at 1%-20%, and as the infection of secondary Other diseases, the death meeting causing is larger, and what aviculture was caused is very harmful.
Avian pneumovirus genome is the strand RNA of non-segmented negative, length is about 14kb, can be by its classics according to its Nucleotide and aminoacid sequence be divided into 4 hypotypes such as A, B, C, D, wherein A hypotype and B hypotype are in world wide and are widely current, C hypotype is separated at Colorade USA in 1997, and D hypotype exists only in France.Its genome encoding has 8 kinds of albumen, i.e. N-P-M-F-M2-SH-G-L.Wherein F is fusion rotein, is these virus major antigen structural protein, has adsorption.
Real-time fluorescence quantitative PCR is the gene Amplification Technologies that a kind of PCR of melting and DNA probe hybridization technique are integrated, it reaches by the variation of real-time detection PCR product fluorescent signal the object that detects cause of disease nucleic acid, that this technology has is easy and simple to handle, visual result, susceptibility are high, high specificity, the advantage such as reproducible, has become the important method that animal pathogenic detects.SYBR Green I real-time fluorescence quantitative RT-PCR is easier than Taqman fluorescence quantitative RT-RCR technology, and price is lower, but the specificity detecting is lower than Taqman fluorescence quantitative RT-RCR technology.When setting up SYBR Green I real-time fluorescence quantitative RT-PCR, eliminate the impact of primer dimer, to carry out solubility curve analysis, during for single peak, specificity is better.Equally, this technology can also directly be calculated the content of corresponding cause of disease sample according to the typical curve of setting up from the fluorescence threshold detecting, in clinical detection with on to the evaluation of disease process, have very high practical value.
At present, also do not apply the research report of SYBR Green I real-time fluorescence quantitative RT-PCR detection and identification avian pneumovirus.
Summary of the invention
An object of the present invention is to provide a kind of primer pair for detection of avian pneumovirus.
Primer pair for detection of avian pneumovirus provided by the present invention, is comprised of primer 1 and primer 2, and described primer 1 is the single stranded DNA shown in sequence 1 in sequence table, and described primer 2 is the single stranded DNA shown in sequence 2 in sequence table.
Wherein, sequence 1 is comprised of 20 Nucleotide; Sequence 2 is comprised of 20 Nucleotide.
Second object of the present invention is to provide a kind of reagent for detection of avian pneumovirus.
Reagent for detection of avian pneumovirus provided by the present invention, is comprised of described primer pair, SYBR Green I real-time fluorescence quantitative RT-PCR amplification buffer and water.
Described SYBR Green I real-time fluorescence quantitative RT-PCR amplification buffer, comprises dNTPs, Mg
2+, SYBRGreen I, archaeal dna polymerase, ThermoScript II and RNA enzyme inhibitors.
In one embodiment of the invention, in described SYBR Green I real-time fluorescence quantitative RT-PCR amplification buffer (being designated as buffer A), described dNTPs, described Mg
2+, described archaeal dna polymerase, described ThermoScript II and described RNA enzyme inhibitors proportioning be: 0.1 μ mol:0.25 μ mol:5U:200U:40U.Further, described SYBR Green I real-time fluorescence quantitative RT-PCR amplification buffer, for coming from the Dalian precious biotechnology Takara of company limited product: One Step SYBR PrimeScript RT-PCR Kit II(catalog number: RR086A).More concrete, described SYBR Green I real-time fluorescence quantitative RT-PCR amplification buffer, for by described One Step SYBR PrimeScript RT-PCR Kit II(catalog number: the 2 * One step SYBR RT-PCR buffer4(RR086A) includes 10mM dNTP Mixture, 25mM Mg
2+sYBRGreen I etc.) and PrimeScript1Step Enzyne Mix2 (including Takara Ex Taq HS5U/ μ L, PremeScript RTase ThermoScript II 200U/ μ L, RNA enzyme inhibitors 40U/ μ L), the ratio that is 10:1 according to volume ratio mix after gained solution.Certainly, use the analogous products of other companies also passable.
In another embodiment of the present invention, described SYBR Green I real-time fluorescence quantitative RT-PCR amplification buffering (being designated as buffer B), is specifically comprised of reverse transcription damping fluid and SYBR fluorescent PCR damping fluid.Wherein, in described reverse transcription damping fluid, contain described dNTPs, described Mg
2+, described ThermoScript II and described RNA enzyme inhibitors; Described dNTPs, described Mg
2+, described ThermoScript II and described RNA enzyme inhibitors proportioning be 0.02 μ mol:0.1 μ mol:2.5U:20U.More concrete, described reverse transcription damping fluid is by AMV ThermoScript II (5U/ μ L, Dalian precious biotechnology company limited, catalog number: D2620), RNA enzyme inhibitors (40U/ μ L, Dalian precious biotechnology company limited, catalog number: D2313A), random primer (50 μ mol/ μ L, Dalian precious biotechnology company limited, catalog number: D3802), 10mM dNTPs Mix (four kinds of bases, Dalian precious biotechnology company limited, catalog number: D4030RA), 5 * RTbuffer is (containing 25mM Mg
2+, in AMV ThermoScript II, include this buffer, Dalian precious biotechnology company limited, catalog number: D2620), the ratio that is 1:1:2:4:8 according to volume ratio is mixed rear gained solution.In described SYBR fluorescent PCR damping fluid, contain described dNTPs, described Mg
2+, described archaeal dna polymerase and described SYBRGreen I; Described dNTPs, described Mg
2+with the proportioning of described archaeal dna polymerase be 0.01 μ mol:0.025 μ mol:5U.More concrete, described SYBR fluorescent PCR damping fluid, for SYBR Premix Ex Taq II(2 *) (include 10mM dNTP Mixture, 25mM Mg
2+, 5U/ μ LTakara Ex Taq HS, SYBRGreen I, Dalian precious biotechnology company limited, catalog number: RR820A).Certainly, use the analogous products of other companies also passable.
The 3rd object of the present invention is to provide a kind of test kit for detection of avian pneumovirus.
Test kit for detection of avian pneumovirus provided by the present invention, contain following a) and b):
A) avian pneumovirus positive plasmid;
B) described primer pair or described reagent.
In described test kit, described avian pneumovirus positive plasmid is the plasmid of the 753-849 position Nucleotide containing sequence 3 in ordered list.
Further, the recombinant plasmid of described avian pneumovirus positive plasmid for obtaining after DNA fragmentation shown in the 753-849 position Nucleotide of sequence in sequence table 3 is connected with pMD18-T carrier.
At described primer pair, or described reagent, or in described test kit, the mol ratio of described primer 1 and described primer 2 is 1:1.
The 4th object of the present invention is to provide following c) or application d).
C) application of described primer pair in the described reagent of preparation or described test kit;
D) described primer pair, or described reagent, preparation detect or auxiliary detection testing sample in whether contain the application in the product of avian pneumovirus.
At d) in described application, in described detection or auxiliary detection testing sample, whether contain avian pneumovirus and can be and use described primer pair, or described reagent, or described test kit carries out the reaction of SYBR Green I real-time fluorescence quantitative RT-PCR to described testing sample.
Described annealing temperature of carrying out the reaction of SYBR Green I real-time fluorescence quantitative RT-PCR can be 60 ℃-62 ℃ (as 60 ℃).
Further, the program of utilizing described SYBR Green I real-time fluorescence quantitative RT-PCR amplification buffer (buffer A) to carry out described SYBR Green I real-time fluorescence quantitative RT-PCR reaction specifically can be: 42 ℃ of 15min; 95 ℃ of 2min; Then by 95 ℃ of sex change 15s, 60 ℃ of annealing, extend 20s and carry out 40 circulations; Finally in 40 ℃, within 10 seconds, finish reaction.The program of utilizing described SYBR Green I real-time fluorescence quantitative RT-PCR amplification buffer (buffer B) to carry out described SYBR Green I real-time fluorescence quantitative RT-PCR reaction specifically can be: first carry out reverse transcription: 25 ℃ of 10min; 42 ℃ of 60min; 95 ℃ of 5min; Then carry out the amplification of SYBR Green I real-time fluorescence PCR: 95 ℃ of sex change 15s; 60 ℃ of annealing are extended 20s and are carried out 40 circulations; Finally in 40 ℃, within 10 seconds, finish reaction.
The temperature transition rate of the described SYBR of carrying out Green I real-time fluorescence quantitative RT-PCR reaction is 20 ℃/s, carries out fluorescent signal detection when the extension of each circulation finishes.
At d) in described application, while whether containing avian pneumovirus in described detection or auxiliary detection testing sample, the described primer 1 in described primer pair and the final concentration of described primer 2 in reaction system all can be 0.5 μ mol/ μ L.
In the present invention, utilize described SYBR Green I real-time fluorescence quantitative RT-PCR amplification buffer (buffer A) to carry out the system of described SYBR Green I real-time fluorescence quantitative RT-PCR reaction specific as follows: 2 * One step SYBR RT-PCR buffer4(includes 10mM dNTP Mixture, 25mM Mg
2+, SYBRGreen I etc.) and 10 μ L; PrimeScript1Step Enzyne Mix2 (including Takara Ex Taq HS5U/ μ L, PremeScript RTase ThermoScript II 200U/ μ L, RNA enzyme inhibitors 40U/ μ L) 1 μ L; Template (RNA or DNA) 2 μ L(approximately 20 μ g); Described primer 1 and the final concentration of described primer 2 in reaction system are 0.5 μ mol/ μ L; DEPC water is supplied 20 μ L.
In the present invention, the system of utilizing described SYBR Green I real-time fluorescence quantitative RT-PCR amplification buffer (buffer B) to carry out described SYBR Green I real-time fluorescence quantitative RT-PCR reaction is specially following reverse transcription system and SYBR Green I real-time fluorescence PCR amplification system:
Reverse transcription system (20 μ L): AMV ThermoScript II (5U/ μ L, Dalian precious biotechnology company limited, catalog number: D2620) 0.5 μ L; RNA enzyme inhibitors (40U/ μ L, Dalian precious biotechnology company limited, catalog number: D2313A) 0.5 μ L; Random primer (50 μ mol/ μ L, Dalian precious biotechnology company limited, catalog number: D3802) 1 μ L; 10mM dNTPs Mix (four kinds of bases, Dalian precious biotechnology company limited, catalog number: D4030RA) 2 μ L; 5 * RTbuffer is (containing 25mM Mg
2+, in AMV ThermoScript II, include this buffer, Dalian precious biotechnology company limited, catalog number: D2620) 4 μ L; Template (RNA) 2 μ L(approximately 20 μ g); DEPC water is supplied 20 μ L.
SYBR Green I real-time fluorescence PCR amplification system (20 μ L): SYBR Premix Ex Taq II(2 *) (include 10mM dNTP Mixture, 25mM Mg
2+, 5U/ μ L Takara Ex Taq HS, SYBRGreen I, Dalian precious biotechnology company limited, catalog number: RR820A) 10 μ L; Template (cDNA of reverse transcription gained or viral DNA) 2 μ L(approximately 20 μ g); Described primer 1 and the final concentration of described primer 2 in reaction system are 0.5 μ mol/ μ L; DEPC water is supplied 20 μ L.
At d) in described application, described testing sample can, from healthy animal or dead animal, can be DNA and/or RNA.
Experiment showed, that tool of the present invention has the following advantages:
1) detection time is short
The present invention, by Primer Express3.0 software, designs the primer of amplification avian pneumovirus, the dimer between the primer designing by compare of analysis, hairpin structure etc., and final design has gone out 1 pair of primer.Set up the SYBR Green I real-time fluorescent quantitative RT-PCR method of avian pneumovirus.The method is without carrying out electrophoresis, only need the time of approximately 30 minutes to complete amplification, and can directly by computer, carry out observing response result, and conventional RT-PCR method needs within 3.5 hours, complete amplified reaction at least, then need to spend 2 hours and carry out gel electrophoresis and carry out observations.
2) detection sensitivity is high
The SYBR Green I real-time fluorescent quantitative RT-PCR method of setting up carries out quantitatively the content of avian pneumovirus in sample, the sensitivity of its detection is very high, can detect limits the quantity of is the avian pneumovirus plasmid of 10 copies, conventional PCR is 1000 copies, and detected result is more responsive than conventional PCR method.The discriminating that can be used for avian pneumovirus with the test kit that this technology is set up detects, significant to the Accurate Diagnosis of avian pneumovirus and prevention.
3) detect easyly, price is relatively low
The fluorescence quantifying PCR method that adopts SYBR Green I to set up in real time, is the two strands occurring while adding SYBR Green I in conjunction with amplification in damping fluid, reaches the object of detection.The primer dimer forming when amplification can affect detected result, therefore will carry out solubility curve analysis, and amplification condition is optimized, and eliminates the generation of primer dimer.The method that adopts SYBR Green I fluorescence dye, easier than Taqman detecting probe method, price is relatively low.
Accompanying drawing explanation
Fig. 1 is the typical curve that SYBR Green I real-time quantitative RT-PCR method detects avian pneumovirus.In figure, each point corresponds to 1:1.0 * 10
7copy number/μ L; 2:1.0 * 10
6copy number/μ L; 3:1.0 * 10
5copy number/μ L; 4:1.0 * 10
4copy number/μ L; 5:1.0 * 10
3copy number/μ L; 6:1.0 * 10
2copy number/μ L; 7:1.0 * 10
1copy number/μ L.
Fig. 2 is the solubility curve that SYBR Green I real-time quantitative RT-PCR method detects avian pneumovirus.Wherein, 1: blank is (with equivalent RNase Free dH
2o replaces template); 2:1.0 * 10
7copy number/μ L; 3:1.0 * 10
6copy number/μ L; 4:1.0 * 10
5copy number/μ L; 5:1.0 * 10
4copy number/μ L; 6:1.0 * 10
3copy number/μ L; 7:1.0 * 10
2copy number/μ L; 8:1.0 * 10
1copy number/μ L.
Fig. 3 is the specificity curve that SYBR Green I real-time quantitative RT-PCR method detects avian pneumovirus.Wherein, 1: avian pneumovirus APV/MN; 2: newcastle disease virus (Lasota); 3:H9 subtype avian influenza virus; 4: avian infectious bronchitis virus (Mass41); 5: avian encephalomyclitis virus (AE Van); 6: Avianreovirus (Reo S1133); 7: chicken Marek's disease (MDV) vaccine virus; 8: avian infectious laryngotracheitis virus (Beijing Strain); 9: chicken poison Mycoplasma (MG); 10: avian escherichia coli (E.coli O
2); 11:dH
2o.
Fig. 4 is the sensitivity detected result that SYBR Green I real-time quantitative RT-PCR method detects avian pneumovirus.Wherein, 1:1.0 * 10
7copy number/μ L; 2:1.0 * 10
6copy number/μ L; 3:1.0 * 10
5copy number/μ L; 4:1.0 * 10
4copy number/μ L; 5:1.0 * 10
3copy number/μ L; 6:1.0 * 10
2copy number/μ L; 7:1.0 * 10
1copy number/μ L; 8:1.0 * 10
0copy number/μ L; 9: blank.
Fig. 5 is repeated curve.Wherein, 1 and 2:1.0 * 10
4copy number/μ L; 3 and 4:1.0 * 10
2copy number/μ L; 5: blank is (with equivalent RNase Free dH
2o replaces template).
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
In following embodiment, materials more used and reagent are as follows:
Lightcycler2.0 quantitative real time PCR Instrument (Roche company product).DNA segment reclaims test kit and plasmid extracts test kit in a small amount purchased from BioDev company; PMD18-T test kits etc. are purchased from Dalian precious biotechnology (TaKaRa) company; TIANamp virus genom DNA/RNA extracts test kit purchased from Tian Gen biochemical technology company limited.
Avian pneumovirus APV/MN strain: be recorded in " Xie Zhiqin etc.; the visual amplification of ring mediated isothermal (LAMP) detects the foundation of avian pneumovirus method. Chinese Animal Quarantine; 2013,30(3): 48-51 " literary composition, public Ke Cong Veterinary Institute of Guangxi Zhuang Autonomous Region obtains;
Newcastle disease virus (Lasota): be recorded in " Xie Zhixun etc., the research of newcastle disease vegetables oil emulsion seedling. Chinese Preventive Veterinary Medicine report, 2000, (04): 23-27 " literary composition, public Ke Cong Veterinary Institute of Guangxi Zhuang Autonomous Region obtains;
H9 subtype avian influenza virus: be documented in " multiple reverse transcriptional PCR rapid detection is differentiated the foundation of H9 subtype avian influenza virus method. Chinese Amphixenosis's journal; 2006; (09): 858-860 " and a literary composition, public Ke Cong Veterinary Institute of Guangxi Zhuang Autonomous Region obtains;
Avian encephalomyclitis virus (AE Van), avian infectious bronchitis virus (Mass41), avian infectious laryngotracheitis virus (Beijing Strain), chicken poison Mycoplasma (MG) are preserved by this chamber; Wherein, avian encephalomyclitis virus (AE Van) be recorded in " Zhang Erqin; Zhao Xinli; Zhao Zhenhua etc. avian encephalomyclitis virus Van Roekel strain capsid protein gene prokaryotic expression and ELISA thereof detect research. animal medicine progress; the 01st phase in 2005 " literary composition, see " avian encephalomyclitis virus Van Roekel strain " in literary composition; Avian infectious bronchitis virus (Mass41) be recorded in " Li Binghong. the fowl pox vaccine of restructuring IBV Mass41 strain S1 gene. animal and veterinary scientific and technical information, the 04th phase in 2003 " literary composition, see " IBV Mass41 " in literary composition; Avian infectious laryngotracheitis virus (Beijing Strain) be recorded in " Yuan Kehu; Zhang Manfu. clone and the sequential analysis of Infectious Laryngotracheitis Virus Beijing strain thymidine kinase gene. Journal of Agricultural Biotechnology; 04 phase in 2002 " literary composition, see " Infectious Laryngotracheitis Virus Beijing strain " in literary composition; Chicken poison Mycoplasma (MG) be recorded in " He Shijun, Huang Ye, Tang Shunfa. chicken poison mycoplasma (MG) immune protection countermeasure 23 phases of .2005 " literary composition, see " chicken poison mycoplasma (MG) " in literary composition;
Avianreovirus (Reo S1133) and avian escherichia coli (E.coli O2): be recorded in " Zhiqin Xie(thanks to will duty) etc., Reverse transcriptase polymerase chain reaction to detect avian Encephalomyelitis virus.Avian Disease (U.S.'s poultry diease magazine), 2005, 49:227-230 " and " Xie Zhiqin etc., apply two temperature multiple round pcrs and differentiate avian infectious bronchitis virus and avian infectious laryngotracheitis virus. Guangxi science, 2001, 8(2): 152-153 ", public Ke Cong Veterinary Institute of Guangxi Zhuang Autonomous Region obtains,
Chicken Marek's disease (MDV) vaccine virus is purchased from Nanjing Cimmeria Animal Health Care Products Corporation.
The design of embodiment 1, primer is with synthetic
According to the conserved sequence of avian pneumovirus F gene in GenBank (GenBank:AF187154, in sequence table, sequence 3), adopt Primer Express3.0 software, design a pair of Auele Specific Primer (table 1).
Table 1 primer and oligonucleotide sequence
Embodiment 2, SYBR Green I real-time fluorescence quantitative RT-PCR detect
One, SYBR Green I real-time fluorescence quantitative RT-PCR detection method determines
1, the preparation of sample to be tested
With reference to TIANamp virus genom DNA/RNA, extract test kit specification sheets, extract the RNA of avian pneumovirus APV/MN, newcastle disease virus (Lasota), H9 subtype avian influenza virus, avian infectious bronchitis virus (Mass41), avian encephalomyclitis virus (AE Van), Avianreovirus (Reo S1133); Extract avian infectious laryngotracheitis virus (Beijing Strain), chicken Marek's disease (MDV) vaccine virus, chicken poison Mycoplasma (MG) and avian escherichia coli (E.coli O
2) DNA.
2, the preparation of standard substance
The cDNA of the avian pneumovirus APV/MN that the step 1 of take obtains carries out pcr amplification as template, and reaction system is 50 μ L:2 * Premix Taq
tMthe mix25 μ L(Dalian precious biotechnology Takara of company limited product, catalog number: RR003A, containing 10 μ M dNTPs, Mg
2+, 5U Taq enzyme), cDNA4 μ L, each 0.5 μ L of the APV upstream and downstream primer (APV1 in table 1 and APV2) of 50pmol/ μ L, sterilizing ultrapure water adds to 50 μ L.Response procedures is 94 ℃ of denaturation 5min, then enters 94 ℃ of 1min, 55 ℃ of 1min, and 72 ℃ of 1min, carry out 35 circulations altogether; 72 ℃ are extended 10min, finally stop at 4 ℃.
PCR product, through 2% agarose gel electrophoresis, reclaims object segment rear clone to pMD18-T carrier, and recombinant plasmid send Dalian Bao Sheng Bioisystech Co., Ltd to check order.Sequencing result is shown containing the recombinant plasmid of the 753-849 position Nucleotide of sequence 3 in ordered list positive, by its called after pMD18-APV.
Using pMD18-APV plasmid as positive criteria product, according to document " Vaitomaa; J.; Rantala A.; Halinen K.; Rouhiainen L.; Tallberg P., Mokelke L. & Sivonen K. (2003) Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes.Applied and Environmental Microbiology.69:7289-7297. " calculate copy number, result copy number is 2.4 * 10
10copy/μ l.
3, typical curve determines and solubility curve analysis
Determining of quantitative fluorescent PCR typical curve, carries out quantitatively, then by 10 times, being diluted to 10 to plasmid pMD-APV DNA
0-10
7copy number/μ L, carries out the amplification of SYBR Green I real-time fluorescence quantitative PCR, Criterion curve using the copy number of dilution as template.Amplification reaction system is that (the Dalian precious biotechnology Takara of company limited product, production number RR820A, includes 5U/ μ L Takara Ex Taq HS, 10mM dNTP Mixture, 25mM Mg to 20 μ L:2 * SYBR Premix Ex Taq II
2+, SYBRGreen I etc.) and 10 μ L, plasmid pMD-APV DNA1 μ L(approximately 20 μ g), concentration is APV upstream primer and each 1 μ L of downstream primer (APV1 in table 1 and APV2) of 10 μ mol/ μ L, RNase Free dH
2o supplies 20 μ L.Blank is set simultaneously (with equivalent RNase Free dH
2o replaces template).Then carry out the amplification of typical curve, amplification program is 95 ℃ of 15s, and then 95 ℃ of 20s, 60 ℃ of 20s carry out 40 circulations, 95 ℃ of 0s then, and 20 ℃/s, 65 ℃ of 20s, 20 ℃/s, 95 ℃ of 0s, 0.1 ℃/s carries out solubility curve analysis, finally enters 40 ℃ of 10s and finishes.
As shown in Figure 1, as can be seen from the figure, each point in a straight line, shows that typical curve is good to typical curve.
As shown in Figure 2, as can be seen from the figure, solubility curve only has single peak to occur to solubility curve, shows only to amplify positive peak, does not have primer dimer or other non-positive peak to occur, is specific amplification.
4, the reaction system of SYBR Green I real-time fluorescence quantitative RT-PCR and the establishment of amplification program
1) single step reaction method
Reaction system (20 μ L): 2 * One step SYBR RT-PCR buffer4(includes 10mM dNTP Mixture, 25mM Mg
2+, SYBRGreen I etc.) and 10 μ L; PrimeScript1Step Enzyne Mix2 (including Takara Ex Taq HS5U/ μ L, PremeScript RTase ThermoScript II 200U/ μ L, RNA enzyme inhibitors 40U/ μ L) 1 μ L; Template (RNA or DNA) 2 μ L(approximately 20 μ g); Concentration is APV upstream primer and each 1 μ L of downstream primer (APV1 in table 1 and APV2) of 10 μ mol/ μ L, and the final concentration of two primers in reaction system is respectively 0.5 μ mol/ μ L; DEPC water is supplied 20 μ L.In reaction system, the composition except primer and template all comes from the Dalian precious biotechnology Takara of company limited product: One Step SYBR PrimeScript RT-PCR Kit II(catalog number: RR086A).
In above-mentioned reaction system, dNTPs, Mg
2+, archaeal dna polymerase, ThermoScript II and RNA enzyme inhibitors proportioning be specially: 0.1 μ mol:0.25 μ mol:5U:200U:40U.
Response procedures: 42 ℃ of 15min; 95 ℃ of 2min; Then by 95 ℃ of sex change 15s, 60 ℃ of annealing, extend 20s and carry out 40 circulations; Finally in 40 ℃, within 10 seconds, finish reaction.Wherein, the temperature transition rate of carrying out the reaction of SYBR Green I real-time fluorescence quantitative RT-PCR is 20 ℃/s, carries out fluorescent signal detection when the extension of each circulation finishes.
Under 530nm exciting light, fluoresce, be used for detecting avian pneumovirus.
2) two-step reaction method
The reverse transcription of the first step: RNA
By step 1 gained avian pneumovirus APV/MN RNA and the synthetic cDNA of other viral RNA reverse transcription, specific as follows: for every kind of virus, all to set up following reverse transcription system, total reaction volume is 20 μ L, template---avian pneumovirus APV/MN RNA or other viral RNA 2 μ L(approximately 20 μ g), AMV ThermoScript II (5U/ μ L, Dalian precious biotechnology company limited, catalog number: D2620) 0.5 μ L, RNA enzyme inhibitors (40U/ μ L, Dalian precious biotechnology company limited, catalog number: D2313A) 0.5 μ L, random primer (50 μ mol/ μ L, the Dalian precious biotechnology Takara of company limited product, catalog number: D3802) 1 μ L, 10mM dNTPs Mix (Dalian precious biotechnology company limited, catalog number: D4030RA) 2 μ L, 5 * RT buffer(is containing 25mM Mg
2+, in AMV ThermoScript II, there is this buffer, Dalian precious biotechnology company limited, catalog number: D2620) 4 μ L, DEPC water is supplied 20 μ L, instantaneous centrifugal, in 25 ℃ of 10min, 42 ℃ of 60min, 95 ℃ of 5min, 4 ℃ of end, obtain each viral cDNA.
In above-mentioned reaction system, dNTPs, Mg
2+, ThermoScript II and RNA enzyme inhibitors proportioning be specially: 0.02 μ mol:0.1 μ mol:2.5U:20U.
Second step: SYBR Green I real-time fluorescence quantitative PCR amplification
Reaction system (20 μ L): SYBR Premix Ex Taq II(2 *) (include 10mM dNTP Mixture, 25mM Mg
2+, 5U/ μ L Takara Ex Taq HS, SYBRGreen I, Dalian precious biotechnology company limited, catalog number: RR820A) 10 μ L; Template (viral DNA that the cDNA of the first step reverse transcription gained or step 1 obtain) 2 μ L(approximately 20 μ g); Concentration is APV upstream primer and each 1 μ L of downstream primer (APV1 in table 1 and APV2) of 10 μ mol/ μ L, and the final concentration of two primers in reaction system is respectively 0.5 μ mol/ μ L; Sterilizing DEPC water is supplied 20 μ L.
In above-mentioned reaction system, dNTPs, Mg
2+be specially with the proportioning of archaeal dna polymerase: 0.01 μ mol:0.025 μ mol:5U.
The program of SYBRGreen I real-time fluorescence quantitative PCR reaction is specially: by 95 ℃ of sex change 15s, 60 ℃ of annealing, extend 20s and carry out 40 circulations; Finally in 40 ℃, within 10 seconds, finish reaction.The temperature transition rate of carrying out the reaction of SYBR Green I real-time fluorescence quantitative RT-PCR is 20 ℃/s, carries out fluorescent signal detection when the extension of each circulation finishes.
Under 530nm exciting light, fluoresce, be used for detecting avian pneumovirus.
Two, the specific test of SYBR Green I real-time fluorescence quantitative RT-PCR
According in above-mentioned steps 1 2) the SYBR Green I fluorescent quantitative RT-PCR method set up of two-step reaction method carries out specific test, the strain template of participating in the experiment is newcastle disease virus (Lasota) RNA of avian pneumovirus APV/MN RNA and contrast, H9 subtype avian influenza virus RNA, avian infectious bronchitis virus (Mass41) RNA, avian infectious laryngotracheitis virus (Beijing Strain) DNA, avian encephalomyclitis virus (AE Van) RNA, Avianreovirus (Reo S1133) RNA, chicken Marek's disease (MDV) vaccine virus DNA, chicken poison Mycoplasma (MG) DNA, avian escherichia coli (E.coli O
2) DNA, with H
2o is as negative control.
Under 530nm exciting light, detect the specific result of avian pneumovirus, as shown in Figure 3,1: avian pneumovirus APV/MN; 2: newcastle disease virus (Lasota); 3:H9 subtype avian influenza virus; 4: avian infectious bronchitis virus (Mass41); 5: avian encephalomyclitis virus (AE Van); 6: Avianreovirus (Reo S1133); 7: chicken Marek's disease (MDV) vaccine virus; 8: avian infectious laryngotracheitis virus (Beijing Strain); 9: chicken poison Mycoplasma (MG); 10: avian escherichia coli (E.coli O
2); 11:dH
2o.As can be seen from the figure, 1 has the specificity fluorescent curve of corresponding virus, and 2-11 does not all have specificity fluorescent curve, confirms that designed primer amplification avian pneumovirus specificity is good, detects strain no cross reaction with other.Therefore, step 1 can be used for identifying by the SYBR Green I real-time fluorescence quantitative RT-PCR detection method that APV upstream primer and downstream primer (APV1 in table 1 and APV2) are set up whether unknown sample has the existence of avian pneumovirus nucleic acid.
Illustrate, the judgement of SYBR Green I real-time fluorescence quantitative RT-PCR reaction result is as follows:
If under 530nm exciting light, reaction result is straight line, negative; Reaction result is S type curve, positive;
Result is under 530nm exciting light, and S type curve appears in the reaction of avian pneumovirus sample, and other control sample of participating in the experiment is straight line, judges that present method specificity is good.
Three, the sensitivity test of SYBR Green I real-time fluorescence quantitative RT-PCR
With the pMD-APV plasmid of preparation in 10 times of serial dilution above-mentioned steps 1, obtaining copy number is 1 * 10
7-1 * 10
0the serial dilutions of copy/μ L (arranges the dH with equivalent RNase Free using the diluent that contains different plasmid copy numbers as template simultaneously
2the blank of O replacement template) carry out the amplification of SYBR Green I fluorescence quantitative RT-RCR and conventional pcr amplification.Wherein, SYBR Green I fluorescence quantitative RT-RCR adopts in above-mentioned steps 1 2) reaction system and response procedures after the optimization that obtains of two-step reaction method; Reaction system and the response procedures of conventional PCR are as follows: reaction system is 50 μ L, wherein, and 2 * Premix Taq
tMthe mix25 μ L(Dalian precious biotechnology Takara of company limited product, catalog number: RR003A, containing 10 μ M dNTPs, Mg
2+, 5U Taq enzyme), pMD-APV plasmid 4 μ L, each 0.5 μ L of the APV upstream and downstream primer (APV1 in table 1 and APV2) of 50pmol/ μ L, sterilizing ultrapure water adds to 50 μ L.Response procedures is 94 ℃ of denaturation 5min, then enters 94 ℃ of 1min, 55 ℃ of 1min, and 72 ℃ of 1min, carry out 35 circulations altogether; 72 ℃ are extended 10min, finally stop at 4 ℃.
SYBR Green I fluorescence quantitative RT-RCR detects, the result under 530nm exciting light, as shown in Figure 4,1:1.0 * 10
7copy number/μ L; 2:1.0 * 10
6copy number/μ L; 3:1.0 * 10
5copy number/μ L; 4:1.0 * 10
4copy number/μ L; 5:1.0 * 10
3copy number/μ L; 6:1.0 * 10
2copy number/μ L; 7:1.0 * 10
1copy number/μ L; 8:1.0 * 10
0copy number/μ L; 9: blank is (with equivalent RNase Free dH
2o replaces template).Fluorescence curve from figure, detection 10 copies to avian pneumovirus still have fluorescence curve, show that SYBR Green I fluorescence quantitative RT-PCR detecting method is 10 copies to the sensitivity of avian pneumovirus, than highly sensitive 100 times of conventional PCR method (detectability is only 1000 copies).In addition, the present inventor has carried out duplicate detection three times, and the result of duplicate detection is consistent.
Four, the replica test of SYBR Green I real-time fluorescence quantitative RT-PCR
According in above-mentioned steps 1 2) the SYBR Green I fluorescent quantitative RT-PCR method set up of two-step reaction method, the pMD-APV plasmid DNA of choosing preparation in the above-mentioned steps 1 of two different concns (arranges the dH with equivalent RNase Free as template simultaneously
2o replaces the blank of template), each template concentrations is done two repetitions, by calculating the standard deviation (S) of Ct value and batch interior repeatability that the variation coefficient (CV) is verified fluorescence quantitative RT-RCR; The pMD-APV plasmid DNA that alternative is got a concentration, as template, is carried out fluorescent PCR amplification, and interval 7d repeats once, carries out altogether 3 times and repeats, and then calculates the variation coefficient (CV) of Ct value, checking fluorescence quantitative RT-RCR batch between repeated.
Choose 1.0 * 10
4copy/μ L and 1.0 * 10
2the pMD-APV plasmid of bis-concentration of copy/μ L is made revision test, and the Ct value of its gained is respectively 15.75,15.88 and 19.03,19.29, and its Ct value variation coefficient is all less than 5%; Choose 1.0 * 10
4the pMD-APV plasmid of copy/μ L, carried out fluorescent PCR mensuration totally three times at interval of 7 days, and the variation coefficient of its Ct value is also less than 5%.
Result shows, the SYBR Green I fluorescent quantitative RT-PCR method that step 14 is set up is reproducible, specifically as shown in Figure 5, and 1 and 2:1.0 * 10
4copy number/μ L; 3 and 4:1.0 * 10
2copy number/μ L; 5: blank is (with equivalent RNase Free dH
2o replaces template).
The detection of embodiment 3, clinical pathological material of disease
Sample to be tested is the swollen head collected of different areas, Guangxi chicken houses and the sick chicken lung that has respiratory symptom and tracheae totally 54 parts (being numbered C1-C54), extracts respectively total RNA of these pathological material of diseases.
Using total RNA of 54 parts of pathological material of diseases extracting respectively as template, according in step 14 in embodiment 2 2) the SYBR Green I fluorescent quantitative RT-PCR method set up of two-step reaction method detects.
If the reaction result under 530nm exciting light is S type curve, in sample to be tested, contain avian pneumovirus; If reaction result is straight line, in sample to be tested, do not contain avian pneumovirus.
Detected result is as follows:
After measured, under 530nm exciting light, the sample that occurs S type curve in sample to be checked has 2 parts (being numbered C1 and C2), 52 parts, sample (being numbered C3-C54) for straight line, illustrate in testing sample and have 2 parts (being numbered C1 and C2) for the avian pneumovirus positive, 52 duplicate samples (being numbered C3-C54) are negative for avian pneumovirus, do not contain avian pneumovirus.
The present inventor extracts above 54 parts of (being numbered C1-C54) pathological material of diseases respectively after total RNA simultaneously, by reverse transcription, obtain cDNA, gained cDNA is adopted to the further exactness that confirms SYBR Green I fluorescence quantitative RT-PCR detecting method provided by the present invention of method of carrying out sequencing for avian pneumovirus genome (sequence 3).Sequencing result shows in testing sample, have 2 parts (being numbered C1 and C2) positive for avian pneumovirus, and 52 duplicate samples (being numbered C3-C54) are for avian pneumovirus feminine gender, in full accord with SYBR Green I fluorescence quantitative RT-PCR detecting method result.
Claims (10)
1. for detection of the primer pair of avian pneumovirus, primer 1 and primer 2, consist of, described primer 1 is the single stranded DNA shown in sequence 1 in sequence table, and described primer 2 is the single stranded DNA shown in sequence 2 in sequence table.
2. for detection of the reagent of avian pneumovirus, by primer pair claimed in claim 1, SYBR Green I real-time fluorescence quantitative RT-PCR amplification buffer and water, formed.
3. reagent according to claim 2, is characterized in that: described SYBR Green I real-time fluorescence quantitative RT-PCR amplification buffer, comprises dNTPs, Mg
2+, SYBR Green I, archaeal dna polymerase, ThermoScript II and RNA enzyme inhibitors.
4. reagent according to claim 3, is characterized in that: described SYBR Green I real-time fluorescence quantitative RT-PCR amplification buffer is following buffer A or buffer B:
In described buffer A, described dNTPs, described Mg
2+, described archaeal dna polymerase, described ThermoScript II and described RNA enzyme inhibitors proportioning be: 0.1 μ mol:0.25 μ mol:5U:200U:40U;
Described buffer B, is comprised of reverse transcription damping fluid and SYBR fluorescent PCR damping fluid; In described reverse transcription damping fluid, containing proportioning is described dNTPs, the described Mg of 0.02 μ mol:0.1 μ mol:2.5U:20U
2+, described ThermoScript II and described RNA enzyme inhibitors; In described SYBR fluorescent PCR damping fluid, containing proportioning is described dNTPs, the described Mg of 0.01 μ mol:0.025 μ mol:5U
2+with described archaeal dna polymerase, also contain described SYBRGreen I simultaneously.
5. for detection of the test kit of avian pneumovirus, contain following a) and b):
A) avian pneumovirus positive plasmid;
B) arbitrary described reagent in primer pair described in claim 1, or claim 2-4.
6. test kit according to claim 5, is characterized in that: described avian pneumovirus positive plasmid is the plasmid of the 753-849 position Nucleotide containing sequence 3 in ordered list.
7. test kit according to claim 6, is characterized in that: the recombinant plasmid of described avian pneumovirus positive plasmid for obtaining after DNA fragmentation shown in the 753-849 position Nucleotide of sequence in sequence table 3 is connected with pMD18-T carrier.
8. arbitrary described reagent in primer pair according to claim 1, or claim 2-4, or arbitrary described test kit in claim 5-7, is characterized in that: the mol ratio of described primer 1 and described primer 2 is 1:1.
Following c) or application d) 9.:
C) primer pair claimed in claim 1 arbitrary described reagent in preparation claim 2-4, or the application in arbitrary described test kit in claim 5-7;
D) arbitrary described reagent in primer pair claimed in claim 1, or claim 2-4, or arbitrary described test kit in claim 5-7, preparation detect or auxiliary detection testing sample in whether contain the application in the product of avian pneumovirus.
10. application according to claim 9, it is characterized in that: d) in described application, in described detection or auxiliary detection testing sample, whether contain avian pneumovirus for using primer pair claimed in claim 1, or arbitrary described reagent in claim 2-4, or in claim 5-7, arbitrary described test kit carries out the reaction of SYBR Green I real-time fluorescence quantitative RT-PCR to described testing sample;
The annealing temperature of the described SYBR of carrying out Green I real-time fluorescence quantitative RT-PCR reaction is 60 ℃-62 ℃.
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| CN102943130A (en) * | 2012-12-04 | 2013-02-27 | 广西壮族自治区兽医研究所 | LAMP (Loop-medicated isothermal amplification) detection kit for metapneumovirus |
| CN103103289A (en) * | 2013-01-15 | 2013-05-15 | 中国农业科学院哈尔滨兽医研究所 | Fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) kit applied to avian pneumovirus C-subgroup specificity detection, and application of fluorescent quantitative RT-PCR kit |
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| CN102943130A (en) * | 2012-12-04 | 2013-02-27 | 广西壮族自治区兽医研究所 | LAMP (Loop-medicated isothermal amplification) detection kit for metapneumovirus |
| CN103103289A (en) * | 2013-01-15 | 2013-05-15 | 中国农业科学院哈尔滨兽医研究所 | Fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) kit applied to avian pneumovirus C-subgroup specificity detection, and application of fluorescent quantitative RT-PCR kit |
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