CN103409559B - RT-PCR kit for detection of poultry source pedigree H3N2 subtype canine influenza virus and application thereof - Google Patents
RT-PCR kit for detection of poultry source pedigree H3N2 subtype canine influenza virus and application thereof Download PDFInfo
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Abstract
The invention discloses an RT-PCR kit for detection of poultry source pedigree H3N2 subtype canine influenza virus and application thereof. The invention provides a primer pair for assisting detection of poultry source pedigree H3N2 subtype canine influenza virus. The nucleotide sequence of one primer is shown as a SEQ ID No.1, and the nucleotide sequence of the other primer is shown as a SEQ ID No. 2. Detection of poultry source pedigree H3N2 subtype canine influenza virus by the kit provided by the present invention is faster, and has higher singularity, sensibility and sensitivity than a traditional method. The kit can detect virus allantoic fluid as well as clinically acquired samples, and is easy for popularization as a useful tool for diagnosis and epidemiological investigation of poultry source pedigree H3N2 subtype canine influenza virus.
Description
Technical field
The present invention relates to a kind of RT-PCR kit and the application thereof that detect fowl source pedigree H3N2 hypotype canine influenza virus.
Background technology
Dog influenza is the high degree in contact sexually transmitted disease of the dog caused by canine influenza virus.Suffer from dog and often show the cold symptoms such as cough, pneumonia.In recent years, the report that canine influenza virus infects increases rapidly, and dog is the important companion animals of the mankind, and the health of dog is more and more subject to people's attention.In addition, the influenza virus in dog can increase the chance infecting people by the intimate contact of people-dog.Therefore, canine influenza virus not only affects the health of dog, also constitutes serious threat to public health security.
Existing bibliographical information, dog can infect fowl source pedigree H3N2 hypotype canine influenza virus, horse source pedigree H3N8 hypotype canine influenza virus, H1N1virus, the seasonal human influenza virus of H3N2 hypotype.Wherein fowl source pedigree H3N2 hypotype canine influenza virus extensively exists in dog group, all detects the influenza infection dog of this hypotype in Asia and America.In China dog group, Major Epidemic is fowl source pedigree H3N2 hypotype dog influenza, and south China and northern area separating for several times are to the canine influenza virus of this hypotype.Therefore, set up a kind of method that can detect fowl source pedigree H3N2 hypotype canine influenza virus to have very important significance.
Traditional canine influenza virus detection method needs to carry out the tests such as viral separation and Culture and blood clotting suppression, and this method wastes time and energy, and can not make quick diagnosis.And due to the antigenic variation of virus, usually cause conventional serological method to detect virus, occur undetected situation.Therefore, clinically in the urgent need to a kind of can the detection method of rapid differential diagnosis fowl source pedigree H3N2 hypotype canine influenza virus, so that guarantee can Timeliness coverage and treatment dog influenza, to preventing canine influenza be very popular and human infection has great importance.
Summary of the invention
The object of this invention is to provide a kind of detect fowl source pedigree H3N2 hypotype canine influenza virus RT-PCR kit and primer special and application thereof.
A kind of primer pair for auxiliary detection fowl source pedigree H3N2 hypotype canine influenza virus (or a kind of primer pair for auxiliary detection H3N2 hypotype canine influenza virus) provided by the present invention, article one, the nucleotide sequence of primer is as shown in SEQ IDNo.1, and the nucleotide sequence of another primer is as shown in SEQ ID No.2.
A kind of RT-PCR kit for auxiliary detection fowl source pedigree H3N2 hypotype canine influenza virus (or a kind of RT-PCR kit for auxiliary detection H3N2 hypotype canine influenza virus) provided by the present invention, this test kit is made up of RNA reverse transcription system and PCR reaction system.
Described RNA reverse transcription system is made up of reverse transcription buffer, RNA enzyme inhibitors, dNTP, ThermoScript II, DEPC process water and Uni12 primer.
The trade name of described reverse transcription buffer is 5 × Reaction Buffer, purchased from fermentas company.
The trade name of described RNA enzyme inhibitors is Ribolock
tMrnase Inhibitor, purchased from fermentas company, catalog number is EO0381.
The trade name of described dNTP is dNTP Mix, and purchased from fermentas company, catalog number is R0191.
The trade name of described ThermoScript II is RevertAid
tMreverse Transcriptase, purchased from fermentas company, catalog number is EP0441.
The trade name of described DEPC process water be DEPC-treated water purchased from fermentas company, catalog number is R0601.
The sequence of described Uni12 primer is 5 '-AGCAAACGACC-3 '.
Described PCR reaction system is by ddH
2o, pcr amplification damping fluid and above-mentioned primer pair composition.
The trade name of described pcr amplification damping fluid is 2 × EasyTaq PCR SuperMix, and purchased from Beijing Quanshijin Biotechnology Co., Ltd, catalog number is AS111.
The application in the product of preparation auxiliary detection fowl source pedigree H3N2 hypotype canine influenza virus of above-mentioned primer pair or mentioned reagent box also belongs to protection scope of the present invention.
The detection utilizing test kit of the present invention to carry out fowl source pedigree H3N2 hypotype canine influenza virus has the following advantages:
1, discriminating detection is carried out with the fowl source pedigree H3N2 hypotype canine influenza virus of time to current popular of 4-5 hour, more quick than traditional method.
2, special detection fowl source pedigree H3N2 hypotype canine influenza virus, and can effectively detect the classical type strain of fowl source pedigree H3N2 hypotype canine influenza virus and popular variant up-to-date in recent years, there is good spreadability and versatility.In addition, detection sensitivity is very high, and 1 × 10
1tCID
50the viral sample of/100 μ l and above concentration all can be detected the specific amplified band of 489bp.
3, not only can detect viral allantoic fluid, also can detect the sample of clinical acquisitions.
4, simple to operate, easy popularization becomes the useful testing tool of the pedigree H3N2 hypotype canine influenza virus diagnosis of fowl source and epidemiology survey.
RT-PCR kit of the present invention is utilized to carry out the detection of fowl source pedigree H3N2 hypotype canine influenza virus to the dog of diagnosis morbidity in time, flu-prevention is very popular, and the epidemiological surveillance carrying out extensive fowl source pedigree H3N2 hypotype canine influenza virus has positive effect.
Accompanying drawing explanation
Fig. 1 is specific detection.
Fig. 2 is sensitivity Detection.
Fig. 3 is sensitivity technique.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The material used in following embodiment and preparation method as follows:
Trizol is purchased from invitrogen company, and catalog number is 15596-026.
Uni12 primer sequence is: 5 '-AGCAAACGACC-3 ', is the synthesis of Beijing Qing Ke Bioisystech Co., Ltd.
Ribolock
tMrnase Inhibitor is purchased from fermentas company, and catalog number is EO0381.
DNTP Mix is purchased from fermentas company, and catalog number is R0191.
RevertAid
tMreverse Transcriptase is purchased from fermentas company, and catalog number is EP0441, includes the 250mM Tris-HCl of 5 × Reaction Buffer(pH8.3,250mM KCl, 20mM MgCl
2, 50mM DTT).
DEPC-treated water is purchased from fermentas company, and catalog number is R0601.
2 × EasyTaq PCR SuperMix is purchased from Beijing Quanshijin Biotechnology Co., Ltd, and catalog number is AS111, comprises the reaction buffer of EasyTaq DNA Polymerase, dNTPs and optimization, and concentration is 2 ×.
Dual anti-PBS solution compound method is as follows: Na
2hPO
412H
202.9g, KH
2pO
40.2g, NaCl8g, KCl0.2g, add distilled water and be settled to 1L, adds 10,000 units of Penicillin and Streptomycin sulphate after autoclaving.
Distilled water negative controls: get sterilizing distilled water 250 μ l, be placed in 1.5ml sterile centrifugation tube, add 750 μ l lysates (the 0.1M sodium-acetate buffer of guanidine thiocyanate 0.8M, ammonium thiocyanate 0.4M, glycerine 5%, phenol 38%), thermal agitation mixes, and room temperature places 5min.
Fowl source pedigree H3N2 canine influenza virus (A/canine/Beijing/364/2009) is disclosed in document " Sun Y.; SunS.; Ma J., Tan Y., Du L.; Shen Y.; Mu Q., Pu J., Lin D.; and Liu J.Identificationand characterization of avian-origin H3N2canine influenza viruses in northernChina during2009-2010.Virology435 (2), 301-307 (2013) ".
Seasonal H3N2 human influenza virus (A/Jiangxi/262/2005) is disclosed in document " Sun Y., Bi Y., Pu J.; Hu Y., Wang J., Gao H.; Liu L.; Xu Q., Tan Y., Liu M.; Guo X.; Yang H., andLiu J.Guiea pig model for evaluating the potential public health risk of swineand avian influenza viruses.Plos One5 (11), e15537 (2010) ".
Horse source pedigree H3N8 canine influenza virus (A/canine/California/70645-4/2006) is disclosed in document " Rivailler P.; Perry A.; Jang Y.; Davis T.; Chen M.; Dubovi J., and Donis O.Evolution of canine and equine influenza (H3N8) viruses co-circulating between2005and2008.Virology408 (1), 71-79 (2010) ".
H1N1 canine influenza virus (A/canine/Beijing/cau9/2009) is disclosed in document " Lin Y.; Sun S., Du L., Ma J.; Fan L.; Pu J., Sun Y., Zhao J.; Sun H.; and Liu, J.Natural andexperimental infection of dogs with pandemic H1N1/2009influenza virus.Journal of General Virology93,119-123 (2012) ".
7 strain fowl sources pedigree H3N2 canine influenza virus A/canine/Beijing/364/2009 (H3N2), A/canine/Beijing/253/2009 (H3N2), A/canine/Beijing/305/2009 (H3N2), A/canine/Beijing/420/2010 (H3N2), A/canine/Beijing/511/2010 (H3N2), A/canine/Liaoning/1578/2010 (H3N2), A/canine/Liaoning/1585/2010 (H3N2) is at document " Sun Y., Sun S., Ma J., Tan Y., Du L., Shen Y., Mu Q., Pu J., Lin D., andLiu J..Identification and characterization of avian-origin H3N2canineinfluenza viruses in northern China during2009-2010.Virology435 (2), 301-307 (2013) " in be disclosed.
Positive swab: clinical identification is nose swab or the throat swab of the dog infecting fowl source pedigree H3N2 hypotype canine influenza virus.4 DEG C of preservations, sent test in laboratory in 24 hours.
Negative swab: clinical identification is nose swab or the throat swab of the dog not infecting fowl source pedigree H3N2 hypotype canine influenza virus.4 DEG C of preservations, sent test in laboratory in 24 hours.
Swab process: swab is immersed in the dual anti-PBS solution of 1ml, fully twist, discard swab after wringing out, 4 DEG C, the centrifugal 5min of 8000rpm, gets supernatant liquor 250 μ l, is placed in 1.5ml sterile centrifugation tube, add 750 μ l lysates again, thermal agitation mixes, and room temperature places 5min.
The design that embodiment 1, special primer are right
Utilize primer premier5.0 design for the primer of fowl source pedigree H3N2 hypotype canine influenza virus HA gene high conservative regional gene.
Primer is as follows:
Upstream primer H3N2-HA-601F:5 '-CCAAGCACTAATCAAGAACAAACC-3 ' (SEQ ID No.1)
Downstream primer H3N2-HA-1089R:5 '-TACCATCCCTTCCCATCCATTTTCT-3 ' (SEQ ID No.2)
The specificity of embodiment 2, detection fowl source pedigree H3N2 hypotype canine influenza virus
Detect sample be fowl source pedigree H3N2 canine influenza virus (A/canine/Beijing/364/2009), seasonal H3N2 human influenza virus (A/Jiangxi/262/2005), horse source pedigree H3N8 canine influenza virus (A/canine/California/70645-4/2006), H1N1 canine influenza virus (A/canine/Beijing/cau9/2009), process after negative swab sample and distilled water negative controls.
One, Trizol method extracts total serum IgE
(1) get each sample 300 μ l, often pipe adds 900 μ l Trizol, puts upside down mixing gently 10 times.
(2) add 200 μ l chloroforms, put upside down mixing 10 times, ice bath 5-10min, period puts upside down mixing gently.4 DEG C, the centrifugal 15min of 13000rpm.
(3) get 700 μ l supernatant liquors and move into new centrifuge tube, add the Virahol of equivalent, put upside down mixing gently, after mixing, place 15min, 4 DEG C, the centrifugal 15min of 13000rpm for-20 DEG C.
(4) abandon supernatant, the adherent alcohol slowly adding 1ml 75% DEPC process, add and gently to take two turns afterwards.4 DEG C, the centrifugal 5min of 13000rpm.
(5) abandon supernatant, first blot residual liquid with yellow rifle head, wink from rear, then blots residual liquid with white rifle head, is placed in air-dry 5min on ice.
Two, reverse transcription synthesis cDNA
(1) add 11 μ l DEPC-treated water and dissolve RNA in pipes, add the Uni12 primer of 1 μ l 20 μMs immediately, wink from, after 70 DEG C of water-bath 5min, ice swashs 1min immediately.
(2) in each pipe, add following reagent successively, form 20 μ lRT reaction systems.
Wink from, be put in 37 DEG C of water-bath 1h, afterwards 70 DEG C of water-bath 5min, 4 DEG C of preservations.
This step obtains reverse transcription product.
Three, pcr amplification
Every tube reaction system is 25 μ l, and reaction system is as follows:
Solution in pipe is mixed gently, and performs mark, carry out PCR.
PCR program is as follows:
94 DEG C of 5min; 94 DEG C of 30s, 59 DEG C of 30s, 72 DEG C of 30s, 30 circulations; 72 DEG C of 10min; 4 DEG C of 24h.
Four, agarose gel electrophoresis
Prepare 2% sepharose, add the every 100ml coagulant liquid of gelsafe(and add 5 μ l).Respectively get 5 μ lPCR products and Marker loading, electrophoresis in 1 × TAE electrophoretic buffer, use Ultraviolet Detector observations.If there is the amplified band of 489bp in test sample, show in sample containing fowl source pedigree H3N2 canine influenza virus.
Electrophoresis result as shown in Figure 1.
M represents Trans DNA Marker I (700bp, 600bp, 500bp, 400 bp, 300bp, 200bp, 100bp).1,2,3,4,5,6 fowl source pedigree H3N2 canine influenza virus (A/canine/Beijing/364/2009), seasonal H3N2 human influenza virus (A/Jiangxi/262/2005), horse source pedigree H3N8 canine influenza virus (A/canine/California/70645-4/2006), H1N1 canine influenza virus (A/canine/Beijing/cau9/2009), negative swab sample and distilled water negative controls is represented respectively.
Fig. 1 shows, fowl source pedigree H3N2 canine influenza virus (A/canine/Beijing/364/2009) sample is visible 489bp amplified band after testing, and seasonal H3N2 human influenza virus (A/Jiangxi/262/2005), horse source pedigree H3N8 canine influenza virus (A/canine/California/70645-4/2006), H1N1 canine influenza virus (A/canine/Beijing/cau9/2009), negative swab sample and distilled water negative controls are all without 489bp amplified band.
Result shows, the detection utilizing test kit of the present invention to carry out fowl source pedigree H3N2 hypotype canine influenza virus has specificity.
The susceptibility of embodiment 3, detection fowl source pedigree H3N2 hypotype canine influenza virus
Fowl source pedigree H3N2 hypotype canine influenza virus detects sample and is respectively: A/canine/Beijing/364/2009 (H3N2), A/canine/Beijing/253/2009 (H3N2), A/canine/Beijing/305/2009 (H3N2), A/canine/Beijing/420/2010 (H3N2), A/canine/Beijing/511/2010 (H3N2), A/canine/Liaoning/1578/2010 (H3N2), A/canine/Liaoning/1585/2010 (H3N2), A/canine/Beijing/1215/2012 (H3N2), A/canine/Beijing/0108/2013 (H3N2).
One, Trizol method extracts total serum IgE
Extract and detect sample total serum IgE, method is with the step one of embodiment 2.
Two, reverse transcription synthesis cDNA
Method is with the step 2 in embodiment 2.
Three, pcr amplification
Method is with the step 3 in embodiment 2.
Four, agarose gel electrophoresis
Method is with the step 4 in embodiment 2.
Electrophoresis result as shown in Figure 2.
M represents Trans DNA Marker I (700bp, 600bp, 500bp, 400bp, 300bp, 200bp, 100bp).1, 2, 3, 4, 5, 6, 7, 8, 9 are respectively strain A/canine/Beijing/364/2009 (H3N2), A/canine/Beijing/253/2009 (H3N2), A/canine/Beijing/305/2009 (H3N2), A/canine/Beijing/420/2010 (H3N2), A/canine/Beijing/511/2010 (H3N2), A/canine/Liaoning/1578/2010 (H3N2), A/canine/Liaoning/1585/2010 (H3N2), A/canine/Beijing/1215/2012 (H3N2), the virus liquid of A/canine/Beijing/0108/2013 (H3N2).
Fig. 2 shows, 9 strain fowl source pedigree H3N2 hypotype canine influenza virus samples are through all visible 489bp amplified band of above-mentioned detection.
Result shows, the detection utilizing test kit of the present invention to carry out fowl source pedigree H3N2 hypotype canine influenza virus has susceptibility.
Embodiment 4, detect fowl source pedigree H3N2 hypotype canine influenza virus sensitivity
One, the preparation of each diluent
Fowl source pedigree H3N2 hypotype canine influenza virus (A/canine/Beijing/364/2009) is diluted by dual anti-PBS solution, makes concentration and be respectively 1 × 10
5, 1 × 10
4, 1 × 10
3, 1 × 10
2, 1 × 10
1, 1 × 10
0tCID
50the viral dilution liquid of/100 μ l.
Two, Trizol method extracts total serum IgE
Extract the total serum IgE of each viral dilution liquid, extracting method is with the step one of embodiment 2.
Three, reverse transcription synthesis cDNA
Method is with the step 2 in embodiment 2.
Four, pcr amplification
Method is with the step 3 in embodiment 2.
Five, agarose gel electrophoresis
Method is with the step 4 in embodiment 2.
Electrophoresis result as shown in Figure 3.
M represents Trans DNA Marker I (700bp, 600bp, 500bp, 400bp, 300bp, 200bp, 100bp).1,2,3,4,5,6 to be respectively virus concentration be 1 × 10
5, 1 × 10
4, 1 × 10
3, 1 × 10
2, 1 × 10
1, 1 × 10
0tCID
50the virus liquid of/100 μ l.
Fig. 3 shows, 1 × 10
1tCID
50the virus liquid sample of/100 μ l and above concentration all can be observed the amplified band of 489bp.
Result shows, utilizes test kit of the present invention to carry out the detection sensitivity of fowl source pedigree H3N2 hypotype canine influenza virus very high.
The detection of embodiment 5, clinical sample
One, Trizol method extracts total serum IgE.
Respectively the positive after process and negative swab and distilled water negative controls are extracted total serum IgE, extracting method is with the step one of embodiment 2.
Two, reverse transcription synthesis cDNA
Method is with the step 2 in embodiment 2.
Three, pcr amplification
Method is with the step 3 in embodiment 2.
Four, agarose gel electrophoresis
Method is with the step 4 in embodiment 2.
Result shows, and clinical identification is the band that positive swab samples amplification obtains 489bp, and clinical identification is that negative swab samples does not increase and obtains any band, and distilled water negative controls does not increase and obtains any band.Prove to utilize test kit of the present invention to carry out the detected result of fowl source pedigree H3N2 hypotype canine influenza virus reliable.
Claims (3)
1., for a primer pair for auxiliary detection fowl source pedigree H3N2 hypotype canine influenza virus, the nucleotide sequence of a primer is as shown in SEQ ID No.1, and the nucleotide sequence of another primer is as shown in SEQ ID No.2.
2., for a RT-PCR kit for auxiliary detection fowl source pedigree H3N2 hypotype canine influenza virus, this test kit is made up of RNA reverse transcription system and PCR reaction system;
Described RNA reverse transcription system is made up of reverse transcription buffer, RNA enzyme inhibitors, dNTP, ThermoScript II, DEPC process water and Uni12 primer;
The trade name of described reverse transcription buffer is 5 × Reaction Buffer, purchased from fermentas company;
The trade name of described RNA enzyme inhibitors is Ribolock
tMrnase Inhibitor, purchased from fermentas company, catalog number is EO0381;
The trade name of described dNTP is dNTP Mix, and purchased from fermentas company, catalog number is R0191;
The trade name of described ThermoScript II is RevertAid
tMreverse Transcriptase, purchased from fermentas company, catalog number is EP0441;
The trade name of described DEPC process water be DEPC-treated water purchased from fermentas company, catalog number is R0601;
The sequence of described Uni12 primer is 5 '-AGCAAACGACC-3 ';
Described PCR reaction system is by ddH
2o, primer pair composition described in pcr amplification damping fluid and claim 1;
The trade name of described pcr amplification damping fluid is 2 × EasyTaq PCR SuperMix, and purchased from Beijing Quanshijin Biotechnology Co., Ltd, catalog number is AS111.
3. primer pair according to claim 1, test kit according to claim 2 application in the product of preparation auxiliary detection fowl source pedigree H3N2 hypotype canine influenza virus.
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| CN105821158A (en) * | 2016-04-11 | 2016-08-03 | 公安部南京警犬研究所 | RT-LAMP primer combination and kit for detecting canine influenza virus as well as application of RT-LAMP primer combination |
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| CN102304591B (en) * | 2011-10-08 | 2013-06-05 | 中国农业大学 | PCR (polymerase chain reaction) primer pair for identifying H3 subtype avian influenza virus and application thereof |
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