CN103540680A - Dual reverse transcription-polymerase chain reaction (RT-PCR) detection method for identifying H9N2 subtype avian influenza virus - Google Patents
Dual reverse transcription-polymerase chain reaction (RT-PCR) detection method for identifying H9N2 subtype avian influenza virus Download PDFInfo
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Abstract
The invention discloses a dual reverse transcription-polymerase chain reaction (RT-PCR) detection method for identifying an H9N2 subtype avian influenza virus. A pair of primers is designed respectively according to HA genes and NA genes of the H9N2 subtype of avian influenza virus (AIV), the HA genes and NA genes of the H9N2 subtype AIV in a sample can be subjected to specific amplification, and lengths of target fragments are respectively 700bp (HA) and 423bp (NA). According to the method, cross reaction is avoided in subtype AIV such as H3N8, H4N6 and H5N8, Newcastle disease virus and infectious bronchitis virus of chicken; the lowest detection amount of allantoic fluid of the virus is 1*103.25EID50/100uL; compared with conventional methods such as hemagglutination inhibition of virus and a neuraminidase inhibition test, the method has the advantage that the coincidence rate of the identification result is 100 percent. A rapid, specific and sensitive detection means is provided for identifying the H9N2 subtype AIV. The detection method can be used for rapidly diagnosing diseases caused by the H9N2 subtype AIV and has good application prospects in aspects of clinical diagnosis and epidemiological investigation.
Description
Technical field
The present invention relates to a kind of dual RT-PCR detection method of the H9N2 of evaluation subtype avian influenza virus.
Background technology
H9N2 subtype avian influenza virus is a kind of influenza virus of low pathogenicity, H9N2 hypotype AIV China Guangdong in 1994 is by separated first, and for over ten years, H9N2 subtype avian influenza is in rising trend in China, particularly, when concurrent or secondary bacterium infect, can cause serious financial loss.H9N2 hypotype AIV China extensively exists at present, is the main AIV hypotype that affects China's aviculture.Have a strong impact on the development of China's aviculture.Research shows, H9N2 hypotype AIV provides internal gene within 1997, causing the H5N1 hypotype AIV of Mao flu.AIV is except infected poultry for H9N2 hypotype, also can make people be infected.Therefore, set up a kind of for H9N2 subtype avian influenza virus detection method, can identify quickly and accurately H9N2 subtype avian influenza virus, again can be for the epidemiology survey of H9N2 subtype avian influenza virus.
Traditional hemagglutination-inhibition test (HI), neuraminidase inhibition test (NI) etc. need serum and the antigen of multiple hypotype, and especially NI test, wastes time and energy, and can not in time virus be identified or be made diagnosis.Also there is no at present the H9 of infected by influenza and the molecular biology method that N2 hypotype detects simultaneously.
Summary of the invention
The dual RT-PCR detection method that the object of this invention is to provide a kind of H9N2 of evaluation subtype avian influenza virus.
Two PCR primer pairs applying in method provided by the present invention, each primer pair is comprised of two single stranded DNAs respectively, and described four single stranded DNAs are the single stranded DNAs shown in sequence 1 in sequence table, sequence 2, sequence 3, sequence 4.
Two PCR primer pairs applying in method provided by the present invention, can be used for characterization or assistant identification H9N2 subtype avian influenza virus reagent or test kit, or for the preparation of diagnosis or auxiliary diagnosis H9N2 subtype avian influenza virus reagent or test kit.
The reagent or the test kit that contain described PCR primer pair all belong to protection scope of the present invention.
The preparation method of two described PCR primer pairs also belongs to protection scope of the present invention.This preparation method specifically can comprise the step that four single stranded DNAs in two described PCR primer pairs are packed separately respectively.
The preparation method of the test kit of described evaluation or assistant identification H9N2 subtype avian influenza virus also belongs to protection scope of the present invention.After this preparation method specifically comprises the steps: four single stranded DNAs described in two described PCR primer pairs to pack separately respectively, be packaged in same reagent box with at least one material in following substances: archaeal dna polymerase and 4 kinds of dNTP (dATP, dTTP, dCTP, dGTP).
In dual RT-PCR detection method provided by the present invention, PCR reaction system and optimum cycle condition also belong to protection scope of the present invention.
Dual RT-PCR detection method provided by the present invention can detect H9N2 subtype avian influenza virus.
Dual RT-PCR detection method provided by the present invention can detect the H9N2 subtype avian influenza virus of different sources, is especially applicable to detecting the H9N2 subtype avian influenza virus of Ji Yuan
Dual RT-PCR detection method provided by the present invention is for detection of H9N2 subtype avian influenza virus high specificity, and sensitivity can reach 1 * 10
4.25eID50/100 μ L.Suppress to compare with the ordinary method such as neuraminidase inhibition test with viral blood clotting, qualification result coincidence rate is 100%.For the evaluation of H9N2 hypotype AIV provides a kind of quick, special, responsive detection means, can be used for the quick diagnosis that H9N2 hypotype AIV causes disease, aspect clinical diagnosis and epidemiology survey, there is good application prospect.
Accompanying drawing explanation
Fig. 1 is H9N2 hypotype AIV dual RT-PCR amplification.Wherein, M is Trans5K DNA Marker; 1 and 2 are H9N2 subtype avian influenza virus.
Fig. 2 is H9N2 hypotype AIV dual RT-PCR specific test.Wherein, M:Trans DNA MarkerI; 1:H9N2 subtype avian influenza virus; 2: newcastle disease virus; 3:H3N8 subtype avian influenza virus; 4:H4N6 subtype avian influenza virus; 5:H5N1 subtype avian influenza virus; 6:H5N8 subtype avian influenza virus; 7: avian infectious bronchitis virus.
Fig. 3 is the sensitivity test of H9N2 hypotype AIV dual RT-PCR.M:Trans DNA Marker I; 1-5: be respectively 10
0, 10
-1, 10
-2, 10
-3, 10
-4the sample of dilution H9N2 subtype avian influenza virus, viral level is followed successively by 1 * 10
7.25eID50/100 μ L, 1 * 10
6.25eID50/100 μ L, 1 * 10
5.25eID50/100 μ L, 1 * 10
4.25eID50/100 μ L, 1 * 10
3.25eID50/100 μ L.
Fig. 4 is the application of H9N2 hypotype AIV dual RT-PCR.M:Trans DNA Marker I; 1-10 is the H9N2 hypotype AIV of China Agricultural University's animal influenza research department isolation identification.11-18 is the allantoic fluid that the sick chicken of H9N2 hypotype AIV is organized inoculated into chick embryo gained.
Embodiment
The experimental technique using in following embodiment is ordinary method if no special instructions.
In following embodiment, material used, reagent etc., all can obtain if no special instructions from commercial channels.
Strain used in following embodiment is preserved and is provided by China Agricultural University's animal influenza research department.
The design of embodiment 1, primer, synthetic and screening
According to the sequence of the HA of H9N2 subtype avian influenza virus and NA gene, by DNAman software, compare, find out the conserved regions of HA and NA gene, conserved regions sequence for HA and NA gene designs respectively Auele Specific Primer, and on Genbank, carry out BLAST and detect compare of analysis, with Oligo4.0 software analysis upstream and downstream primer, whether mate, the qualified primer of analysis is served to the synthetic portion in Hai Shenggong biotechnology limited liability company Beijing synthetic.Synthesized respectively 5 pairs of primers for HA and NA gene, with this laboratory, the separated H9N2 hypotype AIV preserving screens, and according to amplification efficiency, determines primer pair combination.Upstream primer H9-F:5 '-CATAATGGGATGCTGTGTGC-3 ' (SEQ ID NO:1) for H9 gene; Downstream primer H9-R:5 '-CACCTTTTTCGGTCTGACATTG-3 ' (SEQ ID NO:2); Upstream primer N2-F:5 '-ATGGAACTTGTGCCGTAG-3 ' (SEQ ID NO:3) for N2 gene; Downstream primer N2-R:5 '-CATAACCTGAGCGTGAAT-3 ' (SEQ ID NO:4).Annealing temperature is 48 ℃.The length of object segment is respectively 700bp (HA) and 423bp (NA).
The foundation of embodiment 2, dual RT-PCR method
1, the extraction of total RNA
1) the chick embryo allantoic liquid 300 μ L that get respectively infection H9N2 subtype avian influenza virus A/Chicken/Shandong/ZB/2007, A/Chicken/Shandong/6/96 are in the 1.5mL centrifuge tube without RNA enzyme, add 900 μ L Trizol reagent (Invitrogen), both ratios are 1: 3, put upside down gently and mix 10 times, nucleoprotein complex body is dissolved completely;
2) add 200 μ L trichloromethanes (chloroform), put upside down and mix 10 times, ice bath is placed 5 minutes, during put upside down and mix gently; 4 ℃ of centrifugal 15min of 12000rpm.
3) water (approximately 700 μ L) is moved to one new in RNA enzyme 1.5mL centrifuge tube, add equivalent Virahol, put upside down and mix for several times, place after 10 minutes centrifugal 15 minutes of 4 ℃ of 12000rpm, supernatant discarded for-20 ℃;
4) use 1mL75% without RNA enzyme ice washing with alcohol, instantaneous centrifugal, abandon supernatant, notice that the other RNA precipitation outwells, with yellow rifle head, blot, instantaneous centrifugal, then blot with white rifle head, put drying precipitated 5～10min on ice, make ethanol volatilization;
5) by 11 μ L DEPC water dissolution for dry RNA, with or-80 ℃ save backup.
2, the synthetic cDNA of reverse transcription
With total RNA sample that reverse transcription test kit (K1622, Fermentas) obtains step 1 respectively, carry out reverse transcription, obtain cDNA.
1) Oligo primer (20pmol/ μ L) 1 μ L is added in the 11 μ L DEPC water containing RNA, instantaneous centrifugal, after 65 ℃ of water-bath 5min, be placed in 5min on ice, make RNA linearizing.
2) reverse transcription system 20 μ L:Oligo primers (20pmol/ μ L) 1 μ L; Total RNA11 μ L; Reaction (5 *) 4 μ L; 10mMdNTP Mix 2 μ L; Ribolock
tMrnase Inhibitor (20U/ μ L) 1 μ L; Revert Aid
tMm-MuLVReverse Transcriptase (200U/ μ L) 1 μ L.
3) step 1) after completing, add successively all the other the 4 kinds of compositions in reverse transcription system, mix, instantaneous centrifugal.Reaction conditions: 37 ℃ of water-bath 1h, 70 ℃ of 5min, 4 ℃ of preservations.
3, the optimization of dual RT-PCR condition
Adopting cumulative volume is the reaction system of 25 μ L: 2 * PCR MIX buffer (Beijing Quanshijin Biotechnology Co., Ltd, its composition is Easy Taq RNA polymerase, dNTPS and reaction buffer), 12.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 1 primer 1 μ L; Concentration is 20pmol/ μ L sequence table sequence 2 primer 1 μ L; Concentration is 20pmol/ μ L sequence table sequence 3 primer 1.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 4 primer 1.5 μ L; ddH
2o complements to 25 μ L.
The sex change of dual RT-PCR, annealing, elongating temperature and time, cycle index etc. are optimized.
Dual RT-PCR optimum cycle condition is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 48 ℃ of annealing 35s, 72 ℃ are extended 45s, from second step, start to carry out 34 circulations, and then 72 ℃ are extended 10min.
4, electrophoresis
Get respectively PCR product 5 μ L and carry out electrophoresis with 1.5% sepharose.The results are shown in Figure the object band that 1,2 strain H9N2 subtype avian influenza virus all amplifies 700bp and 423bp, conform to expection product size.
5, PCR product order-checking
Double PCR product is reclaimed, send Beijing Hua Da Gene science limited-liability company to carry out sequencing.
HA, NA gene amplification sequencing fragment result are carried out to BLAST in NCBI influenza database, and the homology of the 4th fragment HA gene of the strains such as HA gene order and A/Chicken/Shandong/A1/2009 (H9N2), A/Chicken/Zhejiang/611/2011 (H9N2) is 99%.The homology of the 6th fragment NA gene of the strains such as NA gene order and A/Chicken/Shandong/A1/2009 (H9N2), A/Chicken/Yangzhou/11/2010 (H9N2) is 99%.
The specific test of embodiment 3, dual RT-PCR detection method
1, the extraction of total RNA
Respectively the chick embryo allantoic liquid of a strain H9N2 subtype avian influenza virus, a strain H3N8 subtype avian influenza virus, a strain H4N6 subtype avian influenza virus, a strain H5N1 subtype avian influenza virus, a strain H5N8 subtype avian influenza virus, a strain newcastle disease virus, a strain avian infectious bronchitis virus is extracted to total RNA, method is with embodiment 2.
2, the synthetic cDNA of reverse transcription
With total RNA sample that reverse transcription test kit (K1622, Fermentas) obtains step 1 respectively, carry out reverse transcription, obtain cDNA.
Reaction system, reaction conditions and method are with embodiment 2.
3, pcr amplification detects the specificity of dual RT-PCR method
With determined primer pair in embodiment 1, be combined as primer, the cDNA sample that step 2 is obtained carries out pcr amplification.
Reaction system is with embodiment 2.
Reaction conditions is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 48 ℃ of annealing 35s, 72 ℃ are extended 45s, from second step, start to carry out 34 circulations, and then 72 ℃ are extended 10min.
Electrophoresis.Get respectively PCR product 5 μ L and carry out electrophoresis with 1.5% sepharose.The results are shown in Figure two object bands that 2, H9N2 subtype avian influenza virus amplifies 700bp and 423bp.And the allantoic fluid sample of newcastle disease virus, H3N8 subtype avian influenza virus, H4N6 subtype avian influenza virus, H5N1 subtype avian influenza virus, H5N8 subtype avian influenza virus and avian infectious bronchitis virus is all without PCR object band.Result shows, dual RT-PCR detection method provided by the present invention has very strong specificity, can detect specifically H9N2 subtype avian influenza virus.
The sensitivity of embodiment 4, dual RT-PCR detection method detects
1, the mensuration of H9N2 hypotype AIV viral level
Get the allantoic fluid that infects H9N2 subtype avian influenza virus A/Chicken/Shandong/ZB/2007, adopt Reed-Muench method to carry out viral EID
50mensuration, obtain the toxic amount of this virus in chick embryo allantoic liquid.Its viral level is 1 * 10
7.25eID50/100 μ L.
2, the preparation of different extent of dilution allantoic fluids
Above-mentioned viral allantoic fluid is carried out to 10
0, 10
-1, 10
-2, 10
-3, 10
-4etc. different dilutions, obtain viral level and be respectively 1 * 10
7.25eID50/100 μ L, 1 * 10
6.25eID50/100 μ L, 1 * 10
5.25eID50/100 μ L, 1 * 10
4.25eID50/100 μ L, 1 * 10
3.25the sample of EID50/100 μ L.
3, total RNA of different extent of dilution allantoic fluids extracts
Method is with embodiment 2.
4, the synthetic cDNA of reverse transcription
Reaction system, reaction conditions and method are with embodiment 2.
5, pcr amplification detects the sensitivity of dual RT-PCR method
Reaction system is with embodiment 2.Reaction conditions is with embodiment 3.
Electrophoresis.Get respectively PCR product 5 μ L and carry out electrophoresis with 1.5% sepharose.The results are shown in Figure 3,10
-3during dilution, still can amplify two obvious object bands, the method is 1 * 10 to the limit of identification of viral allantoic fluid
4.25eID50/100 μ L, sensitivity is higher, and susceptibility is stronger.
The application of embodiment 5, dual RT-PCR detection method
1, the collection of sample and processing
8 parts of the tissue samples of the H9N2 subtype avian influenza virus infected chicken that the ordinary method of learning from else's experience is identified, weigh, and the tissue grinder of putting sterilizing grinds to form homogenate, add the suspension of making 10%-20% (W/V) containing dual anti-PBS.Proceed in sterilizing centrifuge tube, through 4 ℃ of centrifugal 10min of 12000r/min, get supernatant liquor and be inoculated in 10 age in days SPF chick embryo allantoic cavities, collect the dead and not dead chick embryo allantoic liquid of 24-96h.
2, the extraction of total RNA
8 samples that the allantoic fluid that 10 strain H9N2 subtype avian influenza virus are infected and step 1 obtain extract respectively total RNA, and method is with embodiment 2.
3, the synthetic cDNA of reverse transcription
Reaction system, reaction conditions and method are with embodiment 2.
4, pcr amplification
Reaction system is with embodiment 2.Reaction conditions is with embodiment 3
Electrophoresis.Get respectively PCR product 5 μ L and carry out electrophoresis with 1.5% sepharose.The results are shown in Figure 4.The chick embryo allantoic liquid sample of 10 strain H9N2 subtype avian influenza virus and 8 sick chicken tissues has all amplified two object bands, size conforms to expection, show dual RT-PCR detection method provided by the present invention, qualification result is compared with ordinary methods such as blood clotting inhibition, neuraminidase inhibition tests, and coincidence rate is 100%.
Claims (7)
1. identify or the PCR primer pair composition of assistant identification H9N2 subtype avian influenza virus for one kind, by two PCR primer pairs, formed, each primer pair is comprised of two single stranded DNAs respectively, it is characterized in that: described four single stranded DNAs are the single stranded DNAs shown in SEQ ID NO:1 in sequence table, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4.
2. two PCR primer pairs as claimed in claim 1 application in characterization or assistant identification H9N2 subtype avian influenza virus reagent or test kit.
3. two PCR primer pairs as claimed in claim 1 application in preparation diagnosis or auxiliary diagnosis H9N2 subtype avian influenza virus reagent or test kit.
4. a PCR reagent for evaluation or assistant identification H9N2 subtype avian influenza virus, is characterized in that: in described PCR reagent, contain two PCR primer pairs as claimed in claim 1.
5. a test kit for evaluation or assistant identification H9N2 subtype avian influenza virus, is characterized in that: in described test kit, contain PCR reagent as claimed in claim 4.
6. a preparation method for two PCR primer pairs as claimed in claim 1, is characterized in that: described preparation method comprises the step that four single stranded DNAs described in two PCR primer pairs claimed in claim 1 are packed separately respectively.
7. the preparation method of a test kit as claimed in claim 5, after comprising the steps: four single stranded DNAs described in two PCR primer pairs claimed in claim 1 to pack separately respectively, be packaged in same reagent box with at least one material in following substances: archaeal dna polymerase and following 4 kinds of dNTP:dATP, dTTP, dCTP, dGTP.
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| CN104004859A (en) * | 2014-06-19 | 2014-08-27 | 广西壮族自治区兽医研究所 | H9 subtype avian influenza virus and N2 subtype avian influenza virus dual-purpose RT-PCR detection kit and primer set thereof |
| CN105861757A (en) * | 2016-06-17 | 2016-08-17 | 广西壮族自治区兽医研究所 | Double RT-PCR kit for H9 subtype avian influenza viruses and H6 subtype avian influenza viruses and application of double RT-PCR kit |
| CN107326103A (en) * | 2017-08-28 | 2017-11-07 | 聊城大学 | A kind of triple RT PCR specificity amplification primers groups and the RT PCR detection methods of triple identifications |
| CN110628954A (en) * | 2019-10-31 | 2019-12-31 | 广西壮族自治区兽医研究所 | H9N2 subtype avian influenza virus double-fluorescence RT-LAMP detection primer group, kit and application |
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| CN112831596A (en) * | 2019-11-22 | 2021-05-25 | 华农（肇庆）生物产业技术研究院有限公司 | Method for detecting content of H9N2 subtype avian influenza virus by using digital PCR |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104004859A (en) * | 2014-06-19 | 2014-08-27 | 广西壮族自治区兽医研究所 | H9 subtype avian influenza virus and N2 subtype avian influenza virus dual-purpose RT-PCR detection kit and primer set thereof |
| CN105861757A (en) * | 2016-06-17 | 2016-08-17 | 广西壮族自治区兽医研究所 | Double RT-PCR kit for H9 subtype avian influenza viruses and H6 subtype avian influenza viruses and application of double RT-PCR kit |
| CN107326103A (en) * | 2017-08-28 | 2017-11-07 | 聊城大学 | A kind of triple RT PCR specificity amplification primers groups and the RT PCR detection methods of triple identifications |
| CN110669868A (en) * | 2019-10-10 | 2020-01-10 | 山东省农业科学院家禽研究所 | H9N2 subtype avian influenza virus amplification primer group, visual detection kit and application thereof |
| CN110628954A (en) * | 2019-10-31 | 2019-12-31 | 广西壮族自治区兽医研究所 | H9N2 subtype avian influenza virus double-fluorescence RT-LAMP detection primer group, kit and application |
| CN110628954B (en) * | 2019-10-31 | 2023-01-24 | 广西壮族自治区兽医研究所 | H9N2 subtype avian influenza virus duplex fluorescence RT-LAMP detection primer group, kit and application |
| CN112831596A (en) * | 2019-11-22 | 2021-05-25 | 华农（肇庆）生物产业技术研究院有限公司 | Method for detecting content of H9N2 subtype avian influenza virus by using digital PCR |
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Application publication date: 20140129 |