CN103571974A - RT-PCR (Reverse Transcription-Polymerase Chain Reaction) primer for detecting poultry-like type H1N1 swine influenza virus and kit - Google Patents
RT-PCR (Reverse Transcription-Polymerase Chain Reaction) primer for detecting poultry-like type H1N1 swine influenza virus and kit Download PDFInfo
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Abstract
The invention discloses an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) primer for detecting poultry-like type H1N1 swine influenza virus and a kit. The RT-PCR primer comprises two pairs: primers for H1 gene and primers for N1 genes, wherein the nucleotide sequence of an upstream primer Hu on the primers for the H1 gene is 5'-GACTCTAGATTTCCATGACCTT-3', and the nucleotide sequence of a downstream Hd is 5'-ACCCATTAGAACACATCCAGAA-3'; the nucleotide sequence of an upstream primer Nu on the primers for the N1 gene is 5'-TCCAAGGGGGATGTGTTTG-3', and the nucleotide sequence of a downstream primer Nd is 5'-GACCAAGCGACTGACTCAA-3'; the kit comprises the primer. The RT-PCR primer and the kit are adopted for detecting the poultry-like type H1N1 swine influenza virus and have the advantages of high sensitivity, strong specificity, fastness and convenience and the like.
Description
Technical field
The invention belongs to animal virus molecular biosciences detection technique field, be specifically related to RT-PCR primer and the test kit of detection type fowl type H1N1 swine influenza virus.
Background technology
H1Nl hypotype swine influenza virus (Swine influenza virus, SIV) be one of important pathogen ，Wei orthomyxoviridae family, the A type Influenza Virus of porcine influenza (Swine influenza, SI), by 8 segmented compositions of genome, the 10-11 kind of encoding altogether albumen.The HINl hypotype porcine influenza U.S. in 1918 reports at first, and Shope is in nineteen thirty this subtype virus of isolation identification for the first time, and this is also to make a definite diagnosis first swine influenza virus, after this constantly propagates, and now in five continents, has all found its trace.This virus can infected pigs, causes that acute, hot respiratory organs transmissible disease occurs pig, mainly take burst respiratory system and to lapse to syndrome be rapidly feature, be one of porcine respiratory syndrome syndrome disease.Because having parent to airway epithelial cell, swine influenza virus has a liking for characteristic, can cause damage to it, the sickness rate of swinery can be up to 100% clinically, case fatality rate 1%～4%, as follow secondary, the polyinfection of other respiratory system diseases, can cause the degree of injury of swinery and case fatality rate significantly to increase.At present, from pig body, being separated to influenza virus has the virus of a plurality of hypotypes such as H1N1, H1N2, H3N2, H1N7, H4N6, H3N6, H5N1 and H9N2, can cause porcine influenza symptom and propagate and mainly contain 3 hypotypes in current swinery, H1N1, H3N2 and H1N2, and H1N1 is in the ascendance in swine influenza virus, the swine influenza virus hypotype major part being separated to all over the world is in recent years all H1Nl.The research such as Sreta finds that H1N1 virulence is stronger compared with other swine influenza virus, and the injury of lung degree of infection wean piglets is more serious than H3N2's, the health that greatly endangers swinery.It should be noted that, having been reported the U.S. is that H1N2 swine influenza virus infects people's case in 10 routine H1N1 hypotypes and 1 example of in December, 2005 to 2009 year appearance in February, wherein confirm that 9 examples have direct contact history with pig, originate from Mexican 21 century Influenza A H1N1 in March, 2009 and be very popular, make this subtype influenza virus further be subject to the attention of height.
H1N1 porcine influenza is different according to virogene fragment source, can be divided into classic type H1N1 porcine influenza, class fowl type H1Nl porcine influenza and the human-like H1Nl porcine influenza of class.Three class H1N1 swine influenza viruses are the popularity in different steps is different all over the world.
Later 1970s Continent of Europe break out H1N1 porcine influenza event, the cause of disease causing is by wild duck, to be passed to the complete avian influenza virus of swinery.This virus detects in 1979 Nian Hungary first, and each gene segment is all from the H1N1 influenza virus in fowl, and the antigenicity of this viroid and heredity are all different from classic H1N1 virus, proves that H1N1 subtype avian influenza virus is transmitted to swinery across kind.Therefore, this viroid is called to class fowl type HINl swine influenza virus.After this H1N1 swine influenza virus in fowl source is generally propagated in European countries and area, replaces just gradually classic type H1N1 swine influenza virus and becomes the Major Epidemic strain of H1N1 subtype virus in swinery.There is restructuring in this viral genome in the near future, within 1984, popular surface gene HA, the NA of containing of European swinery is from people H3N2, H1N2 hypotype, other 6 internal gene fragments come from the new strain H3N2 of restructuring of class fowl H1Nl hypotype, from then on as the cause of disease of zoonosis, so far popular; The appearance of fowl source, Europe H1N1 porcine influenza strain, substituting gradually classic type H1N1 hypotype SIV becomes main epidemic isolates, and causes wide-scale distribution, and the selective advantage that embodies to a certain extent fowl source H1N1 hypotype swine influenza virus is more obvious than classic H1N1 hypotype SIV.Within 1992, in the swinery of Hong Kong, be separated to the porcine influenza H1N1 in fowl source, 2006 there is class fowl H1N1 hypotype swine influenza virus in China's Mainland, and popular leading role is played the part of in beginning in swinery, to China in 2008 from sick swinery again isolation identification to go out 10 strain Europe be class fowl type H1N1 swine influenza virus, mean that class fowl type H1N1 hypotype swine influenza virus and classic type H1N1 hypotype swine influenza virus are common popular in Chinese swinery, formed the Yi Ge Asia branch of Eurasian class fowl type.But class fowl type H1N1 porcine influenza does not still appear in North America swinery, this strain only continues to propagate in Eurasia, does not report, also the Eurasian porcine influenza H1NI virus of name outside Eurasia.
Within 1986, European Region crowd infects class fowl swine influenza virus event, so far, and the case of existing 8 class fowl H1N1 hypotypes and the triple recombinant virus infections of class fowl H3N2.New Zealand's childhood infection in 1993 the H3N2 hypotype recombinant influenza patient ，Bing Cong lung that is separated to from pig be separated to the H1N1 hypotype swine influenza virus in fowl source.In December, 2010, China's Mainland one childhood infection Eurasia be class fowl type H1NI SIV infected children case, in monitoring following closely, in the pig body of patient family's cultivation, be separated to this virus, and be also separated to the swine influenza virus high with this strain homology from local pig slaughtering field sample, illustrate that this viral prevalence is in local swinery, become patient's the source of infection for this reason.Class fowl type H1N1 influenza virus may pass through the adaptation in pig body, then propagates to the mankind and cause popular in crowd.Therefore set up effective diagnostic method significant to control H1Nl hypotype porcine influenza.
Summary of the invention
The RT-PCR primer and the test kit that the object of this invention is to provide detection type fowl type H1N1 swine influenza virus, have higher sensitivity and specificity, and testing cost is low, has good application prospect.
Technical scheme of the present invention is:
The RT-PCR primer of detection type fowl type H1N1 swine influenza virus, described primer is two pairs: for the primer of H1 gene with for the primer of N1 gene, the described upstream primer Hu nucleotides sequence for H1 gene primer is classified 5 '-GACTCTAGATTTCCATGACCTT-3 ' as, and downstream primer Hd nucleotides sequence is classified 5 '-ACCCATTAGAACACATCCAGAA-3 ' as; The described upstream primer Nu nucleotides sequence for N1 gene primer is classified 5 '-TCCAAGGGGGATGTGTTTG-3 ' as, and downstream primer Nd nucleotides sequence is classified 5 '-GACCAAGCGACTGACTCAA-3 ' as.
The test kit of the above detection type fowl type H1N1 swine influenza virus, described test kit comprises above-described primer.
The test kit of the above detection type fowl type H1N1 swine influenza virus, described test kit also comprises: RNA extracts reagent, RT reaction reagent, PCR reaction reagent, negative control and positive control.
Described RNA extracts reagent and comprises: Trizol lysate, chloroform, Virahol and 75% DEPC ethanol;
Described RT reaction reagent comprises: 5 * buffer damping fluid, dNTPs, HIR, Uni12;
Described PCR reaction reagent comprises: Taq DNA enzyme, 10 * buffer damping fluid, dNTPs, MgCl2 and cDNA;
Described negative control comprises: DEPC water;
Described positive control comprises: class fowl type H1N1 swine influenza virus.
Described class fowl type H1N1 swine influenza virus can be class fowl type H1N1 porcine influenza strain, as: the separated class fowl type H1N1 porcine influenza strain of preserving of Guangxi Zhuang Autonomous Region animal epidemic prevention and control centralab.
Concrete principle of the present invention is the homology of class fowl type H1N1 swine influenza virus, application OLIG6.0 software, and the Auele Specific Primer of synthetic two pairs of target nucleotide sequences for H gene and N gene, amplifies specific object band by RT-PCR method.Test method and process are as follows:
1. design of primers:
In PCR reaction, there are two primers, i.e. 5 ' end primer and 3 ' primer.During design primer, take a DNA single chain as benchmark (often take message sense as benchmark), 5 ' end primer is identical with a bit of DNA sequence dna being positioned on fragment 5 ' end to be amplified; 3 ' end primer is complementary with a bit of DNA sequence dna that is positioned at fragment 3 to be amplified ' end.Primer length is generally about 16-25bp, between primer, in primer without complementary sequence.By foot and mouth disease A type genome sequence is compared to analysis, select the fragment without secondary structure and high conservative, design multipair primer, as follows by mating, screen optimum combination of primers:
H1 upstream region of gene primer Hu sequence: 5 '-GACTCTAGATTTCCATGACCTT-3 ';
Downstream primer Hd sequence: 5 '-ACCCATTAGAACACATCCAGAA-3 '.
N1 upstream region of gene primer Nu sequence: 5 '-TCCAAGGGGGATGTGTTTG-3 ';
Downstream primer Nd sequence: 5 '-GACCAAGCGACTGACTCAA-3 '.
2. the foundation of reaction system and optimization:
The sample to be checked of deactivation is extracted to virus genome RNA with the extracting method of Trizol nucleic acid extraction agent, after packing, be stored in respectively-20 ℃ standby.
The optimization of 2.1 primer consumptions, in the situation that other conditions of reaction system are identical, adds system from 0.1 μ L to 1.8 μ L respectively by the consumption of primer pair, and interpretation of result comparison by experiment determines that optimum primer consumption is 0.4 μ L.
The optimization of 2.2 density of magnesium chloride interpretation of result comparison by experiment in the situation that other conditions of reaction system are identical, the concentration of determining magnesium chloride is that 25mM is best.
The optimization result comparative analysis by experiment of 2.3 ThermoScript II (M-MLV), selects 200U/ μ L ThermoScript II as reaction system optimum amount.
The optimization result comparative analysis by experiment of 2.4Taq archaeal dna polymerase (Taq enzyme), selects 5U/ μ L enzyme as reaction system optimum amount.
The optimization of 2.5dNTPs concentration, by using the dNTPs of different concns to detect, is selected 10mM during reverse transcription, select 2.5mM as reaction system optimum amount during pcr amplification.
2.6 reverse transcription primers are stipulated according to < < high pathogenic avian influenza Prevention Technique standard > >, avian influenza virus RT-PCR test is Uni12:5 '-AGCAAAAGCAGG-3 ' with primer, and primer concentration is 20pmol.
Utilize the ultimate density of above-mentioned primer and each composition, finally determine that the RT-PCR reaction system adopting is 25 μ L, required each group and respective concentration are in Table 1.
Table 1 detection type fowl type H1N1 swine influenza virus reaction system
3.RT-PCR reaction conditions is preferably as follows:
Reverse transcription system: 42 ℃ of 60min, 95 ℃ of 5min;
Amplification condition: 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 30s, 35 circulations.
The invention has the beneficial effects as follows:
1. primer detection sensitivity provided by the invention can reach 100 copy/ml, illustrates that it has good sensitivity.
2. primer provided by the invention, for the detection sample without class fowl type H1N1 swine influenza virus without amplified band, illustrates that primer pair goal gene has good specificity.
3. the present invention be directed to class fowl type H1N1 swine influenza virus and represent that strain and high conservative region carry out the design of primer, avoided the generation of false negative result.
4. the primer that the present invention designs can detect sample and whether be the class fowl type H1N1 swine influenza virus positive, and carrying out 35 cyclic amplifications only needed about 2 hours, has saved a large amount of artificial time, experiment consumptive material, improves laboratory work efficiency, has reduced testing cost.
Accompanying drawing explanation
Fig. 1 is the RT-PCR amplification figure of detection type fowl type H1N1 swine influenza virus of the present invention.
M:DNA Marker20001, class fowl type H1N1 porcine influenza positive control 2, classic type H1N1 porcine influenza positive control 3, the human-like H1N1 porcine influenza of class positive control 4, negative control 5, class fowl type H1N1 porcine influenza positive 6, classic type H1N1 porcine influenza negative sample 7, the human-like H1N1 porcine influenza of class negative sample 8, negative sample
Embodiment
Below in conjunction with embodiment, the invention will be further described, but do not limit the scope of the invention and range of application:
One, detect the test kit of foot and mouth disease A C-type virus C
A test kit for detection type fowl type H1N1 swine influenza virus, comprises following reagent, refers to table 2, and primer is this laboratory designed, designed, and other reagent all can be bought from the market.
The test kit of table 2 detection type fowl type H1N1 swine influenza virus
Two, the using method of test kit
The using method of detection type fowl type H1N1 swine influenza virus test kit, comprises the following steps:
1. the extraction of viral RNA
1. get in sample to be checked, positive control, the negative control EP pipe that respectively 500 μ L pollute without RNA enzyme in 1.5ml sterilizing, add equivalent Trizol lysate to carry out cracking, fully vibration, room temperature is placed 10 minutes;
2. add 300 μ L chloroforms, shake up and place 10 minutes;
3. 12000 revs/min, low-temperature and high-speed whizzer is centrifugal 15 minutes;
4. slowly take out supernatant liquid 600 μ L and be positioned over new EP pipe;
5. add equivalent Virahol to mix gently, place 30 minutes for-20 ℃;
6. 12000 revs/min, low-temperature and high-speed whizzer is centrifugal 15 minutes, abandoning supernatant;
7. with 75% DEPC ethanol, draw 1ml washing precipitation in pipe, fully inhale and abandon washing;
8. EP pipe is inverted on thieving paper, blots liquid as far as possible, be positioned in 37 ℃ of incubators 15 minutes, in pipe, take out during without the visible globule;
9. add 25 μ L DEPC water ,-20 ℃ save backup.
2.RT schedule of operation
Get 5 * buffer damping fluid, 5 μ L, dNTPs2 μ L, M-MLV0.5 μ L, HIR0.5 μ L, Uni122 μ L, RNA15 μ L, is placed in aforesaid liquid in same EP pipe, is put in PCR instrument by following procedure operation: 42 ℃ of 60min, 95 ℃ of 5min after mixing.
3.PCR schedule of operation
Each 0.4 μ L of upstream primer Hu, Nu, each 0.4 μ L of downstream primer Hd, Nd, Taq DNA enzyme 0.2 μ L, 10 * buffer damping fluid, 2.5 μ L, dNTPs2 μ L, MgCl22 μ L, DEPC water 11.7 μ L, cDNA5 μ L, aforesaid liquid is placed in same EP pipe, after mixing, be put in PCR instrument by following procedure operation, amplification condition: 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 30s, 35 circulations.
4. result is judged
There is 326bp(H1 gene in positive control) and 212bp(N1 gene) amplified band, negative control is current without taking out of at 326bp and 212bp amplified band corresponding position, and experimental result is set up.
Test-results refers to Fig. 1.There is 326bp(H1 gene in test sample) and 212bp(N1 gene) amplified band, show that test sample is class fowl type H1N1 swine influenza virus positive; Otherwise negative.Present method susceptibility is high, high specificity, fast and convenient, testing cost is low, has good market application foreground.
Animal epidemic prevention and control center, <110> Guangxi Zhuang Autonomous Region
RT-PCR primer and the test kit of <120> detection type fowl type H1N1 swine influenza virus
<160> 4
<210> 1
<211> 22
<212> DNA
The sequence of <213> synthetic
<400> 1
GACTCTAGAT TTCCATGACC TT 22
<210> 2
<211> 22
<212> DNA
The sequence of <213> synthetic
<400> 2
ACCCATTAGA ACACATCCAG AA 22
<210> 3
<211> 19
<212> DNA
The sequence of <213> synthetic
<400> 1
TCCAAGGGGG ATGTGTTTG 19
<210> 4
<211> 19
<212> DNA
The sequence of <213> synthetic
<400> 2
GACCAAGCGA CTGACTCAA 19
Claims (6)
1. the RT-PCR primer of detection type fowl type H1N1 swine influenza virus, it is characterized in that, described primer is two pairs: for the primer of H1 gene with for the primer of N1 gene, the described upstream primer Hu nucleotides sequence for H1 gene primer is classified 5 '-GACTCTAGATTTCCATGACCTT-3 ' as, and downstream primer Hd nucleotides sequence is classified 5 '-ACCCATTAGAACACATCCAGAA-3 ' as; The described upstream primer Nu nucleotides sequence for N1 gene primer is classified 5 '-TCCAAGGGGGATGTGTTTG-3 ' as, and downstream primer Nd nucleotides sequence is classified 5 '-GACCAAGCGACTGACTCAA-3 ' as.
2. a test kit for detection type fowl type H1N1 swine influenza virus, is characterized in that, described test kit comprises primer claimed in claim 1.
3. the test kit of detection type fowl type H1N1 swine influenza virus according to claim 2, is characterized in that, described test kit also comprises: RNA extracts reagent, RT reaction reagent, PCR reaction reagent, negative control and positive control.
4. the test kit of detection type fowl type H1N1 swine influenza virus according to claim 3, is characterized in that:
(1) described RNA extracts reagent and comprises: Trizol lysate, chloroform, Virahol and 75% DEPC ethanol;
(2) described RT reaction reagent comprises: 5 * buffer damping fluid, dNTPs, HIR, Uni 12;
(3) described PCR reaction reagent comprises: Taq DNA enzyme, 10 * buffer damping fluid, dNTPs, MgCl
2and cDNA;
(4) described negative control comprises: DEPC water;
(5) described positive control comprises: class fowl type H1N1 swine influenza virus.
5. the test kit of detection type fowl type H1N1 swine influenza virus according to claim 4, is characterized in that, described class fowl type H1N1 swine influenza virus is class fowl type H1N1 porcine influenza strain.
6. a using method for detection type fowl type H1N1 swine influenza virus test kit as described in claim 4 or 5, comprises the following steps:
(1) extraction of viral RNA:
Get in sample to be checked, positive control, the negative control EP pipe that respectively 500 μ L pollute without RNA enzyme in 1.5ml sterilizing, add equivalent Trizol lysate to carry out cracking, fully vibration, room temperature is placed 10 minutes; Add 300 μ L chloroforms, shake up and place 10 minutes; 12000 revs/min, low-temperature and high-speed whizzer is centrifugal 15 minutes; Slowly take out supernatant liquid 600 μ L and be positioned over new EP pipe; Add equivalent Virahol to mix gently, place 30 minutes for-20 ℃; Centrifugal 15 minutes of 12000 revs/min, low-temperature and high-speed whizzer, abandoning supernatant; DEPC ethanol with 75% is drawn 1ml washing precipitation in pipe, fully inhales and abandons washing; EP pipe is inverted on thieving paper, blots liquid as far as possible, be positioned in 37 ℃ of incubators 15 minutes, in pipe, take out during without the visible globule; Add 25 μ L DEPC water ,-20 ℃ save backup;
(2) RT schedule of operation:
Get 5 * buffer damping fluid, 5 μ L, dNTPs 2 μ L, M-MLV 0.5 μ L, HIR 0.5 μ L, Uni 12 2 μ L, RNA 15 μ L, aforesaid liquid is placed in same EP pipe, after mixing, is put in PCR instrument by following procedure operation: 42 ℃ of 60 min, 95 ℃ of 5 min;
(3) PCR schedule of operation:
Each 0.4 μ L of upstream primer Hu, Nu, each 0.4 μ L of downstream primer Hd, Nd, Taq DNA enzyme 0.2 μ L, 10 * buffer damping fluid, 2.5 μ L, dNTPs 2 μ L, MgCl
22 μ L, DEPC water 11.7 μ L, cDNA 5 μ L, are placed in aforesaid liquid in same EP pipe, are put in PCR instrument by following procedure operation, amplification condition after mixing: 94 ℃ of 30s, 56 ℃ of 30 s, 72 ℃ of 30 s, 35 circulations.
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Cited By (1)
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| CN104450955A (en) * | 2014-11-12 | 2015-03-25 | 中国农业科学院哈尔滨兽医研究所 | Fluorescence quantification RT-PCR (reverse transcription-polymerase chain reaction) detection kit of Eurasian avian-like type H1N1 swine influenza virus and application thereof |
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