CN103898237A - Degenerate primer RT-PCR detection method for eimeria viruses - Google Patents
Degenerate primer RT-PCR detection method for eimeria viruses Download PDFInfo
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Abstract
The invention discloses a degenerate primer RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method for eimeria viruses. A pair of brand-new degenerate primers is provided, and specific degenerate primer RT-PCR amplification is performed on the eimeria viruses by using reverse transcription and touchdown PCR techniques. The degenerate primer RT-PCR detection method is capable of effectively detecting the existence of an eimeria tenella virus and an eimeria stiedae virus, and good in specificity (an electrophoretic band is single). The degenerate primer RT-PCR detection method is used for detecting the eimeria viruses for the first time, and is capable of quickly and accurately identifying whether a coccidium sample to be detected contains a virus-carrying strain or not.
Description
Technical field
The present invention discloses a kind of degenerated primer RT-PCR detection method for Eimeria coccidia virus, and a pair of brand-new degenerated primer is provided, and application reverse transcription and touchdown PCR technology are carried out specific degenerated primer RT-PCR amplification to Eimeria coccidia virus.Applied technology for degenerated primer designs, reverse transcription, touchdown PCR (TD-PCR), belong to molecular Biological Detection technical field.
Background technology
Coccidiosis of chicken (Coccidiosis) is to be caused by one or more Eimeria coccidias, and the very serious parasitosis of harm.As a kind of cytozoon, eimeria tenella causes the massive losses of global aviculture, the loss of annual about 8,000,000,000 dollars.First diplornavirus was found in 1948, after this in protozoon, has been found again dsRNA virus in fungi.Protozoon virus is that a class is present in the virus particle in former polypide, is mostly double-stranded RNA (dsRNA) virus, spherical 20 bodies, and diameter 30-50nm, has rna dependent rna polymerase activity.Aspect coccidia virus research, be reported in Eimeria Tenella, Si Shi eimeria tenella, Nissl eimeria tenella, Bu Shi eimeria tenella, huge eimeria tenella, murder by poisoning eimeria tenella and E.acervulina and had virus like particle or viral sample nucleic acid.But it is not at present still fully aware of about the numerous characteristics of coccidia virus.
In the research of eimeria tenella, find whether the virulence of coccidia and its carry virus and have dependency.At the early-stage for eimeria tenella virus research at present, a large amount of research also concentrates on the electron microscopic observation to virus like particle.At present the detection method of coccidia virus be there is no to standard.Traditional method is the existence of electron microscopic observation virus like particle, but this method cost is high, and recall rate is low.Another kind method is to detect viral nucleic acid, but due to the polyinfection often of natural infection case, take viruliferous worm strain ratio low, particularly in the situation that having other biological material contamination, the sensitivity of its detection is very low, and false drop rate is high, the judgement of result is not had to specific standards.Therefore develop a kind of sensitive, accurately, cost method low and that all Eimeria coccidia virus is carried out to versatility detection, be necessary.
Degenerated primer PCR is because genetic code has degeneracy, most amino acid has a more than codon to encode, and homology isoprotein often has higher aminoacid sequence conservative property in some important section, so can probably infer with the conserved regions of work gene by known multiple homologies unknown goal gene, and design the degenerated primer with degeneracy site with this and carry out pcr amplification.Therefore degenerated primer PCR has the certain versatility of homology with work gene amplification.The design of degenerated primer is the committed step of degenerated primer PCR success or not.Obtaining can be for the object nucleic acid fragment detecting, and must use the method for RT-PCR is DNA by RNA amplification.Classical inverse responsive transcription adopts single downstream primer to carry out for ssRNA.There is no at present the report for detection of the degenerated primer RT – PCR detection method of Eimeria coccidia virus.
Summary of the invention
The object of the invention is openly a kind of degenerated primer RT-PCR detection method for Eimeria coccidia virus, for degenerated primer RT-PCR detection method, solve to there is no at present and detected the difficult problem that eimeria tenella belongs to viral method, the low variation property in the method application territory, high conserved region, use degenerated primer to detect Eimeria coccidia virus, reach specificity and combine with versatility, provide new feasible way for detecting Eimeria coccidia virus.
Comprise the following steps:
1) under the condition of STE damping fluid, use phenol chloroform to carry out extracting to the coccidia material through liquid nitrogen grinding, obtain total nucleic acid, the DNA in total nucleic acid is via DNaseI digestion process, and that obtains RNA slightly carries product;
2) this is slightly carried product and directly carries out reverse transcription (RT) as template, because coccidia viral genome is dsRNA, so reverse transcription is used CF and two degenerated primers of CR to carry out simultaneously;
Upstream primer: CF:5 '-CBGCIGCTBYIGTVGCH-3 '
Downstream primer: CR:5 '-GTVGRCTCDATCCARAASHAD-3 '
Above CF primer has used I(xanthoglobulin), replace N(N=A/T/C/G);
Wherein annex base code:
R=A/G、S=G/C、Y=C/T、V=A/G/C、H=A/C/T、D=A/G/T、B=G/C/T、N=A/G/C/T；
3) it is dsDNA that the cDNA obtaining via reverse transcription uses PCR method amplification, agarose gel electrophoresis detected result.
Due to the comparatively difficulty of reverse transcription of dsRNA, use the multiple ThermoScript II of multiple companies by contrast, finally determined PrimeScript Reverse Transcriptase (2680Q) ThermoScript II, PCR uses TaKaRa LA Taq (RRO2MQ) nucleic acid polymerase.All ingredients used in the present invention is cheap, the object fragment that can effectively increase simultaneously, and amplified production specificity is good, and electrophoresis result easily judges.
positively effect of the present invention is:degenerated primer RT-PCR method can effectively detect Eimeria Tenella virus and Si Shi eimeria tenella virus exists, and specificity is good, and electrophoretic band is single; Versatility is good, can carry out general detection to Eimeria coccidia virus; Highly sensitive, a small amount of object nucleic acid: 0.05ng/ μ l can obtain detected result.For the method detecting for Eimeria coccidia virus first, can identify fast and accurately in coccidia sample to be checked and whether contain and take viral worm strain.
Accompanying drawing explanation
Accompanying drawing 1 is upstream primer CF, downstream primer CR schema.
Accompanying drawing 2 is that template ribonucleic acid concentration gradient is preferred.
Accompanying drawing 3 is that primer concentration grads is preferred.
Accompanying drawing 4 is that TaKaRa La Taq enzyme concn gradient is preferred.
Accompanying drawing 5 is that dNTP concentration gradient is preferred.
Accompanying drawing 6 is that the method for degenerated primer RT-PCR detects Eimeria Tenella virus.
Accompanying drawing 7 is that the method for degenerated primer RT-PCR detects Si Shi eimeria tenella virus.
Embodiment
For further illustrating the application of the present invention in Eimeria coccidia virus detects, describe with the following example, but application of the present invention is not limited to embodiment.
the design of primer and synthetic
One, material: molecular biology software MEGA4, DNAMAN and NCBI network resource.
Two, method and result:
Sequence obtain: by Bu Shi eimeria tenella virus (
e. brunettirNA virus 1) genome analysis, to choose coat gene as target sequence, and from GenBank public database, obtain this gene order, accession number is: NC_002701.Use coat protein sequence for addressing inquires to sequence, on NCBI, carry out BlustP comparison, obtain 330 to 346 (IALGTAAAVAHCDPLVP), the albumen region of 435 to 446 (PFFWIEPTTVLP) two high conservatives, the nucleotide sequence of the corresponding position of all the other reference sequences is downloaded separately, obtain Gremmeniella abietina RNA virus L2, Gremmeniella abietina RNA virus L1, Helicobasidium mompa totivirus 1-17, Helminthosporium victoriae virus, Beauveria bassiana RNA virus 1, Tolypocladium cylindrosporum virus 1, 1 seven mycovirus of Aspergillus foetidus slow virus, and the sequence of 4 one leishmania viruses of Leishmania RNA virus 1 –.
Design of primers: the sequence obtaining, after cluster comparison, is chosen appropriate area and designed primer.(seeing accompanying drawing 1).Degeneracy site is used degeneracy base, when degeneracy site is N, uses I(xanthoglobulin).
Primer is synthetic: by the synthetic primer CF of biotech firm, CR.
Upstream primer: CF:5 '-CBGCIGCTBYIGTVGCH-3 '
Downstream primer: CR:5 '-GTVGRCTCDATCCARAASHAD-3 '
the preparation of nucleic acid samples
One, material: Eimeria Tenella, 2 × STE damping fluid (Ph8.0), water-saturated phenol, trichloromethane, beta-mercaptoethanol, 10%SDS, 70% ethanol, Virahol, supercentrifuge.
Two, method and result:
1) Eimeria Tenella egg capsule fragmentation: by approximately 10
7coccidia to be checked carries out liquid nitrogen grinding, is dissolved in 1ml2 × STE(pH8.0 after grinding) damping fluid.
2) protein extracting: add 30 μ l beta-mercaptoethanols in above-mentioned coccidia solution, 0.6ml water-saturated phenol, 0.4ml trichloromethane, 140 μ l10%SDS.Fully homogenate 5 minutes.Under 4 ℃ of conditions, centrifugal 5 minutes of 12000r/min, gets supernatant.
3) total nucleic acid is obtained: supernatant and isopyknic cold isopropanol mix, and-80 ℃ leave standstill 10 minutes, under 4 ℃ of conditions, and centrifugal 15 minutes of 15000r/min, precipitation is cleaned with 70% cold alcohol solution, under 4 ℃ of conditions, centrifugal 15 minutes of 15000r/min.After precipitation is dry, be dissolved in 200 μ lddH
2o.
4) DNA digestion: get above-mentioned total nucleic acid solution 172 μ l, add DNaseI(TaKaRa, 2270A) 8 μ l, 10*DNase I Buffer 20 μ l.37 ℃ digest 30 minutes, and 80 ℃ make enzyme deactivation for 5 minutes.
5) RNA obtains: get above-mentioned solution, add equal-volume cold isopropanol ,-80 ℃ leave standstill 10 minutes, under 4 ℃ of conditions, and centrifugal 15 minutes of 15000r/min, precipitation is cleaned with 70% cold alcohol solution, under 4 ℃ of conditions, centrifugal 15 minutes of 15000r/min.After precipitation is dry, be dissolved in 100 μ lddH
2o.Centrifugal 15 minutes of 15000r/min, gets supernatant again, abandons precipitation.
degenerated primer RT-PCR detects Eimeria Tenella virus
One, material: Eimeria Tenella RNA crude extract, PrimeScript Reverse Transcriptase (2680Q) ThermoScript II test kit, TaKaRa LA Taq (RRO2MQ) nucleic acid polymerization enzyme reagent kit, agarose, ethidium bromide solution, Biorad PCR instrument.
Two, method and result:
1) 1st-Stand cDNA is synthetic: in Microtube, prepare following mixed solution:
99 ℃ of sex change 1 minute, proceed to immediately-40 ℃ of urgency and freeze.
The amount of template ribonucleic acid is carried out gradient experiment, is divided into six gradients, is respectively 50ng/ μ l, 5ng/ μ l, 0.5ng/ μ l, 0.05ng/ μ l, 0.005ng/ μ l, 0.0005ng/ μ l.When template ribonucleic acid concentration is 0.05ng/ μ l, PCR result still can clear interpretation.(seeing accompanying drawing 2)
In above-mentioned reaction solution, add following component:
30 ℃ of reactions 10 minutes, 42 ℃ of reactions 45 minutes, 70 ℃ of sex change 15 minutes, ice bath.
2) Touchdown-PCR (TD-PCR) amplification reaction system:
TD-PCR amplified reaction program is:
1、 94℃ 5min
2、 95℃ 1min
3,65 ℃ → 56 ℃ 1min (1 ℃ of each circulation landing)
4、 72℃ 1min Go to 2 10times
5、 95℃ 1min
6、 56℃ 1min
7、 72℃ 1min Go to 5 35times
8、 72℃ 10min
9、 Hold 4℃。
3) groping of Touchdown-PCR condition
(1) final concentration of upstream and downstream primer (CF and CR) in system arranges gradient and is: 1mM, 0.6mM, 0.2mM.This interval primer concentration is 1mM and 0.6mM, and object fragment has obtained effective amplification.Consider sensitivity and cost, choosing 0.6mM is primer final concentration.(see accompanying drawing 3; M:Marker D 2000, the concentration of Lane1-6:RNA template is 50ng/ μ l, 5ng/ μ l, 0.5ng/ μ l, 0.05ng/ μ l, 0.005ng/ μ l, 0.0005ng/ μ l.When template ribonucleic acid concentration is 0.05ng/ μ l, PCR result still can clear interpretation.）
(2) final concentration of TaKaRa La Taq in system arranges gradient and is: 0.1U/ μ l, 0.05U/ μ l, 0.025U/ μ l, 0.0125U/ μ l, 0.00625U/ μ l, 0.003125U/ μ l.Within the scope of the final concentration of 0.1U/ μ l to 0.00625U/ μ l, object fragment has all obtained effective amplification, and with 0.05U/ μ l amplification efficiency for the highest, choosing 0.05U/ μ l is TaKaRa La Taq final concentration.(seeing accompanying drawing 4)
(3) final concentration of dNTP in system arranges gradient and is: 0.4mM, 0.2mM, 0.1mM, 0.05mM, 0.025mM, 0.0125mM.Within the scope of this, object fragment has all obtained effective amplification, but under 0.025mM concentration and lower concentration condition, object band is obviously dimmed, and considering cost and sensitivity are optimum concn therefore select 0.05mM dNTP.(seeing accompanying drawing 5)
(4) detect: use above-mentioned groped condition to tender detection Eimeria Tenella virus.It is 0.05ng/ μ l that RT-PCR reaction is used template ribonucleic acid concentration.System program is:
1st-Stand cDNA is synthetic: in Microtube, prepare following mixed solution:
99 ℃ of sex change 1 minute, proceed to immediately-40 ℃ of urgency and freeze.
In above-mentioned reaction solution, add following component:
30 ℃ of reactions 10 minutes, 42 ℃ of reactions 45 minutes, 70 ℃ of sex change 15 minutes, ice bath.
Touchdown-PCR (TD-PCR) amplification reaction system:
TD-PCR amplified reaction program is:
1、 94℃ 5min
2、 95℃ 1min
3,65 ℃ → 56 ℃ 1min (1 ℃ of each circulation landing)
4、 72℃ 1min Go to 2 10times
5、 95℃ 1min
6、 56℃ 1min
7、 72℃ 1min Go to 5 35times
8、 72℃ 10min
9、 Hold 4℃。
5) agarose electrophoresis detects PCR result: the TAE sepharose of preparation 1% concentration, uses DL2000 DNA Marker, ethidium bromide staining, the object band of observation 400bp size.(seeing accompanying drawing 6).Be aim sequence through this fragment that checks order, the amplification of Eimeria Tenella virus distinguished sequence, this sequence length is 408bp, the homology 32% of the protein of its coding and Bu Shi eimeria tenella virus homology segment, and can be identified and belong to Totivirus-coat superfamily.(seeing sequence table 1).
degenerated primer RT-PCR method detects Si Shi eimeria tenella virus
One, material: Si Shi eimeria tenella RNA slightly carries product, PrimeScript Reverse Transcriptase (2680Q) ThermoScript II test kit, TaKaRa LA Taq (RRO2MQ) nucleic acid polymerization enzyme reagent kit, agarose, ethidium bromide solution, Biorad PCR instrument.
Two. method and result:
1) RT-TD-PCR: take Si Shi eimeria tenella RNA crude extract as template, detect the existence of viral specific band.It is 0.05ng/ μ l that RT-PCR reaction is used template ribonucleic acid concentration.
System program is:
1st-Stand cDNA is synthetic: in Microtube, prepare following mixed solution:
99 ℃ of sex change 1 minute, proceed to immediately-40 ℃ of urgency and freeze.
In above-mentioned reaction solution, add following component:
30 ℃ of reactions 10 minutes, 42 ℃ of reactions 45 minutes, 70 ℃ of sex change 15 minutes, ice bath.
Touchdown-PCR (TD-PCR) amplification reaction system:
TD-PCR amplified reaction program is:
1、 94℃ 5min
2、 95℃ 1min
3,65 ℃ → 56 ℃ 1min (1 ℃ of each circulation landing)
4、 72℃ 1min Go to 2 10times
5、 95℃ 1min
6、 56℃ 1min
7、 72℃ 1min Go to 5 35times
8、 72℃ 10min
9、 Hold 4℃。
2) agarose electrophoresis detects PCR result: the TAE sepharose of preparation 1% concentration, uses DL2000 DNA Marker, ethidium bromide staining, the object band of observation 400bp size.(seeing accompanying drawing 7).Be aim sequence through this fragment that checks order, the amplification of Si Shi eimeria tenella virus distinguished sequence, this sequence length is 402bp, the homology 27% of the protein of its coding and Bu Shi eimeria tenella virus homology segment.(seeing sequence table 2)
In sum, degenerated primer RT-PCR method can effectively detect Eimeria Tenella virus and Si Shi eimeria tenella virus exists, and specificity is good, and cost is low.It is the special survey detection method of the first Eimeria virus of exploitation at present.
Sequence table
SEQ no.1
<110> Jilin University
<120>
<140>
<160> 1
<210> 1
<211> 407
<212> DNA
<213> Eimeria Tenella virus coat(E.tenella virus coat)
<400> 1
ACAGCTGCTG CGGTGGCTCA GGCAGGTACA GTAGTGACGA CTGATACCGG GACCACCAGG 60
CCCATAGTCG TCTACGGCAG GATGACTCCT CTCATGGCGC GCGGCGTCCG TCCGGCCGAC 120
CCTGCGATAG TGGGCGACGC CGATCGGTAC GCACGTGATC AGGAGCAGTA TAGGTTGGAC 180
TGCACCGAGC ACGTCAACTT ACTCCGCGCC CCGCTGCATG GAGCCTACAC GGAAATGGCC 240
GAGTACTACC TCCCAGCGTT AGCCAACATT TTTCGGATGT CGCGGGCGGC AGAGGAGACA 300
CGGCCAGCTC ACACATGGTT GTCTCAAGCG GCCCCATCGG TCCTCGAAAA CTATGATAAC 360
CGACACCTGC TGAAGAACGA TGCTGTGGCG CCTTTCTTTT GGATTGAA 407
SEQ no.2
<110> Jilin University
<120>
<140>
<160> 1
<210> 1
<211> 402
<212> DNA
<213> Si Shi eimeria tenella virus coat(E.stiedai virus coat)
<400> 1
ACAGCAGCTG CTGTCGCCCA CTGCGCCCCG TTAGTCTACA CTGGGCTGGG CACTAGCTGC 60
CCTAGTGTGG CATACGGGCG CCTGGCGCCG GAATATCAGC ACCAGAAACG TATGGAAGAC 120
TTTGAAACCC CCTTCCTGGA TGCTTCGACG CATGCAAAAG ACGTTGTGTC GGCGCGGACC 180
CAGCACGTCG AGATATTACG CCAAGCGTTG GAGCACTCCT TCTTCGAGTT CGGGGCTCTA 240
TACCTACCAG CCATCGCTTC GTTTTGGGGG TTCTCACCTA CGGGAGAGCG GGAACAGGCC 300
TGTCGCCAAT GGCCTTGGGA CGCTTTGGGG TCGGTGCTAG ACGATTGCCA CAACAGACAC 360
CTAATTAAGG GTGAAAGCAT AGCCCCGTTC TTCTGGATTG AG 402
Claims (1)
1. for the degenerated primer RT-PCR detection method of Eimeria coccidia virus, comprise the following steps:
1) under the condition of STE damping fluid, use phenol chloroform to carry out extracting to the coccidia material through liquid nitrogen grinding, obtain total nucleic acid, the DNA in total nucleic acid is via DNaseI digestion process, and that obtains RNA slightly carries product, is nucleic acid samples to be checked;
2) this nucleic acid samples to be checked, directly as template, uses PrimeScript Reverse Transcriptase (2680Q) ThermoScript II to carry out reverse transcription (RT); Because coccidia viral genome is dsRNA, so reverse transcription is used CF and two degenerated primers of CR to carry out (classical inverse responsive transcription only uses single downstream primer) simultaneously;
Upstream primer: CF:5 '-CBGCIGCTBYIGTVGCH-3 '
Downstream primer: CR:5 '-GTVGRCTCDATCCARAASHAD-3 '
Above CF primer has used I(xanthoglobulin), replace N(N=A/T/C/G);
Wherein annex base code: R=A/G, S=G/C, Y=C/T, V=A/G/C, H=A/C/T, D=A/G/T, B=G/C/T, N=A/G/C/T;
3) it is dsDNA that the cDNA obtaining via reverse transcription uses TaKaRa LA Taq (RRO2MQ) nucleic acid polymerase to carry out pcr amplification, agarose gel electrophoresis detected result (the single band of about 400bp size).
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| CN104962568A (en) * | 2015-06-24 | 2015-10-07 | 吉林大学 | RNA transfection system with exogenous genes expressed in Eimeria stiedai bodies |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1706966A (en) * | 2004-06-04 | 2005-12-14 | 何以丰 | Typing and quantitative gene detection method of human papillomavirus |
| CN102140555A (en) * | 2011-04-07 | 2011-08-03 | 厦门出入境检验检疫局检验检疫技术中心 | Degenerate primers for universal reverse transcription-polymerase chain reaction (RT-PCR) detection of cowpea mosaic virus and fabavirus, detection method and application thereof |
| CN102382907A (en) * | 2011-11-10 | 2012-03-21 | 中华人民共和国大榭出入境检验检疫局 | Degenerate reverse transcription-polymerase chain reaction (RT-PCR) detection reagent and kit for hantavirus group |
| CN103224999A (en) * | 2013-04-25 | 2013-07-31 | 中华人民共和国大榭出入境检验检疫局 | Degenerate primer for detecting virus belonging to virus family Arenaviruses and RT-PCR detection method |
| CN103290144A (en) * | 2013-05-29 | 2013-09-11 | 江苏省疾病预防控制中心 | Primer for detecting H7N9 subtype avian influenza virus infected by humans though RT-LAMP (reverse transcription loop-mediated isothermal amplification) and application of primer |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1706966A (en) * | 2004-06-04 | 2005-12-14 | 何以丰 | Typing and quantitative gene detection method of human papillomavirus |
| CN102140555A (en) * | 2011-04-07 | 2011-08-03 | 厦门出入境检验检疫局检验检疫技术中心 | Degenerate primers for universal reverse transcription-polymerase chain reaction (RT-PCR) detection of cowpea mosaic virus and fabavirus, detection method and application thereof |
| CN102382907A (en) * | 2011-11-10 | 2012-03-21 | 中华人民共和国大榭出入境检验检疫局 | Degenerate reverse transcription-polymerase chain reaction (RT-PCR) detection reagent and kit for hantavirus group |
| CN103224999A (en) * | 2013-04-25 | 2013-07-31 | 中华人民共和国大榭出入境检验检疫局 | Degenerate primer for detecting virus belonging to virus family Arenaviruses and RT-PCR detection method |
| CN103290144A (en) * | 2013-05-29 | 2013-09-11 | 江苏省疾病预防控制中心 | Primer for detecting H7N9 subtype avian influenza virus infected by humans though RT-LAMP (reverse transcription loop-mediated isothermal amplification) and application of primer |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962568A (en) * | 2015-06-24 | 2015-10-07 | 吉林大学 | RNA transfection system with exogenous genes expressed in Eimeria stiedai bodies |
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