CN103898237B - Degenerate primer RT-PCR detection method for Eimeria coccidia virus - Google Patents
Degenerate primer RT-PCR detection method for Eimeria coccidia virus Download PDFInfo
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Abstract
The present invention discloses a kind of degenerate primer RT-PCR detection method for Eimeria coccidia virus, provides a pair of completely new degenerate primer, expands using the degenerate primer RT-PCR that reverse transcription and touchdown PCR technology carry out specificity to Eimeria coccidia virus.Degenerate primer RT-PCR method of the present invention can effectively detect Eimeria tenella poison and Si Shi eimeria tenella virus exists, and specificity is good (electrophoretic band is single).Whether the method to be used for Eimeria coccidia viral diagnosis for the first time can fast and accurately identify in coccidia sample to be checked containing the worm strain for taking virus.
Description
Technical field
The present invention discloses a kind of degenerate primer RT-PCR detection method for Eimeria coccidia virus, provides a pair
Completely new degenerate primer carries out the degenerate primer of specificity using reverse transcription and touchdown PCR technology to Eimeria coccidia virus
RT-PCR amplification.Applied technology designs for degenerate primer, reverse transcription, touchdown PCR (TD-PCR), belongs to molecular biology inspection
Survey technology field.
Background technique
Chicken coccidiasis (Coccidiosis) is to be caused by one or more Eimeria coccidias, and endanger extremely serious
Parasitic disease.As a kind of cytozoon, eimeria tenella leads to the massive losses of global aviculture, and every year about 8,000,000,000
The loss of dollar.Diplornavirus was found first in fungi in 1948, hereafter had found dsRNA virus in protozoon again.
Protozoon virus is that one kind is present in the intracorporal virion of protozoon, is mostly that double-stranded RNA (dsRNA) is viral, spherical 20 face bodies, directly
Diameter 30-50nm has rna dependent rna polymerase activity.In terms of coccidia virus research, be reported in Eimeria Tenella,
Si Shi eimeria tenella, Nissl eimeria tenella, Bu Shi eimeria tenella, huge eimeria tenella, poison eimeria tenella with
And there are virus like particle or virus-like nucleic acid in E.acervulina.But still not about the numerous characteristics of coccidia virus at present
It is fully aware of.
In the research of eimeria tenella, it is found that the pathogenicity of coccidia with it whether carry virus that there are correlations.At present
At the early-stage for eimeria tenella virus research, a large amount of research also focuses on the Electronic Speculum observation to virus like particle.At present
Standard there is no to the detection method of coccidia virus.Traditional method is the presence of Electronic Speculum observation virus like particle, however the method
It is at high cost, and recall rate is low.Another method is detection viral nucleic acid, but since natural infection case often mixes sense
Dye, takes that viruliferous worm strain ratio is low, and the sensitivity that especially it is detected in the case where there is other biological material contamination is very
It is low, and false detection rate is high, to the no specific standards of the judgement of result.Therefore develop it is a kind of sensitive, accurate, at low cost and
To the method that all Eimeria coccidia viruses carry out versatility detection, it is necessary.
Degenerate primer PCR is because genetic code has degeneracy, and most amino acid has more than one codon
It encodes, and homologous isoprotein is often in some important sections conserved amino acid sequences with higher, so to not
Know that target gene can probably speculate by the conserved region of known multiple homologous same work genes, and is designed with this with degeneracy position
The degenerate primer of point carries out PCR amplification.Therefore degenerate primer PCR has certain versatility of homologous same work gene magnification.
The design of degenerate primer is the committed step of degenerate primer PCR success or not.The purpose nucleic acid segment for detection is obtained, it must
It must the use of the method for RT-PCR be DNA by RNA amplification.Traditional reverse transcription reaction for ssRNA using single downstream primer into
Row.It there is no degenerate primer RT-PCR detection method report for detecting Eimeria coccidia virus at present.
Summary of the invention
The purpose of the present invention is disclosing a kind of degenerate primer RT-PCR detection method for Eimeria coccidia virus, it is
Degenerate primer RT-PCR detection method, solve the problems, such as there is no at present detects eimeria tenella category viral methods, and this method is answered
With the low variation property in high conserved region domain, Eimeria coccidia virus is detected using degenerate primer, reach specificity with
Versatility combines, and provides new feasible method for detection Eimeria coccidia virus.
The following steps are included:
1) under conditions of STE buffer, the coccidia material Jing Guo liquid nitrogen grinding is stripped using phenol chloroform, is obtained
Total nucleic acid is taken, for the DNA in total nucleic acid via DNaseI digestion process, obtain RNA slightly mentions product；
2) this slightly mentions product and carries out reverse transcription (RT) directly as template, because coccidia viral genome is dsRNA,
Reverse transcription is carried out simultaneously using two degenerate primers of CF and CR；
Upstream primer: CF:5 '-CBGCIGCTBYIGTVGCH-3 '
Downstream primer: CR:5 '-GTVGRCTCDATCCARAASHAD-3 '
The above CF primer has used I(hypoxanthine), instead of 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；
It 3) the use of PCR method amplification is dsDNA, agarose gel electrophoresis detection knot via the obtained cDNA of reverse transcription
Fruit.
Since the reverse transcription of dsRNA is more difficult, by comparing a variety of reverse transcriptase using multiple companies, finally determine
PrimeScript Reverse Transcriptase (2680Q) reverse transcriptase, PCR use TaKaRa LA Taq
(RRO2MQ) nucleic acid polymerase.Various reagents used in the present invention are cheap, while can effectively expand target fragment, amplification
Product specificity is good, and electrophoresis result easily determines.
The positive effect of the present invention is: degenerate primer RT-PCR method can effectively detect Eimeria tenella poison and
Si Shi eimeria tenella virus exists, and specificity is good, and electrophoretic band is single；Versatility is good, can be to Eimeria globidiosis
Poison carries out general detection；High sensitivity, a small amount of purpose nucleic acid: 0.05ng/ μ l can obtain testing result.To be used for Amy for the first time
Whether you belong to the method for coccidia viral diagnosis, can fast and accurately identify in coccidia sample to be checked containing the worm strain for taking virus.
Detailed description of the invention
Attached drawing 1 is upstream primer CF, downstream primer CR design drawing.
Attached drawing 2 is that template ribonucleic acid concentration gradient is preferred.
Attached drawing 3 is that primer concentration grads are preferred.
Attached drawing 4 is that TaKaRa La Taq enzyme concentration gradient is preferred.
Attached drawing 5 is that dNTP concentration gradient is preferred.
The method that attached drawing 6 is degenerate primer RT-PCR detects Eimeria tenella poison.
The method that attached drawing 7 is degenerate primer RT-PCR detects Si Shi eimeria tenella virus.
Specific embodiment
To further illustrate application of the present invention in Eimeria coccidia viral diagnosis, said with the following example
Bright but of the invention application is not limited to embodiment.
Embodiment 1
The design and synthesis of primer
One, material: molecular biology software MEGA4, DNAMAN and NCBI Internet resources.
Two, method and result:
Sequence obtains: by Bu Shi eimeria tenella virus (E. brunetti RNA virus 1) genome analysis,
Coat gene is chosen as target sequence, and obtains this gene order, accession number from GenBank public database are as follows: NC_
002701.The use of coat protein sequence is to address inquires to sequence, BlustP comparison is carried out on NCBI, obtains 330 to 346
(IALGTAAAVAHCDPLVP), 435 to 446 (PFFWIEPTTVLP) two highly conserved protein domains, remaining is referred to
The nucleic acid sequence of the corresponding position of sequence is individually downloaded, 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, Aspergillus foetidus slow 1 seven mycoviruses of virus, and
The sequence of 1-4 Leishmania virus of Leishmania RNA virus.
Design of primers: the sequence obtained chooses appropriate area and designs primer after cluster is compared.(see attached drawing 1).
Degeneracy site uses degeneracy base to use I(hypoxanthine when degeneracy site is N).
Primer synthesis: by biotech firm synthetic primer CF, CR.
Upstream primer: CF:5 '-CBGCIGCTBYIGTVGCH-3 '
Downstream primer: CR:5 '-GTVGRCTCDATCCARAASHAD-3 '
Embodiment 2
The preparation of nucleic acid samples
One, material: Eimeria Tenella, 2 × STE buffer (Ph8.0), water-saturated phenol, chloroform, β-sulfydryl second
Alcohol, 10%SDS, 70% ethyl alcohol, isopropanol, supercentrifuge.
Two, method and result:
1) Eimeria Tenella egg capsule is broken: will about 107Coccidia to be checked carries out liquid nitrogen grinding, be dissolved in after grinding 1ml2 ×
STE(pH8.0) buffer.
2) protein extracts: 30 μ l beta -mercaptoethanols, 0.6ml water-saturated phenol, 0.4ml being added in above-mentioned coccidia solution
Chloroform, 140 μ l10%SDS.Sufficiently homogenate 5 minutes.Under the conditions of 4 DEG C, 12000r/min is centrifuged 5 minutes, takes supernatant.
3) total nucleic acid obtains: supernatant and isometric cold isopropanol mix, under the conditions of -80 DEG C stand 10 minutes, 4 DEG C,
15000r/min is centrifuged 15 minutes, and precipitating is cleaned with 70% cold alcohol solution, and under the conditions of 4 DEG C, 15000r/min is centrifuged 15 minutes.
200 μ lddH are dissolved in after precipitating is dry2O。
4) DNA digests: taking above-mentioned 172 μ l of total nucleic acid solution, DNaseI(TaKaRa, 2270A is added) 8 μ l, 10*
DNase I Buffer 20μl.37 DEG C digest 30 minutes, and 80 DEG C inactivate enzyme in 5 minutes.
5) RNA is obtained: above-mentioned solution is taken, isometric cold isopropanol is added, under the conditions of -80 DEG C stand 10 minutes, 4 DEG C,
15000r/min is centrifuged 15 minutes, and precipitating is cleaned with 70% cold alcohol solution, and under the conditions of 4 DEG C, 15000r/min is centrifuged 15 minutes.
100 μ lddH are dissolved in after precipitating is dry2O.15000r/min is centrifuged 15 minutes again, takes supernatant, abandons precipitating.
Embodiment 3
Degenerate primer RT-PCR detects Eimeria tenella poison
One, material: Eimeria Tenella RNA crude extract, PrimeScript Reverse Transcriptase
(2680Q) Reverse Transcriptase Reagents 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 synthesized: following mixed liquor is prepared in Microtube:
99 DEG C are denaturalized 1 minute, are transferred to -40 DEG C of anxious jellies immediately.
The amount of template ribonucleic acid carries out gradient experiment, is divided into six gradients, respectively 50ng/ μ l, 5ng/ μ l, 0.5ng/ μ l,
0.05ng/μl, 0.005ng/μl, 0.0005ng/μl.When template ribonucleic acid concentration is that 0.05ng/ μ l, PCR result still can be clear
Clear interpretation.(see attached drawing 2)
Following component is added in above-mentioned reaction solution:
30 DEG C are reacted 10 minutes, and 42 DEG C are reacted 45 minutes, and 70 DEG C are denaturalized 15 minutes, ice bath.
2) Touchdown-PCR (TD-PCR) amplification reaction system:
TD-PCR amplified reaction program are as follows:
1、 94℃ 5min
2、 95℃ 1min
3,65 DEG C → 56 DEG C 1min (each circulation is landed 1 DEG C)
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) Touchdown-PCR condition is groped
(1) gradient is arranged in final concentration of the upstream and downstream primer (CF and CR) in system are as follows: 1mM, 0.6mM, 0.2mM.This area
Between primer concentration be 1mM and 0.6mM, target fragment obtains effective amplification.Comprehensively consider sensitivity and cost, chooses
0.6mM is primer final concentration.(see attached drawing 3;The concentration of M:Marker D 2000, Lane1-6:RNA template is 50ng/ μ l,
5ng/μl, 0.5ng/μl, 0.05ng/μl, 0.005ng/μl, 0.0005ng/μl.When template ribonucleic acid concentration is 0.05ng/ μ
L, PCR result still can clear interpretations.)
(2) gradient is arranged in final concentration of the TaKaRa La Taq in system are as follows: 0.1U/ μ l, 0.05U/ μ l, 0.025U/ μ
l,0.0125U/μl,0.00625U/μl,0.003125U/μl.Within the scope of the final concentration of 0.1U/ μ l to 0.00625U/ μ l, mesh
Segment all obtain effective amplification, and using 0.05U/ μ l amplification efficiency as highest, selection 0.05U/ μ l is TaKaRa La Taq
Final concentration.(see attached drawing 4)
(3) gradient is arranged in final concentration of the dNTP in system are as follows: 0.4mM, 0.2mM, 0.1mM, 0.05mM, 0.025mM,
0.0125mM.Target fragment all obtains effective amplification within this range, but in 0.025mM concentration and lower concentration condition
Under, purpose band is obviously dimmed, considering cost and sensitivity, therefore selects 0.05mM dNTP for optium concentration.(see attached drawing
5)
(4) it detects: using above-mentioned groped condition to tender detection Eimeria tenella poison.RT-PCR reaction makes
It is 0.05ng/ μ l with template ribonucleic acid concentration.System program are as follows:
1st-Stand cDNA synthesis: following mixed liquor is prepared in Microtube:
99 DEG C are denaturalized 1 minute, are transferred to -40 DEG C of anxious jellies immediately.
Following component is added in above-mentioned reaction solution:
30 DEG C are reacted 10 minutes, and 42 DEG C are reacted 45 minutes, and 70 DEG C are denaturalized 15 minutes, ice bath.
Touchdown-PCR (TD-PCR) amplification reaction system:
TD-PCR amplified reaction program are as follows:
1、 94℃ 5min
2、 95℃ 1min
3,65 DEG C → 56 DEG C 1min (each circulation is landed 1 DEG C)
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: preparing the TAE Ago-Gel of 1% concentration, uses DL2000 DNA
Marker, ethidium bromide staining observe the purpose band of 400bp size.(see attached drawing 6).It is purpose sequence that the segment, which is sequenced,
The amplification of Eimeria tenella poison distinguished sequence, this sequence length are 408bp, protein and Bu Shi the Amy that of coding
The homology 32% of coccidia virus homology segment, and can be identified and belong to Totivirus-coat superfamily.(see sequence table 1)
Embodiment 4
Degenerate primer RT-PCR method detects Si Shi eimeria tenella virus
One, material: Si Shi eimeria tenella RNA slightly mentions product, PrimeScript Reverse Transcriptase
(2680Q) Reverse Transcriptase Reagents kit, TaKaRa LA Taq (RRO2MQ) nucleic acid polymerization enzyme reagent kit, agarose, ethidium bromide
Solution, Biorad PCR instrument.
Two, methods and result:
1) RT-TD-PCR: using Si Shi eimeria tenella RNA crude extract as template, the presence of viral specific band is detected.
RT-PCR reaction is 0.05ng/ μ l using template ribonucleic acid concentration.
System program are as follows:
1st-Stand cDNA synthesis: following mixed liquor is prepared in Microtube:
99 DEG C are denaturalized 1 minute, are transferred to -40 DEG C of anxious jellies immediately.
Following component is added in above-mentioned reaction solution:
30 DEG C are reacted 10 minutes, and 42 DEG C are reacted 45 minutes, and 70 DEG C are denaturalized 15 minutes, ice bath.
Touchdown-PCR (TD-PCR) amplification reaction system:
TD-PCR amplified reaction program are as follows:
1、 94℃ 5min
2、 95℃ 1min
3,65 DEG C → 56 DEG C 1min (each circulation is landed 1 DEG C)
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: preparing the TAE Ago-Gel of 1% concentration, uses DL2000 DNA
Marker, ethidium bromide staining observe the purpose band of 400bp size.(see attached drawing 7).It is purpose sequence that the segment, which is sequenced,
The amplification of Si Shi eimeria tenella virus distinguished sequence, this sequence length are 402bp, protein and Bu Shi the Amy that of coding
The homology 27% of coccidia virus homology segment.(see sequence table 2)
In conclusion degenerate primer RT-PCR method can effectively detect Eimeria tenella poison and Si Shi Amy that ball
Parasitosis poison exists, and specificity is good, at low cost.It is the dedicated survey detection method of the first Eimeria virus developed at present.
SEQ no.1
<110>Jilin University
<120>
<140>
<160> 1
<210> 1
<211> 407
<212> DNA
<213>Eimeria tenella poison 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. a kind of degenerate primer RT-PCR detection method of the Eimeria coccidia virus for non-disease diagnosing and treating purpose,
The following steps are included:
1) under conditions of STE buffer, the coccidia material Jing Guo liquid nitrogen grinding is stripped using phenol chloroform, is obtained total
Nucleic acid, the DNA in total nucleic acid is via DNaseI digestion process, and obtain RNA slightly mentions product, nucleic acid samples as to be checked；
2) nucleic acid samples to be checked are inverted directly as template using PrimeScript Reverse Transcriptase
It records enzyme and carries out reverse transcription (RT)；Because coccidia viral genome is dsRNA, reverse transcription uses two degenerate primers of CF and CR
It carries out simultaneously；
Upstream primer: CF:5 '-CBGCIGCTBYIGTVGCH-3 '；
Downstream primer: CR:5 '-GTVGRCTCDATCCARAASHAD-3 '；
The above CF primer has used I(hypoxanthine), instead of 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) carrying out PCR amplification using TaKaRa LA Taq nucleic acid polymerase via the obtained cDNA of reverse transcription is dsDNA, fine jade
Sepharose electrophoresis detection result.
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| 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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2014
- 2014-01-09 CN CN201410009964.2A patent/CN103898237B/en not_active Expired - Fee Related
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 |
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