CN107034313A - The RT PCR detection primers and RT PCR detection methods of a pair of Sai Nika paddy virus - Google Patents
The RT PCR detection primers and RT PCR detection methods of a pair of Sai Nika paddy virus Download PDFInfo
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- CN107034313A CN107034313A CN201710327027.5A CN201710327027A CN107034313A CN 107034313 A CN107034313 A CN 107034313A CN 201710327027 A CN201710327027 A CN 201710327027A CN 107034313 A CN107034313 A CN 107034313A
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Abstract
The present invention relates to the detection technique field of swine disease poison, and in particular to the RT PCR detection primers and detection method of a pair of Sai Nika paddy virus.The nucleotide sequence of the primer such as SEQ ID NO：1 and SEQ ID NO：Shown in 2；The RT PCR detection methods include：Design the primer described in synthesis；Sample rna is extracted, is saved backup；Using the sample rna of gained as template ribonucleic acid, RT PCR reactions are carried out using the primer；Interpretation testing result.The present invention program enriches the technological means of the Viral diagnosis, and specificity and sensitivity are good, and detection speed is fast, with good application value.
Description
Technical field
The present invention relates to the detection technique field of swine disease poison, and in particular to the RT-PCR detections of a pair of Sai Nika paddy viruses are drawn
Thing and RT-PCR detection method.
Background technology
Sai Nika paddy virus (Seneca Valley virus, SVV) belongs to Picornaviridae
(picornaviridae) Senecavirus Tobamovirus, is single strand plus RNA virus, full-length genome about 7.28kb, comprising 5 '
UTR, 3 ' UTR and in single ORF between the two, there are VPg albumen covalent bonds at 5 ' ends, and there are polyA tails at 3 ' ends.SVV is initial
It is to be found in PER.C6 cell cultures for 2002, blister disease occur in the swinery in the slaughterhouse of the U.S. one in 2007 faces
Bed symptom, exclude after testing it is various can cause the cause of disease of blister, it is final to assert that SVV is original of causing a disease.But since then have no other
The report of morbidity.
Until 2014~2015 years, Brazil reports it and blister disease, the disease is broken out in the swinery for being related to six states
There are three principal characters：(1) there is blister and polymerism erosion on sow nose and coronary band；Newborn piglet in (2) four ages in days
Acute death is up to 30%~70%；(3) self limiting Burst duration about 1~2 week.Official's diagnosis finds the water of mandatory report
Blister disease is feminine gender, and only SVV is positive, sequence analysis find its be 87.6% with SVV-001 plant of nucleotide similarity~
98.5%, amino acid similarity is 95%~99.4%, and it is the pathogenic original of this Brazilian blister illness outbreak to illustrate SVV, and
Reported for work first outside North America for SVV.The U.S. also there occurs a lot of zonal infection in 2014~2015 years.In recent years it is existing
Report display also occurs in that SVV infection within Chinese territory, but there is presently no fast and effectively detection means accordingly, causes one
Denier has Epidemic outbreak of disease, it is impossible to timely make a definite diagnosis cause of disease, so that prophylactico-therapeutic measures can not timely and effectively be taken, brings huge to aquaculture
Big loss.
It can be seen that, prior art problem is urgently to be resolved hurrily.
The content of the invention
In consideration of it, being necessary to provide a pair of viral RT-PCR detection primers of Sai Nika paddy and RT-PCR regarding to the issue above
Detection method, the primer specificity is good, sensitivity is high and amplified fragments are small, and whole RT-PCR detection process can be complete in 2h
Into Buddhist nun's card paddy virus of leaving the boundary can specifically being detected, available for test in laboratory and clinical assistant diagnosis, with important reality meaning
Justice.
The object of the invention is achieved through the following technical solutions：
The RT-PCR detection primers of a pair of Sai Nika paddy virus, its nucleotides sequence is classified as：
SVVUP：5’-GATGTATAAACCTTCTC-3’(SEQIDNO：1)
SVVDN：5’-ATTGTAAGTGCCAAGAG-3’(SEQIDNO：2).
A kind of RT-PCR detection method of Sai Nika paddy virus, step includes：
(1), the design synthesis primer (SEQIDNO：1 and SEQIDNO：2)；
(2) sample rna, is extracted, -20~-80 DEG C of freezen protectives are standby；
(3), by the sample rna of gained in (2) as template ribonucleic acid, RT-PCR is carried out using the primer of gained in (1) anti-
Should；
(4), the RT-PCR products obtained by electroresis appraisal (3), and judging result.
Further, storage temperature is -70 DEG C in the sample rna that the step (2) is extracted.
Further, in the RT-PCR reactions, 12.5 μ l reaction systems are：
2×1StepBuffer：6.25μl；PrimeScript1StepEnzymeMix：0.5μl；SVVUP(10μmol/l)
(SEQIDNO：1)：0.25μl；SVVDN(10μmol/l)(SEQIDNO：2)：0.25μl；Template ribonucleic acid：0.5μl；Nuclease-free water
Complement to 12.5 μ l.
Further, in the RT-PCR reactions, response procedures are：
4 DEG C or 16 DEG C are finally cooled to, 4 degree are typically cooled to, the interim preservation of sample also can be at 4 degree, if but being temporarily stored into
In PCR instrument, 4 DEG C can condensing water droplet, it is bad to PCR instrument, be now set to 16 degree of effects for preserving protection PCR instrument, it is and 16 degree short
Shi Baocun influences little to PCR primer.
Further, in the RT-PCR reactions, response procedures are：
Finally it is cooled to 4 DEG C.
Further, the electroresis appraisal in the step (4) is identified for 1% agarose gel electrophoresis.
Beneficial effect of the present invention：
Primer specificity of the present invention is good, sensitivity is high and amplified fragments are small, and whole RT-PCR detection process can be in 2h
Complete, Buddhist nun's card paddy virus of leaving the boundary can be specifically detected, available for test in laboratory and clinical assistant diagnosis, with important reality
Meaning.
The invention provides a kind of method of detection Sai Nika paddy virus, the technological means of the Viral diagnosis is enriched, together
When this method specificity and sensitivity it is good, detection speed is fast, with good application value.
Brief description of the drawings
Fig. 1 Sai Nika paddy virus RT-PCR method specific test results:M is DNAMarker2000, and 1 is positive control,
2 be negative control, and 3~7 be respectively aftosa, swine fever, pseudo- mad dog, blue ear, annulus vaccine sample.
Fig. 2 Sai Nika paddy virus RT-PCR method sensitivity test results:M is DNAMarker2000, and 1 is negative control,
2 be positive control, and 3~7 be respectively the sample of 10 times of gradient dilutions of positive control, and its dilution gradient is followed successively by：10-1、10-2、
10-3、10-4、10-5。
The Sai Nika paddy Viral diagnosis results of Fig. 3 clinical samples：M is DNAMarker2000, and 1 is positive control, and 2 be the moon
Property control, 3 and 4 be clinical sample.
Embodiment
In order to which problem solved by the invention, the technical scheme used and the effect reached is better described, now tie
Close specific embodiment and related data is expanded on further.It should be noted that present invention is implemented including but not limited to following
Example and combinations thereof embodiment.
Embodiment one
1st, design of primers：It is domestic at that time not set up common also because Sai Nika paddy virus is newfound Causative virus
PCR detection techniques, the upper gene orders that can be found of GenBank are also extremely limited, and we are by only Sai Nika paddy on GenBank
The gene order of viral SVV001 plants and 11-55910-3 plants is compared, and by its a variety of viral gene sequence with same Viraceae
Row are compared, and find out its conservative region, select SVV001 plants of 1336~1662 genetic fragments as target, then
BLAST further determines that its conservative.Pair for amplification mesh is designed on the conserved sequence by Oligo6.0 primer-design softwares
Band be 327bp primer SVVUP/SVVDN, primer sequence is as follows：
SVVUP：5’-GATGTATAAACCTTCTC-3’(SEQIDNO：1)
SVVDN：5’-ATTGTAAGTGCCAAGAG-3’(SEQIDNO：2).
2nd, the extraction of sample rna：Using AxyGen DNA/RNA Mini Kits, extracted according to operation instructions
Template ribonucleic acid, -70 DEG C save backup.
3rd, RT-PCR reacts：Using TaKaRa One step RT-PCR kit, according to operation instructions with step (2)
The sample rna extracted carries out RT-PCR reactions as template ribonucleic acid.12.5 μ l reaction systems are as follows：
| Sequence number | Component | Volume (μ l) |
| 1 | 2×1StepBuffer | 6.25 |
| 2 | PrimeScript1StepEnzymeMix | 0.5 |
| 3 | SVVUP(10μM)(SEQIDNO：1) | 0.25 |
| 4 | SVVDN(10μM)(SEQIDNO：2) | 0.25 |
| 5 | Template ribonucleic acid | 0.5 |
| 6 | Nuclease-free water | Complement to 12.5 |
Optimized response procedures are as follows：Reverse transcription：50 DEG C, 30min；Pre-degeneration：95 DEG C, 2min；(denaturation：94 DEG C,
30s；Annealing：56 DEG C, 30s；Extension：72 DEG C, 45s) 30 circulations of amplification；After extend：72 DEG C, 10min；Finally it is cooled to 4 DEG C.
4th, the judgement of testing result：RT-PCR products are identified through 1% agarose gel electrophoresis, are if size can be amplified
327bp single goal band, then judge the sample as Sai Nika paddy virus-positives.
The specific assay of embodiment two
According to the RT-PCR reaction systems and response procedures described in embodiment one, sample form RNA is changed into a mouthful hoof respectively
Epidemic disease, swine fever, pseudo- mad dog, blue ear, annulus vaccine sample, the Sai Nika paddy disease with this test in laboratory positive and through sequencing identification
Malicious positive (the viral pathological material of disease RNA sample that CH-01-2015 plants (KT321458) is named as after i.e.) is positive control, is entered
Row RT-PCR is reacted, and RT-PCR products are identified through 1% agarose gel electrophoresis, and electrophoresis result is as shown in figure 1, result shows only positive
Property the single band of control swimming lane 327bp place's appearance, illustrate that this method specificity is good.
The sensitivity test of embodiment three
According to the RT-PCR reaction systems and response procedures described in embodiment one, with the positive control RNA in embodiment two
10 are pressed as positive control, and by positive RNA-1、10-2、10-3、10-4、10-510 times of gradient dilutions are carried out, to distill
Water is negative control, carries out RT-PCR reactions, and RT-PCR products are identified through 1% agarose gel electrophoresis, electrophoresis result such as Fig. 2 institutes
Show.As a result dilution 100 (10 is shown-2) times band is still more apparent, dilution 1000 (10-3) still have unobvious but macroscopic again
Band, shows that this method sensitivity is higher.
Example IV clinical sample is detected
RT-PCR reactions are carried out according to the RT-PCR reaction systems described in embodiment one and response procedures, with embodiment two
Positive control RNA as positive control, using distilled water as negative control, generation Sai Nika paddy virosis doubtful to two is faced
Bed sample is detected that the extraction of template ribonucleic acid is with reference to embodiment one.RT-PCR products are identified through 1% agarose gel electrophoresis, electric
Result of swimming is as shown in Figure 3.As a result yin and yang attribute control establishment is shown, two samples are SVV positive.
Embodiment described above only expresses the several embodiments of the present invention, and it describes more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
<110>Guangdong Wen'S Foodstuffs Group Co., Ltd.
<120>The RT-PCR detection primers and RT-PCR detection method of a pair of Sai Nika paddy virus
<160> 2
<170> Oligo 6.0
<210> 1
<211> 17
<212> DNA
<213>Artificial sequence
<400> 1
gatgtataaa ccttctc 17
<210> 2
<211> 17
<212> DNA
<213>Artificial sequence
<400> 2
attgtaagtg ccaagag 17
Claims (7)
1. the RT-PCR detection primers of a pair of Sai Nika paddy virus, it is characterised in that the nucleotide sequence of the primer such as SEQ
ID NO：1 and SEQ ID NO：Shown in 2.
2. a kind of RT-PCR detection method of Sai Nika paddy virus, it is characterised in that operating procedure includes：
(1), the primer described in design synthesis claim 1；
(2) sample rna, is extracted, -20~-80 DEG C of freezen protectives are standby；
(3) sample rna of gained in (2), is subjected to RT-PCR reactions as template ribonucleic acid using the primer of gained in (1)；
(4), the RT-PCR products obtained by electroresis appraisal (3), and judging result.
3. detection method according to claim 2, it is characterised in that temperature is preserved in the sample rna that the step (2) is extracted
Spend for -70 DEG C.
4. detection method according to claim 2, it is characterised in that the RT-PCR reactions in the step (3), reactant
It is to be：
2×1 Step Buffer：6.25μl；
PrimeScript 1 Step Enzyme Mix：0.5μl；
SVVUP(10μmol/l)(SEQ ID NO：1)：0.25μl；
SVVDN(10μmol/l)(SEQ ID NO：2)：0.25μl；
Template ribonucleic acid：0.5μl；
Nuclease-free water complements to 12.5 μ l.
5. detection method according to claim 2, it is characterised in that the RT-PCR reactions in the step (3), reaction interval
Sequence is：
Reverse transcription：50 DEG C, 30min；
Pre-degeneration：95 DEG C, 2~5min；
After extend：72 DEG C, 10min；
Finally it is cooled to 4 DEG C or 16 DEG C.
6. detection method according to claim 5, it is characterised in that the response procedures are：Reverse transcription：50 DEG C,
30min；
Pre-degeneration：95 DEG C, 2min；
After extend：72 DEG C, 10min；
Finally it is cooled to 4 DEG C.
7. detection method according to claim 2, it is characterised in that the electroresis appraisal in the step (4) is 1% agar
Sugared gel electrophoresis identification.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107513524A (en) * | 2017-09-30 | 2017-12-26 | 中牧实业股份有限公司 | One plant of pig Sai Neijia paddy virus stain and its application |
| CN107937617A (en) * | 2017-12-28 | 2018-04-20 | 广州维佰生物科技有限公司 | Detect the RT LAMP primer compositions thing and its kit and method of Sai Neijia paddy viruses |
| CN107937618A (en) * | 2017-12-29 | 2018-04-20 | 中国检验检疫科学研究院 | The droplet numeral RT PCR detection primers of A types Senecan virus and probe and its application |
| CN108085323A (en) * | 2018-01-12 | 2018-05-29 | 金宇保灵生物药品有限公司 | The whole genome sequence and its amplimer of Sai Nika paddy viruses SVV/CH/NM/2016 |
| CN108192898A (en) * | 2018-01-12 | 2018-06-22 | 金宇保灵生物药品有限公司 | The whole genome sequence and its amplimer of Sai Nika paddy viruses SVV/CH/ZZ/2016 |
| CN108300811A (en) * | 2018-04-18 | 2018-07-20 | 滕建芬 | The RT-PCR detection kit and its detection method of one boar Sai Nika paddy viruses |
| CN108384894A (en) * | 2018-05-03 | 2018-08-10 | 中国农业科学院兰州兽医研究所 | Taqman real-time fluorescence quantitative RT-PCRs kit for detecting Sai Nika paddy viruses and its application |
| CN108467904A (en) * | 2018-05-24 | 2018-08-31 | 华南农业大学 | Detect RT-LAMP primer sets, kit and the application of Sai Nika paddy viruses |
| CN108707697A (en) * | 2018-07-10 | 2018-10-26 | 南京农业大学 | The LAMP detection primer pair and detection method of Sai Nika paddy viruses |
| CN110093320A (en) * | 2019-03-27 | 2019-08-06 | 华南农业大学 | GD-SVA-2018 plants of pig Senecan virus and its application |
| CN110257554A (en) * | 2018-03-12 | 2019-09-20 | 金宇保灵生物药品有限公司 | The real-time fluorescence quantitative PCR detection kit and its primer special and TaqMan probe of Seneca Valley virus |
| CN111394367A (en) * | 2020-03-24 | 2020-07-10 | 中国农业科学院兰州兽医研究所 | Selcarinovirus recombinant nucleic acid, recombinant vaccine strain, and preparation methods and applications thereof |
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| CN107513524A (en) * | 2017-09-30 | 2017-12-26 | 中牧实业股份有限公司 | One plant of pig Sai Neijia paddy virus stain and its application |
| CN107513524B (en) * | 2017-09-30 | 2021-02-09 | 中牧实业股份有限公司 | Swine Saxifraga Valley virus strain and application thereof |
| CN107937617A (en) * | 2017-12-28 | 2018-04-20 | 广州维佰生物科技有限公司 | Detect the RT LAMP primer compositions thing and its kit and method of Sai Neijia paddy viruses |
| CN107937618A (en) * | 2017-12-29 | 2018-04-20 | 中国检验检疫科学研究院 | The droplet numeral RT PCR detection primers of A types Senecan virus and probe and its application |
| CN108085323A (en) * | 2018-01-12 | 2018-05-29 | 金宇保灵生物药品有限公司 | The whole genome sequence and its amplimer of Sai Nika paddy viruses SVV/CH/NM/2016 |
| CN108192898A (en) * | 2018-01-12 | 2018-06-22 | 金宇保灵生物药品有限公司 | The whole genome sequence and its amplimer of Sai Nika paddy viruses SVV/CH/ZZ/2016 |
| CN110257554A (en) * | 2018-03-12 | 2019-09-20 | 金宇保灵生物药品有限公司 | The real-time fluorescence quantitative PCR detection kit and its primer special and TaqMan probe of Seneca Valley virus |
| CN108300811A (en) * | 2018-04-18 | 2018-07-20 | 滕建芬 | The RT-PCR detection kit and its detection method of one boar Sai Nika paddy viruses |
| CN108384894A (en) * | 2018-05-03 | 2018-08-10 | 中国农业科学院兰州兽医研究所 | Taqman real-time fluorescence quantitative RT-PCRs kit for detecting Sai Nika paddy viruses and its application |
| CN108467904A (en) * | 2018-05-24 | 2018-08-31 | 华南农业大学 | Detect RT-LAMP primer sets, kit and the application of Sai Nika paddy viruses |
| CN108707697A (en) * | 2018-07-10 | 2018-10-26 | 南京农业大学 | The LAMP detection primer pair and detection method of Sai Nika paddy viruses |
| CN110093320A (en) * | 2019-03-27 | 2019-08-06 | 华南农业大学 | GD-SVA-2018 plants of pig Senecan virus and its application |
| CN111394367A (en) * | 2020-03-24 | 2020-07-10 | 中国农业科学院兰州兽医研究所 | Selcarinovirus recombinant nucleic acid, recombinant vaccine strain, and preparation methods and applications thereof |
| CN111394367B (en) * | 2020-03-24 | 2021-05-14 | 中国农业科学院兰州兽医研究所 | Selcarinovirus recombinant nucleic acid, recombinant vaccine strain, and preparation methods and applications thereof |
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Address after: 527499 9 East Causeway North Road, Xinxing County new town, Yunfu, Guangdong Applicant after: Winson food group Limited by Share Ltd Address before: 527499 9 East Causeway North Road, Xinxing County new town, Yunfu, Guangdong Applicant before: Guangdong Wens Foodstuff Group Co., Ltd. |
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Application publication date: 20170811 |