CN107937617A - Detect the RT LAMP primer compositions thing and its kit and method of Sai Neijia paddy viruses - Google Patents
Detect the RT LAMP primer compositions thing and its kit and method of Sai Neijia paddy viruses Download PDFInfo
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Abstract
The invention discloses a kind of the RT LAMP primer compositions thing and its kit and method of detection Sai Neijia paddy viruses；Aim to provide a kind of high sensitivity, the SVA detection primers and method of high specificity, its technical solution include being made of positive inner primer SVA FIP, reverse inner primer SVA BIP, positive outer primer SVA F3, reverse outer primer SVA B3, forward direction ring primer SVA LF and reverse ring primer SVA LB；Detection method is：1) RNA of virus is extracted in sample；2) using the RNA of extraction as template, utilize claim 1) described in SVA RT LAMP primer composition things, establish RT LAMP amplification systems, ring mediated isothermal amplification carried out under 60 DEG C of 65 DEG C of constant temperatures；3) detected using deaou 308c constant-temperature fluorescence detectors；Belong to technical field of biological.
Description
Technical field
The invention discloses a kind of Primer composition, is a kind of RT- of detection Sai Neijia paddy viral (SVA) specifically
LAMP primer composition thing, the invention also discloses the kit and method of detection Sai Neijia paddy virus；Belong to Measurement for Biotechnique
Field.
Background technology
Sai Neijia paddy virus (SVA) is a kind of RNA virus newly going out, can causing pig bubble, belongs to micro ribonucleic acid disease
Malicious section, Sai Neijia paddy Tobamovirus, full-length genome about 7.2Kb.Clinically occur and aftosa, vesiculovirus with hog snout mirror and hoof
Bubble that stomatitis, swine pox are difficult to differentiate between, symptom of festering；Piglet is also with symptoms such as spiritual depressed, drowsiness, diarrhea.The disease
Separated first on pig in 1988, after be used for always study tumor disease.SVA diseases were just identified as until 2007
Poison.Occur in Brazil and wide-scale distribution just draws attention.PCR detections Schweineseuche, pig blisters, vesicular stomatitis and blister
Property rash be feminine gender, and SVA for the positive, it is thus regarded that SVA can cause primary blister disease.The disease does not cause a disease pig
Property, but piglet can be caused dead.Sai Neijia paddy virus causes live pig salivation symptom to continue to grow very much one period according to statistics.SVA
Antibody is detected with pig, mouse and ox, and not yet have been reported that confirms that SVA can infect people and trigger clinical symptoms at present
(Poirier et al.,2013)。
Because be clinically difficult to aftosa, vesicular stomatitis, swine pox distinguish, laboratory rely primarily on RT-PCR,
The technology for detection such as ELISA, but operating procedure is complicated, the instrument and equipment of dependence costly.So need to establish a kind of new, fast
Fast, practical, sensitive detection technique.
The content of the invention
In view of the above-mentioned problems, first purpose of the present invention is to provide a kind of RT-LAMP of detection Sai Neijia paddy viruses and draws
Compositions；Second object of the present invention is to provide a kind of kit of the detection Sai Neijia paddy viruses of high sensitivity, this hair
The 3rd bright purpose is the method for detecting Sai Neijia paddy viruses.
For this reason, first technical solution provided by the invention is such：
A kind of RT-LAMP Primer compositions of detection Sai Neijia paddy viruses, including by positive inner primer SVA-FIP, reversely
Inner primer SVA-BIP, positive outer primer SVA-F3, reverse outer primer SVA-B3, forward direction ring primer SVA-LF and reverse ring primer
SVA-LB is formed；Primer SVA-F3 nucleotide sequences such as SEQ ID NO：Shown in 1；
Primer SVA-B3 nucleotide sequences such as SEQ ID NO：Shown in 2；
Primer SVA-FIP nucleotide sequences such as SEQ ID NO：Shown in 3；
Primer SVA-BIP nucleotide sequences such as SEQ ID NO：Shown in 4；
Primer SVA-LF nucleotide sequences such as SEQ ID NO：Shown in 5；
Primer SVA-LB nucleotide sequences such as SEQ ID NO：Shown in 6.
Second technical solution of the present invention is to provide the RT-LAMP detection reagent box of SVA a kind of, and the kit is containing above-mentioned
RT-LAMP Primer compositions.
Latter solution provided by the invention is a kind of method of detection Sai Neijia paddy viruses, is comprised the following steps：
1) RNA of virus is extracted from sample；
2) take the RNA that step 1) is extracted to be added in RT-LAMP reaction solutions to mix, amplification system is established, at 60 DEG C -65 DEG C
Ring mediated isothermal amplification is carried out under constant temperature；
3) in the amplified production for obtaining fluorescent dye addition step 2), examined using deaou-308c constant-temperature fluorescence detectors
Survey.
Further, a kind of method of above-mentioned detection Sai Neijia paddy virus, the RT-LAMP reaction solutions are：Cumulative volume
For 25 μ L, 2.5 μ L of reaction buffer；MgSO41.5μL；dNTP Mix 10mM 3.5μL；Primer mix 1μL；4 μ of glycine betaine
L；RNA 2.0μL；1 μ L of Bst 2.0DNA polymerases；0.5 μ L of reverse transcriptase；0.5 μ L of fluorescent dye.Further, above-mentioned one
The method of kind detection Sai Neijia paddy viruses, the MgSO4Concentration is 100mM, and dNTP Mix concentration is 10mM；Beet alkali concn
For 5M；Reverse transcriptase concentrations are 1U/ μ L；Fluorescent dye concentration is 5mM.
Further, the method for above-mentioned a kind of detection Sai Neijia paddy virus, the ring mediated isothermal amplification program are 63
DEG C amplification 1h.
Compared with prior art, technical solution provided by the invention has following technological merit：
1st, technical solution provided by the invention uses ring mediated isothermal amplification (Loop-mediated isothermal
Ampli-fication, RT-LAMP) technology, dependent on can identify on target sequence that the primer of 6 specific regions and a kind of tool solve
Revolve function and make the Bst archaeal dna polymerases that target sequence is in ring mediated isothermal amplification, under isothermal conditions can efficiently, quickly, specifically
Ground expands target sequence.
2nd, technical solution provided by the invention uses LAMP to expand mode as ring mediated isothermal amplification, and amplification efficiency is high, by
What it is in LAMP identifications is 6 specific sites on nucleic acid, so its high specificity.
3rd, technical solution LAMP provided by the invention only only needs water-bath i.e. in 60 DEG C of -65 DEG C of progress constant-temperature amplifications, process
Can, it is not required that special instrument, therefore it is very easy；And since LAMP is constant-temperature amplification, it is not necessary to reverse transcription step and PCR
The time of instrument heating and cooling, so proliferation time is short.
4th, after adding fluorescent material in technical solution LAMP reactions provided by the invention, when synthesizing substantial amounts of DNA, in fluorescence
It can be seen that amplification curve, amplification procedure is very clear, and drastically increases sensitivity on constant-temperature amplification instrument.
5th, Technical Design specific primer provided by the invention, the specificity of more virus verification the method, to RT-
LAMP technology detected value is compared with RT-PCR detected values, is examined its repeatability and stability, is established suitable for existing
The quick determination method of field detecting Sai Neijia paddy viruses.
Brief description of the drawings
Sepharose electrophoresis result figure when Fig. 1 is RT-PCR measure.
Fig. 2 is that RT-PCR and RT-LAMP measure testing results compare figure；
Fig. 3 is specific detection result figure.
Embodiment
The claim of the present invention is described in further detail with reference to embodiment, is for the application
The information disclosed in detail, carries out according to this area conventional technical means.
Embodiment 1
A kind of RT-LAMP Primer compositions of detection Sai Neijia paddy viruses provided by the invention, including by positive inner primer
SVA-FIP, reverse inner primer SVA-BIP, positive outer primer SVA-F3, reverse outer primer SVA-B3, positive ring primer SVA-LF
Formed with reverse ring primer SVA-LB；
Primer SVA-F3 nucleotide sequences such as SEQ ID NO：Shown in 1；
Primer SVA-B3 nucleotide sequences such as SEQ ID NO：Shown in 2；
Primer SVA-FIP nucleotide sequences such as SEQ ID NO：Shown in 3；
Primer SVA-BIP nucleotide sequences such as SEQ ID NO：Shown in 4；
Primer SVA-LF nucleotide sequences such as SEQ ID NO：Shown in 5；
Primer SVA-LB nucleotide sequences such as SEQ ID NO：Shown in 6.
Embodiment 2
The RT-LAMP detection reagent box of SVA of the present invention a kind of, RT-LAMP primer combination of the kit containing embodiment 1
Thing.
Embodiment 3
The method of detection Sai Neijia paddy viruses provided by the invention, used material are as follows：
DNA/RNA nucleic acid extraction kit TGUide Virus DNA/RNA Kit kits (give birth to by article No. #Q5724 Tiangengs
Change Science and Technology Ltd.)
Nucleic acid automatic extracting instrument T-Guide OSE-M48 (Tiangeng biochemical technology Co., Ltd)
Constant-temperature fluorescence detector DEAOU-308C (Guangzhou Deaou Biotechnology Co., Ltd.)
(the WarmStart DNA Polymerase article No. #M5038L NEW ENGLANG of Bst2.0 polymerases Bst 2.0
BioLaBs companies)
Adlerika 100mM (the MgSO of 100mM4Solution article No. #B1003S NEW ENGLANG BioLaBs are public
Department)
10 × reaction buffer Isothermal Amplification Buffer (article No. #B0537S NEW ENGLANG
BioLaBs companies), component：20mM Tris-HCl, 10mM (NH4) 2SO4,2mM MgSO4,50mM KCl, 0.1%Tween
20, pH 8.8 25 DEG C of@.
DNTP Mix (the green skies biotech companies of article No. D7373)
Glycine betaine (article No. BCBS087V SIGMA companies)
AMV reverse transcriptase (AMV Rerverse Transcriptase, article No. M510A Promega company)
9 fluorescent dye (SYTO of SYTOTM9green fluorescent nucleic acid stain, article No.
S34854invitrogen companies)
One-step method reverse transcription PCR kit PrimeScriptTMOne Step RT-PCR Kit Ver.2 kit (goods
Number #RR055A TAKARA companies)
One-step method PCR kit for fluorescence quantitative One Step PrimeScriptTMRT-PCR Kit(Perfect Real
Time) kit (article No. #RR064A TAKARA companies)
Wherein：RNA virus
Using total nucleic acid extracts kit (TGuide virusDNA/RNA Kit), full-automatic nucleic acid extraction machine pair is utilized
The cell supernatant of SVA viruses has been connect, nucleic acid extraction has been carried out with the cell liquid of VSV, FMDV, SVDV of cell culture, has extracted
Put -80 DEG C of preservations.
RT-LAMP detection methods comprise the following steps：
1) primer is designed
The complete genome sequence of 50 SVA is downloaded from NCBI, is compared, picks out opposite conserved sequence (accession number:
DQ641257.1 the online website of lamp design of primers) is used：http://primerexplorer.jp/e/ designs primer, and Lamp draws
Thing：
2) take the RNA that step 1) is extracted to be added in RT-LAMP reaction solutions to mix, amplification system is established, in 63 DEG C of constant temperature
Under the conditions of carry out ring mediated isothermal amplification, after amplification80 DEG C of inactivation enzymatic activity 20min；
The RT-LAMP reaction solutions are：
3) detected using deaou-308c constant-temperature fluorescence detectors, parameter setting：Time 60min assay intervals 30s temperature
63 DEG C of threshold values set 3-6min variances multiple 10.
In order to verify preparatory reliability, the specificity of the method for the application offer, the skill of the application offer is given below
Art scheme and conventional RT-PCR, fluorescence quantitative RT-RCR detection comparative experimental example.
1) RT-PCR methods and fluorescence quantitative RT-RCR method
RT-PCR：(primer that laboratory is used by oneself)
Sense primer：GCTGTAAAAACCTTCTC(1337-1353)
Anti-sense primer：ATAGTATGTGCCAAGAG(1648-1664)
Fluorescence quantitative RT-RCR (is quoted：The separation identification of Zhao Xiao Asias pig Sai Neijia paddy viral (svv) and Study on Pathogenicity
[R] Guangzhou：Agricultural University Of South China 2016：27-32)
Sense primer：GAGCTTCAATCTCCTAGA
Sense primer：GTGTCATCATTCTCGTTAG
Probe：CAGACATTCGAGCCAAGCAACAA (5`6-FAM, 3`BHQ1)
2) RT-LAMP and fluorescence quantitative RT-RCR method sensitivity technique
RNA carries out continuous ten times of dilutions, and 10 are carried out with RT-PCR method0、101、102、103、104、105、106、107、108,
And do negative sample, detection sensitivity, after take product to see Fig. 1 into row agarose gel electrophoresis, testing result.
By 10 in RNA dilution gradients5、106、107、108、109, and negative sample is done, using the sensitive of RT-LAMP methods
Property, and compared with RT-PCR measure, testing result is shown in Fig. 2.
3) specific detection
By SVA positives nucleic acid, VSV, FMDV, SVDV feminine gender nucleic acid and ddH2O does specificity relatively, and testing result is shown in Fig. 3.
4) repeatability detection
10,10 are selected respectively1、104、106、108, the RNA templates of 5 concentration；The RNA templates of different time extracting are selected,
Carry out the experiment of 6 repetitions respectively by established RT-LAMP detection methods, verify its repeatability and stability.
5) clinical sample detects sample RT-PCR, fluorescence quantitative RT-RCR, RT-LAMP with SVA doubtful to 35
Detect respectively, testing result is shown in Table 1.
Clinical sample is as follows through three kinds of RT-LAMP, conventional RT-PCR, fluorescence quantitative RT-RCR method testing results：
By above-mentioned experiment it can be proved that technical scheme designs specific primer, more virus verification the method
Specifically
Property, RT-LAMP technology for detection value is compared with RT-PCR detected values, examines its repeatability with stablizing
Property, establish the quick determination method suitable for Site Detection Sai Neijia paddy viruses.
Above content is that a further detailed description of the present invention in conjunction with specific preferred embodiments, it is impossible to is assert
The specific implementation of the present invention is confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of not departing from present inventive concept, some simple deduction or replace can also be made, should all be considered as belonging to the present invention's
Protection domain.
Sequence table
<110>Tie up one hundred bio tech ltd in Guangzhou
<120>Detect the RT-LAMP Primer compositions and its kit and method of Sai Neijia paddy viruses
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>Sai Neijia paddy virus (Seneca virus A)
<400> 1
tatccacggc tcgatcca 18
<210> 2
<211> 20
<212> DNA
<213>Sai Neijia paddy virus (Seneca virus A)
<400> 2
ccggttacgt cttcaaaggt 20
<210> 3
<211> 43
<212> DNA
<213>Sai Neijia paddy virus (Seneca virus A)
<400> 3
aaggcacgct aaggcctagc tttttggcat gatcccccta gca 43
<210> 4
<211> 45
<212> DNA
<213>Sai Neijia paddy virus (Seneca virus A)
<400> 4
gacggcctag tcgtgtcggt tttttgccag aggctgtatc gaaag 45
<210> 5
<211> 20
<212> DNA
<213>Sai Neijia paddy virus (Seneca virus A)
<400> 5
acagttcccg ctgtagctcg 20
<210> 6
<211> 24
<212> DNA
<213>Sai Neijia paddy virus (Seneca virus A)
<400> 6
aggtagcaca tacaactatg caga 24
Claims (6)
1. a kind of RT-LAMP Primer compositions of detection Sai Neijia paddy viruses, it is characterised in that including by positive inner primer SVA-
FIP, reverse inner primer SVA-BIP, positive outer primer SVA-F3, reverse outer primer SVA-B3, forward direction ring primer SVA-LF and instead
Formed to ring primer SVA-LB；Each primer nucleotide sequences are as follows：
Primer SVA-B3 nucleotide sequences such as SEQ ID NO：Shown in 2；
Primer SVA-FIP nucleotide sequences such as SEQ ID NO：Shown in 3；
Primer SVA-BIP nucleotide sequences such as SEQ ID NO：Shown in 4；
Primer SVA-LF nucleotide sequences such as SEQ ID NO：Shown in 5；
Primer SVA-LB nucleotide sequences such as SEQ ID NO：Shown in 6.
2. a kind of kit of detection Sai Neijia paddy viruses, it is characterised in that the kit is containing the RT- described in claim 1
LAMP primer composition thing.
A kind of 3. method of detection Sai Neijia paddy viruses, it is characterised in that comprise the following steps successively：
1) RNA of virus is extracted from sample；
2) using the RNA of extraction as template, utilize claim 1) described in SVA RT-LAMP Primer compositions, establish RT-
LAMP amplification systems, carry out ring mediated isothermal amplification under 60 DEG C of -65 DEG C of constant temperatures；
3) detected using deaou-308c constant-temperature fluorescence detectors.
4. the method for a kind of detection Sai Neijia paddy viruses according to claim 3, it is characterised in that the RT-LAMP is anti-
The liquid is answered to be：Cumulative volume is 25 μ L, 2.5 μ L of reaction buffer；MgSO4 1.5μL；dNTP Mix 3.5μL；Primer mix 1μ
L；4 μ L of glycine betaine；RNA 2.0μL；2.0 archaeal dna polymerases of Bst, 1 μ L；0.5 μ L of reverse transcriptase；0.5 μ L of fluorescent dye.
5. the method for a kind of detection Sai Neijia paddy viruses according to claim 4, it is characterised in that the MgSO4 is dense
It is 10mM to spend for 100mM, dNTP Mix concentration；Beet alkali concn is 5M；Reverse transcriptase concentrations are 1U/ μ L；Fluorescent dye concentration
For 5mM.
A kind of 6. method of detection Sai Neijia paddy viruses according to claim 3, it is characterised in that the ring mediated isothermal
Amplification program is 63 DEG C of amplification 1h.
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| 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 |
| CN110229933A (en) * | 2019-06-24 | 2019-09-13 | 派生特（福州）生物科技有限公司 | A kind of primer sets, kit and application for the viral RT-Nested PCR detection of pig card inside competition |
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| 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 |
| CN108467904B (en) * | 2018-05-24 | 2021-11-19 | 华南农业大学 | RT-LAMP primer group for detecting seneca valley virus, kit and application |
| CN110229933A (en) * | 2019-06-24 | 2019-09-13 | 派生特（福州）生物科技有限公司 | A kind of primer sets, kit and application for the viral RT-Nested PCR detection of pig card inside competition |
| CN110229933B (en) * | 2019-06-24 | 2022-06-07 | 派生特（福州）生物科技有限公司 | Primer group and kit for nested RT-PCR (reverse transcription-polymerase chain reaction) detection of porcine Saikovia virus and application of primer group and kit |
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