CN102329892A - Hyper-branched rolling cycle amplification detection method of soft-shelled turtle iridovirus - Google Patents
Hyper-branched rolling cycle amplification detection method of soft-shelled turtle iridovirus Download PDFInfo
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Abstract
The invention discloses a hyper-branched rolling cycle amplification detection method of soft-shelled turtle iridovirus, and the method is characterized by comprising the following steps: (1) designing a padlock probe and a pair of universal primers aiming at the connection sequence of the padlock probe; (2) preparing a padlock probe connection reaction system for performing connection reaction; and (3) preparing an HRCA (hyper-branched rolling cycle amplification) reaction system for performing HRCA amplification reaction, and finally detecting HRCA reaction products. The method has the following advantages: the method has higher sensitivity, specificity and convenience compared with the PCR (polymerase chain reaction) detection method of the soft-shelled turtle iridovirus in the prior art; furthermore, the method is simple and easy to operate, the detection method is fast and efficient, and 2-4 hours can be saved in comparison with the conventional PCR detection.
Description
Technical field
The present invention relates to the detection method of Trionyx sinensis (Wiegmann) irido virus, especially relate to a kind of using hyper-branched rolling circle amplification detection method of Trionyx sinensis (Wiegmann) irido virus.
Background technology
Trionyx sinensis (Wiegmann) is a kind of aquatic Reptilia of preciousness, and meat flavour is delicious, and is nutritious, has important researching value.Along with the raising of living standards of the people, market demand increases to some extent, and it is flourish that Trionyx sinensis (Wiegmann) is propagated industry artificially.Because the high-density intensive culture mode of Trionyx sinensis (Wiegmann) adds that the transaction of Trionyx sinensis (Wiegmann) commodity and seedling is frequent, various diseases frequently takes place, and the serious threat of soft-shelled turtle disease China and supported the soft-shelled turtle industry, and a year financial loss reaches several hundred million units, has restricted further developing of foster soft-shelled turtle industry.Therefore, diseases prevention and treatment are research directions of Trionyx sinensis (Wiegmann) aquaculture.The pathogenic agent that causes the Trionyx sinensis (Wiegmann) contagious disease mainly contains bacterium, fungi, parasite, virus etc., and often is several kinds of cause of disease polyinfections.To the first three cause of disease, the culturist has had the prevention and the treat-ment of a cover comparative maturity.Yet the research of virus disease also is in the starting stage.Research report about the Trionyx sinensis (Wiegmann) virus disease is very few.In recent years, the soft-shelled turtle disease that is caused by virus is obvious ascendant trend, causes great financial loss for the Trionyx sinensis (Wiegmann) aquaculture.Up to the present; The Trionyx sinensis (Wiegmann) virus of finding in China has 7 kinds; Be rhabdovirus, type reovirus, type adenovirus, the Trionyx sinensis (Wiegmann) virus of similar ribonucleic acid virus, the Trionyx sinensis (Wiegmann) spherical viruses of similar calicivirus, Trionyx sinensis (Wiegmann) irido virus; Chen Zaixian etc. be separated in the body of Trionyx sinensis (Wiegmann) the irido virus that a strain diameter is 120-160nm (Soft-she1led turtle iridovirus, STIV).The prevention and control of disease press for sets up quick, special, diagnostic techniques accurately.
Existing Trionyx sinensis (Wiegmann) irido virus (Soft-shelled turtle iridovirus, detection method STIV) mainly contains routine diagnostic methods such as serology detection and colloid gold immune detection, and these methods operations are complicated, sense cycle is long, detection sensitivity is low; Based on the round pcr of target DNA cloning as sophisticated molecular detecting method; (Fan Wanhong etc. 2007) are identified in the detection that has been used for this virus, but conventional round pcr is to detecting the amplification of target, having increased the risk of cross infection; And sensitivity is on the low side, can't detect disease timely and accurately; And above-mentioned detection method often needs specific equipment, laboratory and molecular biology Specialty Experiment personnel operation, when actual field is operated, has bigger limitation.
The using hyper-branched rolling circle amplification method (hyperbranched rolling circle amplification, HRCA) because of its high specific, highly sensitive, it is fast and convenient etc. that advantage is increasing is applied in molecular biology fundamental research and the actual detected.This technology (1h) at short notice increases in a large number, and amplification times reaches 10
7(Thomas et al.1999), sensitivity is high, can detect single molecules level (Zhang et al.2001).HRCA technical security, quick, efficient, highly sensitive and do not have equipment and technical limitation have the irreplaceable advantage of other technologies, and the HRCA technology has become the research focus, extensively applies to medicine, herding and animal doctor, fields such as agricultural.But seldom see the report of HRCA in aquatic animal cause of disease context of detection, the present invention is applied to the HRCA technology detection of STIV for the first time.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method based on using hyper-branched rolling circle amplification technology for detection Trionyx sinensis (Wiegmann) irido virus, to realize that the Trionyx sinensis (Wiegmann) irido virus is carried out quick, special, easy scene to be detected.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: a kind of using hyper-branched rolling circle amplification detection method of Trionyx sinensis (Wiegmann) irido virus may further comprise the steps:
(1) padlock probe is synthetic: from gene pool, choose one section main capsid protein MCP (major capsid protein) (accession number is EU627010) gene order that the Trionyx sinensis (Wiegmann) irido virus is exclusive; From the MCP gene order, choose the specific recognition district of 20 bases respectively as padlock probe 5 ' end and 3 ' end; 5 ' terminal bases and 3 ' terminal bases are continuous on the MCP gene order; From plasmid vector pNAK1, choose one section sequence; With the linkage section part of the nucleotide sequence that forms with 5 ' terminal bases and 3 ' terminal bases complementary base replacement back in this sequence as padlock probe; Carry out the synthetic of padlock probe then, the nucleotide sequence of described padlock probe is following:
5’-CTGCGTCACTCCCGTCGTGGtggactgctgaatccgttagccagcagccgcctccttattatcacttattcaggcgtagcaccagTCCGTCGGCTCCAATTACAC-3’；105
(2) according to a pair of universal primer CF1 of the linkage section sequences Design of padlock probe and CF2, sequence is following:
CF1：5’-CTGGTGCTACGCCTGAATAAGTGA-3’，24
CF2：5’-GCTGAATCCGTTAGCCAGCAG-3’；21
(5) ligation of padlock probe:
The final concentration of ligation system is respectively: padlock probe is respectively 100pM-10 μ M; 10 * T4DNA LigaseBuffer, 1 μ l, sample template DNA 2 μ l add distilled water to reacting TV 10 μ l; The condition that connects is: behind 94 ℃ of following sex change 5min; Add 1U/ μ l T4 DNA Ligase again, under 16 ℃ condition, reaction 10-60min;
(4) HRCA reaction system amplification
The final concentration of HRCA reaction system is respectively: dNTPs 0.4mM, CF10.4 μ M, CF20.4 μ M, pH 8.8deTris-HCl 20mM, KCl 10mM, MgSO
46.5mM, (NH
4)
2SO4 10mM, 0.1%Triton x-100, big fragment of 8U Bst archaeal dna polymerase and ligation product 2 μ L; Adding distilled water to reaction system TV is 25 μ L; Above-mentioned reaction system is carried out amplified reaction, and the amplified reaction temperature is 61-65 ℃, and the amplified reaction time is 10-60min;
(5) detection of HRCA reaction product
HRCA amplified reaction product is observed amplified band through agarose gel electrophoresis, and the product sequence of HRCA distributes discontinuous on length, sees stepped distribution from gel electrophoresis figure.Wherein the minimum band of length is the distance between primers F 1 and the F2; Thereafter the stripe size of every big one-level all increases the size of a padlock probe on the basis of previous stage stripe size; Through observing the brightness of electrophoretic band, can judge the optimum condition of connection and HRCA reaction.If the stepped distribution of band then represent that the detected result of STIV of this sample is positive, on the contrary amplified band do not had or amplified band is non-stepped distribution, then negative.
The ligation optimum condition is under 16 ℃ condition in the step (3), reaction 10min.
HRCA amplified reaction optimum condition is 61 ℃ of reaction 20min in the step (4).
Compared with prior art, the invention has the advantages that:
(1) hypersensitivity: HRCA has the amplification ability stronger than PCR, can in 1h, carry out 10 to target sequence
9Amplification, this highly sensitive characteristic makes it can detect single molecules level at short notice.
(2) high sequence-specific: the detection method of PCR-based technology all is the amplification to target DNA; This has increased the probability of crossed contamination; What increase in the HRCA detection method is not target dna sequence dna but the sequence of padlock probe; Target DNA only provides complementary sequence for the cyclisation padlock probe, thereby reduces the risk that reaction is polluted to greatest extent.Padlock probe has very high specificity and single base mismatch recognition capability; When utilizing padlock probe to detect target sequence; Because being connected of probe 5 ' end and 3 ' end needs two sections of probe recognition sequence and target sequence complementary fully, when mispairing existed, the ligation of probe just can't be accomplished; Just can not increased the high specific in therefore having guaranteed to detect yet.
(3) diversity: the padlock probe of cyclisation can increase through universal primer; Universal primer can wait many different padlock probes of efficient ground amplification; Overcome reactant and amplified production in other liquid phase amplification methods such as PCR react to each other and interference, multiplex PCR in problem such as primer competition, realize the amplification and the multivariate detection of detection signal.
(4) simple to operation: the HRCA nucleic acid amplification carries out under isothermal condition, does not need the thermally denature of template, long-time temperature cycle.Do not need special thermal cycling instrument, can in simple water-bath, carry out.As in system, adding SYBR Green I optical dye, can judge through the change in fluorescence range estimation, make detection more convenient.
(5) rapidly and efficiently: the rolling circle amplification technology is the external nucleic acid amplification technologies of a kind of exponential, can be in 1h target sequence be carried out 109 times amplification, detects than conventional PCR and saves 2～4h.
Description of drawings
Fig. 1 detects the electrophorogram of the sensitivity of SITV for the HRCA method; M:GeneRuler 100bp DNA Ladder Plus; 1,2,3,4,5,6 sample template concentrations (copies/ μ l) is respectively 10
5, 10
4, 10
3, 10
2, 10
1, 10
07: distilled water is made blank;
Fig. 2 is the sensitive electrophorogram that conventional PCR method detects STIV; M:GeneRuler 100bp DNA Ladder Plus; 1,2,3,4,5,6 sample template concentrations (copies/ μ l) is respectively 10
5, 10
4, 10
3, 10
2, 10
1, 10
07: distilled water is made blank;
Fig. 3 is the specificity test electrophorogram of HRCA method; M:GeneRuler 100bp DNA Ladder Plus; 1: the Trionyx sinensis (Wiegmann) irido virus; 2: WSSV; 3: Singapore's grouper irido virus; 4: the swimming crab reovirus; 5: no template.
Embodiment
Embodiment describes in further detail the present invention below in conjunction with accompanying drawing.
1, preparation SITV genomic DNA sample template and standard model
1.1 the extraction of Trionyx sinensis (Wiegmann) irido virus DNA
1.1.1 the purifying of virus
The tissue that 20 grams are infected shreds, and places the 200mlTNE damping fluid, and 8000r/min, 4 ℃ of centrifugal 20min get supernatant.Under 4 ℃, the centrifugal 1h of 35000g.Abandon supernatant, except that the black circle of deposition and deposition.Be resuspended in the 5mlTM damping fluid.Spend the night, the deposition that will spend the night is blown and beaten evenly gently, and 8000r/min, 4 ℃ of centrifugal 20min get supernatant, and under 4 ℃, the centrifugal 1h of 35000g, the gained white precipitate is purified virus.
1.1.2 the extraction of purified virus DNA
With 500 microlitre TE damping fluids virus is transferred in the centrifuge tube fully.Xiang Guanzhong adds 125 microlitre 10%SDS, and to make its final concentration be 2%.In the EP pipe, add Proteinase K, making its final concentration is 1mg/mL.50 ℃～60 ℃ water-bath 1～2h, intermittently concussion.To wherein adding isopyknic saturated phenol upset 30min, 4 ℃, 12000rpm, 20min.Get the upper strata water sample, add in the new EP pipe, add isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing 15min, 40 ℃, 12000rpm, 15min.Get supernatant, add the equal-volume chloroform: primary isoamyl alcohol (24: 1) mixing 15min, 4 ℃, 12000rpm, 15min.Get supernatant, the NaAc and the absolute ethyl alcohol (in 20 ℃ of refrigerators) of 2 times of volume precoolings that add the 3M of 1/10 volume are put in-20 ℃ of refrigerators, and deposition is spent the night.4 ℃ of solution, 10000rpm, the 15min of overnight treatment is centrifugal.Add 75% alcohol, washed twice is removed salt ion, and room temperature is placed tasteless until alcohol.Subsequent use with-20 ℃ of preservations of 20 μ l pure water dissolving.
1.2 the preparation of standard model
STIV genomic dna with said extracted is the sample template, carries out pcr amplification with primer STIV-F (+)/STIV-R, and pcr amplification reaction is 25 μ L systems, comprises 10mM Tris-HCl (pH 8.3), 50mM KCl, 1.5mM MgCl
2, 0.8mM dNTPs, 0.2 μ M primer STIV-F (+), 0.2 μ M primer STIV-R, STIV genomic dna 1.0 μ L, 5U/ μ L rTaq archaeal dna polymerase; React behind 94 ℃ of preparatory sex change 2min cyclic amplification 30 times: 94 ℃ of sex change 30s, 55 ℃ of renaturation 30s, 72 ℃ of reaction 1.5min, 72 ℃ of extension 10min after the loop ends; Amplified production is after 1% (w/v) agarose gel electrophoresis separates, and the amplified band of expection size is with QIAquick Gel Extraction purifying and recovering, through spectrophotometric determination, gained PCR product is carried out gradient dilution as standard substance after quantitative.
2, the using hyper-branched rolling circle amplification detection method of Trionyx sinensis (Wiegmann) irido virus
2.1 material and method
2.1.1 material
The big fragment of Bst archaeal dna polymerase (containing 10 * damping fluid) is available from New England Biolabs company; DNTPs, rTaq archaeal dna polymerase, T4DNA Ligase (containing 10 * damping fluid), GeneRuler 100bp DNA Ladder Plus are available from TaKaRa company, and padlock probe and universal primer CF1/CF2 are by the handsome Bioisystech Co., Ltd in Shanghai.
2.1.2 method
(1) padlock probe is synthetic: from gene pool, choose one section main capsid protein MCP (major capsid protein) (accession number is EU627010) gene order that the Trionyx sinensis (Wiegmann) irido virus is exclusive; From the MCP gene order, choose the specific recognition district of 20 bases respectively as padlock probe 5 ' end and 3 ' end; 5 ' terminal bases and 3 ' terminal bases are continuous on the MCP gene order; Padlock probe 5 ' end and 3 ' end respectively has 20 bases on target DNA, to be close to and complete complementary pairing; From plasmid vector pNAK1, choose one section sequence; With the linkage section part of the nucleotide sequence that forms with 5 ' terminal bases and 3 ' terminal bases complementary base replacement back in this sequence as padlock probe, carry out the synthetic of padlock probe then, the nucleotide sequence of this padlock probe is following:
5’-CTGCGTCACTCCCGTCGTGGtggactgctgaatccgttagccagcagccgcctccttattatcactt?attcaggcgtagcaccagTCCGTCGGCTCCAATTACAC-3’；105
(2) according to a pair of universal primer CF1 of the linkage section sequences Design of padlock probe and CF2, sequence is following:
CF1：5’-CTGGTGCTACGCCTGAATAAGTGA-3’，24
CF2：5’-GCTGAATCCGTTAGCCAGCAG-3’；21
(3) ligation of padlock probe:
The final concentration of ligation system is respectively: padlock probe is respectively 100pM; 10 * T4DNA Ligase Buffer, 1 μ l removes the sample template DNA 2 μ l of method for preparing, adds distilled water water to reacting TV 10 μ l; The condition that connects is: behind 94 ℃ of following sex change 5min; Add 1U/ μ l T4 DNA Ligase again, under 16 ℃ condition, reaction 10min;
(4) HRCA reaction system amplification
The final concentration of HRCA reaction system is respectively: dNTPs 0.4mM, CF1 0.4 μ M, CF20.4 μ M, pH 8.8de Tris-HCl 20mM, KCl 10mM, MgSO
46.5mM, (NH
4)
2SO4 10mM, 0.1%Triton x-100, big fragment of 8U Bst archaeal dna polymerase and ligation product 2 μ L; Adding distilled water to reaction system TV is 25 μ L; Above-mentioned reaction system is carried out amplified reaction, and the amplified reaction temperature is 61 ℃, and the amplified reaction time is 20min;
(5) detection of HRCA reaction product
HRCA amplified reaction product is observed amplified band through agarose gel electrophoresis, and the product sequence of HRCA distributes discontinuous on length, sees stepped distribution from gel electrophoresis figure.Wherein the minimum band of length is the distance between primers F 1 and the F2; Thereafter the stripe size of every big one-level all increases the size of a padlock probe on the basis of previous stage stripe size; Through observing the brightness of electrophoretic band, can judge the optimum condition of connection and HRCA reaction.If the stepped distribution of band then represent that the detected result of STIV of this sample is positive, on the contrary amplified band do not had or amplified band is non-stepped distribution, then negative.
Except that the foregoing description 1, padlock probe can also be 1nM, 10nM; 100nM; 1 μ M, 10 μ M, the time of the ligation of padlock probe can also be 20min, 30min, 40min, 50min, 60min; The temperature of HRCA reaction system amplified reaction can also be 62 ℃, 63 ℃, 64 ℃, 65 ℃, all right 10min of the time of amplified reaction, 30min, 40min, 50min, 60min.
The present invention is based on the sensitivity determination of HRCA technology for detection STIV method
1, the preparation of standard substance: the concentration of the DNA sample that extracts with spectrophotometric determination is diluted to 10
10Copies/ μ L is as standard substance.Get the standard substance that prepare and be diluted to 10
0-10
5Copies/ μ L is respectively as template.
2, use optimum condition method that embodiment 1 draws that the template of each concentration is detected, thereby draw sensitivity of the present invention, carry out conventional PCR in addition and detect, it is right to carry out remolding sensitivity.
2.1 the connection of padlock probe
The ligation system is following: padlock probe 0.1 μ M, T4DNA Ligase 1U/ μ l, 10 * T4DNA Ligase Buffer, 1 μ l; Sample template DNA 2 μ l add water to reaction TV 10 μ l, and the condition of connection is: behind 94 ℃ of following sex change 5min; Add 1U/ μ l T4 DNA Ligase again; Under 16 ℃ condition, reaction 10, ligation liquid is for use.
2.2HRCA reaction
The final concentration of reaction system is respectively: dNTPs 0.4mM, CF1 0.4 μ M, CF20.4 μ M, Tris-HCl (pH8.8) 20mM, KCl 10mM, MgSO
46.5mM, (NH4)
2SO
410mM, 0.1%Triton x-100, the big fragment of 8U Bst archaeal dna polymerase (New England Biolabs) is connected product with 2 μ L, and adding distilled water, to make the reaction system TV be 25 μ L.Reaction conditions is: 61 ℃, and 20min.
2.3PCR reaction
The PCR reaction is 25 μ L systems, comprises 10mM Tris-HCl (pH 8.3), 50mM KCl, 1.5mM MgCl
2, 0.8mM dNTPs, 0.2 μ M primer STIV-F (+), 0.2 μ M primer STIV-R, STIV genomic dna 1.0 μ L, 5U/ μ L rTaq archaeal dna polymerase; React behind 94 ℃ of preparatory sex change 2min cyclic amplification 30 times: 94 ℃ of sex change 30s, 55 ℃ of renaturation 30s, 72 ℃ of reaction 1.5min, 72 ℃ of extension 10min after the loop ends, wherein the PCR primer is following to being STIV-F/STIV-R (extension increasing sequence is 926bp) sequence:
STIV-F：5’-TTTGTCAAGGAGCACTACCC-3’
STIV-R：5’-ACGGGATCTACCGCAAGG-3’；
Detected result shows that the detected minimum template amount of HRCA method institute ability is near 10
1Copy, and be limited to 10 under the detection of conventional PCR
3Therefore, drawn by sensitivity experiment, the conventional PCR method of the remolding sensitivity of HRCA method has higher sensitivity, result such as Fig. 1, shown in Figure 2.
Embodiment 3
The present invention is based on the specific assay of rolling circle amplification technology for detection STIV method
With the STIV genomic dna as positive control; The virus that other gets 3 kinds of different degrees of correlation is carried out the specificity checking of this research; Be WSSV (white spot syndrome virus; WSSV), (Singapore grouperiridovirus, SGIV) with the swimming crab reovirus, wherein distilled water is as negative control for Singapore's grouper irido virus.Amplified production detects through agarose gel electrophoresis respectively.Fig. 3 is seen in result's demonstration, explains that HRCA detection method provided by the invention can guarantee the specific detection to STIV, not with other correlated virus generation cross reactions.
The foregoing description is a detailed description of the invention, and protection scope of the present invention is not limited to the described technical scheme of above-mentioned embodiment, and should be as the criterion with the described protection domain of claim.
Claims (3)
1. the using hyper-branched rolling circle amplification detection method of a Trionyx sinensis (Wiegmann) irido virus is characterized in that may further comprise the steps:
(1) padlock probe is synthetic: from gene pool, choose one section main capsid protein MCP gene order that the Trionyx sinensis (Wiegmann) irido virus is exclusive; From the MCP gene order, choose the specific recognition district of 20 bases respectively as padlock probe 5 ' end and 3 ' end; 5 ' terminal bases and 3 ' terminal bases are continuous on the MCP gene order; From plasmid vector pNAK1, choose one section sequence; With the linkage section part of the nucleotide sequence that forms with 5 ' terminal bases and 3 ' terminal bases complementary base replacement back in this sequence as padlock probe, carry out the synthetic of padlock probe then, the nucleotide sequence of described padlock probe is following:
5’-CTGCGTCACTCCCGTCGTGGtggactgctgaatccgttagccagcagccgcctccttattatcacttattcaggcgtagcaccagTCCGTCGGCTCCAATTACAC-3’；105
(2) according to a pair of universal primer CF1 of the linkage section sequences Design of padlock probe and CF2, sequence is following:
CF1：5’-CTGGTGCTACGCCTGAATAAGTGA-3’，24
CF2：5’-GCTGAATCCGTTAGCCAGCAG-3’；21
(5) ligation of padlock probe:
The final concentration of ligation system is respectively: padlock probe is respectively 100pM-10 μ M; 10 * T4 DNA Ligase Buffer, 1 μ l, sample template DNA 2 μ l add distilled water to reacting TV 10 μ l; The condition that connects is: behind 94 ℃ of following sex change 5min; Add 1U/ μ l T4 DNA Ligase again, under 16 ℃ condition, reaction 10-60min;
(4) HRCA reaction system amplification
The final concentration of HRCA reaction system is respectively: dNTPs 0.4mM, CF1 0.4 μ M, CF2 0.4 μ M, pH 8.8de Tris-HCl 20mM, KCl 10mM, MgS0
46.5mM, (NH
4)
2S04 10mM, 0.1%Triton x-100, big fragment of 8U Bst archaeal dna polymerase and ligation product 2 μ L; Adding distilled water to reaction system TV is 25 μ L; Above-mentioned reaction system is carried out amplified reaction, and the amplified reaction temperature is 61-65 ℃, and the amplified reaction time is 10-60min;
(5) detection of HRCA reaction product
HRCA amplified reaction product is observed amplified band through agarose gel electrophoresis, if the stepped distribution of band then represent that the detected result of STIV of this sample is positive, on the contrary amplified band do not had or amplified band is non-stepped distribution, then negative.
2. the using hyper-branched rolling circle amplification detection method of a kind of Trionyx sinensis (Wiegmann) irido virus according to claim 1 is characterized in that: the ligation optimum condition is reacted 10min under 16 ℃ condition in the step (3).
3. the using hyper-branched rolling circle amplification detection method of a kind of Trionyx sinensis (Wiegmann) irido virus according to claim 1 is characterized in that: HRCA amplified reaction optimum condition is 61 ℃ of reaction 20min in the step (4).
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| CN114231647A (en) * | 2021-11-23 | 2022-03-25 | 广西壮族自治区食品药品检验所 | Method for detecting food-borne pathogenic bacteria staphylococcus aureus by using rolling circle isothermal amplification technology based on visualization method and application thereof |
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