CN103173574B - The method of SVCV is detected based on liquid-phase chip - Google Patents
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Abstract
The invention discloses a kind of method detecting SVCV based on liquid-phase chip.The invention provides the special primer pair of assistant identification SVCV, be made up of the single strand dna shown in the sequence 2 of the single strand dna shown in the sequence 1 of sequence table and sequence table.The present invention also protects the primed probe composition of assistant identification SVCV, is made up of with probe T1 described special primer; The nucleotide sequence of described probe T1 is as shown in the sequence 3 of sequence table.Special primer provided by the invention is adopted to have good specificity to qualification SVCV.Adopt primed probe composition provided by the invention in conjunction with liquid-phase chip qualification SVCV, have that specificity is good, highly sensitive, easy and simple to handle, required time is short, free from environmental pollution, there is not health threat to people, the advantage of high throughput testing can be carried out.The present invention is applicable to detecting hydrocoles of passing in and out very much.
Description
Technical field
The present invention relates to the primer pair of assistant identification SVCV, primed probe composition and their application, more specifically relate to the method detecting SVCV based on liquid-phase chip.
Background technology
Spring viremia (spring viraemia of carp, SVC) has another name called carp spring virus disease, is a kind of acute, hemorrhagic transmissible disease, for one of epidemic disease must be declared by OIE (OIE).Spring viremia is caused by SVCV (Spring viraemia ofcarp virus, SVCV) virus.SVCV is also known as carp rhabdovirus (Rhabdovirus carpio).
SVCV can infect the multiple fish such as bright and beautiful carp, silver carp, bighead, crucian, grass carp and pike, but the most responsive to carp, and carp is the topmost host of SVCV.In the cultivation of fishing ground, the carp at any age all can suffer from SVC, and water temperature fluctuated when Winter-Spring replaces reduces carp immunizing power, therefore easily breaks out in spring.There is the clinical disease such as acute ascites and systemic bleeding in the fish infecting SVCV.SVCV invades carp by water through the gill, about 20 days sequela of hiding in fish body.In low water temperature, virus can be survived about 11 weeks in infected carp blood, namely occurs the viremia period of persistence.SVC is popular in whole Europe and North America, popular in expanding to Asia in recent years, forms larger threat to China's culture fishery.
The detection method of current fishes virus mainly comprises cytodiagnosis technology, immunology diagnosis technology, diagnostic technique in molecular biology three major types.Cytodiagnosis technology mainly comprises employing cell cultures isolated viral, tissue pathological slice and electron microscopic observation, and its complex operation, sense cycle are long and sensitivity is low.Immunology diagnosis technology mainly comprises Immunofluorescence test, immune dot blot, have high specificity, advantage that susceptibility is high, but step is quite loaded down with trivial details, is not suitable for detecting a large amount of sample.Diagnostic technique in molecular biology mainly comprises polymerase chain reaction (PCR), fast, sensitive, but need to carry out agarose gel electrophoresis and by ethidium bromide staining observations, ethidium bromide is carcinogens, have harm to human body and environment, cross-contamination issue is more serious.
Summary of the invention
The object of this invention is to provide the primer pair of assistant identification SVCV, primed probe composition and their application, more specifically relate to the method detecting SVCV based on liquid-phase chip.
The invention provides the special primer pair of assistant identification SVCV, be made up of the single strand dna shown in the sequence 2 of the single strand dna shown in the sequence 1 of sequence table and sequence table.
The present invention also protects the primed probe composition of assistant identification SVCV, is made up of with probe T1 described special primer; The nucleotide sequence of described probe T1 is as shown in the sequence 3 of sequence table.
Described special primer to or described primed probe composition can be used for the test kit preparing assistant identification SVCV.
Described special primer to or described primed probe composition can be used for assistant identification SVCV.
The present invention also protects a kind of test kit of assistant identification SVCV, comprise described special primer to or described primed probe composition.
The present invention also protects a kind of method of assistant identification SVCV, comprises the steps: with the total serum IgE of virus to be measured for template, with described special primer to carrying out RT-PCR amplification; If described RT-PCR amplification obtains amplified production, virus to be measured is the SVCV of candidate; If described RT-PCR amplification does not obtain amplified production, virus to be measured is the non-SVCV of candidate.The size of described amplified production can be between 100-250bp, specifically can be 153bp.Described virus to be measured can be SVCV, infectious hematopoietic necrosis's poison, grass carp hemorrhage virus (GCHV), viral haemorrhagic septicaemia virus or infectious pancreatic necrosis virus.
The present invention also protects the method for another kind of assistant identification SVCV; comprise the steps: with the total serum IgE of virus to be measured as template; hybridize with described probe T1 after carrying out RT-PCR amplification with described special primer; if results of hybridization is positive virus to be measured is the SVCV of candidate, if results of hybridization is negative virus to be measured is the non-SVCV of candidate.Described virus to be measured can be SVCV, infectious hematopoietic necrosis's poison, grass carp hemorrhage virus (GCHV), viral haemorrhagic septicaemia virus or infectious pancreatic necrosis virus.
The method of described assistant identification SVCV specifically comprises the steps (Luminex):
(1) with the total serum IgE of virus to be measured for template, with described special primer to carrying out RT-PCR amplification;
(2) microsphere suspensions is prepared
1. by the fluorescence-encoded micro-beads vortex oscillation 20s of surface carboxyl groups, 75 μ L(are got about containing 3 × 10
6individual microballoon) be placed in 1.5mL centrifuge tube, the centrifugal 1min of 10000g, abandons supernatant;
2. in step centrifuge tube 1., add the 50 μ L0.1mol/L N-Methylimidazole aqueous solution (pH4.5), whirlpool mixes, ultrasonication 30-60 second (power of 400W, often ultrasonic 9 seconds stop 6 seconds);
3. in step centrifuge tube 2., add probe T1 described in 1.0nmol, whirlpool mixes;
4. in step centrifuge tube 3., add 2.5 μ L Carbodiimide solution (preparation method of Carbodiimide solution: sterilizing pure water 10mg carbodiimide powder being added 1.0mL, matching while using) fully to mix, room temperature lucifuge hatches 30min;
5. in step centrifuge tube 4., add 2.5 μ L Carbodiimide solution (preparation method of Carbodiimide solution is the same) fully to mix, room temperature lucifuge hatches 30min;
6. in step centrifuge tube 5., add the 1.0mL0.02g/100mL Tween-20 aqueous solution, mixing, the centrifugal 1min of 10000g, abandons supernatant;
7. in step centrifuge tube 6., add the 1.0mL0.1g/100mL SDS aqueous solution, mixing, the centrifugal 1min of 10000g, abandons supernatant;
8. in step centrifuge tube 7., the 100 μ L0.1mol/L N-Methylimidazole aqueous solution (pH4.5) are added, resuspended, obtain microsphere suspensions;
(3) microsphere suspensions that the product of step (1) and step (2) obtain is carried out hybridization and be experimental group, replace the product of step (1) to be control group with TE damping fluid; Hybridization system is mixed rear 98 DEG C of sex change 10min, then at the temperature identical with hybridization temperature, hatch 15min, the centrifugal 2min of 18000g, abandons supernatant; Then add the SA-PE that 50 μ L 1 × TMAC hybridization solutions are diluted to 500 times of volumes, 48 DEG C-54 DEG C centrifugal 2min of (being preferably 52 DEG C) hybridization 15min, 18000g, abandon supernatant; Then add 50 μ L 1 × TMAC hybridization solutions, whirlpool mixing makes microballoon resuspended, is placed in BD FACSArray liquid-phase chip instrument and analyzes; If LQRR >=3, detected result is positive; If LQRR < 2, detected result is negative; LQRR=MFIS/MFIB, MFIS are the fluorescence intensity median of experimental group, and MFIB is the fluorescence intensity median of control group.
The response procedures of RT-PCR amplification: 50 DEG C of 30min; 94 DEG C of 2min; 94 DEG C of 30sec, 58 DEG C of 30sec, 72 DEG C of 30sec, 30 circulations; 72 DEG C extend 5min; 4 DEG C of insulations.
Liquid-phase chip is a kind of novel Fast Detection Technique, and collecting type cell art, nanometer fluorescent microspheres, fluorescent signal digital processing and traditional chemical luminescence technology are integrated, and has the advantage that required sample size is few, sense cycle is short.
Special primer provided by the invention is adopted to have good specificity to qualification SVCV.Adopt primed probe composition provided by the invention in conjunction with liquid-phase chip qualification SVCV, except good specificity, also have highly sensitive, easy and simple to handle, required time is short, free from environmental pollution, there is not health threat to people, the advantage of high throughput testing can be carried out.The present invention is applicable to detecting hydrocoles of passing in and out very much.
Accompanying drawing explanation
Fig. 1 is the electrophorogram in embodiment 2.
Fig. 2 is hybridization temperature when being 52 DEG C, the output collection of illustrative plates of BD FACSArray liquid-phase chip instrument.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.
N-Methylimidazole (TE), carbodiimide purchase U.S. company BD.DL2000marker is purchased from the precious biotech firm in Dalian.RNA extracts test kit (TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver4.0): the precious biotech firm in Dalian, article No. is DV819A.RT-PCR kit (PrimeScript RT-PCR Kit): the precious biotech firm in Dalian, article No. is DRR014S.The fluorescence-encoded micro-beads of surface carboxyl groups: U.S. company BD, article No. is 558578.TMAC(temtrameihyl-ammdnium chloride): Sigma Co., USA, article No. is 87718; According to product description preparation 1 × TMAC hybridization solution and 1.5 × TMAC hybridization solution.SA-PE(streptavidin-phycoerythrin): Sigma Co., USA, article No. is S6940.Alphamager
tM2200 gel imaging systems: AP company of the U.S..Grads PCR amplification instrument (Eppendorf Mastercycler Gradient): German Eppendorf company.BD FACSArray liquid-phase chip instrument: U.S. company BD.
SVCV (SVCV), RNA viruses, reference: Fu Feng; Liu's Polygonum; Yellow Kinetix; Progress [J] Chinese aquatic science .2006 (02) of Cai Shengli SVCV (SVCV).
Infectious hematopoietic necrosis's poison (IHNV), RNA viruses: reference: Yue Zhiqin; Liu's Polygonum; The real-time quantitative RT-PCRs such as Liang Chengzhu detect the foundation and application [J] of fishes infectious hematopoietic necrosis virus's method. hydrobiont journal .2008 (01).
Grass carp hemorrhage virus (GCHV) (GCRV), RNA viruses, reference: Li Jun; Wang Tiehui; The progress [J] of grass carp hemorrhage virus (GCHV) such as after Lu Ren. Oceanologia et Limnologia Sinica .1999 (04).
Viral haemorrhagic septicaemia virus (VHSV), RNA viruses, reference: Ni Sui; Yu Xiaowei; Wang Jianping etc.; Application Reverse-transcription nested-polymerase chain reaction detects fishes virus haemorrhagic septicaemia virus (VHSV) [J] Oceanologia et Limnologia Sinica 2009 (07).
Infectious pancreatic necrosis virus (IPNV), RNA viruses, reference: Xu Ye; Duan Hongan; Week, firm real-time fluorescence quantitative RT-PCR detected the Agriculture of Anhui science of setting up 2011 (11) of the sick viral methods of fishes infectious Pancreatic Necrosis.
Embodiment 1, special primer are to the design with specific probe
Compared by a large amount of sequence alignment and expanding effect, be designed for amplification SVCV glycoprotein encoding gene special primer to and specific probe.
Special primer is to following (target sequence is 153bp):
The sequence 1 of F1(sequence table): 5 '-TTGGGATGACTGGGAGTT-3 ';
The sequence 2 of R1(sequence table): 5 '-GGGATAATATCGGCTTGG-3 '.
The nucleotide sequence (sequence 3 of sequence table) of specific probe is as follows:
5’-TGTATATAAAGGAAAGGATGGGAAG-3’。
Embodiment 2, application special primer are to assistant identification SVCV
Respectively SVCV, infectious hematopoietic necrosis's poison, grass carp hemorrhage virus (GCHV), viral haemorrhagic septicaemia virus and infectious pancreatic necrosis virus is tested as follows as virus to be measured:
1, RNA extraction test kit is adopted to extract the total serum IgE of virus to be measured.
2, the total serum IgE obtained with step 1, for template, adopts the primer pair of F1 and R1 composition, adopts RT-PCR kit, grads PCR amplification instrument carries out RT-PCR amplification, obtains RT-PCR amplified production.
RT-PCR amplification reaction system: in 0.2mL PCR reaction tubes, add each 2.5mmol/L of 10 × RT-PCR buffer2.5 μ L, dNTP(successively) 2.0 μ L, 10pmol/ μ L F1 1 μ L, 10pmol/ μ L R1 1 μ L, Inhibiter0.5 μ L, AMV XL0.5 μ L, AMV Taq (5U/ μ L) 0.25 μ L, 25mmol/L MgCl
25.0 μ L, total serum IgE 5.0 μ L(are about 300ng), add distilled water and make reaction volume reach 25 μ L.In above reaction system, except primer, total serum IgE and distilled water, other component is the component of RT-PCR kit.
The response procedures of RT-PCR amplification: 50 DEG C of 30min; 94 DEG C of 2min; 94 DEG C of 30sec, 58 DEG C of 30sec, 72 DEG C of 30sec, 30 circulations; 72 DEG C extend 5min; 4 DEG C of insulations.
3, RT-PCR amplified production step 2 obtained carries out 2% agarose gel electrophoresis.
Electrophorogram is shown in Fig. 1, and swimming lane 1 to swimming lane 5 is followed successively by SVCV, infectious hematopoietic necrosis's poison, grass carp hemorrhage virus (GCHV), viral haemorrhagic septicaemia virus and infectious pancreatic necrosis virus, and swimming lane M is DL2000marker.With the total serum IgE of SVCV for template, adopt the primer pair of F1 and R1 composition to carry out RT-PCR, obtain size amplified production between 100-250bp, other four kinds of viruses all do not obtain any amplified production.Checked order by the amplified production of SVCV, sequencing result shows, the size of amplified production is 153bp.
Embodiment 3, application primed probe composition assistant identification SVCV
One, the preparation of primer and probe
Be prepared as follows primer and probe:
F2：5’-biotin-TTGGGATGACTGGGAGTT-3’；
R2：5’-biotin-GGGATAATATCGGCTTGG-3’。
Probe T1:5 '-NH
2-TGTATATAAAGGAAAGGATGGGAAG-3 '.
F2 carries out biotin labeling at the 5 ' end of F1 to obtain, and R2 carries out biotin labeling at the 5 ' end of R1 to obtain.Probe T1 is that 5 ' end of Single-stranded DNA fragments shown in the sequence 3 of sequence table carries out amination and modifies and obtain.
Two, the foundation of liquid-phase chip detection system
1, RNA is adopted to extract the total serum IgE of test kit extraction SVCV.
2, the total serum IgE obtained with step 1, for template, adopts the primer pair of F2 and R2 composition, adopts RT-PCR kit, grads PCR amplification instrument carries out RT-PCR amplification, obtains RT-PCR amplified production.
RT-PCR amplification reaction system: in 0.2mL PCR reaction tubes, add each 2.5mmol/L of 10 × RT-PCR buffer2.5 μ L, dNTP(successively) 2.0 μ L, 10pmol/ μ L F2 1 μ L, 10pmol/ μ L R2 1 μ L, Inhibiter0.5 μ L, AMV XL0.5 μ L, AMV Taq (5U/ μ L) 0.25 μ L, 25mmol/L MgCl
25.0 μ L, total serum IgE 5.0 μ L(are about 300ng), add distilled water and make reaction volume reach 25 μ L.In above reaction system, except primer, total serum IgE and distilled water, other component is the component of RT-PCR kit.
The response procedures of RT-PCR amplification: 50 DEG C of 30min; 94 DEG C of 2min; 94 DEG C of 30sec, 58 DEG C of 30sec, 72 DEG C of 30sec, 30 circulations; 72 DEG C extend 5min; 4 DEG C of insulations.
3, oligonucleotide probe and microballoon covalent coupling
1. the fluorescence-encoded micro-beads vortex oscillation 20s of surface carboxyl groups is fully disperseed to make it, get 75 μ L(about containing 3 × 10
6individual microballoon) be placed in 1.5mL centrifuge tube, the centrifugal 1min of 10000g, abandons supernatant.
2. in step centrifuge tube 1., add the 50 μ L0.1mol/L N-Methylimidazole aqueous solution (pH4.5), whirlpool mixes, ultrasonication 30-60 second (power of 400W, often ultrasonic 9 seconds stop 6 seconds).
3. in step centrifuge tube 2., add 1.0nmol probe T1, whirlpool mixes.
4. in step centrifuge tube 3., add 2.5 μ L Carbodiimide solution (preparation method of Carbodiimide solution: sterilizing pure water 10mg carbodiimide powder being added 1.0mL, matching while using) fully to mix, room temperature lucifuge hatches 30min.
5. in step centrifuge tube 4., add 2.5 μ L Carbodiimide solution (preparation method of Carbodiimide solution is the same) fully to mix, room temperature lucifuge hatches 30min.
6. in step centrifuge tube 5., add the 1.0mL0.02g/100mL Tween-20 aqueous solution, mixing, the centrifugal 1min of 10000g, abandons supernatant.
7. in step centrifuge tube 6., add the 1.0mL0.1g/100mL SDS aqueous solution, mixing, the centrifugal 1min of 10000g, abandons supernatant.
8. in step centrifuge tube 7., add the 100 μ L0.1mol/L N-Methylimidazole aqueous solution (pH4.5), resuspended microballoon (probe is coupled to microsphere surface), obtains microsphere suspensions, and 2-8 DEG C keeps in Dark Place, stand-by.
4, hybridize
The hybridization system of experimental group: RT-PCR amplified production that the microsphere suspensions obtained by 1.5 μ L steps 3,33 μ L1.5 × TMAC hybridization solutions, 14.5 μ L TE damping fluids and 2.5 μ L steps 2 obtain (i.e. 25 microlitre RT-PCR amplified productions 1/10th) forms.
The hybridization system of blank group: replace RT-PCR amplified production with isopyknic TE damping fluid, other is with the hybridization system of experimental group.
In 98 DEG C of sex change 10min after hybridization system being mixed, then at the temperature identical with hybridization temperature, hatch 15min, the centrifugal 2min of 18000g, abandons supernatant; Then add the SA-PE that 50 μ L 1 × TMAC hybridization solutions are diluted to 500 times of volumes, under selected temperature, hybridize the hybridization temperature that 15min(adopts 48 DEG C, 50 DEG C, 52 DEG C, 54 DEG C or 56 DEG C respectively), the centrifugal 2min of 18000g, abandons supernatant; Then add 50 μ L1 × TMAC hybridization solutions, whirlpool mixing makes microballoon resuspended, is placed in BD FACSArray liquid-phase chip instrument and analyzes.
The experimental group of each hybridization temperature arranges three re-treatments, and the blank group of each hybridization temperature arranges three re-treatments.
In each re-treatment: the fluorescence-encoded micro-beads participating in counting is all more than 100, and show that the fluorescence-encoded micro-beads quantity for calculating is effective, the fluorescence intensity median (MFI) produced is credible; The MFI value of the fluorescence-encoded micro-beads of 3 blank groups is all less than 500, and test can carry out result judgement.
Adopt the hybridization temperature of 48 DEG C, the MFI of blank group process is 169, and the MFI of experimental group process is 6945 ± 23.1.
Adopt the hybridization temperature of 50 DEG C, the MFI of blank group process is 171, and the MFI of experimental group process is 7013 ± 18.7.
Adopt the hybridization temperature of 52 DEG C, the MFI of blank group process is 166, and the MFI of experimental group process is 7345 ± 17.6.
Adopt the hybridization temperature of 54 DEG C, the MFI of blank group process is 177, and the MFI of experimental group process is 6866 ± 28.3.
Adopt the hybridization temperature of 56 DEG C, the MFI of blank group process is 176, and the MFI of experimental group process is 2916 ± 14.6.
According to the liquid-phase chip result criterion of Luminex corporate policy, the qualitative ratio result of liquid-phase chip (Luminex qualitative ratio result, LQRR) equal the MFI(MFIS of experimental group treatment group) with the average MFI(MFIB of blank group) ratio, formula is: LQRR=MFIS/MFIB.If LQRR >=3, detected result is positive; If 2≤LQRR < 3, be then doubtful, need detect further; If LQRR < 2, detected result is negative.
Result shows: the hybridization temperature adopting 48 DEG C-54 DEG C, and the MFI amplitude of fluctuation of experimental group process is little, and when adopting the hybridization temperature higher than 54 DEG C, the MFI of experimental group process declines violent, and best hybridization temperature is 52 DEG C.
When hybridization temperature is 52 DEG C, Fig. 2 is shown in by the output collection of illustrative plates of BD FACSArray liquid-phase chip instrument.In Fig. 2: A: the quantity of fluorescence-encoded micro-beads and scope; B: the hybridization temperature adopting 52 DEG C, the relative intensity of fluorescence of experiment treatment group under Red-A, Yellow-A passage; C: the hybridization temperature adopting 52 DEG C, experiment treatment group relative intensity of fluorescence under Red-A passage; D: the hybridization temperature adopting 52 DEG C, experiment treatment group relative intensity of fluorescence under Yellow-A passage.Can observe from Fig. 2, the RT-PCR amplified production being coupled at probe T1 on microballoon and SVCV all has fluorescent signal under Red-A, Yellow-A sense channel, shows that liquid-phase chip successfully constructs.
Three, the repeatability checking of liquid-phase chip detection system
Repeat three step 2, hybridization temperature all adopts 52 DEG C, calculates the variation coefficient detecting the MFI obtained between each batch.Result shows, and the variation coefficient, within 2%, shows that the method for step 2 has good repeatability.
Four, the specificity verification of liquid-phase chip detection system
Respectively SVCV, infectious hematopoietic necrosis's poison, grass carp hemorrhage virus (GCHV), viral haemorrhagic septicaemia virus and infectious pancreatic necrosis virus is carried out step 2 as virus to be measured, hybridization temperature all adopts 52 DEG C, the results are shown in Table 1.
Table 1 specific detection result
| Sample | MFI | LQRR | Result |
| SVCV | 7946±21.4 | 45.93 | + |
| IHNV | 189±10.7 | 1.09 | － |
| GCRV | 193±6.3 | 1.15 | － |
| VHSV | 233±9.1 | 1.34 | － |
| IPNV | 196±4.8 | 1.13 | － |
| MFIB | 173 |
Result shows, the method for step 2 is positive to the detected result of SVCV, and is feminine gender to other four kinds viral detected results, has good specificity.
Five, liquid-phase chip detection system sensitivity checking
1, RNA is adopted to extract the total serum IgE of test kit extraction SVCV.
2, the total serum IgE obtained with step 1, for template, adopts the primer pair of F2 and R2 composition, adopts RT-PCR kit, grads PCR amplification instrument carries out RT-PCR amplification, obtains RT-PCR amplified production.
RT-PCR amplification reaction system: in 0.2mL PCR reaction tubes, add each 2.5mmol/L of 10 × RT-PCR buffer2.5 μ L, dNTP(successively) 2.0 μ L, 10pmol/ μ L F2 1 μ L, 10pmol/ μ L R2 1 μ L, Inhibiter0.5 μ L, AMV XL 0.5 μ L, AMV Taq (5U/ μ L) 0.25 μ L, 25mmol/L MgCl
25.0 μ L, total serum IgE 5.0 μ L(are containing about 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg or 10fg RNA), add distilled water and make reaction volume reach 25 μ L.In above reaction system, except primer, total serum IgE and distilled water, other component is the component of RT-PCR kit.
The response procedures of RT-PCR amplification: 50 DEG C of 30min; 94 DEG C of 2min; 94 DEG C of 30sec, 58 DEG C of 30sec, 72 DEG C of 30sec, 30 circulations; 72 DEG C extend 5min; 4 DEG C of insulations.
3, oligonucleotide probe and microballoon covalent coupling
With 3 of step 2.
4, hybridize
With 4 of step 2.Hybridization temperature adopts 52 DEG C.
The results are shown in Table 2, limit of identification is 10pg.
Table 2 sensitivity technique result
| Add-on × 0.1 of total serum IgE when RT-PCR is initial | Average fluorescent strength value (MFI) | LQRR | Result |
| 10ng | 5731±52.3 | 30.32 | + |
| 1ng | 3627±11.9 | 19.19 | + |
| 100pg | 2317±29.3 | 12.25 | + |
| 10pg | 876±25.6 | 4.63 | + |
| 1pg | 365±13.4 | 1.93 | － |
| 100fg | 289±12.9 | 1.52 | － |
| 10fg | 239±19.6 | 1.26 | － |
| 1fg | 192±8.7 | 1.01 | － |
| MFIB | 189 |
Embodiment 4, application primed probe composition assistant identification SVCV (oligonucleotide probe and microballoon covalent coupling adopt ordinary method)
1, with embodiment 3 step 21.
2, with embodiment 3 step 22.
3, oligonucleotide probe and microballoon covalent coupling
1. with embodiment 3 step 21 1..
2. with embodiment 3 step 21 2..
3. with embodiment 3 step 21 3..
4. in step centrifuge tube 3., add 5.0 μ L Carbodiimide solution (preparation method of Carbodiimide solution: sterilizing pure water 10mg carbodiimide powder being added 1.0mL, matching while using) fully to mix, room temperature lucifuge hatches 30min.
5. with embodiment 3 step 21 6..
6. with embodiment 3 step 21 7..
7. with embodiment 3 step 21 8..
4, hybridize
With 4 of the step 2 of embodiment 3, hybridization temperature adopts 52 DEG C.
MFI is 4894 ± 15.6.
Claims (4)
1. the special primer pair of assistant identification SVCV, is made up of the single strand dna shown in the sequence 2 of the single strand dna shown in the sequence 1 of sequence table and sequence table.
2. the primed probe composition of assistant identification SVCV, is made up of with probe T1 special primer described in claim 1; The nucleotide sequence of described probe T1 is as shown in the sequence 3 of sequence table.
3. special primer described in claim 1 to or claim 2 described in the application of primed probe composition in the test kit of preparation assistant identification SVCV.
4. the test kit of assistant identification SVCV, comprise special primer described in claim 1 to or claim 2 described in primed probe composition.
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