CN105861746A - Method for rapidly detecting early-stage PEDV infection based on nanogold label amplification technology - Google Patents

Method for rapidly detecting early-stage PEDV infection based on nanogold label amplification technology Download PDF

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CN105861746A
CN105861746A CN201610231624.3A CN201610231624A CN105861746A CN 105861746 A CN105861746 A CN 105861746A CN 201610231624 A CN201610231624 A CN 201610231624A CN 105861746 A CN105861746 A CN 105861746A
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pedv
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童德文
黄勇
邢娜
赵晓民
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Northwest A&F University
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Abstract

The invention provides a method for enriching viruses from faeces rapidly and efficiently, amplifying signals through gold nanoparticles crosslinked through specific DNA labels and then rapidly detecting early-state PEDV infection with a PCR. According to the method, an PRF1a gene located in a PEDV whole genome is adopted as a target sequence, specific nucleic acid probes are designed, second-segment probes which are high in specificity, great in virus enriching capability and easy to amplify and detect are screened out of the specific nucleic acid probes and optimized, and by means of functionalized magnetic particles and nanogold particles of the second-segment probes, a PCR detection technology based on nanoparticle PEDV specificity is established; meanwhile, by means of the technical method, the technical breakthrough of conducting a direct hybridization reaction between faeces sample lysate and the functionalized magnetic particles is achieved, the step of purifying a sample nucleic acid is completely omitted, specificity and sensitiveness are improved, meanwhile, the detection step is simplified, and detection time is shortened. By means of the method, time and labor are saved, cost is low, and in addition, the animal virus infection level can be prompted accurately.

Description

A kind of quickly detect PEDV early infection based on nm of gold label amplifying technique Method
Technical field
The present invention relates to one from early infection swine excrement, quickly detect Porcine Epidemic Diarrhea The method that poison (PEDV) low dosage infects, is specifically related to a kind of based on the amplification of nm of gold label Technology quickly detects the method for PEDV early infection, belongs to veterinary science detection technique field.
Background technology
Along with the development of large scale of pig farm industry, the sick impact on pig industry of pig virus diarrhoea is increasingly Seriously, cause huge economic loss to the pig industry of China, simultaneously also to these infectious diseases Rapid&Early diagnosis and preventing and treating bring new challenge.
The cause of disease of pig virus diarrhoea specifically includes that Porcine epidemic diarrhea virus (PEDV), pig TGE (TGEV), porcine rotavirus (PoRV), wherein PEDV is in recent years Present, in China, the trend of breaking out, become the main pathogen that harm pig industry develops in a healthy way.Pig is flowed Row diarrhoea is a kind of high degree in contact chitling road transmission caused by Porcine epidemic diarrhea virus Disease, sick pig, mainly with severe diarrhea, vomits and is dehydrated as Clinical symptoms.The pig at any age is equal Susceptible, the incidence of disease of suckling pig, feeder pig or growing and fattening pigs is up to 100%, and especially lactation is young Pig is injured the most serious.Due to PED and transmissible gastroenteritis of swine, porcine rotavirus infection etc. Clinical symptoms and pathological change and similar, cannot be right according only to clinical symptoms and pathological change PED makes and making a definite diagnosis.
Pig epidemic diarrhea disease still lacks effective radical cure means at present, mainly by prevention monitoring And isolation processing.Traditional detection method currently for Porcine epidemic diarrhea virus (PEDV) Including: virus be separately cultured, In-stitu hybridization, ELISA, indirect immunofluorescence, RT-PCR etc..These methods the most in various degree there is complex operation, time and effort consuming, special Property is poor, sensitiveness is low or the shortcoming such as detection range limitation.Detection PEDV sub-gene group at present The real-time PCR method of S, M, N, ORF3 is it has been established that but use real-time PCR Method quantitatively virus expends higher, and detection sensitivity is only capable of improving tens of than common RT-PCR Times, therefore limited by financial cost in production practices.It addition, it was found that use pin To PEDV full-length genome and sub-gene group primer, by RT-PCR and real-time PCR During method quantitatively virus, Viral Quantification result is significantly different, therefore for being positioned at the full base of PEDV Because of the rdrp gene design specific probe of group, and carry out the most quickly detection, can be more accurate The most internal virus titer is infected in the most real reaction, thus the preventing and treating for this disease provides real Time, instruct accurately.
Summary of the invention
It is an object of the invention to for deficiency of the prior art, it is provided that one saves time, laborsaving, The method quickly detecting PEDV early infection based on nm of gold label amplifying technique of low cost.
For achieving the above object, the invention discloses following technical scheme:
A kind of method quickly detecting PEDV early infection based on nm of gold label amplifying technique, Comprise the steps:
The design of S1 PEDV specific dna probe and screening:
According to be uploaded to NCBI PEDV different strains use DNASTAR or VECTOR NTI 9.0 software carries out Multiple sequence alignments, according to the ORF1a of coding replicase 16 sections of specific dna probes of gene conserved regions design, by designed nucleic acid probe respectively It is marked on magnetic particle and nanogold particle, forms magnetic bead and the nanogold particle of functionalization, and It is optimized and screens；
The screening of functionalization probe:
16 sections of functionalization magnetic bead probe sequences are:
16 sections of functional gold nanoparticles particle probe sequences are:
The screening process of functionalization magnetic bead is as follows: by 16 sections of designed specific dna probes Mark magnetic particle respectively, form MMPs-p1, p2, p3, p4, p5, p6, p7, p8, p9, p10, P11, p12, p13, p14, p15, p16, prepare the magnetic particle of PEDV specific probe mark Comprise the following steps that described, by the magnetic bead of functionalization with viral RNA in hybridization buffer Hatch 30min, Magneto separate, detect with the specific RT-PCR of PEDV for 40 DEG C；
The screening process of functionalized nano gold grain is as follows: by 16 sections of designed specific cores Acid probe marking nano gold grain respectively, forms AuNPs-oligo1, oligo2, oligo3, oligo4, oligo5,oligo6,oligo7,oligo8,oligo9,oligo10,oligo11oligo12,oligo13, Oligo14, oligo15, oligo16, prepare the tool of the AuNPs of PEDV specific probe mark Body step is as described below, by the nanogold particle of different probe functionalization with viral RNA miscellaneous Friendship buffer solution hatches 40min for 50 DEG C, after centrifugation, specific with PEDV RT-PCR detects.
The functionalization magnetic bead probe sequence determined through screening is Probe 1:
5’NH2-T15GCCTCAGAATAGTATGAGACGGCTTC；(76-101)
The functional gold nanoparticles particle probe sequence determined through screening is Oligo 2:
5’SH-T15CCTGTAGATAGTAGCAAGTGGCTCAGACCGTGATG GAATGGA；(626-651)
S2 prepare PEDV specific probe mark magnetic particle:
Specific probe sequence is:
5’NH2-T15GCCTCAGAATAGTATGAGACGGCTTC；
S3 prepare specific signals probe mark nanogold particle:
Specific probe sequence is:
5’SH-T15CCTGTAGATAGTAGCAAGTGGCTCAGACCGTGATG GAATGGA；
Magnetic particle and the nanogold particle of S4PEDV probe mark are sick with PEDV in fecal sample The hybridization reaction of poison RNA and the wash-out of target dna label:
S4.1 takes fecal sample and mixes with PBS vortex, and 4,000g are centrifuged 10min goes Except ight soil residue；
S4.2 takes the PEDV fecal sample supernatant 500 μ L after process and 500 μ L lysates extremely In 1.5mL centrifuge tube, boiling 15min and make viral RNA discharge, 4 DEG C, 13400g is centrifuged 5min, takes supernatant standby, and wherein, lysate is: 10mmol/L Tris-HC1,1mmol/L EDTA, 15mmol/L NaCl, 0.5% SDS, pH 8.0；
In S4.3 fecal sample lysate supernatant in step S4.2, after adding 2 μ L marks Magnetic particle MMPs-p1 and 110 μ L hybridization buffers mixing, 40 DEG C hybridization 30min, Add the nanogold particle AuNPs-Oligo 2 after 2 μ L marks after hybridization to mix, 50 DEG C Hybridization 40min, it is thus achieved that AuNPs-RNA-MMPs compound, wherein, hybridization buffer: 20 × SSC, 1% Tween-20 and 2% SDS；
S4.4 takes 1mL TE buffer solution every time, and Magneto separate is washed 2-3 time, removes the hybridization of residual Buffer solution, unconjugated probe and DNA label；
S4.5 is combined with AuNPs-RNA-MMPs with 100 DTT eluents newly configured for μ L Thing at room temperature acts on 10min, takes supernatant after Magneto separate 3min, and wherein, DTT elutes Liquid is: 0.5mol/L DTT, 10mM Tris-HCl, 1mM EDTA, pH 7.5；
S4.6 adds NaAc and the anhydrous second of 2 times of volumes of 1/10 volume at elution supernatant Alcohol, precipitates 30min；
S4.7 is under the conditions of 4 DEG C, and 13400g is centrifuged 10min, abandons supernatant, adds 75% second Alcohol washes twice；
S4.8 is under the conditions of 4 DEG C, and 13400g is centrifuged 10min, abandons supernatant, natural air drying, Add 10 μ L ddH2O dissolving DNA label；
S5PCR detection and the DNA label of PEDV specific bond:
S5.1 is with luciferase reporter gene carrier as template, and PCR amplification obtains linker DNA Fragment
Upstream primer F:TGGCATTCCGGTACTGTTGG,
Downstream primer R:AGGAGAATAGGGTTGGCACC,
Use 50 μ l systems: 10 × buffer 5 μ L, dNTP 4 μ L, upstream primer 1 μ L, Downstream primer 1 μ L, Taq enzyme 0.5 μ l, template 0.5 μ L, ddH2O 38.0μL；
Response procedures is as follows: 94 DEG C of denaturations 4min；94 DEG C of sex change 1min, 54.8 DEG C Annealing 30s, 72 DEG C extend 60s, totally 30 circulations；72 DEG C extend 10min；
The linker DNA fragment that will obtain with the amplification of luciferase reporter gene support template PCR Cut glue to reclaim, and be soaked in 4 DEG C of preservations in TE；
S5.2 with the DNA label of linker DNA fragment and wash-out as template,
Upstream primer: GATGCACATATCGAGGTGG,
Downstream primer: AAGTGTCCACATACGCAC,
Use 25 μ l systems: 10 × buffer 2.5 μ L, dNTP 2 μ L, upstream primer 0.5 μ L, Downstream primer 0.5 μ L, Taq enzyme 0.2 μ L, template includes linker DNA fragment 14.5ng, The DNA label 10 μ L, ddH purified2O adds to 25 μ L；
Response procedures is as follows: 94 DEG C of denaturations 5min；94 DEG C of sex change 1min, 45 DEG C of companies Meeting 2min, 72 DEG C extend 50s, totally 5 circulations；94 DEG C of sex change 40s, 57 DEG C of annealing 30s, 72 DEG C extend 35s, totally 30 circulations；72 DEG C extend 10min；
Take 25 μ L pcr amplification products carry out electrophoresis with the Ago-Gel of 1.0% and use gel to become As instrument is observed and record result.
Wherein, in described step S2, prepare the magnetic particle of PEDV specific probe mark Specifically comprise the following steps that
S2.1 draws the unmarked magnetic bead that 50 μ L concentration are 10mg/mL and divides to centrifuge tube, magnetic From 2min, supernatant discarded；
S2.2 adds 100 μ L TE buffer solutions in centrifuge tube and shakes up, and Magneto separate sucks supernatant；
S2.3 repeats step S2.2 once；
The buffer solution of 100 μ L 100mM MES, pH 4.8 is added in centrifuge tube by S2.4, profit Magnetic bead is cleaned twice by the method for Magneto separate, then resuspended with 50 μ L above-mentioned MES buffer solution Magnetic bead；
S2.5 to 20 μ L 1.25M EDC-100mM MES, pH 4.8 solution in add 10 μ L concentration is the PEDV specific probe of 100 μm ol/L, adds 20 μ L H2O, makes Final volume reaches 50 μ L, and wherein, specific probe sequence is:
5’NH2-T15GCCTCAGAATAGTATGAGACGGCTTC；
Nucleic acid-EDC solution in step S2.5 is joined in the magnetic bead of step S2.4 by S2.6, After mixing more than incubated at room 3h or overnight, Magneto separate, supernatant discarded；
100 μ L TT buffer solutions are added in the magnetic bead after step S2.6 processes by S2.7, shake up, Hatch more than 30min, Magneto separate, supernatant discarded, wherein, TT buffer solution: 1M Tris-HCl, PH 8.0,0.1% Tween-20；
S2.8 repeats step S2.7 twice；
Magnetic bead is resuspended in 50 μ L TE buffer solutions to magnetic bead final concentration of 10 by S2.9 Mg/mL, saves backup in 4 DEG C.
Wherein, in described step S3, the nanogold particle of preparation specific signals probe mark Specifically comprise the following steps that
S3.1 take the nano-Au solution 1.0mL, 7500g of 10nmol/L, diameter 15nm from Heart 50min, removes supernatant, resuspended with 100 μ L aseptic deionized waters；
It is 100 μm ol/L specific probes that S3.2 adds 3 μ L concentration, specific signals probe Final concentration of 3 μm ol/L, incubated at room 16h, wherein, specific probe sequence is:
5’SH-T15CCTGTAGATAGTAGCAAGTGGCTCAGACCGTGATG GAATGGA；
S3.3 adds 1mol/L NaCl and 0.1mol/L, the PB of pH 7.2 in three times, extremely The final concentration of NaCl and PB is respectively 0.1mol/L and 10mmol/L, fully mixes, room Temperature hatches more than 48h；
S3.4 with 7500g eccentric cleaning 50min, cleans two by 0.01mol/L PBS solution Secondary, remove supernatant, the 0.01mol/L PBS with 100 μ L is resuspended, and 4 DEG C store for future use, Wherein, PBS solution: 135mM NaCl, 2.7mM KCl, 1.5mM KH2PO4,8mM K2HPO4,pH 7.2。
Summary of the invention technical scheme is improved:
1. the present invention encodes replicase protein gene ORF1a conservative region, warp based on PEDV Cross a series of screening criteria, as continuously less than three continuous print bases (A/T/G/C), GC Content more than 40% etc., is assessed its binding ability, hybridization difficulty and mismatch rate, be there are 16 Section nucleotide sequence, and through a series of probe screening tests, finally choose MMPs-p1 and AuNPs-Oligo2 is capture PEDV nucleic acid and the specific probe of enriching virus signal.
2., in the present invention, after fecal specimens is scrubbed, directly mixes with lysate and boil, excrement Just sample cracking supernatant can be directly added into functionalization magnetic bead and hybridization buffer, without warp Cross the purifying settling step of nucleic acid, substantially reduce the detection time, and further increase detection Sample size and susceptibility.
The most having declared invention is to examine for TGEV in suspected infection animal blood sample Surveying, the present invention can detect the existence of PEDV virus, detection from the fecal sample after processing Susceptibility can reach even more than blood sample.
In sum, one disclosed by the invention quickly detects based on nm of gold label amplifying technique The method of PEDV early infection, has the advantages that
1. for the design of PEDV specific dna probe, screen and optimize, this side can be improved Method specific, sensitiveness and repeatability.
2. need not move through extraction RNA from ight soil, then the tedious steps inverted.
3., after fecal sample cracking, lysate can directly carry out miscellaneous with the magnetic bead of probe functionalization Hand over reaction, it is not necessary to deposition and purification viral nucleic acid from lysate, step is the easiest.
4. infect in early days at PEDV, in the case of virus titer is low, can from ight soil quickly Enriching virus efficiently, and the most further by specific DNA label coupling Sample of nucleic acid signal is amplified by gold nano grain, needed for making sample of nucleic acid reach PCR detection Nucleic acid-templated concentration, have save time, the feature such as laborsaving, low cost.
Accompanying drawing explanation
Fig. 1 is the method quickly detecting PEDV early infection based on nm of gold label amplifying technique Flow chart；
Fig. 2 is quickly to detect the spirit of PEDV early infection method based on nm of gold label amplifying technique Basis of sensitivity analysis nucleic acid electrophoresis figure；
Fig. 3 is quickly to detect PEDV early infection method spy based on nm of gold label amplifying technique Specific analysis nucleic acid electrophoresis figure；
Fig. 4 is difference in functionality magnetic bead and nanogold particle capture nucleic acid ability contrast.
Detailed description of the invention
The present invention is according to being positioned at PEDV full-length genome, and the ORF1a gene of coding replicase sets Count specific probe, can more accurate response infectious virus particle levels.Although it addition, Method based on DNA label detection TGEV in early days blood sample is it has been established that but we Method can detect the PEDV in swine excrement, and this method is according to ORF1a gene conserved regions simultaneously Devise 16 sections of specific probes, by series of experiments thus screening and optimization specific By force, enriching virus and the amplification high 2 sections of nucleic acid probes of ability, improve the spy of the method further The opposite sex, sensitiveness and repeatability.Meanwhile, this technical method is carried out in the enterprising step of detection method Improve, can directly hybridize with functionalization magnetic bead, without through core after sample cracking The deposition and purification step of acid, substantially reduces the detection time.
Refer to Fig. 1, be quickly to detect PEDV sense in early days based on nm of gold label amplifying technique The method flow diagram of dye.
A kind of method quickly detecting PEDV early infection based on nm of gold label amplifying technique, Comprise the steps:
The design of S1 PEDV specific dna probe and screening:
According to being uploaded to National Center for Biotechnology Information (NCBI) the different strains of PEDV use DNASTAR or VECTOR NTI 9.0 Software carries out Multiple sequence alignments, divides according to the ORF1a gene conserved regions of coding replicase Not She Ji 16 sections of specific dna probes, designed nucleic acid probe is marked on magnetic respectively Particulate and nanogold particle, form magnetic bead (MMPs) and the nanogold particle of functionalization (AuNPs), and to it it is optimized and screens；
The screening of functionalization probe:
Wherein, 16 sections of functionalization magnetic bead probe sequences are:
16 sections of functional gold nanoparticles particle probe sequences are:
The screening process of functionalization magnetic bead is as follows: by 16 sections of designed specific dna probes Mark magnetic particle respectively, form MMPs-p1, p2, p3, p4, p5, p6, p7, p8, p9, p10, P11, p12, p13, p14, p15, p16, prepare the magnetic particle of PEDV specific probe mark Comprise the following steps that described, by the magnetic bead of functionalization with viral RNA in hybridization buffer Hatch 30min, Magneto separate, detect with the specific RT-PCR of PEDV for 40 DEG C；
The screening process of functionalized nano gold grain is as follows: by 16 sections of designed specific cores Acid probe marking nano gold grain respectively, forms AuNPs-oligo1, oligo2, oligo3, oligo4, oligo5,oligo6,oligo7,oligo8,oligo9,oligo10,oligo11,oligo12,oligo13, Oligo14, oligo15, oligo16, prepare the tool of the AuNPs of PEDV specific probe mark Body step is as described below, by the nanogold particle of different probe functionalization with viral RNA miscellaneous Friendship buffer solution hatches 40min for 50 DEG C, after centrifugation, specific with PEDV RT-PCR detects.
Test result indicate that: MMP-p1 and MMP-p2 has higher capture PEDV RNA Ability, the nanogold particle of oligo1 and oligo2 mark has and higher is combined with viral nucleic acid Ability.Therefore select MMP-p1 and oligo2-AuNPs for Porcine epidemic diarrhea virus UNDP-PCR detects.
The functionalization magnetic bead probe sequence i.e. determined is Probe 1:
5’NH2-T15GCCTCAGAATAGTATGAGACGGCTTC；(76-101)
The functional gold nanoparticles particle probe sequence determined is Oligo 2:
5’SH-T15CCTGTAGATAGTAGCAAGTGGCTCAGACCGTGATG GAATGGA；(626-651)
S2 prepare PEDV specific probe mark magnetic particle (MMPs):
Prepare following probe sequence mark magnetic particle:
5’NH2-T15GCCTCAGAATAGTATGAGACGGCTTC；
In the present embodiment, prepare the concrete steps of the magnetic particle of PEDV specific probe mark As follows:
S2.1 draws the unmarked magnetic bead that 50 μ L concentration are 10mg/mL and divides to centrifuge tube, magnetic From 2min, supernatant discarded；
S2.2 adds 100 μ L TE (1M Tris Buffer pH 8.0,1mM in centrifuge tube EDTA) buffer solution shakes up, Magneto separate, sucks supernatant；
S2.3 repeats step S2.2 once；
The buffer solution of 100 μ L 100mM MES, pH 4.8 is added in centrifuge tube by S2.4, profit Magnetic bead is cleaned twice by the method for Magneto separate, then resuspended with 50 μ L above-mentioned MES buffer solution Magnetic bead；
S2.5 to 20 μ L 1.25M EDC-100mM MES, pH 4.8 solution in add 10 μ L concentration is the PEDV specific probe of 100 μm ol/L, adds 20 μ L H2O, makes Final volume reaches 50 μ L, and wherein, specific probe sequence is:
5’NH2-T15GCCTCAGAATAGTATGAGACGGCTTC；
Nucleic acid-EDC solution in step S2.5 is joined in the magnetic bead of step S2.4 by S2.6, After mixing more than incubated at room 3h or overnight, Magneto separate, supernatant discarded；
S2.7 is by 100 μ L TT buffer solutions (1M Tris-HCl, pH 8.0,0.1% Tween-20) Add in the magnetic bead after step S2.6 processes, shake up, hatch more than 30min, Magneto separate, Supernatant discarded；
S2.8 repeats step S2.7 twice；
Magnetic bead is resuspended in 50 μ L TE buffer solutions to magnetic bead final concentration of 10 by S2.9 Mg/mL, saves backup (storage life is 6 months) in 4 DEG C.
S3 prepare specific signals probe mark nanogold particle (AuNPs):
Prepare following probe sequence mark nanogold particle:
5’SH-T15CCTGTAGATAGTAGCAAGTGGCTCAGACCGTGATG GAATGGA；
In the present embodiment, the concrete steps of the nanogold particle of preparation specific signals probe mark As follows:
S3.1 take the nano-Au solution 1.0mL, 7500g of 10nmol/L, diameter 15nm from Heart 50min, removes supernatant, resuspended with 100 μ L aseptic deionized waters；
It is 100 μm ol/L specific probes that S3.2 adds 3 μ L concentration, specific signals probe Final concentration of 3 μm ol/L, incubated at room 16h, wherein, specific probe sequence is:
5’SH-T15CCTGTAGATAGTAGCAAGTGGCTCAGACCGTGATG GAATGGA；
S3.3 adds 1mol/L NaCl and 0.1mol/L, the PB of pH 7.2 in three times, extremely The final concentration of NaCl and PB is respectively 0.1mol/L and 10mmol/L, fully mixes, room Temperature hatches more than 48h；
S3.4 0.01mol/L PBS (135mM NaCl, 2.7mM KCl, 1.5mM KH2PO4,and 8mM K2HPO4, pH 7.2) solution with 7500g eccentric cleaning 50min, Cleaning twice, remove supernatant, the 0.01mol/L PBS with 100 μ L is resuspended, 4 DEG C of storages Standby (storage life is 6 months).
Magnetic particle and the nanogold particle of S4 PEDV probe mark are sick with PEDV in ight soil The hybridization reaction of poison RNA and the wash-out of target dna label:
S4.1 takes fecal sample and mixes with PBS vortex, and 4,000g are centrifuged 10min goes Except ight soil residue；
S4.2 takes the PEDV fecal sample supernatant 500 μ L after process and 500 μ L lysates (10 Mmol/L Tris-HC1,1mmol/L EDTA, 15mmol/L NaCl, 0.5% SDS, PH 8.0) in 1.5mL centrifuge tube, boil 15min and make viral RNA discharge, 4 DEG C, 13400 G is centrifuged 5min, takes supernatant standby；
In S4.3 fecal sample lysate supernatant in step S4.2, after adding 2 μ L marks Magnetic particle MMPs-p1 and 110 μ L hybridization buffer (20 × SSC, 1% Tween-20 With 2% SDS) mixing, 40 DEG C of hybridization 30min, after adding 2 μ L marks after hybridization Nanogold particle AuNPs-Oligo2 mixing, 50 DEG C hybridization 40min, it is thus achieved that AuNPs-RNA-MMPs compound；
S4.4 takes 1mL TE buffer solution every time, and Magneto separate is washed 2-3 time, removes the hybridization of residual Buffer solution, unconjugated probe and DNA label；
S4.5 is with 100 newly configured for μ L DTT eluent (0.5mol/L DTT, 10mM Tris-HCl, 1mM EDTA, pH 7.5) with AuNPs-RNA-MMPs compound in room Act on 10min under temperature, after Magneto separate 3min, take supernatant；
S4.6 adds the NaAc of 1/10 volume and the anhydrous of 2 times of volumes at eluent supernatant Ethanol, precipitates 30min；
S4.7 is under the conditions of 4 DEG C, and 13400g is centrifuged 10min, abandons supernatant, adds 75% second Alcohol washes twice；
S4.8 is under the conditions of 4 DEG C, and 13400g is centrifuged 10min, abandons supernatant, natural air drying, Add 10 μ L ddH2O dissolving DNA label.
S5 PCR detection and the DNA label of PEDV specific bond:
S5.1 is with luciferase reporter gene carrier as template, and PCR amplification obtains linker DNA Fragment
Upstream primer F:TGGCATTCCGGTACTGTTGG (58-77),
Downstream primer R:AGGAGAATAGGGTTGGCACC (967-948),
Use 50 μ l systems: 10 × buffer 5 μ L, dNTP 4 μ L, upstream primer 1 μ L, Downstream primer 1 μ L, Taq enzyme 0.5 μ l, template 0.5 μ L, ddH2O 38.0μL；
Response procedures is as follows: 94 DEG C of denaturations 4min；94 DEG C of sex change 1min, 54.8 DEG C Annealing 30s, 72 DEG C extend 60s, totally 30 circulations；72 DEG C extend 10min；
The linker DNA fragment that will obtain with the amplification of luciferase reporter gene support template PCR Cut glue to reclaim, and be soaked in 4 DEG C of preservations in TE；
S5.2 with the DNA label of linker DNA fragment and wash-out as template,
Upstream primer: GATGCACATATCGAGGTGG,
Downstream primer: AAGTGTCCACATACGCAC,
Use 25 μ l systems: 10 × buffer 2.5 μ L, dNTP 2 μ L, upstream primer 0.5 μ L, Downstream primer 0.5 μ L, Taq enzyme 0.2 μ L, template includes linker DNA fragment 14.5ng, The DNA label 10 μ L, ddH purified2O adds to μ L；
Response procedures is as follows: 94 DEG C of denaturations 5min；94 DEG C of sex change 1min, 45 DEG C of companies Meeting 2min, 72 DEG C extend 50s, totally 5 circulations；94 DEG C of sex change 40s, 57 DEG C of annealing 30s, 72 DEG C extend 35s, totally 30 circulations；72 DEG C extend 10min；
Take the 25 μ L pcr amplification products Ago-Gel of 1.0% carry out electrophoresis and use solidifying Glue imager is observed and record result.
Refer to Fig. 1.PEDV early infection is quickly detected based on nm of gold label amplifying technique Method flow diagram.
Refer to Fig. 2.Fig. 2 embodies and quickly detects PEDV based on nm of gold label amplifying technique Early infection method sensitivity analysis.In figure: (A) is quick based on nm of gold label amplifying technique The fecal sample of detection doubling dilution；(B) excrement of conventional RT-PCR method detection doubling dilution Just sample.
By Fig. 2 obtain it is concluded that quickly detect PEDV based on nm of gold label amplifying technique The method sensitivity of early infection is 25 copies/g, and the sensitivity of conventional RT-PCR method is 104 Copy/g.This invention sensitivity is 400 times of conventional RT-PCR.
Refer to Fig. 3.Fig. 3 embodies and quickly detects PEDV based on nm of gold label amplifying technique Early infection method is specifically analyzed.In figure: Lane M:Trans 2K Plus DNA Marker；Lane 1:PEDV；Lane 2: health pig fecal sample；Lane 3:PCV2；lane 4:PPV；Lane 5:TGEV；Lane 6:PRRSV；Lane 7:CSFV.
By Fig. 3 obtain it is concluded that quickly detect PEDV based on nm of gold label amplifying technique The method of early infection has the strongest specific, with other viruses, cross reaction will not occur.
Refer to Fig. 4.Difference in functionality magnetic bead and nanogold particle capture nucleic acid ability contrast. In figure: (A) M:Trans 2K Plus DNA Marker；1:P1；2:P2；3:P3；4:P4；5:P5； 6:P6；7:P7；8:P8；9:P9；10:P10；11:P11；12:P12；13:P13；14:P14；15: P15；16:P16.(B)M:Trans 2K Plus DNA Marker；1:oligo1；2:oligo2； 3:oligo3；4:oligo4；5:oligo5；6:oligo6；7:oligo7；8:oligo8；9:oligo9；10: oligo10；11:oligo11；12:oligo12；13:oligo13；14:oligo14；15:oligo15； 16:oligo16。
It is concluded that by Fig. 4: P1 and Oligo2 is to have high degree of specificity, enrichment disease Poison ability is strong, be prone to amplification and 2 sections of probes of detection.
The present invention is directed to the design of PEDV specific dna probe, screen and optimize, improve this Method specific, sensitiveness and repeatability.Infecting in early days at PEDV, virus titer is low In the case of, can from ight soil enriching virus quickly and efficiently, and pass through the most further Sample of nucleic acid signal is amplified by the nanogold particle of specific DNA label coupling, makes nucleic acid sample Originally reach the nucleic acid-templated concentration needed for PCR detection, have save time, laborsaving, low cost etc. Feature.And can be with accurate response animal viral infections level.Additionally, after fecal sample cracking, Lysate can directly and the magnetic bead of probe functionalization carries out hybridization reaction, it is not necessary to from lysate Deposition and purification viral nucleic acid, step is the easiest.
The above is only the preferred embodiment of the present invention, it is noted that for this area Those of ordinary skill, under the premise of not departing from the present invention, it is also possible to if the present invention is made Doing and improve and supplement, these improve and supplement, and also should be regarded as protection scope of the present invention.

Claims (3)

1. the side quickly detecting PEDV early infection based on nm of gold label amplifying technique Method, it is characterised in that comprise the steps:
The design of S1PEDV specific dna probe and screening:
According to be uploaded to NCBI PEDV different strains use DNASTAR or VECTOR NTI 9.0 software carries out Multiple sequence alignments, according to the ORF1a of coding replicase 16 sections of specific dna probes of gene conserved regions design, by designed nucleic acid probe respectively It is marked on magnetic particle and nanogold particle, forms magnetic bead and the nanogold particle of functionalization, and It is optimized and screens；
The screening of functionalization probe:
16 sections of functionalization magnetic bead probe sequences are:
16 sections of functional gold nanoparticles particle probe sequences are:
The screening process of functionalization magnetic bead is as follows: by 16 sections of designed specific dna probes Mark magnetic particle respectively, form MMPs-p1, p2, p3, p4, p5, P6, p7, p8, p9, p10, p11, p12, p13, p14, p15, p16, preparation PEDV specific probe mark Magnetic particle comprise the following steps that described, by the magnetic bead of functionalization with viral RNA miscellaneous Friendship buffer solution hatches 30min, Magneto separate for 40 DEG C, examines with the specific RT-PCR of PEDV Survey；
The screening process of functionalized nano gold grain is as follows: by 16 sections of designed specific cores Acid probe marking nano gold grain respectively, forms AuNPs-oligo1, oligo2, oligo3, oligo4, oligo5,oligo6,oligo7,oligo8,oligo9,oligo10,oligo11oligo12,oligo13, Oligo14, oligo15, oligo16, prepare the tool of the AuNPs of PEDV specific probe mark Body step is as described below, by the nanogold particle of different probe functionalization with viral RNA miscellaneous Friendship buffer solution hatches 40min for 50 DEG C, after centrifugation, specific with PEDV RT-PCR detects；
The functionalization magnetic bead probe sequence determined after screening is Probe 1:
5’NH2-T15GCCTCAGAATAGTATGAGACGGCTTC；(76-101)
The functional gold nanoparticles particle probe sequence determined after screening is Oligo 2:
5’SH-T15CCTGTAGATAGTAGCAAGTGGCTCAGACCGTGATG GAATGGA；(626-651)
S2 prepare PEDV specific probe mark magnetic particle:
Specific probe sequence is:
5’NH2-T15GCCTCAGAATAGTATGAGACGGCTTC；
S3 prepare specific signals probe mark nanogold particle:
Specific probe sequence is:
5’SH-T15CCTGTAGATAGTAGCAAGTGGCTCAGACCGTGATG GAATGGA；
In the magnetic particle of S4PEDV probe mark and nanogold particle and fecal sample The hybridization reaction of PEDV viral RNA and the wash-out of target dna label:
S4.1 takes fecal sample and mixes with PBS vortex, and 4,000g are centrifuged 10min goes Except ight soil residue；
S4.2 takes the PEDV fecal sample supernatant 500 μ L after process and 500 μ L lysates extremely In 1.5mL centrifuge tube, boiling 15min and make viral RNA discharge, 4 DEG C, 13400g is centrifuged 5min, takes supernatant standby, and wherein, lysate is: 10mmol/L Tris-HC1,1mmol/L EDTA, 15mmol/L NaCl, 0.5%SDS, pH 8.0；
In S4.3 fecal sample lysate supernatant in step S4.2, after adding 2 μ L marks Magnetic particle MMPs-p1 and 110 μ L hybridization buffers mixing, 40 DEG C hybridization 30min, Add the nanogold particle AuNPs-Oligo 2 after 2 μ L marks after hybridization to mix, 50 DEG C Hybridization 40min, it is thus achieved that AuNPs-RNA-MMPs compound；Wherein, lysate supernatant is The viral nucleic acid obtained in previous step S4.2, whole sequence is uncertain, but has PEDV The conserved sequence of virus；Hybridization buffer: 20 × SSC, 1%Tween-20 and 2%SDS；
S4.4 takes 1mL TE buffer solution every time, and Magneto separate is washed 2-3 time, removes the hybridization of residual Buffer solution, unconjugated probe and DNA label；
S4.5 is combined with AuNPs-RNA-MMPs with 100 DTT eluents newly configured for μ L Thing at room temperature acts on 10min, takes supernatant after Magneto separate 3min, and wherein, DTT elutes Liquid is: 0.5mol/L DTT, 10mM Tris-HCl, 1mM EDTA, pH 7.5；
S4.6 adds NaAc and the anhydrous second of 2 times of volumes of 1/10 volume at elution supernatant Alcohol, precipitates 30min；
S4.7 is under the conditions of 4 DEG C, and 13400g is centrifuged 10min, abandons supernatant, adds 75% second Alcohol washes twice；
S4.8 is under the conditions of 4 DEG C, and 13400g is centrifuged 10min, abandons supernatant, natural air drying, adds Enter 10 μ L ddH2O dissolving DNA label；
S5PCR detection and the DNA label of PEDV specific bond:
S5.1 is with luciferase reporter gene carrier as template, and PCR amplification obtains linker DNA Fragment
Upstream primer F:TGGCATTCCGGTACTGTTGG,
Downstream primer R:AGGAGAATAGGGTTGGCACC,
Use 50 μ l systems: 10 × buffer 5 μ L, dNTP 4 μ L, upstream primer 1 μ L, Downstream primer 1 μ L, Taq enzyme 0.5 μ l, template 0.5 μ L, ddH2O 38.0μL；
Response procedures is as follows: 94 DEG C of denaturations 4min；94 DEG C of sex change 1min, 54.8 DEG C Annealing 30s, 72 DEG C extend 60s, totally 30 circulations；72 DEG C extend 10min；
The linker DNA fragment that will obtain with the amplification of luciferase reporter gene support template PCR Cut glue to reclaim, and be soaked in 4 DEG C of preservations in TE；
S5.2 with the DNA label of linker DNA fragment and wash-out as template,
Upstream primer: GATGCACATATCGAGGTGG,
Downstream primer: AAGTGTCCACATACGCAC,
Use 25 μ l systems: 10 × buffer 2.5 μ L, dNTP 2 μ L, upstream primer 0.5 μ L, Downstream primer 0.5 μ L, Taq enzyme 0.2 μ L, template includes linker DNA fragment 14.5ng, The DNA label 10 μ L, ddH purified2O adds to 25 μ L；
Response procedures is as follows: 94 DEG C of denaturations 5min；94 DEG C of sex change 1min, 45 DEG C of companies Meeting 2min, 72 DEG C extend 50s, totally 5 circulations；94 DEG C of sex change 40s, 57 DEG C of annealing 30s, 72 DEG C extend 35s, totally 30 circulations；72 DEG C extend 10min；
Take the 25 μ L pcr amplification products Ago-Gel of 1.0% carry out electrophoresis and use solidifying Glue imager is observed and record result.
One the most according to claim 1 quickly detects based on nm of gold label amplifying technique The method of PEDV early infection, it is characterised in that in described step S2, prepares PEDV Specifically comprising the following steps that of the magnetic particle of specific probe mark
S2.1 draws the unmarked magnetic bead that 50 μ L concentration are 10mg/mL and divides to centrifuge tube, magnetic From 2min, supernatant discarded；
S2.2 adds 100 μ L TE buffer solutions in centrifuge tube and shakes up, and Magneto separate sucks supernatant；
S2.3 repeats step S2.2 once；
The buffer solution of 100 μ L 100mM MES, pH 4.8 is added in centrifuge tube by S2.4, profit Magnetic bead is cleaned twice by the method for Magneto separate, then resuspended with 50 μ L above-mentioned MES buffer solution Magnetic bead；
S2.5 to 20 μ L 1.25M EDC-100mM MES, pH 4.8 solution in add 10 μ L concentration is the PEDV specific probe of 100 μm ol/L, adds 20 μ L H2O, makes Final volume reaches 50 μ L, and wherein, specific probe sequence is:
5’NH2-T15GCCTCAGAATAGTATGAGACGGCTTC；
Nucleic acid-EDC solution in step S2.5 is joined in the magnetic bead of step S2.4 by S2.6, After mixing more than incubated at room 3h or overnight, Magneto separate, supernatant discarded；
100 μ L TT buffer solutions are added in the magnetic bead after step S2.6 processes by S2.7, shake up, Hatch more than 30min, Magneto separate, supernatant discarded, wherein, TT buffer solution: 1M Tris-HCl, PH 8.0,0.1%Tween-20；
S2.8 repeats step S2.7 twice；
Magnetic bead is resuspended in 50 μ L TE buffer solutions final concentration of to magnetic bead by S2.9 10mg/mL, saves backup in 4 DEG C.
One the most according to claim 1 quickly detects based on nm of gold label amplifying technique The method of PEDV early infection, it is characterised in that in described step S3, preparation is specific Specifically comprising the following steps that of the nanogold particle of signal probe mark
S3.1 take the nano-Au solution 1.0mL, 7500g of 10nmol/L, diameter 15nm from Heart 50min, removes supernatant, resuspended with 100 μ L aseptic deionized waters；
It is 100 μm ol/L specific probes that S3.2 adds 3 μ L concentration, specific signals probe Final concentration of 3 μm ol/L, incubated at room 16h, wherein, specific probe sequence is:
5’SH-T15CCTGTAGATAGTAGCAAGTGGCTCAGACCGTGATG GAATGGA；
S3.3 adds 1mol/L NaCl and 0.1mol/L, the PB of pH 7.2 in three times, extremely The final concentration of NaCl and PB is respectively 0.1mol/L and 10mmol/L, fully mixes, room Temperature hatches more than 48h；
S3.4 with 7500g eccentric cleaning 50min, cleans two by 0.01mol/L PBS solution Secondary, remove supernatant, the 0.01mol/L PBS with 100 μ L is resuspended, and 4 DEG C store for future use, Wherein, PBS solution: 135mM NaCl, 2.7mM KCl, 1.5mM KH2PO4,8mM K2HPO4,pH 7.2。
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