CN116121452A - RT-RPA fluorescent establishment method based on porcine reproductive and respiratory syndrome ORF2a gene - Google Patents
RT-RPA fluorescent establishment method based on porcine reproductive and respiratory syndrome ORF2a gene Download PDFInfo
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Abstract
The invention relates to a method for establishing RT-RPA fluorescence based on porcine reproductive and respiratory syndrome ORF2a gene, which adopts the following primer sequences: forward primer: AGCTTCACGATTTTCAGCAATGGCT; reverse primer (5 '-3'): ACGTAGCATTGGAATTCGCAACCAC; probes (5 '-3'): 5' -AATAGCTGTACATTCCTCCATATTTTCT (HEX-dT) C (THF) G (BHQ 1-dT) TGCAGCTTCTTGT-C3Spacer. By using the primer designed by the invention, after RNA is reversely transcribed into cDNA by reverse transcriptase M-MLV, a target product is amplified by a recombinase mediated amplification (RPA) technology, and a real-time fluorescent quantitative PCR instrument is combined, so that the rapid and accurate detection of PRRSV can be realized. By using the primer, if the PRRSV exists in the sample to be detected, an amplification curve appears in the detection result, and if the PRRSV does not exist in the sample to be detected, the detection result does not peak; the method has the advantages of rapid operation, strong specificity and high sensitivity.
Description
Technical Field
The invention relates to the technical field of detection, in particular to an RT-RPA fluorescent type establishment method based on porcine reproductive and respiratory syndrome ORF2a genes.
Background
The clinical symptoms of porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) are manifested by respiratory disorders of piglets, reproductive disorders of sows, pulmonary bleeding, immunosuppression, etc. Porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV) is a single positive strand RNA virus belonging to the genus arterivirus of the family arterividae, order monoviridae, and is classified into european type (PRRSV-1) and american type (PRRSV-2) according to genotype; the PRRSV full-length genome is approximately 15kb, comprising 10 Open Reading Frames (ORFs), encoding 14 nonstructural proteins and 7 structural proteins. The virus particles are spherical or elliptic with the diameter of 50-80nm, have a capsule membrane and have microvilli-like protrusions on the surface.
The early detection and prevention of PRRS is very critical, and the construction of an RT-RPA fluorescent method capable of rapidly detecting PRRSV has important significance for the detection and prevention of PRRS.
RT-RPA is a detection method combining a recombinase-mediated amplification technology with reverse transcriptase M-MLV and a fluorescent quantitative PCR instrument, RPA is an isothermal amplification technology for amplifying a target sequence under isothermal conditions by using a recombinase-mediated polymerase, RNA is inverted into cDNA under the action of the reverse transcriptase M-MLV, and DNA/cDNA can be amplified in a short time for 10 9 -10 12 The fluorescent groups carried by the amplified products release fluorescent signals, amplification curves with different degrees are displayed according to the concentration of the amplified products and the intensity of the fluorescent signals, and the amplification curves are judged to be positive in 15min from peak to peak. The RPA isothermal amplification technology has become a hot spot in various industries, and the fluorescent quantitative PCR instrument is widely applied in the fields of food safety monitoring, human and animal disease control, plant disease diagnosis and the like, and a plurality of pig farms are also provided with the fluorescent quantitative PCR instrument.
Because PRRSV ORF2a can code a secondary structural protein GP2a, the protein plays a key role in virus invasion binding to a receptor, forward and reverse primers and probes are designed according to a relatively conserved region of the ORF2a sequence, and an RT-RPA fluorescent detection method of the PRRSV ORF2a sequence is constructed, so that the PRRS can be monitored, prevented and controlled rapidly and efficiently, and the health of a pig population and the stable development of the pig breeding industry can be maintained.
Disclosure of Invention
The invention relates to an RT-RPA fluorescent primer and a probe for detecting PRRSV, which comprise an RPA-ORF2a primer pair and an RPA-ORF2a probe, and are characterized in that the nucleotide sequence of an upstream primer of the RPA-ORF2a primer pair is as follows: 5'-AGCTTCACGATTTTCAGCAATGGCT-3', the nucleotide sequence of the downstream primer is: 5'-ACGTAGCATTGGAATTCGCAACCAC-3';
the RPA-ORF2a probe is,
5'-AATAGCTGTACATTCCTCCATATTTTCT(HEX-dT)C(THF)G(BHQ1-dT)TGCAGCTTCTTGT-C3 Spacer。
the invention also provides an RT-RPA amplification reagent for detecting PRRSV, which comprises the primer and the probe.
Further, the RT-RPA amplification reagents also include PrimerFree RehydrationBuffer, DEPC treated water and DNA/RNA samples.
The invention also provides an application of the RT-RPA amplification reagent for detecting PRRSV in detecting PRRSV, which comprises an RT-RPA amplification method,
s1, constructing a 51-mul amplification system, firstly adding PrimerFree Rehydrolation buffer29.5 mul, an upstream primer (10 mu M) 2.1 mul, a downstream primer (10 mu M) 2.1 mul, a probe (10 mu M) 0.6 mul, reverse transcriptase M-MLV 1 mul and DEPC treatment water to 43.5 mul, and adding all the solutions into a detection unit tube with reaction dry powder;
s2, adding 5.0 μl of the treated DNA/RNA sample into the detection unit tube;
s3, adding 2.5 mu l of MgOAc to the tube cover of the detection unit, covering the tube cover (transparent tube cover), reversing upside down, slightly oscillating, fully mixing for 5-6 times, and centrifuging at a low speed for 10S;
s4, placing the detection unit tube into a fluorescence quantitative PCR instrument for fluorescence detection.
Further, the method for constructing the positive plasmid comprises the following steps:
a1, amplifying a target gene: cloning and amplifying ORF2a gene by taking PRRSV full-length plasmid pOK-XH-GD as a template,
forward primer F: TAGTaagcttATGAAATGGGGTCCATG;
reverse primer R: TAGTctcgagCCATGAGTTCAAAAGAAAAGT, PCR amplification was performed with an amplification system of 100. Mu.l: 50. Mu.l of high-fidelity enzyme (PrimeSTAR Max Premix (2X)), 0.5. Mu.l of each of the upstream and downstream primers, ddH 2 O47 μl, DNA 2 μl, melting 94 ℃, annealing 57 ℃, extension 72 ℃,30 cycles, electrophoresis of PCR amplification products on a 1% agarose gel (containing EB nucleic acid dye), electrophoresis in a1 xTAE buffer at 100V for 1h, and recording the electrophoresis results in a gel imaging system.
A2, glue recovery:
a2.1, cutting and weighing the gel containing the target strip, adding XP2 Binding Buffer in a proportion of 1g/ml, and standing at 50-60 ℃ for 7min until the gel is completely melted;
a2.2, sucking 700 mul to a filter column after melting, centrifuging at 12000rpm for 1min, discarding the liquid in a collecting pipe, and repeating the step B2.2 until the liquid is added;
a2.3, 300. Mu.l XP2 Buffer was added and centrifuged at 12000rpm for 1min, and the liquid in the collection tube was discarded.
A2.4, adding 700 μl of SPW Buffer, centrifuging at 12000rpm for 1min, and discarding the liquid in the collection tube;
a2.5 centrifuging the empty filter column at 12000rpm12000 rpm for 2min, placing the filter column in a sterile 1.5ml centrifuge tube, adding preheated Elution Buffer or ddH 2 O15-30 μl to the center of the filter membrane, standing at room temperature for 2min, centrifuging at 12000rpm for 1min, measuring concentration, and preserving at-20deg.C.
A3, connection: the gel recovery product was ligated with the vector pMD 18-T16 overnight, 10. Mu.l system: 1 μl pMD18-T (0.03 pmol/50 ng), DNA to vector molar ratio 1: 2-10, adding sterilized water to 5 μl,5 μl Solution I to obtain a ligation product;
a4, conversion: ligation product with DH-5α1:10 parts of the strain are added into a sterilizing centrifuge tube for ice bath for 30min at 42 ℃ for 90s, then ice bath is carried out for 2min, 500 mu l of antibiotic-free LB liquid medium is added, the mixture is placed in a shaking table for 37 ℃ for culturing 45min at 160rpm, the mixture is centrifuged at 3000rpm for 1min, 400 mu l of supernatant is discarded, the rest bacterial liquid is coated on LB solid medium containing ampicillin, and the mixture is cultured for 16h at 37 ℃;
a5, extracting plasmids: picking up single colony with gun head, throwing gun head into LB liquid culture medium containing ampicillin, placing in a shaking table at 37deg.C, treating at 200rpm for 6 hr, sucking 10 μl of bacterial liquid, adding into 10ml LB liquid culture medium containing ampicillin, culturing for two times, treating at 37deg.C at 200rpm for 16 hr, and extracting plasmid, wherein the steps are as follows:
a5.1, sucking the bacterial liquid into a 1.5ml sterile centrifuge tube at 12000rpm for 1min, and discarding the supernatant;
a5.2, adding 250 μl Buffer P1 (RNase A is added), blowing, mixing, precipitating, and fully lysing the bacterial liquid;
a5.3, adding 250 μl of BufferP2, gently reversing for 8-10 times (the solution becomes viscous and transparent, and the cover is opened for drawing, and the step is controlled within 2-3 min);
a5.4, adding 350 μl Buffer P3, immediately and gently reversing upside down for 8-10 times, and centrifuging at 12000rpm for 10min;
a5.5, placing the adsorption column in a collecting pipe, transferring the supernatant obtained in the steps into the adsorption column, and centrifuging at 12000rpm for 1min without sucking sediment;
a5.6, adding 600 μl of BufferPW2 (absolute ethanol is added) into the adsorption column, centrifuging at 12000rpm for 1min, and discarding the solution;
a5.7, repeating the step B5.6;
a5.8, 12000rpm centrifugal 1min;
a5.9 placing the adsorption column in a new sterile centrifuge tube, adding 30-100 μl of preheated Elution Buffer or ddH 2 O to the center of the membrane, standing at room temperature for 2min, centrifuging at 12000rpm for 1min, discarding the adsorption column, testing the concentration of DNA product, and preserving at-20deg.C to obtain positive plasmid.
Further, the method further comprises the steps of obtaining a plurality of positive particle test samples by diluting positive plasmids, then testing the positive particle test samples by adopting an RT-RPA amplification method, and judging the sensitivity of the RT-RPA amplification method.
Further, it also includes obtaining the strain as a test sample, and then performing a specificity test by RT-RPA amplification method.
In the PRRSV detection process, it includes:
1. extracting PRRSV genome RNA of a sample to be detected by using a nucleic acid extraction kit and combining a Vazyme full-automatic nucleic acid extractor, wherein the extraction steps are as follows:
1.1 processing samples: adding 1ml of sterile PBS buffer solution into the tissue sample, and grinding for later use; adding 1ml of sterile PBS buffer solution into a swab sample, rinsing the swab in a centrifuge tube, and discarding the liquid sample for later use; blood samples can be used directly for nucleic acid extraction. The volume of sample to be extracted was 200. Mu.l, and if not enough PBS was added to 200. Mu.l.
1.2 taking out the pre-packaged reagent, reversely and uniformly mixing for several times to enable the magnetic beads to be resuspended, lightly throwing the pore plate, enabling the reagent and the magnetic beads to be concentrated at the bottom of the pore plate, confirming the direction of the plate before use and carefully tearing off the aluminum foil sealing film (avoiding vibration when tearing off the sealing film and preventing liquid from splashing).
1.3 automated instrument extraction: 200. Mu.l of sample and 20. Mu.l of protease K solution were added to the wells of column 1 and column 7 of the 96-well plate reagent; and (3) placing the 96 deep hole plate into a nucleic acid extraction instrument, loading the nucleic acid extraction instrument into a magnetic rod sleeve, and automatically extracting according to a program.
2. And (3) carrying out isothermal amplification by taking the DNA/RNA of the sample to be detected extracted in the step (1) as a template and the specific primer RPA-ORF2a as an amplification primer.
Amplification system (51 μl): primer Free Rehydration Buffer 29.5.5. Mu.l each of forward and reverse primer (10. Mu.M), 0.6. Mu.l probe (10. Mu.M), 1. Mu.l reverse transcriptase M-MLV, 5. Mu.l template DNA/RNA, 3. Mu.l DEPC treated water, 2.5. Mu.l MgOAc.
Amplification conditions: the temperature is maintained at 39 ℃, the preheating is carried out for 30s, 39 cycles are carried out, and each cycle is 30s.
Sample adding operation: primer Free Rehydration Buffer, forward and reverse primers, probes, templates and DEPC treated water are mixed uniformly, then added into the reaction dry powder, mixed uniformly, added with MgOAc, mixed uniformly by shaking and centrifuged for 10s, and put into a fluorescent quantitative PCR instrument for amplification.
3. And (3) judging a detection result: after the amplification reaction is finished, the peak starting within 15min is an effective positive result according to the amplification curve in the fluorescent quantitative PCR instrument.
The invention has the advantages that: because PRRSV ORF2a can code a secondary structural protein GP2a, the protein plays a key role in virus invasion binding to a receptor, forward and reverse primers and probes are designed according to a relatively conserved region of the ORF2a sequence, and an RT-RPA fluorescent detection method of the PRRSV ORF2a sequence is constructed, so that PRRS can be monitored, prevented and controlled rapidly and efficiently, and the health of a pig population and the stable development of pig farming are maintained.
The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments, as illustrated in the accompanying drawings.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is an electrophoretically imaged picture of the amplified product of PRRSV ORF2a gene.
FIG. 2 is a graph of the detection results of a sensitivity experiment.
FIG. 3 is a diagram showing the results of specificity experiments
Fig. 4 is a partial enlarged view of fig. 3.
FIG. 5 is a nucleotide sequence of an amplified product of PRRSV ORF2a gene.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In a preferred embodiment of the invention, the method for establishing the RT-RPA fluorescent type based on the ORF2a gene of the porcine reproductive and respiratory syndrome comprises the following steps:
1. material
Strains: PRRSV NADC30 pedigree GD strain, PRRSV vaccine strain TJM-F92 (highly pathogenic strain), vaccine strain R98 (classical strain), CSFV vaccine strain (rabbit source), PRV vaccine strain Bartha-K61 strain, PEDV LJX strain, TGEV Purde strain is preserved by Hainan national institute of livestock and veterinary laboratory, hainan province tropical breeding and epidemic disease research emphasis laboratory.
The main reagent comprises: the primer is synthesized by TaKaRa from the division of biological engineering (Shanghai); high fidelity enzyme PrimeSTAR Max Premix (2×) (TaKaRa), pMD18-T (TaKaRa), e.coll DH5 a components Cells (9057) (TaKaRa), dNTP mix (4030Q) (TaKaRa), recombinant RNase Inhibitor (2313Q) (TaKaRa), reverse Transcriptase M-MLV (2641Q) (TaKaRa), DL 2,000dna Marker (3427A) (TaKaRa), gel purification kit (Omega), nucleic acid extraction kit (Vazyme), plasmid extraction kit (Vayzme), RPA-exo (twist dx).
2. Constructing positive plasmids:
2.1, amplifying target genes: the ORF2a gene was amplified by PCR cloning using forward and reverse primers (F: TAGTaagcttATGAAATGGGGTCCATG; R: TAGTctcgagCCATGAGTTCAAAAGAAAAGT) using PRRSV full-length plasmid pOK-XH-GD stored in laboratory at the Proc. Of agricultural sciences, hainan province as a template, and 100. Mu.l of the amplification system: 50. Mu.l of high-fidelity enzyme (PrimeSTAR Max Premix (2X)), 0.5. Mu.l of each of the upstream and downstream primers (10. Mu.M), ddH 2 O47. Mu.l, DNA 2. Mu.l, melting 94 ℃, annealing 57 ℃, extension 72 ℃,30 cycles. The PCR amplified product was electrophoresed on a 1% agarose gel (containing EB nucleic acid dye) at 100V in 1 XTAE buffer for 1h, and the result of electrophoresis was recorded by photographing in a gel imaging system as shown in FIG. 1, and the amplified product had a size of 770bp (nucleotide sequence FIG. 5).
2.2 glue recovery: (1) Cutting and weighing the gel containing the target strip, adding XP2 Binding Buffer at a ratio of 1g/ml, and standing at 50-60deg.C for 7min until the gel is completely melted;
(2) Sucking 700 μl to a filter column after melting, centrifuging at 12000rpm for 1min, discarding the liquid in the collecting tube, and repeating the step (2) until the liquid is completely added;
(3) Mu.l XP2 Buffer was added and centrifuged at 12000rpm for 1min and the collection tube was discarded.
(4) Adding 700 μl of SPW Buffer, centrifuging at 12000rpm for 1min, and discarding the liquid in the collection tube;
(5) Centrifuging the empty filter column at 12000rpm12000 rpm for 2min, placing the filter column in a sterile 1.5ml centrifuge tube, adding preheated EluthBuffer or ddH 2 O15-30 μl to the center of the filter membrane, standing at room temperature for 2min, centrifuging at 12000rpm for 1min, measuring concentration, and preserving at-20deg.C.
2.3 connection: the gel recovery product was ligated with the vector pMD 18-T16 overnight, 10. Mu.l system: 1. Mu.l pMD18-T (0.03 pmol/50 ng), DNA to vector molar ratio 1: 2-10, sterilized water is added to 5. Mu.l, 5. Mu.l Solution I.
2.4 conversion: ligation product with DH-5α1:10 portions are added into a sterilizing centrifuge tube for ice bath 30min at 42 ℃ for 90s, ice bath is carried out for 2min, 500 mu l of LB liquid medium without antibiotics is added, the mixture is placed on a shaking table at 37 ℃ for culturing 45min at 160rpm, centrifugation is carried out at 3000rpm for 1min, 400 mu l of supernatant is discarded, the rest bacterial liquid is coated on LB solid medium containing ampicillin, and the culture is carried out for 16h at 37 ℃.
2.5 plasmid extraction: picking up single colony with gun head, throwing gun head into LB liquid culture medium containing ampicillin, placing in a shaking table at 37deg.C, treating at 200rpm for 6 hr, sucking 10 μl of bacterial liquid, adding into 10ml LB liquid culture medium containing ampicillin, culturing for two times, treating at 37deg.C at 200rpm for 16 hr, and extracting plasmid, wherein the steps are as follows:
(1) Sucking the bacterial liquid into a 1.5ml sterile centrifuge tube at 12000rpm for 1min, and discarding the supernatant;
(2) Adding 250 μl of BufferP1 (RNase A is added), blowing, mixing, precipitating, and fully lysing the bacterial liquid;
(3) Adding 250 μl Buffer P2, gently reversing for 8-10 times (the solution becomes viscous and transparent, and the cover is opened for drawing, and the step is controlled within 2-3 min);
(4) Adding 350 μl Buffer P3, immediately gently reversing upside down for 8-10 times, and centrifuging at 12000rpm for 10min to obtain white flocculent precipitate;
(5) Placing the adsorption column in a collecting pipe, transferring the supernatant of the steps into the adsorption column without sucking sediment, centrifuging at 12000rpm for 1min, and discarding the liquid;
(6) Adding 600 μl of BufferPW2 (anhydrous ethanol is added) into the adsorption column, centrifuging at 12000rpm for 1min, and discarding the solution;
(7) Repeating step (6);
(8) Centrifuging at 12000rpm for 1min;
placing the adsorption column in a new sterile centrifuge tube, adding 30-100 μl of preheated Elutation Buffer or ddH 2 O was placed in the center of the membrane, left standing at room temperature for 2min, centrifuged at 12000rpm for 1min, the adsorption column was discarded, and the DNA product was stored at-20℃after concentration measurement.
2.6 calculation of positive plasmid copy number
According to the formula:
and calculating the copy number of the positive plasmid, wherein the plasmid length is the total length of the vector and the target gene fragment.
3. Sensitivity test
3.1 dilution of positive plasmid according to copy number
The positive plasmid copy number was calculated to be 1.6X10 according to the above formula 11 Ten times dilution by 1.6X10 10 ～1.6×10 1 Selecting copy number 1.6X10 7 ～1.6×10 1 As a sensitivity test sample.
3.2RPA isothermal amplification
3.2.1 amplification System 51. Mu.l, primerFree RehydrologicBuffer 29.5. Mu.l, upstream primer (10. Mu.M) 2.1. Mu.l, downstream primer (10. Mu.M) 2.1. Mu.l, probe (10. Mu.M) 0.6. Mu.l, reverse transcriptase M-MLV 1. Mu.l and DEPC treated water were added to 43.5. Mu.l, and all of the above solutions were added to a detection cell tube with a reaction dry powder;
3.2.2 adding 5.0. Mu.l of the treated DNA/RNA sample to the reaction cell tube;
3.2.3 adding 2.5 mu l of MgOAc to the tube cover of the detection unit, covering the tube cover (transparent tube cover), reversing upside down, slightly oscillating, fully mixing for 5-6 times, and centrifuging at low speed for 10s;
3.2.4 the detection cell tube was placed in a fluorescent quantitative PCR instrument.
3.3 sensitivity test results
Ten times diluted sample copy number was chosen to be 1.6X10 7 ～1.6×10 1 A total of 7 samples were amplified and tested as described above. The results are shown in FIG. 2 (note: copy number 1.6X10 from 1 to 7) 1 Up to 1.6X10 7 The lowest detectable copy number of 1.6X10) 1 Is a sample of (a).
4. Specificity experiments
The PRRSVNADC pedigree GD strain, PRRSV vaccine strain TJM-F92 (homogenic virus strain), vaccine strain R98 (classical strain), CSFV vaccine strain (rabbit source), PRV vaccine strain Bartha-K61 strain, PEDV LJX strain, TGEV Purde strain, and 7 strains were selected as detection samples, and the detection results were shown in FIG. 3, and the results showed that the samples were specific to PRRSV only.
RT-RPA fluorescence detection of clinical samples
160 parts of pig farm serum samples from Haikou City, lin Gao county, qionzhong county and the like of Hainan province, 63 parts of PCR detection positive samples, 97 parts of PCR detection negative samples, 65 parts of RPA fluorescence quantification method detection positive samples, 95 parts of RPA fluorescence quantification method sensitivity of the negative samples is 100%, and the specificity is 97.9%.
Table 1 comparison table of RPA fluorescence method and PCR method for detecting clinical specimens
The principles and embodiments of the present invention have been described in detail with reference to specific examples, which are provided to facilitate understanding of the method and core ideas of the present invention; meanwhile, as those skilled in the art will have variations in the specific embodiments and application scope in accordance with the ideas of the present invention, the present description should not be construed as limiting the present invention in view of the above.
Claims (7)
1. An RT-RPA fluorescent primer and probe for detecting PRRSV, comprising an RPA-ORF2a primer pair and an RPA-ORF2a probe, wherein the nucleotide sequence of the upstream primer of the RPA-ORF2a primer pair is: 5'-AGCTTCACGATTTTCAGCAATGGCT-3' the number of the individual pieces of the plastic,
the nucleotide sequence of the downstream primer is as follows: 5'-ACGTAGCATTGGAATTCGCAACCAC-3', the amplification length is 113bp of the length of the amplification product;
the RPA-ORF2a probe is,
5'-AATAGCTGTACATTCCTCCATATTTTCT(HEX-dT)C(THF)G(BHQ1-dT)TGCAGCTTCTTGT-C3Spacer-3'。
2. an RT-RPA amplification reagent for detecting PRRSV, wherein the RT-RPA amplification reagent comprises the primer and probe of claim 1.
3. The RT-RPA amplification reagent for detecting PRRSV according to claim 2, wherein: the RT-RPA amplification reagents also included PrimerFree RehydrationBuffer, sterile water, reverse transcriptase M-MLV and DNA/RNA samples.
4. Use of an RT-RPA amplification reagent for detecting PRRSV according to claims 1-3, characterized in that: comprising an RT-RPA amplification method,
s1, constructing a 510 μl amplification system, firstly adding PrimerFree Rehydrolation buffer29.5 μl, upstream primer (10 μM) 2.1 μl, downstream primer (10 μM) 2.1 μl, probe (10 μM) 0.6 μl, reverse transcriptase M-MLV 1 μl and DEPC treatment water to 43.5 μl, and adding all the above solutions into a detection unit tube with a reaction dry powder;
s2, adding 5.0 μl of the treated DNA/RNA sample into the detection unit tube;
s3, adding 2.5 mu l of MgOAc to the tube cover of the detection unit, covering the transparent tube cover, reversing upside down, slightly oscillating, fully mixing for 5-6 times, and centrifuging at a low speed for 10S;
s4, placing the detection unit tube into a fluorescence quantitative PCR instrument for fluorescence detection.
5. The use of the RT-RPA amplification reagent for detecting PRRSV according to claim 4, further comprising constructing a positive plasmid, wherein the construction method of the positive plasmid comprises:
a1, amplifying a target gene: cloning and amplifying ORF2a gene by taking PRRSV full-length plasmid pOK-XH-GD as a template,
forward primer F: TAGTaagcttATGAAATGGGGTCCATG;
reverse primer R: TAGTctcgagCCATGAGTTCAAAAGAAAAGT, PCR amplification was performed with an amplification system of 100. Mu.l: 50 μl of high-fidelity enzyme, 0.5 μl of each of the upstream and downstream primers, and ddH 2 O47 μl, DNA 2 μl, melting 94 ℃, annealing 57 ℃, extension 72 ℃,30 cycles, electrophoresis of PCR amplification products on a 1% agarose gel containing EB nucleic acid dye, electrophoresis in a1 xTAE buffer at 100V for 1h, and recording the electrophoresis results in a gel imaging system.
A2, glue recovery:
a2.1, cutting and weighing the gel containing the target strip, adding XP2 Binding Buffer in a proportion of 1g/ml, and standing at 50-60 ℃ for 7min until the gel is completely melted;
a2.2, sucking 700 mul to a filter column after melting, centrifuging at 12000rpm for 1min, discarding the liquid in a collecting pipe, and repeating the step B2.2 until the liquid is added;
a2.3, 300. Mu.l XP2 Buffer was added and centrifuged at 12000rpm for 1min, and the liquid in the collection tube was discarded.
A2.4, adding 700 μl of SPW Buffer, centrifuging at 12000rpm for 1min, and discarding the liquid in the collection tube;
a2.5 centrifuging the empty filter column at 12000rpm12000 rpm for 2min, placing the filter column in a sterile 1.5ml centrifuge tube, adding preheated Elution Buffer or ddH 2 O15-30 μl to the center of the filter membrane, standing at room temperature for 2min, centrifuging at 12000rpm for 1min, measuring concentration, and preserving at-20deg.C.
A3, connection: the gel recovery product was ligated with the vector pMD 18-T16 overnight, 10. Mu.l system: 1 μl pMD18-T (0.03 pmol/50 ng), DNA to vector molar ratio 1: 2-10, adding sterilized water to 5 μl,5 μl Solution I to obtain a ligation product;
a4, conversion: ligation product with DH-5α1:10 parts of the strain are added into a sterilizing centrifuge tube for ice bath for 30min at 42 ℃ for 90s, then ice bath is carried out for 2min, 500 mu l of antibiotic-free LB liquid medium is added, the mixture is placed in a shaking table for 37 ℃ for culturing 45min at 160rpm, the mixture is centrifuged at 3000rpm for 1min, 400 mu l of supernatant is discarded, the rest bacterial liquid is coated on LB solid medium containing ampicillin, and the mixture is cultured for 16h at 37 ℃;
a5, extracting plasmids: picking up single colony with gun head, throwing gun head into LB liquid culture medium containing ampicillin, placing in a shaking table at 37deg.C, treating at 200rpm for 6 hr, sucking 10 μl of bacterial liquid, adding into 10ml LB liquid culture medium containing ampicillin, culturing for two times, treating at 37deg.C at 200rpm for 16 hr, and extracting plasmid, wherein the steps are as follows:
a5.1, sucking the bacterial liquid into a 1.5ml sterile centrifuge tube at 12000rpm for 1min, and discarding the supernatant;
a5.2, adding 250 mul Buffer P1, adding RNase A into the Buffer P1, blowing, uniformly mixing and precipitating to fully crack bacterial liquid;
a5.3, adding 250 μl of BufferP2, gently reversing for 8-10 times, making the solution become viscous and transparent, and opening the cover to draw wires, wherein the step is controlled within 2-3 min);
a5.4, adding 350 μl Buffer P3, immediately and gently reversing upside down for 8-10 times, and centrifuging at 12000rpm for 10min;
a5.5, placing the adsorption column in a collecting pipe, transferring the supernatant obtained in the steps into the adsorption column, and centrifuging at 12000rpm for 1min without sucking sediment;
a5.6, adding 600 mu l of Buffer PW2 into an adsorption column, pre-adding absolute ethyl alcohol into the Buffer PW2, centrifuging at 12000rpm for 1min, and discarding the liquid;
a5.7, repeating the step B5.6;
a5.8, 12000rpm centrifugal 1min;
a5.9 placing the adsorption column in a new sterile centrifuge tube, adding 30-100 μl of preheated Elution Buffer or ddH 2 O to the center of the membrane, standing at room temperature for 2min, centrifuging at 12000rpm for 1min, discarding the adsorption column, testing the concentration of DNA product, and preserving at-20deg.C to obtain positive plasmid.
6. The use of the RT-RPA amplification reagent for PRRSV detection according to claim 5, further comprising obtaining a plurality of positive particle test samples by diluting positive plasmids, then testing the positive particle test samples by RT-RPA amplification method, and judging the sensitivity of RT-RPA amplification method.
7. The use of an RT-RPA amplification reagent for detecting PRRSV according to claim 8, further comprising obtaining a strain as a test sample and then performing a specificity test by an RT-RPA amplification method.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050170335A1 (en) * | 2003-10-09 | 2005-08-04 | Tetracore, Inc. | Detection of PRRSV |
| CN105936944A (en) * | 2016-04-22 | 2016-09-14 | 浙江省检验检疫科学技术研究院 | Triple fluorescent RT-PCR detection kit, primer and probe for CSFV, PRRSV and SIV H1 subtype |
| CN106636459A (en) * | 2016-10-18 | 2017-05-10 | 河北出入境检验检疫局检验检疫技术中心 | Fluorescent RT-RPA specific detection of American porcine reproductive and respiratory syndrome virus |
| CN108929918A (en) * | 2018-07-19 | 2018-12-04 | 华南理工大学 | It is a kind of for detecting the field fast detection method and kit of PRRSV |
| CN109797246A (en) * | 2019-02-23 | 2019-05-24 | 军事科学院军事医学研究院军事兽医研究所 | A kind of quickly detection African swine fever virus RPA amplimer and detection kit |
| CN114736989A (en) * | 2022-04-01 | 2022-07-12 | 中国农业科学院兰州兽医研究所 | Fluorescent RPA detection primer, method and kit for American porcine reproductive and respiratory syndrome virus |
-
2022
- 2022-11-07 CN CN202211383585.0A patent/CN116121452A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050170335A1 (en) * | 2003-10-09 | 2005-08-04 | Tetracore, Inc. | Detection of PRRSV |
| CN105936944A (en) * | 2016-04-22 | 2016-09-14 | 浙江省检验检疫科学技术研究院 | Triple fluorescent RT-PCR detection kit, primer and probe for CSFV, PRRSV and SIV H1 subtype |
| CN106636459A (en) * | 2016-10-18 | 2017-05-10 | 河北出入境检验检疫局检验检疫技术中心 | Fluorescent RT-RPA specific detection of American porcine reproductive and respiratory syndrome virus |
| CN108929918A (en) * | 2018-07-19 | 2018-12-04 | 华南理工大学 | It is a kind of for detecting the field fast detection method and kit of PRRSV |
| CN109797246A (en) * | 2019-02-23 | 2019-05-24 | 军事科学院军事医学研究院军事兽医研究所 | A kind of quickly detection African swine fever virus RPA amplimer and detection kit |
| CN114736989A (en) * | 2022-04-01 | 2022-07-12 | 中国农业科学院兰州兽医研究所 | Fluorescent RPA detection primer, method and kit for American porcine reproductive and respiratory syndrome virus |
Non-Patent Citations (2)
| Title |
|---|
| XIAO-XIAO TIAN等: "Rapid visual detection of porcine reproductive and respiratory syndrome virus via recombinase polymerase amplification combined with a lateral flow dipstick", ARCH VIROL, vol. 167, no. 2, 7 January 2022 (2022-01-07), pages 494, XP038168523, DOI: 10.1007/s00705-021-05349-8 * |
| 张日腾;张乔亚;姜辰龙;刘宗霞;杨茂春;姜平;白娟;: "猪繁殖与呼吸综合征病毒RT-LAMP可视化检测方法的建立与应用", 中国兽医科学, vol. 48, no. 01, 8 January 2018 (2018-01-08), pages 7 - 12 * |
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