CN116042908A - Kit for rapidly detecting combination of RPA amplification and flow-measuring chromatographic immunoassay test strip of African swine fever virus - Google Patents
Kit for rapidly detecting combination of RPA amplification and flow-measuring chromatographic immunoassay test strip of African swine fever virus Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention discloses a kit for rapidly detecting the combination of RPA amplification and a flow-measuring chromatographic immunoassay test strip of African swine fever virus. The kit comprises a primer and a probe for detecting the African swine fever virus, wherein the primer consists of an upstream primer and a downstream primer, and the kit further comprises amplification mixed enzyme for detecting the African swine fever virus, wherein the mixed enzyme contains UNG enzyme. Compared with the prior art, the invention provides a rapid, simple and accurate detection reagent for molecular diagnosis of ASFV, particularly has low requirements on instruments and equipment, has high detection speed by only needing a constant-temperature water bath kettle or a constant-temperature device for about 30min, is suitable for quantitative analysis of scientific research units, is suitable for pathogen detection analysis of various levels of prevention and control units, basic veterinary stations, large and medium farms and the like, and has good application prospects.
Description
Technical Field
The invention belongs to the technical field of molecular biology detection, and particularly relates to a kit for detecting combination of RPA (recombinant polymerase real-time quantitative polymerase chain reaction) and a flow-measurement chromatography immunoassay test strip of African swine fever virus.
Background
The main method for detecting virus nucleic acid is a PCR method, and the conventional PCR detection needs a special thermal cycler and a complicated test procedure, so that the detection needs under the field or field conditions are difficult to meet. In recent years, some nucleic acid isothermal amplification technologies have been developed, which do not require an expensive PCR apparatus, and which can rapidly amplify a large amount of target fragments in a short time, and which have advantages of simplicity, rapidity, high sensitivity, and the like. Among them, the RPA technology is more called a nucleic acid detection technology that can replace PCR. RPA technology relies primarily on three enzymes: recombinant enzymes capable of binding single stranded nucleic acids (oligonucleotide primers), single stranded DNA binding proteins (SSBs), and strand displacement DNA polymerases. The mixture of these three enzymes is also active at normal temperature, with an optimal reaction temperature of about 37 ℃. RPA technology was developed based on the principle of recombinase polymerase-mediated amplification, which mimics DNA replication in organisms. The method can enable the target gene to grow exponentially in a very short time, and can realize real-time monitoring of template amplification if the target gene is matched with a fluorescent marked probe and a fluorescent signal detector. At present, no report is made on a method for detecting African swine fever virus by combining a test strip with isothermal amplification of a recombinase.
The invention patent application with the application publication number of CN 114703177A discloses a pseudorabies virus detection composition, a pseudorabies virus detection method and a pseudorabies virus detection kit combining RPA isothermal amplification and immunochromatography technologies. Meanwhile, the preparation of the nucleic acid template can be realized through high-temperature treatment of the sample, so that the whole detection cost is greatly reduced, and the detection time is shortened.
Disclosure of Invention
The invention establishes and optimizes a simple, cheap and sensitive kit for detecting African swine fever virus (Infection with African swine fever virus, ASFV) by combining recombinase isothermal amplification with an immune colloidal gold test strip and a method for detecting African swine fever virus nucleic acid by using the kit. The RPA technology greatly reduces the detection time, simplifies the reaction procedure, combines the portable real-time fluorescence detection equipment with the lateral flow colloidal gold test strip, so that the RPA technology is suitable for field or field detection and has wide application prospect. The detection of ASFV by using RPA technology and immune colloidal gold has great clinical application value.
In order to solve the problems, the invention adopts the following technical scheme:
the kit for rapidly detecting the combination of RPA amplification and a flow-measuring chromatographic immunoassay test strip of the African swine fever virus comprises an RPA amplification system and the flow-measuring chromatographic immunoassay test strip;
the RPA amplification system comprises a primer and a probe for detecting African swine fever virus, wherein the primer consists of an upstream primer and a downstream primer, and the sequence of the upstream primer is as follows: 5'-GCC GAA GGG AAT GGA TAC TGA GGG AAT AGC AA-3', biotin (Biotin) is marked at the 5 'end of the downstream primer, the sequence of the Biotin is 5' -Biotin-TCC CGA GAA CTC TCA CAA TAT CCA AAC AGC AG-3', and a fluorescent reporter group marked at the 5' end of the probe is FAM; an extension blocking group (C3-spacer, modified by a phosphate group) is added to the 3' end of the probe, and the 30 th position is replaced by Tetrahydrofuran (THF); the sequence of the probe is 5'-6FAM-GAA CAT TAC GTC TTA TGT CCA GAT ACG TG-THF-CCG TGA TAG GAG TGA-Spacer C3-3'.
The RPA amplification system further comprises a mixed enzyme for detecting African swine fever virus, wherein the mixed enzyme comprises UNG enzyme and RPA mixed enzyme mix, and the RPA mixed enzyme mix comprises recombinase, single-stranded DNA binding protein (SSB) and strand displacement DNA polymerase. The UNG enzyme can degrade uracil bases in U-DNA pollutants in a reaction system, then break DNA chains under the condition of subsequent denaturation, and eliminate amplification caused by polluted DNA, so that the specificity and accuracy of an amplification result are ensured; meanwhile, UNG enzyme is inactivated under the denaturation condition, and U-DNA in the new amplified product can not be degraded.
In addition, the RPA amplification system includes a RPA reaction buffer, a positive control, a negative control, and a PBS buffer. Preferably, the positive control is a plasmid containing the sequence shown in SEQ ID NO. 1, and the negative control is DNase-free water.
Further, when the kit disclosed by the invention is used for detecting African swine fever virus, the method comprises the following steps of:
(1) The RPA total system was 50 μl, including:
a. RPA reaction buffer: 27.2 Mu.l;
b. mixed enzyme: 3.5 Mu.l;
c. the concentration of the upstream and downstream primers was 10. Mu. Mol/. Mu.l each 0.7. Mu.l;
d. 10. Mu. Mol/. Mu.l probe 0.7. Mu.l;
e. 5 μl of DNA of the sample to be tested or the tissue grinding supernatant of the sample to be tested;
f. DNase-free water (DEPC water) was made up to 50. Mu.L;
the above components were added to a 0.2. 0.2 ml amplification tube in this order, respectively.
(2) PCR amplification
Adding an RPA system into an amplification tube according to the step (1), centrifuging at 12000rpm for 5-30 seconds, placing the amplification tube into a constant-temperature amplification instrument, reacting at 40 ℃ for 20 minutes, and reading fluorescent signals in real time in the reaction process.
(3) Lateral flow chromatography flow immunity test strip detection
Mixing 10 μl of the amplification product in the step (2) with 90 μl of 0.01M PBS diluent, dripping onto a sample pad of an immune colloidal gold test strip, and observing the result with naked eyes after 2-3 min;
furthermore, the invention also provides application of the kit combining RPA amplification and flow-measurement chromatography immunoassay test strips.
The present invention utilizes recombinase polymerase mediated isothermal amplification (RecombinasePolymerase Amplification, RPA) to rapidly detect ASFV nucleic acids. Specifically, the invention designs a specific primer according to the most conserved P72 gene of ASFV, screens a group of RPA primers from the specific primer, and then carries out RPA amplification reaction after respectively marking the reverse primer and the 5' end of the probe by biotin and hydroxyfluorescein (FAM). In the reaction, a hydroxyfluorescein (FAM) marked probe is specifically hybridized with a biotin marked loop-mediated isothermal amplification technology amplification product and is combined with a colloidal gold marked anti-hydroxyfluorescein antibody to form a ternary complex which is combined on a detection line of a transverse flow test strip with biotin antibody; the unhybridized hydroxyfluorescein labeled probe and the colloidal gold labeled anti-hydroxyfluorescein antibody form a biotin-free binary complex, and are combined on a quality control line; can realize rapid and effective detection of whether the sample contains ASFV nucleic acid.
Compared with the prior art, the invention has the beneficial effects that:
the invention only needs to insert the flow detection chromatography immune test strip into the amplified reaction liquid, and can read the result by naked eyes, the detection technology of the used lateral flow test strip is that the 5' ends of the reverse primer and the probe are respectively marked with the hydroxy Fluorescein (FAM) primer and the biotin, the double-marked nucleic acid isothermal amplification product is obtained through the RPA reaction, the nucleic acid detection test strip absorbing the biotin antibody and the hydroxy fluorescein antibody is inserted into the amplification product, the nucleic acid detection test strip can be combined with the specific antibody marked by colloidal gold and displayed at the detection line, otherwise, the unhybridized primer can not be combined with the specific antibody marked by colloidal gold and displayed at the quality control line, the display result can be observed by naked eyes, the experimental process is greatly shortened, the experimental step is simplified, the detection method is obviously improved in the aspects of accuracy and rapid detection, the electrophoresis step can be omitted, and the fluorescent dye ethidium bromide with high carcinogenicity is not needed.
The RPA primer has high specificity, can rapidly detect whether the detected product contains ASFV nucleic acid in the RPA amplification reaction after being respectively marked by biotin and FAM, and has high sensitivity, high detection speed and strong intuitiveness.
The detection method of the invention uses the double-labeled nucleic acid product detection test strip, and can observe the detection result and rapidly detect ASFV nucleic acid only by 5-10 minutes, thereby greatly saving the detection cost.
The invention can directly detect the ground supernatant of the diseased animal tissue, and does not need to extract the DNA of the sample to be detected. In addition, the invention adopts UNG enzyme to degrade uracil base in U-DNA pollutant in the reaction system, then DNA chain breaks under the condition of subsequent denaturation, and amplification caused by polluted DNA is eliminated, thereby ensuring the specificity and accuracy of the amplification result; meanwhile, UNG enzyme is inactivated under the denaturation condition, and U-DNA in the new amplified product can not be degraded.
Drawings
FIG. 1 shows the detection results of the RPA amplification standard in example 3 of the present invention;
FIG. 2 shows the result of detecting the RPA amplification standard by the immune colloidal gold test strip in the embodiment 3 of the invention;
FIG. 3 shows the detection results of the tissue milling supernatant of an RPA-amplified diseased animal in example 4 of the present invention; the tissue sample used in the figure is the tissue grinding supernatant of the pathogenic animal. FIG. 3a shows the amplification system containing UNG enzyme and FIG. 3b shows the amplification system without UNG enzyme;
FIG. 4 shows the result of detecting the rpA amplified diseased animal tissue supernatant by using the immune colloidal gold test strip in example 4 of the present invention;
FIG. 5 shows the result of detection of RPA amplification specificity in example 5 of the present invention;
FIG. 6 shows the result of detecting RPA amplification specificity by using the immune colloidal gold test strip in example 5 of the present invention.
Detailed Description
The following detailed description of the present invention is provided to facilitate understanding of the technical solution of the present invention, but is not intended to limit the scope of the present invention.
Example 1 design and preparation of primers and probes
Searching ASFV strain genome sequence by referring to GenBank (gene bank), searching the conserved regions on the P72 gene sequence by sequence comparison, selecting the conserved regions, designing and screening a pair of amplification primers, and one probe primer with the following sequence:
the amplification primer sequences are:
an upstream primer: 5'-GCC GAA GGG AAT GGA TAC TGA GGG AAT AGC AA-3' the number of the individual pieces of the plastic,
a downstream primer: 5 '-Biotin-TCC CGA GAA CTC TCA CAA TAT CCA AAC AGC AG-3'.
The probe sequence is as follows: 5'-6FAM-GAA CAT TAC GTC TTA TGT CCA GAT ACG TG-THF-CCG TGA TAG GAG TGA-C3-Spacer-3'.
The primer and the probe sequence are synthesized and marked by da Lian Bao bioengineering Co., ltd, and the 5' end of the probe is marked with a fluorescence reporting group FAM; an extension blocking group (C3-spacer, phosphate group modification) was added to the 3' end of the probe, with the 30 th position being replaced by a Tetrahydrofuran (THF).
Example 2 Assembly of kit for rapid detection of African swine fever Virus RPA amplification in combination with a lateral flow chromatography immunoassay test strip
The following reagents are assembled to obtain the kit for rapidly detecting the combination of RPA amplification and flow-measuring chromatographic immunoassay test strips of African swine fever virus:
1. the mixture (126. Mu.L) of the upstream and downstream primers and probes of example 1 was 10. Mu. Mol/. Mu.L for both the primers and probes.
2. RPA reaction buffer (1632. Mu.L) purchased from TwitDx.
3. Mixed enzyme solutions, including RPA mixed enzyme mix available from TwistDx at a concentration of 500 u/. Mu.L and UNG enzyme (210. Mu.L) at a concentration of 500 u/. Mu.L (the volume ratio of RPA mixed enzyme mix to UNG enzyme is 6:1), were purchased from Daidao Biotechnology Co., ltd.
4. Positive control: 300. Mu.L of standard positive plasmid at a concentration of 1 ng/. Mu.L.
The standard positive plasmid was obtained as follows:
the sequence shown in SEQ ID No. 1 (P72 gene sequence of ASFV) is synthesized, and is connected with a pMD-18T cloning vector to transform competent cells DH5 alpha of the escherichia coli, and the competent cells DH5 alpha are coated on LB medium plates containing 100mg/L ampicillin, and are cultured for 12-16 h at 37 ℃. After screening with blue and white spots, plasmids are extracted by a plasmid extraction kit, and the plasmids with positive sequence determination are named pMD-627, namely the standard positive plasmids.
5. Negative control: 570. Mu.L of DNase-free water (DEPC water).
6. Immune colloidal gold test strips were prepared in the references (Wan Y, shi Z, peng, et al Development and application of a colloidal-gold dual immunochromatography strip for detecting African swine fever virus antibodies [ J ]. Applied Microbiology and Biotechnology, 2022, 106 (2): 799-810.).
7. Dilution liquid: PBS, pH7.6, 5400. Mu.L.
Example 3 application of the kit of example 2 in the detection of ASFV Standard
1. Preparation of standards
The pMD-627 standard positive plasmid was diluted 10-fold to give: 10 -2 ～10 -9 RPA was performed in triplicate for each gradient.
2. Sample detection
(1) The RPA total system was 50. Mu.l:
a. RPA reaction buffer: 27.2 Mu.l;
b. mixed enzyme: 3.5 Mu.l;
c. the concentration of the upstream and downstream primers was 10. Mu. Mol/. Mu.l each 0.7. Mu.l;
d. 10. Mu. Mol/. Mu.l probe 0.7. Mu.l;
e. DNase-free water 12.2. Mu.L.
The above components were added to a 0.2. 0.2 ml amplification tube in this order, respectively.
(2) 5 μl of the gradient diluted standard substance was added to the amplification tube, and the mixture was centrifuged at 12000rpm for 5 to 30 seconds, and the amplification tube was placed in an isothermal amplification apparatus at 40℃for 20 minutes, while negative and positive controls were established. Directly observing the amplification result on a constant temperature amplification instrument or observing the detection result by naked eyes by an immune colloidal gold detection method.
3. Analysis of results
(1) Based on the RPA amplification result judgment standard: in the case of no amplification curve of the NTC (negative control), the sample to be tested has a distinct amplification curve, indicating that the sample to be tested has ASFV nucleic acid.
(2) Direct visual interpretation criteria:
1) Negative (-). Only a dark red band appears on the quality control line, no dark gray band appears in the detection line, and the detected sample is proved to have no ASFV nucleic acid;
2) Positive (+): two dark red bands appeared, one in the detection line and the other in the quality control line, confirming the presence of ASFV nucleic acids in the detected sample.
The results show that the standard is in a dilution gradient 10 -2 ～10 -7 The time-dependent amplification curves were evident (FIG. 1); meanwhile, the RPA amplified sample is detected by an immune colloidal gold test strip, and the dilution gradient is 10 -2 ～10 -6 Bands of deep or light red appear, 10 -7 No banding occurs (fig. 2).
Example 4 use of the kit of example 2 in the detection of tissue milling supernatant of an ASFV infected pathogenic animal
1. The collected diseased animal tissue is ground, and the supernatant is extracted.
2. RPA amplification
(1) RPA total system was 50. Mu.l
a. RPA reaction buffer: 27.2 Mu.l;
b. enzyme mixture: 3.5 Mu.l;
c. the concentration of the upstream and downstream primers was 10. Mu. Mol/. Mu.l each 0.7. Mu.l;
d. 10. Mu. Mol/. Mu.l probe 0.7. Mu.l;
e. DNase-free water 12.2. Mu.L.
The above components were added to a 0.2. 0.2 ml amplification tube in this order, respectively.
A tube of the amplification system without UNG enzyme was also used as a comparative example, which was identical to the RPA amplification system described above except that the enzyme mixture was free of UNG enzyme.
(2) Adding 5 μl of extracted ground supernatant of the animal tissue to be detected into the different amplification tubes, centrifuging at 12000rpm for 5-30 seconds, placing the amplification tubes into an isothermal amplification instrument at 40 ℃ for 20min, and simultaneously setting up a negative control and a positive control (standard positive plasmid pMD-627). Directly observing the amplification result on a constant temperature amplification instrument or observing the detection result by naked eyes by an immune colloidal gold detection method.
3. Analysis of results
(1) Based on the RPA amplification result judgment standard: in the case of no amplification curve of the NTC (negative control), the sample to be tested has a distinct amplification curve, indicating that the sample to be tested has ASFV nucleic acid.
(2) Direct visual interpretation criteria:
1) Negative (-). Only a dark red band appears on the quality control line, no dark gray band appears in the detection line, and the detected sample is proved to have no ASFV nucleic acid;
2) Positive (+): two dark red bands appeared, one in the detection line and the other in the quality control line, confirming the presence of ASFV nucleic acids in the detected sample.
The results showed that ASFV infected tissues had a significantly single amplification curve in the amplification system using UNG enzyme (FIG. 3 a), while the detection line of the immune colloidal gold strip appeared as a single detection line (dark red band) (FIG. 4).
In the amplification system without UNG enzyme, ASFV infected tissues had multiple amplification curves, as shown in fig. 3 b.
Example 5 specific detection of the kit of example 2
1. DNA from ASFV (serum of African swine fever infected animals), CSFV (swine fever) and PRRSV (porcine reproductive and respiratory syndrome virus) samples was extracted using commercial kits.
2. RPA amplification
(1) The RPA total system was 50. Mu.l:
a. RPA reaction buffer: 27.2 Mu.l;
b. enzyme mixture: 3.5 Mu.l;
c. the concentration of the upstream and downstream primers was 10. Mu. Mol/. Mu.l each 0.7. Mu.l;
d. 10. Mu. Mol/. Mu.l of the TwistAmp nfo probe 0.7. Mu.l;
e. DNase-free water 12.2. Mu.L.
The above components were added to a 0.2. 0.2 ml amplification tube in this order, respectively.
(2) The extracted ASFV, CSFV and PRRSV nucleic acids were added into the above-mentioned different amplification tubes respectively, centrifuged at 12000rpm for 5-30 seconds, and the amplification tubes were put into an isothermal amplification apparatus at 40℃for 20min while negative and positive controls were established. Directly observing the amplification result on a constant temperature amplification instrument or observing the detection result with naked eyes by using an immune colloidal gold test strip.
3. Analysis of results
(1) Based on the RPA amplification result judgment standard: in the case of no amplification curve of the NTC (negative control), the sample to be tested has a distinct amplification curve, indicating that the sample to be tested has ASFV nucleic acid.
(2) Direct visual interpretation criteria:
1) Negative (-). Only a dark red band appears on the quality control line, no dark gray band appears in the detection line, and the detected sample is proved to have no ASFV nucleic acid;
2) Positive (+): two dark red bands appeared, one in the detection line and the other in the quality control line, confirming the presence of ASFV nucleic acids in the detected sample.
The result shows that the ASFV sample has obvious amplification curve, and meanwhile, the detection line of the immune colloidal gold test strip has dark red strips; while CSFV and PRRSV did not have amplification curves, no dark red bands appeared on the immune colloidal gold test strips (FIGS. 5, 6).
The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention, so that all equivalent changes or modifications of the structure, characteristics and principles described in the claims should be included in the scope of the present invention.
Claims (8)
1. The kit for rapidly detecting the combination of RPA amplification and a flow-measuring chromatographic immunoassay test strip of the African swine fever virus is characterized by comprising an RPA amplification system and the flow-measuring chromatographic immunoassay test strip;
the RPA amplification system comprises a primer and a probe for detecting African swine fever virus, wherein the primer consists of an upstream primer and a downstream primer, and the sequence of the upstream primer is as follows: 5'-GCC GAA GGG AAT GGA TAC TGA GGG AAT AGC AA-3' the sequence of the downstream primer is 5 '-Biotin-TCC CGA GAA CTC TCA CAA TAT CCA AAC AGC AG-3', and the sequence of the probe is 5'-6FAM-GAA CAT TAC GTC TTA TGT CCA GAT ACG TG-THF-CCG TGA TAG GAG TGA-Spacer C3-3';
the RPA amplification system also comprises a mixed enzyme for detecting African swine fever virus, wherein the mixed enzyme contains UNG enzyme.
2. The kit of claim 1, wherein the concentration of each of the primer and probe is 10 μmol/μl.
3. The kit of claim 1, wherein the RPA amplification system further comprises an RPA reaction buffer, a positive control, and a negative control.
4. The kit according to claim 3, wherein the positive control is a plasmid having the sequence shown in SEQ ID NO. 1, and the negative control is DNase-free water.
5. The kit of claim 1, wherein the lateral flow chromatography immunoassay test strip is an immunoassay colloidal gold test strip.
6. The kit of claim 1, further comprising a PBS buffer.
7. The kit according to claim 1, wherein the mixed enzymes comprise RPA mixed enzyme mix at a concentration of 500 u/. Mu.l and UNG enzyme at a concentration of 500 u/. Mu.l.
8. The kit of claim 7, wherein the volume ratio of RPA mix enzyme mix to UNG enzyme is 6:1.
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