CN113122656A - Foot-and-mouth disease virus nucleic acid extraction-free fluorescent isothermal amplification detection kit - Google Patents
Foot-and-mouth disease virus nucleic acid extraction-free fluorescent isothermal amplification detection kit Download PDFInfo
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Abstract
The invention selects a highly conserved 3D gene segment of foot-and-mouth disease virus as a detection target gene, designs a specific primer and establishes an FMDV specific fluorescent isothermal amplification detection method. The Bst DNA polymerase is adopted, so that the method has the advantages of high amplification efficiency and good specificity; the reaction result is indicated by adopting fluorescent dye, so that accurate judgment is facilitated; the DNA of a sample does not need to be extracted, so that the detection operation is simplified; the whole detection process is fast and only needs 50 min. And the invention can detect a plurality of samples at one time, and is suitable for the detection of a large number of samples. The used fluorescent constant-temperature amplification instrument has low cost; has the advantages of high amplification efficiency and good specificity; can be used for detecting foot-and-mouth disease virus in animal blood and tissues.
Description
Technical Field
The invention relates to a foot-and-mouth disease virus nucleic acid fluorescence isothermal amplification detection technology and a kit, belonging to the technical field of microbial detection.
Background
Foot-and-mouth disease is caused by FMDV (foot-and-mouth disease virus) and is an acute, hot and highly contagious disease occurring in artiodactyl animals such as cattle, sheep, pigs and the like. The foot-and-mouth disease is listed as an animal fulminating infectious disease which needs to be reported by the world animal health organization, and the development of animal husbandry and the related products trade are seriously damaged. Animals with foot and mouth disease are characterized by fever, lameness and blisters on the skin and skin mucosa. Malignant foot-and-mouth disease can also lead to rapid death.
Animals with onset or incubation are the major sources of foot and mouth disease virus infection, which can be transmitted through a variety of routes including air, secretions and excretions, as well as contaminated feed. The control measures for foot-and-mouth disease include: strengthening inactivated vaccine immunization; the epidemic situation monitoring and reporting are enhanced, and an effective active detection, prediction and early warning mechanism is established; establishing a sound emergency mechanism and perfecting an emergency plan; strict quarantine supervision on mobile animals and animal products.
Foot and mouth disease virus detection is an important basis for epidemic disease diagnosis and quarantine. The Virus Infection Associated Antigen (VIAA) agar diffusion test is mainly used for general epidemic investigation and live stock import and export quarantine, can only detect whether animals have foot-and-mouth disease virus infection antibodies, and takes more than one day.
When detecting foot-and-mouth disease viruses in veterinary laboratories of all levels at present, a common RT-PCR method and a fluorescent RT-PCR method are mainly adopted, but the common RT-PCR detection operation easily causes pollution of virus nucleic acid amplification products and false positive; the fluorescence RT-PCR technique requires an expensive fluorescence PCR instrument, and usually requires more than 2 hours to complete the whole detection process from nucleic acid extraction. In recent years, the etiology detection is carried out on the quarantine requirements of animals and products thereof in China, and quarantine proofs are provided according to detection results, so that an FMDV detection technology which is quicker, simpler, more convenient, more accurate and lower in cost and is suitable for field application is required.
The nucleic acid isothermal amplification technology is a new gene detection technology, and has many advantages compared with the widely applied common RT-PCR and fluorescent RT-PCR. The method does not need expensive instruments (constant temperature), is simple to operate, has high speed and high sensitivity, and is widely applied. However, the existing nucleic acid isothermal amplification method mainly depends on the visual observation of the color change of the reaction solution to judge the result, and has strong subjectivity and poor accuracy.
The method adopts the fluorescent agent to dye the double-stranded DNA amplification product, and uses the reader to detect the fluorescent signal to judge the result, thereby improving the accuracy compared with the eye observation method; the invention adopts the extraction-free reaction solution to process and release the virus RNA in the sample, directly carries out the amplification reaction without extracting the RNA in the sample, reduces the operation steps and the cost, shortens the detection time, and eliminates the cross contamination in the nucleic acid extraction process; the invention designs 6 specific primers aiming at the foot-and-mouth disease virus gene, thereby ensuring the specificity of amplification reaction.
The invention comprehensively applies the technologies and finally establishes a novel foot-and-mouth disease virus nucleic acid rapid detection method and a kit. Compared with the PCR and fluorescence PCR detection methods widely used at present, the method has the advantages of high detection speed, high sensitivity, simple and convenient operation, low reagent and equipment cost and the like, is suitable for being used in the fields and basic units of animal breeding, slaughtering, quarantine and monitoring, and has good application prospect.
Disclosure of Invention
The invention aims to establish a foot-and-mouth disease virus fluorescent isothermal amplification detection method and a kit which do not take nucleic acid, so that the detection process is simple and easy, the result interpretation is accurate, expensive instruments are not used, the detection cost is reduced, the total detection time is shortened to 50 minutes, and a rapid, sensitive, specific and low-cost technical means is provided for the foot-and-mouth disease virus detection.
In order to realize the purpose, the invention comprehensively uses Bst DNA polymerase isothermal amplification technology, tissue sample nucleic acid hands-free technology and fluorescent indicator to establish a foot-and-mouth disease virus fluorescent isothermal amplification detection method and develop a kit, and the specific contents are as follows:
1. designing isothermal amplification primers: referring to the nucleotide sequence of the foot-and-mouth disease virus genome, screening 3D gene conserved regions, and designing 6 primers:
FMDF3 ACCCTCGARGCTATC
FMDB3 GCGTCACCGCACAC
FMDFIP TGGACGGCAAGTCCTGTTTTCTCTCCTTTGCACG
FMDBIP TACCGGCGTCTCTTTGATTTTCACCCAACGCAGG
FMDLF CAACTTCTCCTGTATGGTCC
FMDLB TGAGATTCCAAGCTACAGAT
2. preparing a primer solution: diluting artificially synthesized primers FMDF3, FMDB3, FMDFIP and FMDBIP to 20 mu mol/L by DEPC treated water, and diluting artificially synthesized primers FMDLF and FMDLB to 40 mu mol/L by DEPC treated water; the diluted primers are mixed evenly according to the proportion of FMDF3, FMDB3, FMDFIP, FMDBIP, FMDLF and FMDLB =1:1:8:8:2: 2.
3. Preparation of positive control: cloning the nucleotide sequence of the foot-and-mouth disease virus 3D gene by a conventional method, constructing pMD20-T-FMDV vector recombinant plasmids, and carrying out sequence determination on target genes in the plasmids. After the sequencing result is correct, the competent cells of E.coli Top10 were transformed, and the cells were cultured in an expanded manner to extract plasmids as positive controls.
4.10 × preparation of isothermal amplification extraction-free buffer: weighing Trisbase: 24.23g, KCl: 3.73g (NH4)2SO4：7g，MgSO47H2O: 2.465g, Triton X-100: 5mL, Brij-58: 5g, DEPC treated water is added to 400mL, the pH value is adjusted to 8.8 (25 ℃) by hydrochloric acid, and the volume is adjusted to 500 mL. And (4) after autoclaving and cooling, storing at 2-8 ℃ for later use.
5. Preparing a fluorescence isothermal amplification extraction-free reaction solution: taking 2.5 mL of 10 times isothermal amplification extraction-free buffer solution, 5.5 mL of primer solution, 4 mL of 5 mol/L betaine solution, 3.5 mL of 10 mmol/L dNTP mix and 100 mmol/L MgSO41.5 mL of the solution, 0.5 mL of 0.1% SYBR GreenI solution and 4.5 mL of 0.1% DEPC treated water, mixing, filtering with a 0.45 μm filter membrane for sterilization (sterile low temperature in the whole process), packaging into small tubes, and labeling.
Preparation of Bst DNA polymerase: bst DNA polymerase was purchased from outsourced (NEB) at a concentration of 8U/. mu.L, 1000. mu.L/tube. Bst DNA polymerase was quantitatively dispensed aseptically and labeled.
7. Preparation of mineral oil: quantitatively packaging the liquid paraffin oil into small tubes, and labeling.
8. Preparation of negative control: 0.1% DEPC aqueous solution, aseptically packaging, and labeling.
9. The detection method comprises the following steps:
(1) in a 0.2 mL reaction tube, 22. mu.L of the fluorescence isothermal amplification immunoassay extraction reaction solution and 1.0. mu.L of Bst DNA polymerase were added to each tube, and 20. mu.L of mineral oil was added to each tube.
(2) Sample adding: adding 1: 5 diluting the blood or tissue grinding liquid of the tested animal by 2.0 mu L, and setting a positive control tube and a negative control tube at the same time. Covering the pipe cap tightly, performing instantaneous centrifugation, and putting into a special fluorescent constant-temperature amplification instrument or a common fluorescent PCR instrument.
(3) Optimized reaction parameters: FAM channel fluorescence signals are collected every 1min at 64 ℃ for 50min for 50 times. When the Tt (time threshold) value is absent and a characteristic amplification curve is absent, determining that the sample is negative; when the Tt value is less than or equal to 40.0 and a typical amplification curve appears, the sample is judged to be positive; when the Tt value is more than 40.0 and a typical amplification curve appears, the process should be repeated, the result still appears, and the result is judged to be positive, otherwise, the result is judged to be negative.
10. The invention also relates to a kit which comprises the primer pair combination product, and preferably further comprises one or more of a fluorescence isothermal amplification non-extraction reaction solution (containing SYBR GreenI fluorescent agent), Bst DNA polymerase, a negative control and a positive control. Preferably, the Bst DNA polymerase can be added into the fluorescence isothermal amplification non-extraction reaction solution for integrated packaging. The kit of the invention also comprises a positive control and a negative control; the positive control is pMD20-T-FMDV recombinant plasmid; the negative control was no nucleic acid water. The kit provided by the invention can be used for detecting the foot-and-mouth disease virus and can be used for detecting the foot-and-mouth disease virus in animal blood and tissues.
11. The kit verification method comprises the following steps: (1) the positive control plasmid was diluted 10 fold-1～10-6The detection is carried out according to the method in 9, and the sensitivity of the kit is verified to reach 10-3. (2) The kit is used for detecting African swine fever virus nucleic acid, peste des petits ruminants virus nucleic acid, porcine reproductive and respiratory syndrome virus nucleic acid and pseudorabies virus nucleic acid, verifying the specificity of the kit and avoiding positive cross reaction.
The invention selects a highly conserved 3D gene segment of foot-and-mouth disease virus as a detection target gene, designs a specific primer and establishes an FMDV specific fluorescent isothermal amplification detection method. The Bst DNA polymerase is adopted, so that the method has the advantages of high amplification efficiency and good specificity; the reaction result is indicated by adopting fluorescent dye, so that accurate judgment is facilitated; the DNA of a sample does not need to be extracted, so that the detection operation is simplified; the whole detection process is fast and only needs 50 min. And the invention can detect a plurality of samples at one time, and is suitable for the detection of a large number of samples.
Detailed Description
The present invention is described in further detail below, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention.
Example 1 was carried out: primer design and construction of foot-and-mouth disease virus gene pMD20-T-FMDV recombinant plasmid
1. Designing a primer: according to the nucleotide sequence of the foot-and-mouth disease virus genome (see table 1) recorded on GenBank, 3D gene conserved regions are screened, and 12 primers in total of 6 pairs are designed, compared and screened.
TABLE 1 foot-and-mouth disease reference strains
| Viral strain name | GenBank accession number | Viral strain name | GenBank accession number |
| SEA-97 | LC483874.1 | 154 | MN275118.1 |
| 01-A01Lc | MK341545.1 | K74 | MN116688.1 |
| 01-CapLc | MK341544.1 | NIG | MN103523.1 |
| Dh-301 | MK088170.1 | K19 | MN116694.1 |
| 19-005 | MN250318.1 | K60 | MN116693.1 |
| 18CD-1610.2 | MN250317.1 | LUNG1 | MH559804.1 |
| 18-5490 | MN250316.1 | VES2 | MH559800.1 |
| 18-3766 | MN250315.1 | MUK564 | MN095362.1 |
| 17-19073 | MN250314.1 | MUK359 | MN095359.1 |
| 2186 | MN275121.1 | 349(740) | MN095357.1 |
| 2101 | MN275120.1 | SV2_d1 | LC485147.1 |
| 171 | MN275119.1 | B14-112_A24 | MH426574. |
The nucleotide sequence of the finally determined isothermal amplification primers is as follows:
FMDF3 ACCCTCGARGCTATC
FMDB3 GCGTCACCGCACAC
FMDFIP TGGACGGCAAGTCCTGTTTTCTCTCCTTTGCACG
FMDBIP TACCGGCGTCTCTTTGATTTTCACCCAACGCAGG
FMDLF CAACTTCTCCTGTATGGTCC
FMDLB TGAGATTCCAAGCTACAGAT
pMD20-T-FMDV recombinant plasmid construction: according to the published 3D gene sequence (GenBank: NC 002657.1) of foot-and-mouth disease virus, Shanghai junjiki Biotech Co., Ltd is entrusted to synthesize a 3D gene fragment, and the 3D gene fragment is cloned into a pMD20-T vector to form a pMD20-T-FMDV plasmid. The Top10 bacteria-competent cells (purchased product) were transformed by the conventional method, and the clones were selected on ampicillin-resistant agar plates and identified by the conventional PCR. And (4) extracting plasmids from the bacteria liquid which is identified as positive by PCR, sending the plasmids to a sequencing company for sequencing, and comparing the sequencing result with the sequence of the reference template to be consistent.
Example 2 establishment of fluorescent isothermal amplification reaction System for foot-and-mouth disease Virus
Comparing and analyzing the foot-and-mouth disease virus gene sequence, designing and screening primers, primarily establishing a foot-and-mouth disease virus fluorescent isothermal amplification detection method, and detecting the primer concentration and Mg of the detection method2+The optimal conditions of the detection method are determined by optimally selecting reaction conditions such as concentration, SYBR GreenI fluorescent dye dosage, Bst DNA polymerase dosage, reaction temperature and the like: the reaction system is 25 mu L in total and comprises three parts of fluorescent isothermal amplification reaction liquid, Bst DNA polymerase and a template. Wherein,one reaction solution for detection was composed of 2.5. mu.L of 10 × Thermopolybuffer, 5.5. mu.L of primer solution, 4. mu.L of 5 mol/L betaine solution, 3.5. mu.L of dNTP Mix (10 mmol/L), 1.5. mu.L of MgSO 24(100 mmol/L), 0.5 mu L SYBR GreenI solution and 4.5 mu L DEPC treated water; one Bst DNA polymerase tested consisted of 1. mu.L of Bst DNA polymerase (8U/. mu.L); one template tested consisted of 2.0 μ L of sample; finally, 20. mu.L of mineral oil (liquid paraffin oil) was added to 25. mu.L of the reaction system. The experiment can be carried out using a fluorescence constant temperature amplification instrument or a commonly used fluorescence PCR instrument.
Finally, the amplification conditions of the foot-and-mouth disease virus fluorescence isothermal amplification detection method are determined to be 64 ℃, 50min, and fluorescence is collected once per minute. When the Tt (time threshold) value is absent and a characteristic amplification curve is absent, determining that the sample is negative; when the Tt value is less than or equal to 40.0 and a typical amplification curve appears, the sample is judged to be positive; when the Tt value is more than 40.0 and a typical amplification curve appears, the process should be repeated, the result still appears, and the result is judged to be positive, otherwise, the result is judged to be negative.
Example 3 Assembly of foot-and-mouth disease Virus nucleic acid extraction-free fluorescent isothermal amplification detection kit
1. The components of the kit are prepared according to the following method:
(1) preparing a primer solution: diluting artificially synthesized primers FMDF3, FMDB3, FMDFIP and FMDBIP to 20 mu mol/L by DEPC treated water, and diluting artificially synthesized primers FMDLF and FMDLB to 40 mu mol/L by DEPC treated water; the diluted primers are mixed evenly according to the proportion of FMDF3, FMDB3, FMDFIP, FMDBIP, FMDLF and FMDLB =1:1:8:8:2: 2.
(2) Preparation of positive control: cloning the foot-and-mouth disease virus 3D gene sequence by a conventional method, constructing pMD20-T-FMDV recombinant plasmid, and carrying out sequence determination on the genes in the plasmid. And (3) after the sequencing result is correct, transforming the recombinant plasmid bacteria obtained by the escherichia coli, carrying out amplification culture on the recombinant plasmid bacteria, and extracting the recombinant plasmid. Measuring the content of recombinant plasmid with Nanodrop2000 instrument, calculating the copy number of 5' UTR gene DNA in microliter volume, and diluting with TE buffer solution to 104The copy number/mu L was measured and dispensed in a sterile manner at a rate of 200 mu L/tube as a positive control of the kit.
(3) Preparation of 10 × isothermal amplification extraction-free buffer: weighing Trisbase: 24.23g, KCl: 3.73g (NH4)2SO4：7g，MgSO47H2O: 2.465g, Triton X-100: 5mL, Brij-58: 5g, DEPC treated water is added to 400mL, the pH value is adjusted to 8.8 (25 ℃) by hydrochloric acid, and the volume is adjusted to 500 mL. And (5) sterilizing at high pressure, cooling, and storing at 2-8 ℃ for later use.
(4) Preparing a fluorescence isothermal amplification extraction-free reaction solution: taking 2.5 mL of 10 times isothermal amplification extraction-free buffer solution, 5.5 mL of primer solution, 4 mL of 5 mol/L betaine solution, 3.5 mL of 10 mmol/L dNTP mix and 100 mmol/L MgSO41.5 mL of the solution, 0.5 mL of 0.1% SYBR GreenI solution and 4.5 mL of 0.1% DEPC treated water, mixing, filtering with a 0.45 μm filter membrane for sterilization (sterile low temperature in the whole process), packaging into small tubes, and labeling.
(5) Preparation of Bst DNA polymerase: bst DNA polymerase was purchased from outsourced (NEB Co.), 8U/. mu.L, 1000. mu.L/tube. Bst DNA polymerase was quantitatively dispensed aseptically and labeled.
(6) Preparation of mineral oil: quantitatively packaging the liquid paraffin oil into small tubes, and labeling.
(7) Preparation of negative control: 0.1% DEPC aqueous solution, aseptically packaging, and labeling.
The kit is assembled according to the following requirements:
the fluorescence isothermal amplification non-extraction reaction solution, Bst DNA polymerase, mineral oil, negative control, positive control and the like are packaged by an external packaging box according to the quantity required by the table 2, and labels (including identification names, batch numbers, production dates, storage periods, production unit information and the like) are added.
TABLE 2 fluorescent isothermal amplification detection kit (extraction-free) composition list for foot-and-mouth disease virus
| Components | Specification (specification) |
| Fluorescence isothermal amplification extraction-free reaction solution | 1100 mu L/tube X1 tube |
| Bst DNA polymerase | 50 mu L/tube X1 tube |
| Negative control | 1000 uL/tube X1 tube |
| Positive control | 200 mu L/tube X1 tube |
| Mineral oil | 1000 uL/tube X1 tube |
| Description | 1 part/box |
Example 4: foot-and-mouth disease virus nucleic acid extraction-free fluorescent isothermal amplification detection kit sensitivity verification
The pMD20-T-FMDV recombinant plasmid (DNA content 10) was purified by treating water with DEPC4Copy/. mu.L) as a sensitive quality control sample at 1:10, 1:100, 1:1000, 1:10000, 1:100000 dilutions. Detection and determination were carried out according to the "usage and determination" of the kit. The detection results of the quality control samples at 1:10, 1:100 and 1:1000 are positive, positive or negative at 1:10000 and negative at 1: 100000. And detecting 22 samples which are clinically diagnosed as positive foot-and-mouth disease virus nucleic acid, including animal blood, tonsil and spleen tissues, wherein the detection results are positive.
Example 5: foot-and-mouth disease virus nucleic acid extraction-free fluorescent isothermal amplification detection kit specificity test
The fluorescent isothermal amplification detection kit for the foot-and-mouth disease viruses of 3 batches is adopted to detect the nucleic acids of highly pathogenic porcine reproductive and respiratory syndrome vaccines, pseudorabies viruses, porcine parvoviruses, porcine circovirus type 2, peste des petits ruminants viruses and 10% suspensions of normal animal blood, tonsils and spleen tissues, and no positive cross reaction exists. 45 samples which are clinically diagnosed and identified as negative are detected, the results are all negative, and the specificity is 100%.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Beijing Senkang Biotechnology development Co., Ltd
Fluorescent isothermal amplification detection kit for foot-and-mouth disease virus nucleic acid extraction-free
<130> 2019
<141> 2019-12-31
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> DNA
<213> FMDV sequence
<400> 1
accctcgarg ctatc 15
<210> 2
<211> 14
<212> DNA
<213> FMDV sequence
<400> 2
gcgtcaccgc acac 14
<210> 3
<211> 34
<212> DNA
<213> FMDV sequence
<400> 3
tggacggcaa gtcctgtttt ctctcctttg cacg 34
<210> 4
<211> 34
<212> DNA
<213> FMDV sequence
<400> 4
taccggcgtc tctttgattt tcacccaacg cagg 34
<210> 5
<211> 20
<212> DNA
<213> FMDV sequence
<400> 5
caacttctcc tgtatggtcc 20
<210> 6
<211> 20
<212> DNA
<213> FMDV sequence
<400> 6
tgagattcca agctacagat 20
Claims (4)
1. A fluorescence isothermal amplification kit for quickly detecting foot-and-mouth disease virus nucleic acid extraction comprises 6 specific primers, and nucleotide sequences of the primers are as follows:
FMDF3 ACCCTCGARGCTATC;SEQ ID NO.1;
FMDB3 GCGTCACCGCACAC;SEQ ID NO.2;
FMDFIP TGGACGGCAAGTCCTGTTTTCTCTCCTTTGCACG;SEQ ID NO.3;
FMDBIP TACCGGCGTCTCTTTGATTTTCACCCAACGCAGG;SEQ ID NO.4;
FMDLF CAACTTCTCCTGTATGGTCC;SEQ ID NO.5;
FMDLB TGAGATTCCAAGCTACAGAT;SEQ ID NO.6。
2. the kit according to claim 1, further comprising one or more of a fluorescence isothermal amplification immunoassay extraction reaction solution (containing SYBR GreenI fluorescent staining agent), Bst DNA polymerase, a negative control, a positive control and mineral oil.
3. A preparation method of a foot-and-mouth disease virus nucleic acid extraction-free fluorescence isothermal amplification detection kit specifically comprises the following steps:
(1) preparing a primer solution: synthesizing primers according to the nucleotide sequence of claim 1, wherein the artificially synthesized primers FMDF3, FMDB3, FMDFIP and FMDBIP are diluted to 20 μmol/L with DEPC treated water, and the artificially synthesized primers FMDLF and FMDLB are diluted to 40 μmol/L with DEPC treated water; uniformly mixing the diluted primers according to the proportion of FMDF3 to FMDB3 to FMDFIP to FMDBIP to FMDLF to FMDLB =1:1:8:8:2: 2;
(2) preparing a fluorescence isothermal amplification extraction-free reaction solution: 0.5 mL of 0.1% SYBR GreenI solution, 2.5 mL of 10-fold concentration reaction buffer solution, 5.5 mL of primer solution, 4 mL of 5 mol/L betaine solution, 3.5 mL of 10 mmol/L dNTP mix, and 100 mmol/L MgSO41.5 mL of the solution and 4.5 mL of 0.1 percent DEPC treated water are mixed uniformly, sterilized by a filter membrane and subpackaged into small tubes;
(3) preparation of Bst DNA polymerase: the concentration is 8U/muL, and the mixture is subpackaged according to 1000 muL/tube aseptically;
(4) preparation of positive control: cloning the sequence of the foot-and-mouth disease virus gene by a conventional method, constructing pMD20-T-FMDV vector plasmid, and carrying out sequence determination on 5' UTR gene in the plasmid; after the sequencing result is correct, Top10 bacteria competent cells are transformed, expanded culture is carried out, and plasmids are extracted as positive control;
(5) the optimized reaction conditions are as follows: adding 22. mu.L of fluorescence isothermal amplification extraction-free reaction solution and 1.0. mu.L of Bst DNA polymerase into a reaction tube, adding 20. mu.L of mineral oil, and adding 2.0. mu.L of sample (without pre-extracting nucleic acid); the reaction condition parameters are 64 ℃ and 50min, and the fluorescence signal is collected once per minute.
4. Use of the kit according to claim 1 and claim 2 for the detection of foot and mouth disease virus in animal blood and animal tissue.
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