CN113564276A - Establishment of foot-and-mouth disease virus visual RT-LAMP detection system and method - Google Patents

Abstract

The invention discloses establishment of a visual RT-LAMP detection method for FMDV. The RT-LAMP primers, primer concentrations, reaction time, reaction temperature, color developing agent and the like are screened by taking synthesized in vitro RNA transcripts with 39 3D-T7 sequences as templates. An RT-LAMP detection method of FMDV is established, the total amount is 20 mu L, and the method comprises 15.2 mu L of RT-LAMP buffer solution, 1.5 mu L of BstAMV enzyme mixed solution, 0.04 mu L (100 mu M) of each of outer primers F3 and B3, 0.5 mu L (100 mu M) of each of inner primers FIP and BIP, 0.11 mu L (100 mu M) of each of loop primers Floop and Bloop, 1 mu L (500 mu M) of neutral red and 1 mu L of template. The reaction time was 55min and the reaction temperature was 65 ℃. After the reaction, the reaction tube solution was judged to be positive when it became pink, and negative when it became orange. The method has strong specificity, and does not react with viruses such as BVDV, ORFV, SPPV/GTPV, SVDV, BTV, PRRSV, CSFV, PRV, PPV and PCV, etc. The established FMDV visual RT-LAMP method lays a foundation for basic level field detection so as to achieve the purpose of quickly diagnosing FMDV.

Description

Establishment of foot-and-mouth disease virus visual RT-LAMP detection system and method

Technical Field

The invention belongs to the technical field of foot-and-mouth disease virus detection, and particularly relates to establishment of a foot-and-mouth disease virus visual RT-LAMP detection system and method.

Technical Field

Foot and Mouth Disease (FMD) is a highly infectious vesicular Disease caused by infection of pigs, cattle, sheep and many wild artiodactyls by Foot and Mouth Disease Virus (FMDV). When adult animals are infected, the body temperature is continuously raised, and oral mucosa, hoof and breast skin generate blisters which can die when serious; immature animals may die prematurely due to a series of diseases that occur prior to the production of blisters, such as acute myocarditis, myocardial infarction, etc. Although the foot-and-mouth disease has low fatality rate, the disease incidence is higher, the normal growth and development of infected animals can be seriously influenced, the production capacity is greatly reduced, the healthy development of animal husbandry is seriously harmed, and huge external trade economic loss and prevention and control economic loss can be brought. Therefore, the world animal health organization ranks the zoonosis as an animal A type fulminating infection disease, and the department of agriculture in China also ranks FMD as a type of animal disease.

The foot-and-mouth disease can be divided into seven serotypes according to serology, and the seven serology can be divided into Asia-European types, namely O type, A type, C type and Asia1 type according to regional homology; south Africa type: south africa type 1 (SAT1), south africa type 2 (SAT2), and south africa type 3 (SAT 3). Each serotype can be divided into multiple topotypes (topotypes) again according to FMDV strain gene sequence and region evolutionary relationship analysis.

Foot and mouth disease virus diagnostics can be divided into clinical diagnostics and laboratory diagnostics. The clinical diagnosis can be judged by that the adult animals with the disease have characteristic blister phenomena at the lip, tongue surface, gum, heel, toe, breast and other parts, and the pain is hard to endure. However, young animals often die before the characteristic blister occurs and have a high mortality rate, so laboratory diagnosis is also essential.

Foot-and-mouth disease is determined as a national animal epidemic disease and occupies an important position in diagnosis, prevention and control in China. Therefore, various laboratory diagnosis modes of the system are continuously enriched by scientific researchers. Laboratory diagnostic methods can be divided into three main groups according to the principle of the respective detection method. One is the pathogen separation technology, the 'gold standard' for detecting pathogens by OIE. The second is serological detection technology-detection of pathogens is generally based on the specific binding interactions between antigen-antibody and complement. Thirdly, molecular biology detection technology, which utilizes complementary pairing combination of basic groups to design a primer and then confirms the amplification of the gene segment of the virus.

The loop-mediated isothermal amplification technology is a new nucleic acid amplification method, and millions of times of DNA with few copy numbers can be amplified in a short time and at a constant temperature by means of the unique strand displacement function and the strong thermal stability of strand displacement DNA polymerase-Bst DNA polymerase and 2 pairs of primers or 3 pairs of primers according to the design of a target gene. This method was originally proposed by Notomi, a Japanese scholarer, and was first published in 2000 in the journal of nucleic acids Res. Compared with the conventional nucleic acid diagnosis QPCR and PCR methods, the method can complete the reaction without a specific instrument-a water bath, does not need the changing temperature-constant temperature (generally between 60-65 ℃), greatly shortens the reaction time with the assistance of the loop primer, and can complete the reaction only in 30-60 min. The high conformity and pertinence of the multiple primers to the target region and the characteristics of Bst DNA polymerase enable LAMP to have sensitivity and specificity similar to qPCR, and the sensitivity can be 10-100 times higher than that of a PCR method. The white precipitate generated in the LAMP reaction result can be observed by naked eyes, so the convenient, simple, quick and specific detection method is developed quickly and has extremely wide research and application prospects. For example, the kit is used for detecting various pathogenic microorganisms, embryos, food biosafety and the like.

FMDV is not eliminated in China, and even in recent years, new FMDV epidemic strains are continuously transmitted from foreign countries to China, and south African FMDV strains are at risk of being transmitted to China. Therefore, the ability to quickly detect FMDV is an urgent need. The experiment uses seven serotype strain sequences of the foot-and-mouth disease as research objects, a universal visual RT-LAMP detection method for the foot-and-mouth disease is predicted to be established and optimized, and a new method is provided for the rapid detection of the foot-and-mouth disease in China.

Disclosure of Invention

In order to solve the technical problems, the invention provides a visualized RT-LAMP detection method for foot-and-mouth disease virus, the total FMDV RT-LAMP detection reaction system is 20 mu L, and the detection method comprises 15.2 mu L of RT-LAMP buffer solution, 1.5 mu L of BstAMV enzyme mixed solution, 0.04 mu L (100 mu M) of each of outer primers F3 and B3, 0.5 mu L (100 mu M) of each of inner primers FIP and BIP, 0.11 mu L (100 mu M) of each of loop primers Floop and Bloop, 1 mu L (500 mu M) of neutral red and 1 mu L of template. The reaction time was 55min and the reaction temperature was 65 ℃. After the reaction, the reaction tube solution was judged to be positive when it became pink, and negative when it became orange.

Preferably, the six primers are as follows:

preferably, the six primers are as follows:

preferably, the six primers are as follows:

the method for establishing the foot-and-mouth disease virus visual RT-LAMP detection system comprises the following steps:

(1) screening, synthesis and identification of foot-and-mouth disease virus target gene 3D fragment

Serotype classification and similarity analysis are carried out on FMDV 3D sequences downloaded from GenBank; according to the principle of time proximity, serotype and difference between sequences of the same serotype, the 3 'end of 39 3D sequences is 500bp, the 5' end of the sequences is added with a T7 promoter sequence (5'-TAATACGACTCACTATAGGG-3') of 20bp, and the sequences are sent to Aiji biotechnology Limited, Guangzhou to be synthesized by taking pUC57 of Amp +; primers were designed for the synthetic 3D-T7pUC57 recombinant plasmid to amplify the 3D and T7 synthetic regions and the amplified PCR products were sent for sequencing to identify the correctness of sequence synthesis;

(2) establishment and optimization of FMDV (frequency modulated double-stranded DV) visual RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection method

Making 3D RNA transcript and making verification, then using RT-LAMP universal detection kit to screen multiple sets of designed RT-LAMP primers, finally obtaining three sets of primers with good amplification effect; the RT-LAMP detection method with the FMDV reaction overall system of 20 mu L is established by optimizing and screening the RT-LMAP primer group with the best detection effect on primer concentration, reaction time, reaction temperature and reaction chromogenic indicators (hydroxynaphthol blue, calcein, SYBR green I, neutral red, phenol red and bromothymol blue) in the RT-LAMP reaction system, and comprises 15.2 mu L of RT-LAMP buffer solution, 1.5 mu L of BstAMV enzyme mixed solution, 0.04 mu L (100 mu M) of each of outer primers F3 and B3, 0.5 mu L (100 mu M) of each of inner primers FIP and BIP, 0.11 mu L (100 mu M) of each of loop primers Floop and Bloop, 1 mu L (500 mu M) of neutral red and 1 mu L of template. The reaction time was 55min and the reaction temperature was 65 ℃. After the reaction, the reaction tube solution was judged to be positive when it became pink, and negative when it became orange.

(3) Specificity and sensitivity detection of RT-LAMP method and clinical sample detection

The established RT-LAMP reaction is subjected to cross reaction verification, and the result shows that the method does not have cross reaction with viruses such as BVDV, ORFV, SPPV/GT PV, SVDV, BTV, PRRSV, CSFV, PRV, PPV, PCV and the like; then 10 of 3D DNA and RNA respectively⁷-10⁰The primers/mu L are used as a template to compare RT-/LAMP, RT-/QPCR and RT-/PCR, and as a result, the method can detect 10 percent by using plasmids as the template³The gene is copies/mu L, and the RT-LAMP can detect 10 by taking RNA as a template⁴copies/μL。

Preferably, the step (1) includes the steps of:

1) performing gene homology analysis on sequences of seven serotypes by using biological software MEGA7.0 and DNAStar7.0, and selecting a region with better conservation as an RT-LAMP design primer and an identified region; then, the 3D sequences of 39 serotypes are screened out by using the published time of the sequences, the serotype of the sequences and the difference of the sequences in the same serotype as the principle, and the 3D sequences of the seven serotypes comprise 6 types A, 8 types O, 4 types Asia1, 4 types C, 5 types SAT1, 6 types SAT2 and 6 types SAT 3. The fragment containing the identified region was cut at the 3 'end to a length of 500bp and the T7 promoter sequence was added at the 5' end to 20bp (5'-TAATACGACTCACTAT AGGG-3') for a total of 520bp each. Sending to Guangzhou Egypti biotechnology limited for cloning to pUC57 vector containing Amp + to construct recombinant plasmid, finally providing glycerobacteria containing seven serotype 3D sequences of 39 pieces of 520bp, and naming according to serotype-3D-pUC 57-FMDV;

accession information for 39 seven serotype 3D sequences are shown in the table below;

2) seven FMDV serotypes, 39 glycerol strains, are subjected to amplification culture.

3) Adopts a plasmid extraction kit (AxyPrep)^TMPlasmid Miniprep kit), the 39 recombinant 3D plasmids were extracted and their concentrations were determined, labeled and stored in a-20 ℃ freezer.

4) And PCR amplification identification and sequencing identification of the recombinant plasmid.

Preferably, the step (2) includes the steps of:

1) primer design

Carrying out similarity comparison on FMDV 3D sequences, selecting a region with higher similarity, namely the tail end of the FMDV 3D region, and carrying out primer design by Beijing Meilaibo biotechnology company, wherein each set of primers comprises two outer primers F3 and B3, two loop primers, i.e. a Floop and a BLOop, and two inner primers, i.e. a FIP and a BIP;

2) RNA template preparation

RNA in vitro transcript preparation:

a) conventional PCR amplification of synthetic 3D fragments of interest

b) PCR amplification product gel recovery

c) In vitro transcription

d) RNA cleaning, removing substances such as oligonucleotide, protein and salts in vitro RNA transcripts;

3) RT-LAMP method primer screening

Screening RT-LAMP outer primer

Diluting the synthesized RT-LAMP outer primers F3 and B3 to 10 mu M, verifying whether the RT-LAMP outer primers can be used or not by adopting a PCR method and taking the synthesized FMDV 3D plasmid as a template, and preliminarily detecting the specificity of the RT-LAMP primers; after PCR amplification, detecting the result by electrophoresis; the RT-LAMP external primer PCR amplification system is as follows:

PCR reaction procedure: 95 ℃ for 5 min; (95 ℃, 30s, 56 ℃, 30s, 72 ℃, 30s) X36 cycles; 72 ℃ for 10 min; at 25 ℃, forever;

② screening RT-LAMP primer by adopting fluorescent RT-LAMP universal kit

Preparing an RT-LAMP reaction system according to the following table, taking an in vitro RNA transcript of an FMDV 3D sequence as a template, and selecting a method for comparative substitution of RT-LAMP primers, wherein reaction procedures of 63 ℃, 60min, 80 ℃ and 10min are selected as initial reaction conditions according to the instructions of an RT-LAMP universal kit; detecting a fluorescence signal by using a QPCR instrument, judging the quality of the primer according to the fluorescence signal and a product electrophoretogram, and screening the RT-LAMP primer;

4) optimization of primer content in RT-LAMP method

Outer primer concentration optimization

On the basis of table 6, the amount of each reagent was fixed, and the concentration of the outer primer was optimized; f3, B3 were diluted to 10 μ M and mixed at 1: 1 volume of the mixture is mixed, and the volume is increased by 0.2 mu L each time from 0.2 mu L/tube until the adding amount is 2.0 mu L; repeating the test, and screening out the optimal concentration of the outer primer by fluorescence screening and electrophoresis detection;

inner primer concentration optimization

On the basis of the table 6 and the screened optimal outer primer concentration, the dosage of each reagent is fixed, and the inner primer concentration is optimized; flood, Bloop diluted to 10 μ M and mixed at 1: 1 volume of the mixture is mixed, and the volume is increased by 0.2 mu L each time from 0.6 mu L/tube until the adding amount is 3.0 mu L; repeating the test, performing fluorescence screening and performing electrophoresis detection to screen out the optimal concentration of the inner primer;

loop primer concentration optimization

On the basis of the table 6 and the screened optimal outer primer concentration and optimal inner primer concentration, the dosage of each reagent is fixed, and the concentration of the loop primer is optimized; FIP, BIP were diluted to 50. mu.M and mixed at 1: 1 volume of the mixture is mixed, and the volume is increased by 0.2 mu L each time from 0.4 mu L/tube until the adding amount is 3.0 mu L; repeating the test, performing fluorescence screening and performing electrophoresis detection to screen out the optimal concentration of the inner primer;

5) RT-LAMP method reaction time optimization

Starting from 30min, and after 40min, taking a gradient every 5min, wherein the total number of reaction gradients is 8; then checking the result by 1.5% nucleic acid gel electrophoresis, and screening out the optimal reaction time;

6) temperature optimization

Under the condition that a fixed reaction system and other reagents are not changed, the reactions are respectively carried out at 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃ and 66 ℃, then the results are checked by 1.5 percent nucleic acid gel electrophoresis, and the optimal reaction temperature is screened out;

7) and (4) screening the RT-LAMP method dye.

Infection by Foot and Mouth Disease Virus (FMDV) causes acute fever, high contact, infectious and acute diseases of artiodactyl, and is very easy to cause large-area and large-scale epidemics, which brings huge loss to the development of socioeconomic. FMDV is divided into seven serotypes, namely A, O, C, Asia l, SAT1, SAT2 and SAT3, wherein SAT1, SAT2 and SAT3 mainly exist in south Africa regions, other serotypes mainly prevail in Asia Europe regions, but the south Africa FMDV is at risk of being introduced into China due to the fact that world trade communication is frequent in recent years, and a new A, O lineage strain is discovered domestically. The FMDV diagnosis method which is rapid, simple and convenient and can meet the field detection requirement is the first step of preventing and controlling virus introduction. The Loop-mediated isothermal amplification (LAMP) has the characteristics of high specificity, high sensitivity, simplicity and convenience, and can meet the requirements of rapid detection and field detection.

Sequence alignment is carried out on FMDV genome, and a highly conserved 3D polymerase gene region is selected as a target region for RT-LAMP diagnosis. On the basis of comparative analysis of seven serotype 3D nucleic acid sequence homologies of FMDV, a 500bp region at the 3' end of each of 39 representative FMDV 3D strains is selected, an Amp + pUC57 plasmid is used as a vector to construct a 3D sequence and T7 promoter recombinant plasmid, and the recombinant plasmid is used for subsequent experiments after being identified and analyzed to be correct. And designing a plurality of groups of RT-LAMP primers according to 8 regions of the 3D target gene, wherein each group of primers comprises six primers including outer primers F3 and B3, and loop primers, i.e., a Floop primer, a BLOop primer, an inner primer FIP primer and a BIP primer.

The synthesized in vitro RNA transcript of the recombinant 3D-T7pUC57 plasmid is used as a template, and conditions such as RT-LAMP primer, optimal primer concentration, reaction time, reaction temperature and the like are screened. Finally, proper primers are screened out, and the system is optimized on the basis of the primers. An RT-LAMP detection method with 20 mu L of FMDV reaction overall system is established, and comprises 15.2 mu L of RT-LAMP buffer solution, 1.5 mu L of BstAMV enzyme mixed solution, 0.04 mu L (100 mu M) of each of outer primers F3 and B3, 0.5 mu L (100 mu M) of each of inner primers FIP and BIP, 0.11 mu L (100 mu M) of each of loop primers Floop and Bloop, 1 mu L (500 mu M) of neutral red and 1 mu L of template. The reaction time was 55min and the reaction temperature was 65 ℃. The reaction result of RT-LAMP can be detected by nucleic acid gel electrophoresis, fluorescence signal detection by a fluorescence detection system, and color change before and after RT-LAMP reaction by a color developing agent. The research compares the color development results of color development agents such as hydroxynaphthol blue, calcein, SYBR green I, neutral red, phenol red, bromothymol blue and the like, and finally selects the neutral red as a color development indicator. After the reaction, the reaction tube solution was judged to be positive when it became pink, and negative when it became orange.

The specificity and sensitivity analysis is carried out on the establishment of the FMDVRT-LAMP method, and the result shows that the method does not have cross reaction with viruses such as BVDV, ORFV, SPPV/GTPV, SVDV, BTV, PRRSV, CSFV, PRV, PPV, PCV and the like. And the method can detect 10 by taking plasmid DNA as a template³The copies/mu L, QPCR and PCR methods can respectively detect 10¹And 10²copies/. mu.L; RT-LAMP (reverse transcription loop-mediated isothermal amplification) with RNA as template can detect 10⁴copies/. mu.L. 10 can be detected by the RT-QPCR and RT-PCR recommended by OIE³And 10⁴copies/. mu.L. 39 parts of 3D RAN transcript samples and 44 parts of clinical samples are detected by the established FMDV RT-LAMP method and compared with RT-QPCR and RT-PCR, and the results show that the negative and positive detection rates of the three methods are consistent. Detection limit phase of RT-LAMP and RT-PCR on sensitivityRecently, it corresponds to the virus content detected at RT-qPCR CT value of 32.5. The experiment establishes a universal visual RT-LAMP detection method for FMDV, lays a foundation for basic level field detection, and achieves the purpose of quickly diagnosing FMDV.

Drawings

FIG. 1 is a 3D plasmid in vitro transcription validation graph.

FIG. 2 is a diagram showing the results of the first two or three sets of outer primers.

FIG. 3 is the electrophoretogram of the first, second and third sets of RT-LAMP primers.

FIG. 4 is a graph showing the results of RT-PCR or PCR amplification of nucleic acids from ten viruses.

FIG. 5 is a diagram showing the results of detection of RT-LAMP primer specificity.

FIG. 6 shows FMDVDNA 10⁷-10⁰copies/μ LLAMP assay results.

FIG. 7 is FMDV seven serotype plasmid DNA 10⁷-10⁰copies/μ L QPCR assay results.

FIG. 8 is FMDV seven serotype plasmid DNA 10⁷-10⁰copies/mu LPCR test result chart.

FIG. 9 is FMDVRNA 10⁷-10⁰copies/mu LRT-LAMP detection result graph.

FIG. 10 is an FMDV seven serotype RNA transcript 10⁷-10⁰copies/mu LRT-QPCR detection result graph.

FIG. 11 is an FMDV seven serotype RNA transcript 10⁷-10⁰copies/μ LRT-PCR detection results.

FIG. 12 is a graph showing the results of RT-QPCR assay on 6 clinical specimen stock solutions.

FIG. 13 is a graph showing the results of RT-LAMP detection of 6 clinical sample stock solutions and diluent solutions.

FIG. 14 is a graph showing the results of RT-PCR detection of 6 dilutions of clinical samples

Detailed Description

Example 1: screening, synthesis and identification of foot-and-mouth disease virus target gene 3D fragment

About 1000 FMDV 3D sequences were downloaded from genbank database, removing sequences that were too long in time and whose sequences were incomplete. The remaining 631 complete 3D sequences included 142 type a sequences, 46 type Asia1 sequences, 24 type C sequences, 292 type O sequences, 50 SAT1 sequences, 45 SAT2 sequences, and 28 SAT3 sequences. The sequences of the seven serotypes are subjected to gene homology analysis by using biological software MEGA7.0 and DNAStar7.0, and a region with better conservation is selected as a LAMP design primer and a region to be identified. Then, the 3D sequences of 39 serotypes are screened out by using the published time of the sequences, the serotype of the sequences and the difference of the sequences in the same serotype as the principle, and the 3D sequences of the seven serotypes comprise 6 types A, 8 types O, 4 types Asia1, 4 types C, 5 types SAT1, 6 types SAT2 and 6 types SAT 3. The fragment containing the identified region was cut at the 3 'end to a length of 500bp and the T7 promoter sequence was added at the 5' end to 20bp (5'-TAATACGACTCACTATAGGG-3') for a total of 520bp each. Sent to Guangzhou Egypti Biotechnology Limited to clone into pUC57 vector containing Amp + to construct recombinant plasmid, finally glycerol bacteria containing seven serotypes of 39 520bp 3D sequence were provided and named according to serotype (GenBank number) -3D-PUC 57-FMDV. The accession number information for the 39 seven serotype 3D sequences is shown in table 1.

TABLE 139 accession number information for seven serotype 3D sequences

Type A

O type

Asia1 type

C type

SAT1 type

SAT2 type

SAT3 type

KT968663.1

MF461724.1

MF782478.1

AF274010.1

AY593845.1

AF540910.1

KF647850.1

KC588943.1

KY234502.1

MF372125.1

AJ007572.1

MF678826.1

AY593849.1

KJ820999.1

KC440882.1

KC503937.1

MG372731.1

AY593805.1

MN116689.1

JX014255.1

KX375417.1

MG923580.1

JN998085.1

AY304994.1

AY593810.1

KM268899.1

KJ144918.1

MH053341.1

MN116688.1

MN250318.1

AY593839.1

KJ676543.1

KF647849.1

KY322680.1

AY593823.1

KJ144904.1

MG372727.1

LC456871.1

JQ973889.1

FMDV seven serotype 39-glycerol strain amplification culture and plasmid DNA extraction

Adding a liquid culture medium of LB with resistance of 4mLAmp into a bacteria shaking tube beside an alcohol lamp in a biological safety cabinet, adding 39 glycerol bacteria containing 3D target fragments according to the proportion of 2%, and performing shake culture on a shaking table at 37 ℃ and 220rpm/min for 9-10 h. After shake culture, 500. mu.L of the bacterial liquid is taken out from a bacteria shaking tube in a biological safety cabinet and added into a 1.5mL centrifuge tube with 500. mu.L of 50% glycerol to prepare 25% glycerol bacteria, the marker is clear and the glycerol bacteria is preserved at minus 40 ℃, and the residual bacterial liquid is used for plasmid extraction.

Plasmid extraction step:

firstly, an overnight culture bacterial liquid with the total amount of 1-3mL is sucked into a 1.5mL centrifuge tube by a pipetting gun with the maximum volume of 1mL for two times, and is centrifuged at 12000rpm/min for 1min, and the supernatant is discarded.

Adding 250 mu LBuffer S1 into a 1.5mL centrifuge tube, and blowing and beating suspended thalli by a pipette gun until the solution is uniform. (ensure that Buffer S1 used has been added to RNaseA and stored in a 4 ℃ environment for long term storage)

And thirdly, adding 250 mu LBuffer S2, fully and gently turning the mixture up and down for 4-6 times, uniformly mixing the mixture to fully crack the thalli, and changing the turbid liquid into clear liquid (so as to avoid violent shaking which otherwise causes pollution of genome DNA), wherein the step is not suitable for more than 5 min. (Buffer S2 should be twisted on the bottle cap immediately after use to avoid CO in the air₂The neutralization reaction is carried out with NaOH of Buffer S2 to reduce the bacteriolysis efficiency)

To this, 350. mu.L Buffer S3 was added and the mixture was turned up and down gently 6 to 8 times, and a large amount of white precipitate was observed to be generated, 12000rpm and 10min after centrifugation.

Fifthly, absorbing the supernatant in the previous step, transferring the supernatant into a preparation tube (provided by a kit) placed on a 2mL centrifuge tube, carrying out centrifugation at 12000rpm/min for 1min (the centrifugation step can be repeated once for obtaining more plasmids), and discarding the waste liquid.

Sixthly, adding 500 mu LBufferW1 into a preparation tube, centrifuging at 12000rpm/min for 1min, and discarding the filtrate.

Add 700. mu. LBufferW2 to the preparation tube at 12000rpm, centrifuge for 1min, discard the filtrate, repeat once. (ensure that the BufferW2 used has had the specified volume of absolute ethanol added)

And then the preparation tube and 2mL centrifuge tube at 12000rpm/min, centrifugal 2 min.

The self-skin is prepared by placing the tube into a new 1.5mL centrifuge tube, and slowly adding 30-50 μ L preheated ddH above the center of the tube membrane₂And O, standing at room temperature for 2min, 12000rpm/min, and centrifuging for 1 min. (to increase plasmid concentration centrifugal steps can be repeated once)

PCR amplification identification and sequencing identification of recombinant plasmid

PCR amplification identification of recombinant plasmid

According to the length of the synthesized 3D sequence, a set of primers capable of amplifying the full length of 520bp of the target fragment is designed. 39 strips of 3D plasmid were PCR amplified. The primers are shown in Table 2, and the PCR amplification system of the 3D fragment is shown in Table 3.

TABLE 23D fragment amplification primers

TABLE 3 PCR amplification System

PCR amplification program, 95 deg.C, 5 min; (95 ℃, 30s, 56 ℃, 30s, 72 ℃, 45s) X36 cycles; 72 ℃ for 10 min; 25 ℃ and forever.

Preparation of nucleic acid gel and electrophoresis identification

First, 1g of Agarose (Agarose) was weighed with weighing paper and poured into a 250mL conical flask.

The 1XTAE of 100mL is measured by a cylinder and poured into a conical flask of 250mL, and the conical flask is forcibly shaken for several times and heated by a microwave oven until the liquid is clear. (better discrimination against light)

And pouring the three parts into the filled nucleic acid mould, and waiting for about 30min to be used for electrophoresis. (4 ℃ can accelerate the gel coagulation process) electrophoresis of PCR products

The method comprises the steps of placing prepared electrophoretic glue in an electrophoretic groove.

And then 6-8 mu L of PCR product is spotted in the glue hole, and the temperature is 145V for 20 min.

And observing the result by ultraviolet photographing of the gel imaging system.

Sequencing identification of recombinant positive plasmid

After the PCR identification is correct, 13 PCR products with the sequences of A (KC440882), A (KC588943), C (AJ007572), O (JN998085), SAT1(MF678826), SAT2(AY593849), SAT3(KF647850), Asia1(AY304994), A (MN116688), O (LC456871), C (AY593810), SAT3(MH053341) -3D-pUC57-FMDV are selected and sent to the Simanidae company for sequencing. Sequence alignment was performed on the synthesized sequence and the sequencing results using bioinformatics software dnastar 7.0.

FMDV seven serotype glycerobacteria amplification culture and plasmid DNA extraction

Adopts a plasmid extraction kit (AxyPrep)^TMPlasmid miniprep kit), the 39 recombinant 3D plasmids were successfully extracted, their concentrations were determined, labeled and stored in a-20 ℃ freezer.

This example performed serotype classification and similarity analysis of FMDV 3D sequences downloaded in GenBank. To meet the requirements of laboratory experimental materials, based on the principles of time proximity, serotype and sequence difference between the same serotypes, 39 3D sequences with 500bp at the 3 'end and T7 promoter sequence (5'-TAATACGACTCACTATAGGG-3') at the 5' end were selected and sent to Guangzhou Egyi Biotechnology Limited to synthesize the sequence by using Amp + resistant pUC57 as a vector. Primers designed and amplified for the synthesized 3D-T7pUC57 are used for amplifying the synthesized 3D and T7 regions and the amplified PCR products are sent to be sequenced to identify the correctness of sequence synthesis, and the identification provides a material basis for the next step of experiment to be carried out smoothly.

Example 2

Establishment and optimization of FMDV (frequency modulated double-stranded DV) visual RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection method

The recombinant plasmid of FMDV 3D-T7pUC57 was constructed synthetically by Erysiphe, Guangzhou, and FMDV 3D RNA transcripts were prepared according to the T7 in vitro transcription kit.

Primary biological agents

Prime Script TM One Step RT-PCR Kit Ver.2.0, Prime Script Taq (Ex Taq Version 2.0plus dye), In Vitro Transcription T7 Kit, Takara miniBestagarose Gel DNAextraction Kit ver4.0(Takara Corp.);

Mini kit；

MiniElute clear kit (QIAGEN); hydroxyl naphthol blue, calcein, SYBR green I, neutral red, phenol red, bromothymol blue and other color reagent solutions, Eve green (20X), RT-LAMP universal detection kit (Beijing Meilaibo Biotech company).

Primer design

The FMDV 3D sequences are subjected to similarity comparison, a region with high similarity, namely the tail end of the FMDV 3D region, is selected, primer design is carried out by Beijing Meilaibo biotechnology, and each group of primers comprises two outer primers F3 and B3, two loop primers, Floop and Bloop, and two inner primers, FIP and BIP.

RNA template preparation

The preparation method of the RNA in vitro transcript comprises the following steps:

a) conventional PCR amplification of synthetic 3D fragments of interest

1. A universal amplification primer was designed for the 3D target fragment, and PCR amplification was performed in a 50. mu.L system using the 3D-T7pUC57 plasmid as a template.

2. The amplification program is 95 ℃ for 5 min; (95 ℃, 30s, 55 ℃, 30s, 72 ℃, 40s) X36 cycles; 72 ℃ for 10 min; 25 ℃ and forever.

3. And (3) detecting the PCR product by electrophoresis, confirming whether the size of the strip is correct, carrying out a gel recovery experiment after the size is correct, and collecting the product.

b) PCR amplification product gel recovery

1. The gel block containing the band of interest was cut with a scalpel under a uv lamp and placed in a new 1.5mL centrifuge tube which was marked.

2. The weight of the gum block was weighed, and Buffer GM was added in an amount of 1mg ═ 1 μ L in 4 volumes.

3. Dissolving in water bath at 60 deg.C while shaking gently to accelerate dissolution.

4. After the colloid was completely dissolved, the gel was transferred to an adsorption column in the kit and placed in a 2mL centrifuge tube provided in the kit. 12000rpm/min, centrifugation for 1min, and discarding the filtrate. (if it is desired to increase the recovery rate, this step can be repeated)

5. Add 700. mu.L of BufferW2 at 12000rpm/min, centrifuge for 1min, discard the filtrate, and repeat once.

6. 12000rpm/min, centrifugation for 2 min.

7. The column was placed in a new 1.5mL centrifuge tube and RNase free ddH preheated at 60 ℃ was added to the center of the membrane₂O20-30 μ L, standing for 2 min.

8. 12000rpm/min, centrifugation for 1min, elution of PCR products. (if it is desired to increase the recovery rate, this step can be repeated)

9. The concentration of Nanodrop was measured and stored at-20 ℃ for further use.

c) In vitro transcription

1. Dissolving a reagent: an appropriate amount of ice was prepared, all reagents were dissolved and centrifuged appropriately T7 RNAPLYLERAse and ATP/GTP/UTP/CTP on ice, 10 XTdescription Buffer was placed at room temperature.

2. In vitro transcription systems were formulated at room temperature as in Table 4 below, and the approach of the premix system was used to reduce errors. (10X transfer Buffer can be added in the last order)

TABLE 4 in vitro transcription reaction System

3. Mixing, and slightly centrifuging to collect the mixture.

4. Incubate at 37 ℃ for 2 h. (RNA yield is related to the amount of DNA template added and reaction time, and when a large amount of RNA is required, the amount of DNA template may be increased or incubation time may be prolonged)

5. After the incubation was completed, 2. mu.L of RNase free DNase I was added and digested at 37 ℃ for 30min to remove the DNA template.

d) RNA cleaning (removal of oligonucleotide, protein and salt from in vitro RNA transcript)

1、RNase free ddH₂The volume of the transcribed RNA sample was adjusted to 100. mu.L in a 1.5mL centrifuge tube, 350. mu.L of BufferRLT was added and mixed well.

2. Add 250. mu.L of ethanol (96-100%), mix well and go immediately to step 3.

3. Transferring the sample into a 2mL matched collection tube, centrifuging at 10000rpm/min for 30s, repeating the centrifuging for once, and discarding the filtrate.

4. Add 500 u LBuffer RPE, 10000rpm/min, centrifuge for 30s, discard filtrate.

5. Adding 500 μ L ethanol (80%), centrifuging at 10000rpm/min for 2min, discarding filtrate, uncovering, standing for 2min, 14000rpm/min, and centrifuging for 5 min.

6. The preparation tube was placed in a new 1.5mL centrifuge tube (provided in kit), and 15. mu.L-20. mu.L of the preheated RNase free ddH was added₂And O, standing for 2 min. 14000rpm/min, centrifuging for 1min, eluting RNA, repeating, measuring the concentration by Nanodrop, and storing at-20 ℃ for later use.

RT-LAMP outer primer screening

The synthesized RT-LAMP outer primers F3 and B3 are diluted to 10 mu M, a PCR method is adopted, and the synthesized FMDV 3D plasmid is used as a template to verify whether the RT-LAMP outer primers can be used or not, so that the specificity of the RT-LAMP primers can be preliminarily detected. And (5) detecting the result by electrophoresis after PCR amplification.

TABLE 5 RT-LAMP outer primer PCR amplification System

PCR reaction procedure: 95 ℃ for 5 min; (95 ℃, 30s, 56 ℃, 30s, 72 ℃, 30s) X36 cycles; 72 ℃ for 10 min; 25 ℃ and forever.

Screening RT-LAMP primers by adopting fluorescent RT-LAMP universal kit

An RT-LAMP reaction system is prepared according to the table 6, an in vitro RNA transcript of an FMDV 3D sequence is used as a template, an RT-LAMP primer is compared and replaced, and reaction procedures of 63 ℃, 60min, 80 ℃ and 10min are selected as initial reaction conditions according to the instructions of an RT-LAMP universal kit. And (3) detecting a fluorescence signal by using a QPCR instrument, judging the quality of the primer according to the fluorescence signal and a product electrophoretogram, and screening the RT-LAMP primer.

TABLE 6 Universal RT-LAMP reaction System

The primer is the most important link in the LAMP reaction, and the quality of the primer directly influences the amplification result of the product. The LAMP primers F3, B3, Floop, BLOop, FIP and BIP have different functions in different stages. And different LAMP results of primer concentrations also differ, so the concentration of LAMP primer was screened.

Outer primer concentration optimization

On the basis of Table 6, the amount of each reagent was fixed, and optimization of the concentration of the outer primer was performed. F3, B3 were diluted to 10 μ M and mixed at 1: 1 volume of the mixture was added in 0.2. mu.L increments from 0.2. mu.L/tube to 2.0. mu.L (i.e., the addition range was 0.2. mu.L-2.0. mu.L). And repeating the test, and screening out the optimal concentration of the outer primer by fluorescence screening and electrophoresis detection.

Inner primer concentration optimization

Based on Table 6 and the selected optimal outer primer concentrations, the amounts of the respective reagents were fixed, and inner primer concentration optimization was performed. Flood, Bloop diluted to 10 μ M and mixed at 1: 1 volume of the mixture was added in 0.2. mu.L increments from 0.6. mu.L/tube until the addition was 3.0. mu.L (i.e., the addition ranged from 0.6. mu.L to 3.0. mu.L). And repeating the test, fluorescence screening and electrophoresis detection to screen out the optimal concentration of the inner primer.

Loop primer concentration optimization

Based on Table 6, and the selected optimal outer primer concentration and optimal inner primer concentration, the amounts of the respective reagents were fixed to optimize the loop primer concentration. FIP, BIP were diluted to 50. mu.M and mixed at 1: 1 volume of the mixture was added in 0.2. mu.L increments from 0.4. mu.L/tube until the addition was 3.0. mu.L (i.e., the addition ranged from 0.4. mu.L to 3.0. mu.L). And repeating the test, fluorescence screening and electrophoresis detection to screen out the optimal concentration of the inner primer.

RT-LAMP method reaction time optimization

Starting from 30min, and after 40min, taking a gradient every 5min, wherein the total number of reaction gradients is 8; then checking the result by 1.5% nucleic acid gel electrophoresis, and screening out the optimal reaction time;

temperature optimization

Under the condition that a fixed reaction system and other reagents are not changed, the reactions are respectively carried out at 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃ and 66 ℃, then the results are checked by 1.5 percent nucleic acid gel electrophoresis, and the optimal reaction temperature is screened out;

and (4) screening the RT-LAMP method dye.

Six different chromogenic reagents including hydroxynaphthol blue (HNB), calcein, SYBR green I, Neutral Red (Neutral Red), phenol Red (Cresol Red), bromophenol blue were tested with the A (KC440882) -3D-pUC57 plasmid and screened for their chromogenic effects. Melapbo's 2X U-LAMP buffer and 2 Xunbuffered LAMP MIX buffer (pH adjusted to 8.5) were used as buffer systems. In this experiment, the concentrations and volumes of HNB (1mM), calcein, SYBR green (500. mu.M), neutral Red (500. mu.M), Cresol Red (500. mu.M) and bromophenol blue were verified by the Biotech company of Meilebo, Beijing to be the optimum concentrations and volumes. After preparing the system as shown in tables 7 and 8, the reaction was carried out in a PCR apparatus at 65 ℃ for 55min, at 80 ℃ for 5min, and the results were visually observed.

TABLE 7 HNB/calcein/SYBRgreenLAMP reaction System

TABLE 8 LAMP reaction System of phenol Red/neutral Red/bromophenol blue

Primer design

The primers were divided into two batches, the first primer specific information is shown in Table 9, and the second primer specific information is shown in Table 10. Primer design was done by the Biotech company, Meilaibo, Beijing, and synthesized by the Biotech company, Simian.

TABLE 9 first LAMP primer details

TABLE 10 second batch LAMP detailed information

Template preparation

After successful preparation of T7 in vitro transcripts of 3D plasmid, as shown in FIG. 1, A (KC440882) -3D-pUC57-FMDV was used as an example to verify whether RNA was successfully transcribed in vitro. The size of the in vitro transcribed RNA was 520bp, consistent with the size indicated by RNA standard marker, slightly smaller than the normal DNA band. No bands are shown in the samples treated with no transcriptase and with the addition of the random.

RT-LAMP (reverse transcription loop-mediated isothermal amplification) outer primer screening result

The PCR detection is carried out on 39 3D plasmids by using three sets of external primers of RT-LAMP, the result is as follows, each set of primers can successfully amplify corresponding fragments, which indicates the availability of the external primers of RT-LAMP, but the second set of external primers can obviously show that the amplification result has certain impurity bands which may influence the amplification efficiency.

RT-LAMP primer screening result

After the preliminary screening of RT-LAMP outer primers, the RT-LAMP primers are comprehensively screened, and finally 22 sets of primers are introducedThree sets of RT-LAMP primer groups with good effects are screened out from the substance, and the three sets of primers are renamed as a first set, a second set and a third set respectively. The details of the primers are shown in tables 11, 12 and 13. Seven serotype RNA 10 corresponding thereto⁵The RT-LAMP result of copies/mu L detection is shown in FIG. 2, and each group of primers can successfully amplify seven serotypes, and typical ladder-shaped electrophoresis bands are presented.

TABLE 11 first set of RT-LAMP primers

TABLE 12 second set of RT-LAMP primers

TABLE 13 third set of RT-LAMP primers

In the embodiment, 3D RNA transcripts are prepared and verified, and then RT-LAMP universal detection kits are used for screening a plurality of designed RT-LAMP primers, so that three sets of primers with better amplification effect are finally obtained. The RT-LAMP detection method with the FMDV reaction overall system of 20 mu L is established by optimizing and screening the RT-LMAP primer group with the best detection effect on primer concentration, reaction time, reaction temperature and reaction chromogenic indicators (hydroxynaphthol blue, calcein, SYBR green I, neutral red, phenol red and bromothymol blue) in the RT-LAMP reaction system, and comprises 15.2 mu L of RT-LAMP buffer solution, 1.5 mu L of BstAMV enzyme mixed solution, 0.04 mu L (100 mu M) of each of outer primers F3 and B3, 0.5 mu L (100 mu M) of each of inner primers FIP and BIP, 0.11 mu L (100 mu M) of each of loop primers Floop and Bloop, 1 mu L (500 mu M) of neutral red and 1 mu L of template. The reaction time was 55min and the reaction temperature was 65 ℃. After the reaction, the reaction tube solution was judged to be positive when it became pink, and negative when it became orange.

Example 3: specificity and sensitivity detection of RT-LAMP method and clinical sample detection

In the embodiment, the established RT-LAMP reaction is subjected to cross reaction verification, and the result shows that the method does not have cross reaction with viruses such as BVDV, ORFV, SPPV/GTPV, SVDV, BTV, PRRSV, CSFV, PRV, PPV, PCV and the like. Then 10 of 3D DNA and RNA respectively⁷-10⁰The primers/mu L are used as a template to compare RT-/LAMP, RT-/QPCR and RT-/PCR, and as a result, the method can detect 10 percent by using plasmids as the template³The copies/mu L, QPCR and PCR methods can respectively detect 10¹And 10²copies/. mu.L; RT-LAMP (reverse transcription loop-mediated isothermal amplification) with RNA as template can detect 10⁴copies/. mu.L. 10 can be detected by the RT-QPCR and RT-PCR recommended by OIE³And 10⁴copies/. mu.L. Therefore, the LAMP method is less than 10 times sensitive to DNA than PCR. Sensitivity on RNA was consistent with PCR, 10-fold lower than RT-QPCR.

Plasmids and viruses: recombinant plasmids of A (KC440882), Asia1(MF782478), C (AJ007572), O (KC503937), SAT1(AY593845), SAT2(AF540910), SAT3(KJ820999) -3D-pUC57-FMDV were synthetically constructed by Egyki Biotechnology, Inc., Guangzhou. The cytotoxicities of Orf (ORFV), marsupillaria (SPPV/GTPV), BVDV, CSFV and SVV were provided by Lanzhou veterinary institute; BTV nucleic acids are provided by the lanzhou veterinary institute; FMDV, PPV, PPRV, PCV₃And nucleic acids such as PRRSV are stored in the laboratory.

The main biological reagents are as follows: prime Script TM One Step RT-PCR KitVer.2.0, Prime Script Taq (Ex TaqVersion 2.0plus dye), Takara MiniBestViralRNA/DNAextraction KitVer.5.0(Takara Corp.); one-step RT-QPCR amplification kit (Taqman) probe method, QPCR amplification kit, BstDNA polymerase and AMV reverse transcriptase (Beijing Meilaibo Biotech).

Specific assay

Viral nucleic acid extraction

Viral DNA/RNA extraction protocol:

A200-L centrifugal tube with 1.5mL high pressure is used for treating virus cell toxicity of sheep aphtha (ORFV), cotton pox (SPPV/GTPV), BVDV, CSFV, SVV, etc., and RNase free ddH can be used if it is less than 200-L₂Make up to 200. mu.L in O or PBS.

Adding 200 mu L of BufferVGB, 20 mu L of LProteinase K and 1 mu L of Carrier RNA, fully and uniformly mixing by blowing, adding a water bath kettle which is heated to 56 ℃ in advance, and carrying out water bath for 10 min.

And taking out the centrifuge tube, adding 200 mu L of absolute ethyl alcohol into the mixed solution in the tube, and fully and uniformly mixing.

Fourthly, the Spin Colum provided in the kit is placed in a Collection Tube, and the mixed solution in the centrifugal Tube is transferred to the Spin Colum at 12000rpm and centrifuged for 2 min.

Fifthly, 500 mu LBufferRWA is added into Spin Colum at 12000rpm/min, the mixture is centrifuged for 1min, and the filtrate is discarded.

Sixthly, adding 700 mu LBuffer RWB into Spin Colum, centrifuging at 12000rpm/min for 1min, and discarding the filtrate. (confirm that Buffer RWB had been added to the specified volume of 100% absolute ethanol prior to use, and that Buffer RWB was added uniformly around the Spin Colum tube wall to help wash out the salt attached to the tube wall) and repeated once again.

Spin Chamum was set at 12000rpm/min for 2min, and the Collection Tube was discarded.

In addition, Spin Colum was put in 1.5mL of RNase free collection tube provided in the kit, and 30-50. mu.L of RNase free ddH was added to the center of the Spin Colum membrane₂And O, standing at room temperature for 5 min. 12000rpm/min, centrifugation for 2 min. (this step can be repeated once)

Detection of the DNA/RNA of a triple-point. (since carrierRNA was added during extraction, quantification could not be determined by electrophoresis or absorbance measurement, PCR, RT-PCR, qPCR detection could be performed)

Virus self-identification

Before the established FMDVRT-LAMP detection method is subjected to specificity analysis, the viruses provided by a laboratory need to be self-verified, namely, the successful extraction of virus nucleic acid is ensured. The primers used for identifying the ten viruses such as Orf (ORFV), pimple (SPPV/GTPV), PRV, PCV, PPV, BVDV, CSFV, SVA, BTV and PRRSV are specifically shown in table 14 below, and the FMDV primers were reacted using the third set of the previous screen. The DNA virus Orf (ORFV), cotton pox (SPPV/GTPV), PRV, PCV and PPV reaction systems and reaction programs are shown in Table 15, Table 16 below; the reaction systems and procedures for the RNA viruses FMDV, BVDV, CSFV, SVV, BTV and PRRSV are shown in Table 17 and Table 18 below.

TABLE 14 primer tables for virus self-validation

TABLE 15 PCR reaction systems for ORFV, SPPV/GTPV, PRV, PCV, PPV

TABLE 16 PCR reaction conditions for ORFV, SPPV/GTPV, PRV, PCV, PPV

TABLE 17 RT-PCR reaction systems for BVDV, CSFV, SVV, BTV, PRRSV

TABLE 18 RT-PCR reaction conditions for BVDV, CSFV, SVV, BTV, PRRSV

Determination of specificity of RT-LAMP primers

Detection of 3D-LAMP primers and FMDV 3D RNA 10 by using optimized RT-LAMP response system⁵copies/. mu.L transcripts (one sample randomly selected from 39 samples) and DNA viruses ORFV, SPPV/GTPV, PRV, PCV, PPV, RNA virus FMDV, BVDV, CSFV, SVV, BTV, PRRSV cross-reactivity as shown in Table 19 below. The RT-LAMP reaction conditions are 65 ℃, 55min, 80 ℃ and 5 min. Color development and electrophoresis detection results.

TABLE 19 reaction System for RT-LAMP primer Cross-validation

Sensitivity assay

LAMP reaction sensitivity using DNA as template and QPCR and PCR reaction sensitivity

Seven fresh plasmids of glycerol bacteria such as A (KC440882), Asia1(MF782478), C (AJ007572), O (KC503937), SAT1(AY593845), SAT2(AF540910), SAT3(KJ820999) -3D-pUC57-FMDV are extracted, the concentrations of the plasmids are measured by using Nanodrop software, the copy numbers are respectively calculated, and the plasmids are diluted to 10 times by a method of diluting the plasmids by 10 times⁰copies/. mu.L, 10 of these plasmids were selected⁷-10⁰The sensitivity limit detection was performed using copies/. mu.L as template.

Copy number calculation formula:

average Molecular Weight (MW) dsDNA ═ (number of bases) x (660 daltons/base)

ssRNA ═ (number of bases) X (340 daltons/base)

The plasmid is double-stranded DNA:

(6.02x10²³copy number per mole) x concentration g/mL)/(MW g/mol) i.e. copies/mL

(6.02x10²³Copy number per mole) X concentration g/mL)/(DNA length X660) copies/mL or

(6.02x10²³Copy number l mol) x (ng/. mu.LX 10-⁹) /(DNA length x660) ═ copies/. mu.L.

TABLE 20 LAMP reaction System

LAMP reaction program at 65 deg.C for 55 min; 80 ℃ for 5min

TABLE 21 QPCR and PCR reaction primers

TABLE 22 QPCR reaction System

QPCR reaction procedure: at 95 ℃ for 3 min; (95 ℃, 10s, 55 ℃, 30s, 72 ℃, 30s) X40 cycles.

TABLE 23 PCR reaction System

PCR reaction procedure: 95 ℃ for 5 min; (95 ℃, 30s, 56 ℃, 30s, 72 ℃, 45s) X36 cycles; 72 ℃ for 10 min; 25 ℃ and forever.

RT-LAMP reaction sensitivity taking RNA as template and RT-QPCR and RT-PCR reaction sensitivity

3D plasmids are used as raw materials, 3D RNA transcripts are prepared in vitro, and the reaction sensitivities of RT-LAMP, RT-QPCR and RT-PCR are detected on the basis of RNA. The concentration of the resulting RNA transcripts was measured using a Nanodrop instrument and the copy number was calculated. Diluting RNA to 10 times of 10⁰Selection of 10 of these RNAs⁷-10⁰The sensitivity limit detection was performed using copies/. mu.L as template.

The transcript RNA is single-stranded RNA, and the specific calculation formula is as follows:

(6.02x10²³copy number per mole) X concentration g/mL)/(MW g/mol) i.e. copies/mL

(6.02x10²³Copy number per mole) X concentration g/mL)/(RNA length X330) copies/mL or

(6.02x10²³Copy number/mole) X concentration ng/. mu.LX 10-⁹) L (RNA length x330) ═ copies/. mu.l.

TABLE 24 RT-LAMP reaction System

RT-LAMP reaction program at 65 deg.C for 55 min; 80 ℃ for 5 min.

TABLE 25 RT-QPCR reaction System

RT-QPCR reaction procedure: at 50 ℃ for 30 min; at 95 ℃ for 3 min; (95 ℃, 10s, 55 ℃, 30s, 72 ℃, 30s) X40 cycles.

TABLE 26 RT-PCR reaction System

RT-PCR reaction procedure: at 50 ℃ for 30 min; 95 ℃ for 5 min; (95 ℃, 30s, 56 ℃, 30s, 72 ℃, 45s) X36 cycles; 72 ℃ for 10 min; 25 ℃ and forever.

Detection of clinical samples

The established FMDV visual RT-LAMP is used for detecting clinical positive samples (numbered 1-6) which are prepared by the laboratory and contain 39 parts of seven serotype 3D RNA transcripts and 6 parts of FMDV nucleic acids provided by OIE foot-and-mouth disease reference laboratory, and the laboratory stores Asia1, SAT2 positive antigen two parts and 36 parts of clinical sample nucleic acids (numbered 7-42). And compared with OIE recommendation methods RT-QPCR and RT-PCR.

Cross-reaction validation

Virus self-verification

When the established RT-LAMP method is subjected to specificity analysis, the related viruses need to be identified. As a result, as shown in FIG. 4, the verification result of each viral nucleic acid showed a band of interest at the corresponding position. The amplified RT-PCR or PCR product is sent to Xian engine biology company for sequencing, the sequencing result is subjected to BLAST comparison, the result is consistent with each strain sequence in the gene library, the reliability of the virus nucleic acid is described, and the accuracy of the cross reaction is ensured.

RT-LAMP specificity verification

Detecting A (KC440882) -3D-pUC57-FMDV RNA 10 by using a third set of screened RT-LAMP primers⁵copies/μ L transcript, ten viruses such as ORFV, SPPV/GTPV, PRV, PCV, BVDV, SVV, CSFV, BTV, PRRSV and PPV. Except for the purpose band of FMDV 3D RNA transcripts, the detection results of other virus nucleic acids are negative, which indicates that the specificity of the method is good.

Sensitivity detection using DNA as template

LAMP sensitivity detection

DNA-templated 3D plasmid DNA 10 for seven serotypes⁷-10⁰The copies/mu L is subjected to LAMP detection, and the detection results are 10³copies/. mu.L, as shown in FIG. 6 below, the pink result was positive and the orange result was negative. Positive and negative controls were established.

QPCR sensitivity assay

10 parts of 3D plasmid DNA of seven sera, such as A (KC440882), Asia1(MF782478), C (AJ007572), O (KC503937), SAT1(AY593845), SAT2(AF540910), SAT3(KJ820999) -3D-pUC57-FMDV, and the like⁷-10⁰The sensitivity of copies/μ L was measured by QPCR method, the results are shown in FIG. 7, and plasmid DNA of A, Asia1, C, O, SAT1 and SAT2 were 10¹copies/. mu.L, whereas SAT3 was less effective than the other six serotypes, 10 detected²copies/μL。

PCR sensitivity detection

For A (KC440882), Asia1(MF782478), C (AJ007572), O (KC503937) and SAT1(AY593845), SAT2(AF540910), SAT3(KJ820999) -3D-pUC57-FMDV, etc. seven sera 3D plasmid DNA 10⁷-10⁰Sensitivity detection of PCR method was carried out with co pies/. mu.L, and the results are shown in FIG. 8, and 10 can be detected with A (KC440882) -3D-pUC57-FMDV, Asia1(MF782478) -3D-pUC57-FMDV¹copies/. mu.L, plasmid DNA of other serotypes could be detected 10²copies/μL。

RNA-templated sensitivity assays

RT-LAMP sensitivity detection

In vitro transcript Using RNA 10⁷-10⁰The seven serotypes are subjected to RT-LAMP detection by taking copes/mu L as a template, and the detection results are 10⁴copies/. mu.L, as shown in FIG. 9 below, the pink result was positive and the orange result was negative. Positive and negative controls were established.

RT-QPCR sensitivity assay

10 of RNA transcripts from seven sera, such as A (KC440882), Asia1(MF782478), C (AJ007572), O (KC503937), SAT1(AY593845), SAT2(AF540910), SAT3(KJ820999) -3D-pUC57-FMDV, and the like⁷-10⁰The sensitivity detection of the RT-QPCR method was carried out on copies/μ L, the detection results are shown in the sequence of FIG. 10, and RNA transcripts of A, Asia1, C, O, SAT1 and SAT3 can be detected 10 times³Although the detection effect of SAT2 is relatively disordered, the detection result of the same is 10. mu.L⁴copies/μL。

RT-PCR sensitivity detection

10 of RNA transcripts from seven sera, such as A (KC440882), Asia1(MF782478), C (AJ007572), O (KC503937), SAT1(AY593845), SAT2(AF540910), SAT3(KJ820999) -3D-pUC57-FMDV, and the like⁷-10⁰Sensitivity of copies/. mu.L by RT-PCR is shown in FIG. 11, except that 10 types of FMDV is detected⁴copies/. mu.L, the remaining six serotypes were detected at 10³copies/μL。

Results of clinical sample testing

Three methods of seven serotype 39 RNA transcripts prepared in the laboratory can be known from the previous experiments that the detection results are all positive. The three different detection results of the clinical samples are shown in the following table 27, the detection results of 44 clinical samples, namely RT-PCR, RT-QPCR and RT-LAMP, for Asia1 and SAT2 are positive, the detection results of FMDV nucleic acid samples No. 1-6 provided by OIE foot-and-mouth disease reference laboratories are also positive, the detection results of No. 7-42 are negative, and the RT-LAMP detection method is proved to have better specificity.

Table 2744 test results of clinical samples

Six samples of OIE were run at 1: 10,1: 100,1: 1000, and then detecting according to the RT-LAMP method. The results are as follows, stock solutions 2-5 and 1: 10,1: 100,1: the result of the 1000-diluent RT-LAMP detection is positive. The detection result of No. 1 stock solution is positive, 1: 10,1: 100,1: the result of the 1000-diluent RT-LAMP detection is negative. The stock solutions and the diluted solutions of the six samples were subjected to RT-QPCR and RT-PCR detection, and the detection results are shown in Table 28 below, FIGS. 12, 13 and 14. RT-qPCR No. 1 and No. 6 can detect 10-2, No. 2-5 can detect 10^-3. RT-PCR No. 1 can only detect stock solution, No. 2-6 can detect 10^-3And (4) diluting the solution. The detection results of the three methods show that the detection limits of RT-LAMP and RT-PCR are similar, and the detection limit is equivalent to the virus content detected when the CT value of RT-qPCR is 32.5.

Table 286 CT values corresponding to FMDV nucleic acid sample stock solutions

The RT-LAMP method of FMDV requires detection of the specificity of the RT-LAMP method after the determination of the primer concentration, the reaction time, the reaction temperature and the color developing agent. The established RT-LAMP method is used for detecting Orf (ORFV), cotton pox (SPPV/GTPV), BVDV, CSFV, SVV, BTV, PPV, PRV, PCV and PRRSV, and the RT-LMAP primer has no cross reaction, which indicates that the established RT-LAMP detection method has better specificity.

The invention designs primers aiming at the 3D region of FMDV and screens the primers to finally obtain three sets of primers with better effect. An RT-LAMP reaction system is established, and primer concentration, reaction temperature, reaction time and color developing agents (hydroxynaphthol blue, calcein, SYBR green I, neutral red, phenol red and bromothymol blue) of the RT-LAMP method are screened. The final reaction was 20. mu.L in total, including 15.2. mu.L of RT-LAMP buffer, 1.5. mu.L of BstAMV enzyme mixture, 0.04. mu.L (100. mu.M) of each of outer primers F3 and B3, 0.5. mu.L (100. mu.M) of each of inner primers FIP and BIP, 0.11. mu.L (100. mu.M) of each of loop primers Floop and Bloop, 1. mu.L (500. mu.M) of neutral red, and 1. mu.L of template. The reaction time was 55min and the reaction temperature was 65 ℃. Has no cross specificity reaction with other ten similar and common viruses.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will understand that various changes, modifications and substitutions can be made without departing from the spirit and scope of the invention as defined by the appended claims. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (7)

1. A foot-and-mouth disease virus visualized RT-LAMP detection system is characterized in that the total reaction system is 20 mu L, and comprises 15.2 mu L of RT-LAMP buffer solution, 1.5 mu L of BstAMV enzyme mixed solution, 0.04 mu L (100 mu M) of each of outer primers F3 and B3, 0.5 mu L (100 mu M) of each of inner primers FIP and BIP, 0.11 mu L (100 mu M) of each of loop primers Floop and Bloop, 1 mu L (500 mu M) of neutral red and 1 mu L of template; the reaction time is 55min, and the reaction temperature is 65 ℃; after the reaction, the reaction tube solution was judged to be positive when it became pink, and negative when it became orange.

2. The foot-and-mouth disease virus visual RT-LAMP detection system according to claim 1, characterized in that six primers are as follows:

3. the foot-and-mouth disease virus visual RT-LAMP detection system according to claim 1, characterized in that six primers are as follows:

4. the foot-and-mouth disease virus visual RT-LAMP detection system according to claim 1, characterized in that six primers are as follows:

5. the method for establishing the foot-and-mouth disease virus visual RT-LAMP detection system is characterized by comprising the following steps:

(1) screening, synthesis and identification of foot-and-mouth disease virus target gene 3D fragment

Serotype classification and similarity analysis are carried out on FMDV 3D sequences downloaded from GenBank, 39 sequences of 500bp regions at the 3D 3 'end are selected according to the time proximity and the difference between the serotype and the same serotype sequence as the principle, and a T7 promoter sequence (5'-TAATACGACTCACTATAGGG-3') is added at the 5' end of the sequences and sent to Elegano Biotechnology Limited, Guangzhou to be synthesized by taking pUC57 of Amp + as a vector; primers were designed for synthetic 3D-T7pUC57 to amplify the 3D and T7 synthetic regions and the amplified PCR products were sent to sequencing to identify the correctness of sequence synthesis;

(2) establishment and optimization of FMDV (frequency modulated double-stranded DV) visual RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection method

Making a 3D RNA transcript, verifying, screening multiple sets of designed RT-LAMP primers by using an RT-LAMP universal detection kit, and finally obtaining three sets of primers with better amplification effect; the RT-LMAP primer with the best detection effect is used for optimizing and screening the primer concentration, the reaction time, the reaction temperature and the color indicator (hydroxynaphthol blue, calcein, SYBR green I, neutral red, phenol red and bromothymol blue) in the RT-LAMP reaction system, and the RT-LAMP detection system with the FMDV reaction overall system of 20 mu L is established, and comprises 15.2 mu L of RT-LAMP buffer solution, 1.5 mu L of BstAMV enzyme mixed solution, 0.04 mu L (100 mu M) of each of outer primers F3 and B3, 0.5 mu L (100 mu M) of each of inner primers FIP and BIP, 0.11 mu L (100 mu M) of each of loop primers Floop and Bloop, 1 mu L (500 mu M) of neutral red and 1 mu L of template; the reaction time is 55min, and the reaction temperature is 65 ℃; after the reaction is finished, if the solution in the reaction tube turns to pink, the solution is judged to be positive, and if the solution is orange, the solution is judged to be negative;

(3) specificity and sensitivity detection of RT-LAMP method and clinical sample detection

The established RT-LAMP reaction is subjected to cross reaction verification, and the result shows that the method does not have cross reaction with viruses such as BVDV, ORFV, SPPV/GT PV, SVDV, BTV, PRRSV, CSFV, PRV, PPV, PCV and the like; then 10 of 3D DNA and RNA respectively⁷-10⁰The results of comparison of RT-/LAMP, RT-/QPCR and RT-/PCR using copies/μ L as template showed that 10 could be detected using plasmid as template³The gene is copies/mu L, and the RT-LAMP can detect 10 by taking RNA as a template⁴copies/μL。

6. The method for establishing the foot-and-mouth disease virus visualized RT-LAMP detection system according to claim 5, wherein the step (1) comprises the following steps:

1) performing gene homology analysis on sequences of seven serotypes by using biological software MEGA7.0 and DNAStar7.0, and selecting a region with better conservation as an RT-LAMP design primer and an identified region; then, the 3D sequences of 39 seven serotypes are screened out by using the published timeliness of the sequences, the serotypes of the sequences and the difference of the sequences in the same serotype as the principle, wherein the 3D sequences comprise 6A types, 8O types, 4 Asia1 types, 4C types, 5 SAT1 types, 6 SAT2 types and 6 SAT3 types; cutting a fragment containing the identification region at the 3 'end, wherein the length of the fragment is 500bp, and adding a T7 promoter sequence (5'-TAATACGACTCACTAT AGGG-3') with the length of 20bp at the 5' end, wherein each sequence is 520 bp; sending to Guangzhou Egypti biotechnology limited for cloning to pUC57 vector containing Amp + to construct recombinant plasmid, finally providing glycerol strain containing seven serotype 3D sequences of 39 520bp, and naming according to serotype-3D-pU C57-FMDV;

accession information for 39 seven serotype 3D sequences are shown in the table below;

2) seven FMDV serotypes, 39 glycerol strains, are subjected to amplification culture.

3) Adopts a plasmid extraction kit (AxyPrep)^TMPlasmid Miniprep kit), the 39 recombinant 3D plasmids were extracted, their concentrations were determined, labeled and stored in a-20 ℃ freezer.

4) And PCR amplification identification and sequencing identification of the recombinant plasmid.

7. The method for establishing the foot-and-mouth disease virus visualized RT-LAMP detection system according to claim 5, wherein the step (2) comprises the following steps:

1) primer design

Carrying out similarity comparison on FMDV 3D sequences, selecting a region with higher similarity, namely the tail end of the FMDV 3D region, and carrying out primer design by Beijing Meilaibo biotechnology company, wherein each set of primers comprises two outer primers F3 and B3, two loop primers, i.e. a Floop and a BLOop, and two inner primers, i.e. a FIP and a BIP;

2) RNA template preparation

Preparing RNA in vitro transcripts;

a) conventional PCR amplification of synthetic 3D fragments of interest

b) PCR amplification product gel recovery

c) In vitro transcription

d) RNA cleaning, removing substances such as oligonucleotide, protein and salts in vitro RNA transcripts;

3) RT-LAMP method primer screening

Screening RT-LAMP outer primer

The LAMP outer primers F3 and B3 for synthesizing RT-are diluted to 10 mu M, a PCR method is adopted, and the synthesized FMDV 3D plasmid is used as a template to verify whether the RT-LAMP outer primers can be used or not, so that the specificity of the RT-LAMP primers can be preliminarily detected; after PCR amplification, detecting the result by electrophoresis; the RT-LAMP external primer PCR amplification system is as follows:

PCR reaction procedure: 95 ℃ for 5 min; (95 ℃, 30s, 56 ℃, 30s, 72 ℃, 30s) X36 cycles; 72 ℃ for 10 min; at 25 ℃, forever;

② screening RT-LAMP primer by adopting fluorescent RT-LAMP universal kit

Preparing an RT-LAMP reaction system according to the table 6, taking an in vitro RNA transcript of an FMDV 3D sequence as a template, and selecting a method for comparative substitution of RT-LAMP primers, wherein reaction procedures of 63 ℃, 60min, 80 ℃ and 10min are selected as initial reaction conditions according to the instructions of an RT-LAMP universal kit; detecting a fluorescence signal by using a QPCR instrument, judging the quality of the primer according to the fluorescence signal and a product electrophoretogram, and screening the RT-LAMP primer;

TABLE 6

4) Optimization of primer content in RT-LAMP method

Outer primer concentration optimization

On the basis of table 6, the amount of each reagent was fixed, and the concentration of the outer primer was optimized; f3, B3 were diluted to 10 μ M and mixed at 1: 1 volume of the mixture is mixed, and the volume is increased by 0.2 mu L each time from 0.2 mu L/tube until the adding amount is 2.0 mu L; repeating the test, and screening out the optimal concentration of the outer primer by fluorescence screening and electrophoresis detection;

loop primer concentration optimization

On the basis of the table 6 and the screened optimal outer primer concentration, the dosage of each reagent is fixed, and the concentration of the loop primer is optimized; flood, Bloop diluted to 10 μ M and mixed at 1: 1 volume of the mixture is mixed, and the volume is increased by 0.2 mu L each time from 0.6 mu L/tube until the adding amount is 3.0 mu L; repeating the test, performing fluorescence screening and performing electrophoresis detection to screen out the optimal concentration of the loop primer;

inner primer concentration optimization

On the basis of the optimal loop primer and the outer primer concentrations screened in the table 6, the dosage of each reagent is fixed, and the concentration of the inner primer is optimized; FIP, BIP were diluted to 50. mu.M and mixed at 1: 1 volume of the mixture is mixed, and the volume is increased by 0.2 mu L each time from 0.4 mu L/tube until the adding amount is 3.0 mu L; repeating the test, performing fluorescence screening and performing electrophoresis detection to screen out the optimal concentration of the inner primer;

5) RT-LAMP method reaction time optimization

Starting from 30min, and after 40min, taking a gradient every 5min, wherein the total number of reaction gradients is 8; then checking the result by 1.5% nucleic acid gel electrophoresis, and screening out the optimal reaction time;

6) temperature optimization

Under the condition that a fixed reaction system and other reagents are not changed, the reactions are respectively carried out at 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃ and 66 ℃, then the results are checked by 1.5 percent nucleic acid gel electrophoresis, and the optimal reaction temperature is screened out;

7) and (4) screening the RT-LAMP method dye.

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