CN106929605B - Detection kit, primer and probe capable of simultaneously detecting and identifying foot-and-mouth disease and vesicular stomatitis - Google Patents
Detection kit, primer and probe capable of simultaneously detecting and identifying foot-and-mouth disease and vesicular stomatitis Download PDFInfo
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Abstract
The invention discloses a detection kit, a primer and a probe capable of simultaneously detecting and identifying foot-and-mouth disease and vesicular stomatitis. The primer and the probe are shown in a sequence table SEQ ID NO: 1 to SEQ ID NO: as shown at 14. The invention also discloses a one-step method multiple fluorescence RT-PCR detection kit for simultaneously detecting and identifying the foot-and-mouth disease and the vesicular stomatitis, which is rapid, accurate and convenient to use. The detection method can realize the purposes of one-time sampling and one-time analysis and can simultaneously detect and distinguish 3 important viruses, thereby not only reducing the workload and the cost of detection, but also finishing the detection of epidemic diseases in the shortest time and gaining time for disease prevention and treatment.
Description
Technical Field
The invention relates to a one-step multiplex fluorescence RT-PCR detection kit, primers and probes capable of simultaneously detecting and identifying Foot and Mouth Disease Virus (FMDV), new Jersey vesicular stomatitis virus (VSV-NJ) and Indiana vesicular stomatitis virus (VSV-IND), and aims of realizing one-time sampling and one-time analysis and simultaneously detecting and distinguishing 3 viruses, belonging to the field of inspection and quarantine.
Background
Foot-and-mouth disease virus (FMDV) and Vesicular Stomatitis Virus (VSV) are viral diseases causing infection of various animals such as cattle, sheep, pigs and the like, and are clinically characterized in that oral mucosa, nipple skin and hoof crown skin are vesiculated and eroded, mixed infection occurs, clinical symptoms are similar, identification and diagnosis are difficult, and the virus is the most serious pathogen harming the breeding industry. Foot-and-mouth disease and vesicular stomatitis are regulated by the world animal health Organization (OIE) as pathogens of various animal infections and world-wide spread, and are also important quarantine objects in international trade of animals and animal products in China.
At present, various molecular detection technologies, such as PCR (polymerase chain reaction) technology, fluorescent PCR technology and the like, are established for the pathogens, and play an important role in diagnosis, prevention and control of the diseases. However, most of the established methods aim at single-target detection of a pathogen, the aim of detecting different pathogens can be fulfilled only by repeating the detection for multiple times in actual detection, the detection workload is large, the detection time is long, and the actual requirements of large-scale animal quarantine and quick clearance can not be met. And it is difficult to perform differential diagnosis of mixed infection and similar epidemic diseases which are present in large amounts in actual samples. While a single-target pathogen detection technology is established, veterinary institutions of various countries successively develop multiple PCR capable of detecting multiple viruses, but the application of the traditional PCR technology in multiple detection is limited by the defects of low sensitivity, difficult result judgment and the like.
The research establishes a one-step multiple fluorescence RT-PCR detection and identification method for three viruses including FMDV, VSV-NJ and VSV-IND by utilizing the characteristics of specificity, sensitivity and the like of a fluorescence PCR technology in the aspect of nucleic acid detection, and assembles the detection and identification method into a kit, can realize the purposes of one-time sampling and one-time analysis and can simultaneously detect and distinguish 3 important viruses, thereby not only reducing the workload and the cost of detection, but also completing the detection of epidemic diseases in the shortest time and gaining time for disease control.
Disclosure of Invention
The first purpose of the invention is to provide a multiplex fluorescence RT-PCR primer and a probe which have strong specificity and high sensitivity and simultaneously detect and identify foot-and-mouth disease virus (FMDV), New Jersey type vesicular stomatitis virus (VSV-NJ) and Indiana type vesicular stomatitis virus (VSV-IND).
Another objective of the invention is to provide a rapid, accurate and convenient-to-use one-step multiplex fluorescence RT-PCR detection kit for simultaneously detecting and identifying foot-and-mouth disease virus (FMDV), New Jersey type vesicular stomatitis virus (VSV-NJ) and Indiana type vesicular stomatitis virus (VSV-IND).
In order to achieve the purpose, the invention adopts the following technical scheme:
on the basis of sequence comparison analysis, aiming at a foot-and-mouth disease virus 3D gene, a new Jersey type vesicular stomatitis virus L gene and an Indiana type vesicular stomatitis virus L gene, primers and probes which can cover most of isolates (particularly, primers and probes for the foot-and-mouth disease can realize universal detection of 7 serotypes) and are suitable for multiple detections and can be identified are respectively designed and screened (the sequences are shown in Table 1). The general detection primer pair of each serotype of Foot and Mouth Disease Virus (FMDV) consists of 2 forward primers, 3 reverse primers and 2 probes, the primer pair of the New Jersey vesicular stomatitis virus (VSV-NJ) consists of 2 forward primers, 1 reverse primer and 1 probe, the primer pair of the Indiana vesicular stomatitis virus (VSV-IND) consists of 1 forward primer, 1 reverse primer and 1 probe, the primer mixture in the multiple reaction system is a mixture consisting of the 10 primers, the probes are a mixture consisting of the 4 probes, and the simultaneous detection and identification of the three pathogens can be realized through one-step multiplex fluorescence RT-PCR reaction.
TABLE 1 one-step multiplex fluorescent RT-PCR detection primers and probes for foot and mouth disease viruses and vesicular stomatitis viruses (including New Jersey and Indiana types)
Note: cytosine (C), guanine (G), adenine (A), thymine (T). Degenerate base W represents A or T. The 5 'end of the probe is marked with a luminescent group, and the 3' end of the probe is marked with a quenching group.
A detection method for simultaneously detecting and identifying foot-and-mouth disease viruses and vesicular stomatitis viruses (including New Jersey type and Indiana type) is established by adopting a multiple fluorescence PCR technology, and a kit is assembled.
The one-step multiplex fluorescence RT-PCR detection kit for detecting and identifying foot-and-mouth disease virus, new Jersey type vesicular stomatitis virus and Indiana type vesicular stomatitis virus comprises the multiplex fluorescence RT-PCR detection primer and the probe. Further, the kit may also include materials and reagents necessary to complete the multiplex fluorescent RT-PCR reaction, e.g., reaction buffers, enzymes, positive controls, negative controls, and the like.
In one embodiment of the present invention, the reaction solution is a Multiplex RT-PCR reaction buffer; the enzyme is a Multiplex enzyme; the negative control is sterilized water without nucleic acid; the positive controls are in vitro reverse transcription cRNA of a Foot and Mouth Disease Virus (FMDV)3D gene, a New Jersey vesicular stomatitis virus (VSV-NJ) L gene and an Indiana vesicular stomatitis virus (VSV-IND) L gene respectively.
The invention also provides a one-step multiplex fluorescence RT-PCR detection method for detecting and identifying foot-and-mouth disease viruses, new Jersey vesicular stomatitis viruses and Indiana vesicular stomatitis viruses, which comprises the following steps:
(1) extracting total virus nucleic acid;
(2) preparing an RT-PCR reaction system, wherein the reaction system comprises the multiple fluorescent RT-PCR primers and the probes;
(3) performing one-step multiplex fluorescence RT-PCR reaction;
(4) judging a result;
quality control standard:
the positive controls all have S-shaped amplification curves, the CT value is about 20, and the negative controls have no amplification signals.
When the condition is satisfied, the test is considered to be effective.
Judging the result:
the sample is judged to be positive when the amplification curve exists and the CT value is less than 30;
the amplification curve exists, but the CT value of 30< CT is less than 35, the amplification curve belongs to a suspected interval, and the amplification curve needs to be confirmed again;
if there is no amplification curve or the CT value is greater than 35, the result is judged to be negative.
The invention has the advantages that: 1) multiplicity is as follows: can simultaneously detect and distinguish foot-and-mouth disease virus, new Jersey type vesicular stomatitis virus and Indiana type vesicular stomatitis virus, saves the detection time and cost, and is favorable for timely diagnosis of epidemic diseases, particularly mixed infection. 2) Sensitivity: the multiple fluorescence RT-PCR system can simultaneously detect three pathogens, and the specificity of the multiple fluorescence RT-PCR system is equivalent to that of a single fluorescence RT-PCR. 3) Specifically: the possible interference between pathogens is fully considered when designing the primers and the probes, and the specificity and the exclusivity of the primers and the probes are ensured through screening. 3) The coverage and the universality are good: the primers and probes designed in the invention are designed aiming at the most conserved genes of foot-and-mouth disease virus and vesicular stomatitis virus respectively, for each virus, the primers and probes are multiple, and degenerate primers are included, so that all sequences of the existing virus in Genebank are basically covered. The foot-and-mouth disease virus detection primer and probe contain 7 serotypes of foot-and-mouth disease virus, can realize universal detection, and the vesicular stomatitis virus can identify and distinguish 2 serotypes of New Jersey type and Indiana type. Universal detection between different subtypes or serotypes of each virus can be achieved. 4) Simple and easy automation: all primers and probes are designed in view of the capability of multiplex detection, and the one-step procedure can be used regardless of DNA or RNA viruses, so that the reverse transcription process is omitted, and the time and the labor are saved.
Drawings
FIG. 1: FMDV Single fluorescent RT-PCR Standard Curve.
FIG. 2: VSV-NJ monofluorescence RT-PCR standard curve.
FIG. 3: VSV-IND monofluorescence RT-PCR standard curve.
FIG. 4: FMDV, VSV-NJ and VSV-IND multiplex fluorescence RT-PCR standard curves.
FIG. 5: comparison of FMDV Mono-fluorescent RT-PCR with multiplex fluorescent RT-PCR.
FIG. 6: comparison of VSV-NJ Monofluorescence RT-PCR with multiplex fluorescence RT-PCR.
FIG. 7: comparison of VSV-IND monofluorescent RT-PCR with multiplex fluorescent RT-PCR.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
The methods used in the following examples are conventional methods unless otherwise specified, and the test materials used are commercially available from conventional biochemical reagent suppliers unless otherwise specified.
EXAMPLE 1 composition and use of the kit
1. Preparation composition of kit
TABLE 2 composition of the kit
Wherein 2 XMultiplex RT-PCR buffer and the Multiplex enzyme are purchased from Path-ID. TM. Multiplex one-step RT-PCR kit manufactured by AB.
The primer mixture comprises 2 forward primers and 3 reverse primers of FMDV, 2 forward primers and 1 reverse primer of VSV-NJ, and 1 forward primer and 1 reverse primer of VSV-IND, the primer sequences are shown in Table 1, the working concentration of the mixed primers is 400nM, and the primers are synthesized by Ventto Dalibao biology company.
The FMDV probe mixture comprises 2 probes of FMDV, 1 probe of VSV-NJ and 1 probe of VSV-IND respectively, the sequences of the probes are shown in Table 1, the concentrations of the probes are 200nM respectively, and the probes are synthesized by Ventto Dalibao biology, Inc.
The negative control was sterilized water without nucleic acid.
The FMDV positive control is in-vitro reverse transcription cRNA of a Foot and Mouth Disease Virus (FMDV)3D gene; the VSV-NJ positive control is in-vitro reverse transcription cRNA of a new Jersey vesicular stomatitis virus (VSV-NJ) L gene; the VSV-IND positive control is in vitro reverse transcribed cRNA of the Indiana vesicular stomatitis virus (VSV-IND) L gene.
Preparation of Foot and Mouth Disease Virus (FMDV)3D gene in vitro transcription RNA: recovering RT-PCR amplification product of the O type foot-and-mouth disease virus 3D gene, connecting with pGEM-T vector (purchased from Promega company), transforming JM109 competent cells, extracting plasmid DNA by an alkaline lysis method, obtaining positive recombinant plasmid after PCR and enzyme digestion identification, and naming the plasmid as pGEM-FMDV-3D. After the plasmid was linearized using the purified plasmid as a template, in vitro transcription was performed using the RiboMAXTM Large Scale RNA Production System-T7 kit from Promega; removing the DNA template of the in vitro transcription product by DNAse, extracting by TRIZOL, and determining to obtain the in vitro transcription RNA of the foot and mouth disease virus 3D gene, which is named as FMDV-3D-cRNA; the foot-and-mouth disease virus 3D gene sequence is shown as SEQ ID NO: shown at 15.
Preparation of in vitro transcribed RNA of the L gene of the New Jersey vesicular stomatitis virus (VSV-NJ): the RT-PCR amplification product of the L gene of the new Jersey vesicular stomatitis virus is recovered, the length is 500bp, the RT-PCR amplification product is connected with a pGEM-T vector (purchased from Promega company), JM109 competent cells are transformed, plasmid DNA is extracted by an alkaline lysis method, and a positive recombinant plasmid is obtained after PCR and enzyme digestion identification and is named as pGEM-VSV-NJ-L. After the plasmid was linearized using the purified plasmid as a template, in vitro transcription was performed using the Ribo MAXTM Large Scale RNA Production System-T7 kit from Promega; removing DNA template from the in vitro transcription product by DNAse, extracting by TRIZOL, and measuring to obtain in vitro transcription RNA of the L gene of the New Jersey vesicular stomatitis virus, which is named VSV-NJ-L-cRNA; the gene sequence of the new jersey type vesicular stomatitis virus L is shown as SEQ ID NO: shown at 16.
Preparation of in vitro transcribed RNA of Indiana vesicular stomatitis virus (VSV-IND) L Gene: recovering the RT-PCR amplification product of the Indiana type vesicular stomatitis virus L gene, wherein the length is 500bp, connecting the RT-PCR amplification product with a pGEM-T vector (purchased from Promega company), transforming JM109 competent cells, extracting plasmid DNA by an alkaline lysis method, and obtaining a positive recombinant plasmid after PCR and enzyme digestion identification, wherein the positive recombinant plasmid is named as pGEM-VSV-IND-L. After the plasmid was linearized using the purified plasmid as a template, in vitro transcription was performed using the Ribo MAXTM Large Scale RNA Production System-T7 kit from Promega; removing DNA template from the in vitro transcription product by DNAse, extracting by TRIZOL, and measuring to obtain in vitro transcription RNA of Indiana type vesicular stomatitis virus L gene, named VSV-IND-L-cRNA; the gene sequence of Indiana type vesicular stomatitis virus L is shown as SEQ ID NO: shown at 17.
2. Method of using kit
1) Extraction of viral Total nucleic acids (RNA/DNA)
Viral nucleic acid was extracted using a viral genomic DNA/RNA extraction kit (TIANAmp Virus DNA/RNAKit) as follows:
preparation of Carrier RNA solution:
(1) carrier RNA stock: to the 310 u g Carrier RNA freeze-dried powder in the tube adding 310 u LRNase-Free ddH2O, completely dissolving Carrier RNA to obtain a solution with the final concentration of 1 mu g/mu L, subpackaging, and storing at-20 ℃.
(2) Carrier RNA working solution: calculating the volume of the required buffer solution GB and the Carrier RNA solution according to the number of the samples (the calculation formula is as follows), and reversely and uniformly mixing the buffer solution GB and the Carrier RNA solution to obtain Carrier RNA working solution; to avoid foaming of the solution, vortex shaking was not used.
Calculating the formula:
n×0.22mL＝y mL
y mL×28μL/mL＝zμL
where n is the number of samples extracted at the same time, y is the volume of buffer GB that needs to be added, and z is the volume of Carrier RNA solution that needs to be added.
The method comprises the following operation steps:
(1) add 20. mu.L of Proteinase K to a clean 1.5ml centrifuge tube using a pipette.
(2) To the centrifuge tube, 200. mu.L of plasma/serum/lymph or 25mg of ground tissue (the sample is allowed to equilibrate to room temperature) is added, and if the sample volume is less than 200. mu.L, it can be supplemented with 0.9% NaCl solution.
(3) Add 200. mu.L of Carrier RNA working solution, cover the tube cap, vortex and shake for 15sec and mix.
(4) Incubate at 56 ℃ for 15min and briefly centrifuge to collect liquid attached to the tube walls and caps.
(5) Add 250. mu.L of absolute ethanol, vortex for 15sec, mix thoroughly, and let stand at room temperature (15-25 ℃) for 5 min.
(6) Briefly, centrifugation was performed to collect the liquid adhering to the tube walls and the tube caps.
(7) The solution and flocculent precipitate in the centrifuge tube were transferred to RNase-Free adsorption column CR2 (adsorption column placed in the collection tube), the tube cap was closed, centrifuged at 8,000rpm (. about.6,000 Xg) for 1min, the waste solution was discarded, and the adsorption column was returned to the collection tube.
(8) Carefully open the adsorption column lid, add 500 μ L solution GD (check if absolute ethanol has been added before use), cover the tube lid, centrifuge at 8,000rpm (-6,000 × g) for 1min, discard the waste, and place the adsorption column back into the collection tube.
(9) Carefully open the adsorption column lid, add 600 μ L of solution PW (check if absolute ethanol has been added before use), cover the tube lid, stand for 2min, centrifuge at 8,000rpm (-6,000 Xg) for 1min, discard the waste solution, and place the adsorption column back into the collection tube.
(10) And (5) repeating the step (9).
(11) Carefully open the adsorption column lid, add 500. mu.L of absolute ethanol, cover the tube lid, centrifuge at 8,000rpm (. about.6,000 Xg) for 1min, and discard the waste.
(12) The adsorption column was returned to the collection tube and centrifuged at 12,000rpm (-13,400 Xg) for 3min to completely dry the adsorption membrane and discard the waste.
(13) The column was placed in a new RNase-Free centrifuge tube (1.5ml), the lid of the column was carefully opened and the column was left at room temperature for 3min to completely dry the adsorption film. Dripping 20-150 μ L RNase-freeddH2O into the middle part of the adsorption membrane, covering the cover, and standing at room temperature for 5 min.
(14) The mixture was centrifuged at 12,000rpm (. about.13,400 Xg) for 1min to collect the total viral nucleic acid (DNA or RNA).
2) Fluorescent RT-PCR detection
Respectively taking a detection sample and a positive and negative control, and preparing a reaction system according to the following table 3:
table 3 one-step multiplex fluorescent RT-PCR reaction system: total volume 20uL
After mixing the mixture sufficiently, the template was added finally, centrifuged briefly, and the one-step fluorescent RT-PCR reaction was performed according to the following reaction procedure, as shown in table 4:
TABLE 4 multiplex fluorescent RT-PCR reaction procedure
3) Result description and determination
Quality control standard:
the positive controls all have S-shaped amplification curves, the CT value is about 20, and the negative controls have no amplification signals.
When the condition is satisfied, the test is considered to be effective.
Judging the result:
the sample is judged to be positive when the amplification curve exists and the CT value is less than 30;
the amplification curve exists, but the CT value of 30< CT is less than 35, the amplification curve belongs to a suspected interval, and the amplification curve needs to be confirmed again;
if there is no amplification curve or the CT value is greater than 35, the result is judged to be negative.
Example 2 sensitivity test of kit
1. Material
Foot-and-mouth disease virus and vesicular stomatitis virus are both preserved in the laboratory.
2. Method of producing a composite material
1) Preparation of Positive Standard
The process described in example 1.
2) Quantitative determination of standard
Diluting the prepared in vitro transcription cRNA with sterile water without RNase for 200 times, and measuring the absorbance values (OD) of the cRNA at 260nm and 280nm by using an ultraviolet spectrometer260And OD280) And calculating the concentration and purity of the sample to be detected. Pure RNA: OD of 1.7 ≦260/OD280< 2.0 (protein or phenol contamination is indicated at < 1.7; isothiocyanate may remain at > 2.0). Concentration of RNA sample (. mu.g/. mu.L): OD260Xdilution multiple × 40/1000, and the copy number (Copies/. mu.L) was calculated according to the following formula:
drawing a standard curve:
performing 10-fold gradient dilution on the FMDV in-vitro standard product, performing fluorescence RT-PCR, and making a concentration standard curve by using the reciprocal of the template concentration and a corresponding fluorescence CT value, wherein the PCR amplification efficiency E value, the correlation coefficient R2 value and the Slope value of the curve all fall within an acceptable range, which indicates that the amplification efficiency is good. See fig. 1.
And performing 10-fold gradient dilution on the VSV-NJ in-vitro standard substance, performing fluorescence RT-PCR, and making a concentration standard curve by using the template concentration reciprocal and a corresponding fluorescence CT value, wherein the PCR amplification efficiency E value, the correlation coefficient R2 value and the curve Slope value are all in an acceptable range, which indicates that the amplification efficiency is good. See fig. 2.
And performing 10-fold gradient dilution on the VSV-IND in-vitro standard substance, performing fluorescence RT-PCR, and making a concentration standard curve by using the reciprocal concentration of the template and a corresponding fluorescence CT value, wherein the PCR amplification efficiency E value, the correlation coefficient R2 value and the Slope value of the curve all fall within an acceptable range, which indicates that the amplification efficiency is good. See fig. 3.
After FMDV, VSV-NJ and VSV-IND in vitro standard products are mixed in equal quantity, 10-fold gradient dilution is carried out, multiple fluorescence RT-PCR is carried out, a concentration standard curve is made by using the concentration reciprocal of a template and a corresponding fluorescence CT value, the PCR amplification efficiency E value, the correlation coefficient R2 value and the Slope value of the curve all fall within an acceptable range, and the fact that mutual interference does not exist among multiple PCR and the amplification efficiency is good is shown. See fig. 4.
3) Determination of detection limit of one-step method multiple fluorescence RT-PCR method
The RNA solutions were adjusted to approximately equal concentrations, 10. mu.L of each RNA solution was taken, and the solutions were mixed well to prepare positive standards. After the standard substance is diluted by 10 times of gradient, the fluorescence RT-PCR detection is carried out according to the optimal condition, and the concentration corresponding to the CT value which can be detected by the highest dilution multiple is the lowest detection limit.
3. Results
1) RNA solution copy number assay results
The prepared in vitro transcribed RNA is measured for absorbance values of 260nm and 280nm by an ultraviolet spectrometer and the copy number is calculated, which is detailed in the following table 5.
TABLE 5 determination of RNA purity and content
2) Determination of detection limit of multiplex fluorescence RT-PCR method
The established multiplex fluorescence RT-PCR detection method is used for detecting the 10-time serial dilution positive standard substance, and as a result, the method can stably detect the highest CT value of 35, and the concentration of the corresponding standard substance reaches 1-10 copy number/reaction.
Example 3 specificity test of kit
1 Material
The viruses used in this experiment are shown in Table 6.
TABLE 6 viruses and nucleic acids applied during the course of the specificity test study
| Virus | Origin of origin |
| Foot-and-mouth disease Virus (FMDV) | This laboratory preserves |
| Indiana type vesicular stomatitis virus (VSV-IND) | This laboratory preserves |
| New Jersey type vesicular stomatitis virus (VSV-NJ) | This laboratory preserves |
| Bovine endemic leukemia virus (BVDV) | This laboratory preserves |
| Pseudorabies virus | This laboratory preserves |
| Bluetongue virus (BTV) | This laboratory preserves |
| Transmissible gastroenteritis virus (TGEV) | This laboratory preserves |
2. Method of producing a composite material
2.1 fluorescent RT-PCR detection of other 6 virus nucleic acids in the table is carried out by using primers and probes of foot-and-mouth disease virus, New Jersey type vesicular stomatitis virus and Indiana type foot-and-mouth disease virus respectively to verify the specificity of the primers and the probes.
2.2 the established multiplex fluorescence RT-PCR detection method is used for detecting 7 viruses in the table, and the specificity of the multiplex fluorescence RT-PCR method is verified.
2.3 comparison of Mono-fluorescent RT-PCR with multiplex-fluorescent RT-PCR.
3. Results
3.1 the primers and probes of each virus can only amplify a curve from the nucleic acid of the virus, and the amplification of other virus templates is negative, which indicates that the designed primers and probes have good specificity.
3.2 the established multiple fluorescent RT-PCR method is used for detecting the viruses in the table 6, and the results show that the foot-and-mouth disease virus, the New Jersey type vesicular stomatitis virus and the Indiana type vesicular stomatitis virus have amplification curves, and other viruses have no specific amplification signals, which indicates that the established method has good specificity.
TABLE 7 results of specific detection
3.3 mixing the FMDV, VSV-NJ and VSV-IND in vitro standard products in equal quantity, then carrying out gradient dilution by 10 times, respectively carrying out multiple fluorescence RT-PCR and FMDV single fluorescence PCR, comparing fluorescence CT values corresponding to the same template concentration, solving a regression equation, and solving a correlation coefficient R between the two2The value reaches 0.9995, which indicates that the coincidence between the single fluorescence RT-PCR and the multiple fluorescence RT-PCR is good, the multiple fluorescence RT-PCR does not generate interference, and the amplification efficiency is not influenced. See fig. 5.
Mixing FMDV, VSV-NJ and VSV-IND in vitro standard products in equal quantity, performing gradient dilution by 10 times, performing multiplex fluorescence RT-PCR and VSV-NJ single fluorescence PCR respectively, comparing fluorescence CT values corresponding to the same template concentration, solving a regression equation, and obtaining a correlation coefficient R between the two2The value reaches 0.9998, which indicates that the coincidence between the single fluorescence RT-PCR and the multiple fluorescence RT-PCR is good, the multiple fluorescence RT-PCR does not generate interference, and the amplification efficiency is not influenced. See fig. 6.
Mixing FMDV, VSV-NJ and VSV-IND in vitro standard products in equal quantity, performing gradient dilution by 10 times, performing multiplex fluorescence RT-PCR and VSV-IND single fluorescence PCR respectively, comparing fluorescence CT values corresponding to the same template concentration, solving a regression equation, and obtaining a correlation coefficient R between the two2The value reaches 0.9994, which indicates that the coincidence between the single fluorescence RT-PCR and the multiple fluorescence RT-PCR is good, the multiple fluorescence RT-PCR does not generate interference, and the amplification efficiency is not influenced. See fig. 7.
Example 4: experimental report of clinical sample detection by kit
1. Material
23 parts of known FMDV positive sample, 2 parts of VSV-NJ positive sample, 3 parts of VSV-IND positive sample, 10 parts of BVDV positive sample, 13 parts of TGEV positive sample and 35 parts of other unknown sample which are collected in a laboratory.
2. Method of producing a composite material
And carrying out multiplex fluorescence RT-PCR detection on the samples, and verifying the sensitivity and specificity of the method by using actual samples.
3. Results
The results of the clinical samples are shown in Table 8. As can be seen from Table 8, 23 known FMDV positive samples, 2 known VSV-NJ positive samples and 3 known VSV-IND positive samples were all detected positively, and the coincidence rate was 100%. While 1 FMDV positive sample is detected from 10 known BVDV positive samples, and 2 FMDV positive samples are detected from 13 known TGEV positive samples, which indicates that mixed infection of FMDV exists in the original sample only detecting BVDV and TGEV; in addition, 35 clinical samples were randomly selected for screening and 1 FMDV positive was detected. This shows that the multiple fluorescence RT-PCR method can find non-target pathogens which are not screened by single fluorescence RT-PCR originally, detect mixed infection and avoid omission of detection.
TABLE 8 test results of clinical samples
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.
SEQUENCE LISTING
<110> inspection and quarantine technology center of Beijing entrance and exit inspection and quarantine bureau
<120> detection kit, primer and probe capable of simultaneously detecting and identifying foot-and-mouth disease and vesicular stomatitis
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<170> PatentIn version 3.3
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<213> Artificial Synthesis of primer
<400> 2
ctgggtttta taaacctgtg atggc 25
<210> 3
<211> 21
<212> DNA
<213> Artificial Synthesis of primer
<400> 3
ccacggagat caacttctcc t 21
<210> 4
<211> 22
<212> DNA
<213> Artificial Synthesis of primer
<400> 4
tgccacagag atcaacttct cc 22
<210> 5
<211> 22
<212> DNA
<213> Artificial Synthesis of primer
<400> 5
ccacggaaat caacttctcc tg 22
<210> 6
<211> 19
<212> DNA
<213> artificially synthesized Probe
<400> 6
tctcctttgc acgccgtgg 19
<210> 7
<211> 21
<212> DNA
<213> artificially synthesized Probe
<400> 7
tcctctcctt tgcacgtcgt g 21
<210> 8
<211> 21
<212> DNA
<213> Artificial Synthesis of primer
<400> 8
gctctttatg catgaccctg c 21
<210> 9
<211> 22
<212> DNA
<213> Artificial Synthesis of primer
<400> 9
tgctttttat gcatgacccw gc 22
<210> 10
<211> 20
<212> DNA
<213> Artificial Synthesis of primer
<400> 10
<210> 11
<211> 24
<212> DNA
<213> artificially synthesized Probe
<400> 11
ctggtttgca caccagaaca ttca 24
<210> 12
<211> 22
<212> DNA
<213> Artificial Synthesis of primer
<400> 12
tgatgatgca tgatccwgct ct 22
<210> 13
<211> 21
<212> DNA
<213> Artificial Synthesis of primer
<400> 13
acacwcctcc aatggaaggg t 21
<210> 14
<211> 21
<212> DNA
<213> artificially synthesized Probe
<400> 14
accgggcttg cacagttcta c 21
<210> 15
<211> 1410
<212> DNA
<213> foot-and-mouth disease virus 3D gene sequence
<400> 15
gggttgatcg ttgacaccag agatgtggag gaacgcgtcc acgtgatgcg caaaaccaag 60
ctcgcgccca ccgtggcaca cggtgtgttc aaccctgaat tcgggcctgc tgctctgtcc 120
aacaaggacc cgcgcctgaa agaaggggtc gtccttgacg acgtcatttt ctcgaaacac 180
aagggagata caaggatgtc tgaggaagac aaagcgctgt tccggcgctg tgctgccgac 240
tacgcgtcgc gcctacacag cgtgctaggg acggcaaacg ccccactgag tgtatacgaa 300
gccatcaaag gcgtcgacgg acttgacgcc atggagccag acaccgcacc cggtctcccc 360
tgggctctcc agggaaaacg ccgaggtgcc ctgatcgact tcgaaaacgg tactgtcggg 420
cccgaggttg aagcagcact caagctcatg gaaagccgtg ggtacaaatt cgtctgccaa 480
acctttctga aagacgaaat tcggccgcta gagaaggtgc gcgccggcaa gacgcgcatt 540
gtcgacgttt tgcctgtaga acacattctc tataccagaa tgatgattgg tagattctgt 600
gctcagatgc actcaaacaa cggaccgcaa attggctcag cggtcggttg taaccctgac 660
gttgattggc aaagatttgg cacacatttc gctcagtaca agaacgtgtg ggatgtggac 720
tactcagcct tcgatgcaaa ccactgcagc gatgcgatga acatcatgtt cgaagaggtg 780
ttccgcacgg agtttgggtt ccacccgaat gccgagtgga ttctgaagac tctggttaac 840
acggagcacg cttacgagaa caagcgcatc actgtggaag gtgggatgcc gtccggttgt 900
tccgcaacaa gtatcatcaa cacaattttg aacaacatct acgtgctcta cgctctgcgt 960
aggcactatg agggagttga gctggacacc tacaccatga tctcctatgg agacgacatc 1020
gtggtggcaa gtgactacga cctggacttt gaggctctca agccccactt caagtccctc 1080
ggtcagacca tcactccagc cgacaaaagc gacaaaggtt ttgttcttgg tcactccata 1140
actgatgtca ctttcctcaa aagacacttc cacatggact acggaactgg gttttacaaa 1200
cctgtgatgg cctcgaagac cctcgaggct atcctctcct ttgcacgccg tgggaccata 1260
caggagaagt tgatctccgt ggcaggactc gccgtccact ccggacctga cgagtaccgg 1320
cgtctctttg agcctttcca aggtctcttc gagattccaa gctacagatc actttacctg 1380
cgatgggtga acgccgtgtg cggtaatcac 1410
<210> 16
<211> 500
<212> DNA
<213> New Jersey type vesicular stomatitis virus L Gene sequence
<400> 16
gcgagtgaca tgtgttacaa atgatcaaat tccgacatgt gccaatttga tgtcttcagt 60
ctctaccaac gcattaactg ttgctcattt tgcagaaaac cccataaacg caatgattca 120
atataattat tttgggacct ttgctcgatt attgctcttt atgcatgacc ctgcaataag 180
acattctctg tatacagtcc aggaaaagat acctggtttg cacaccagaa cattcaaata 240
tgctgtgtta tatctagatc cttcaatcgg aggggtgtgt ggtatggcgt tgtctcgatt 300
cttaatcaga gcatttccag atccagtaac agagagcctt tcattctgga aatttattta 360
tgaacatgcc tctgagcctc atcttaaaaa gatggctgta atgtttgggg accctccaat 420
tgccaaattc agaatagagc atatcaataa gttattagag gaccccacct ctttaaacat 480
ctcaatgggg atgagccctg 500
<210> 17
<211> 500
<212> DNA
<213> Indiana type vesicular stomatitis virus L Gene sequence
<400> 17
aattatggaa aaataccgat tttccgtgga gtgattagag ggttagagac caagagatgg 60
tcacgagtga cttgtgtcac caatgaccaa atacccactt gtgctaatat aatgagctca 120
gtttccacaa atgctctcac cgtagctcat tttgctgaga acccaatcaa tgccatgata 180
cagtacaatt attttgggac atttgctaga ctcttgttga tgatgcatga tcctgctctt 240
cgtcaatcat tgtatgaagt tcaagataag ataccgggct tgcacagttc tactttcaaa 300
tacgccatgt tgtatttgga cccttccatt ggaggagtgt cgggcatgtc tttgtccagg 360
tttttgatta gagccttccc agatcccgta acagaaagtc tctcattctg gagattcatc 420
catgtacatg ctcgaagtga gcatctgaag gagatgagtg cagtatttgg aaaccccgag 480
atagccaagt ttcgaataac 500
Claims (6)
1. A group of one-step multiplex fluorescent RT-PCR primers and probes for simultaneously detecting and identifying foot-and-mouth disease virus, New Jersey type vesicular stomatitis virus and Indiana type vesicular stomatitis virus are characterized in that the primers and the probes are respectively:
2 forward primers for detecting foot-and-mouth disease virus, which are shown in a sequence table SEQ ID NO: 1 to SEQ ID NO: 2, 3 reverse primers as shown in a sequence table SEQ ID NO: 3 to SEQ ID NO: 5 and 2 probes shown as SEQ ID NO: 6 to SEQ ID NO: 7 is shown in the specification;
2 forward primers for detecting the new jersey type vesicular stomatitis virus are shown as SEQ ID NO: 8 to SEQ ID NO: 9, 1 reverse primer, shown as SEQ ID NO: 10 and 1 probe, which is shown in a sequence table SEQ ID NO: 11 is shown in the figure;
1 forward primer for detecting Indiana type vesicular stomatitis virus, which is shown as a sequence table SEQ ID NO: 12, 1 reverse primer, shown as SEQ ID NO: 13 and 1 probe, which is shown in a sequence table SEQ ID NO: as shown at 14.
2. The one-step multiplex fluorescent RT-PCR primer and probe as claimed in claim 1, wherein the probe for detecting foot-and-mouth disease virus is labeled with FAM at 5 'end and BHQ1 at 3' end; the probe for detecting the new jersey type vesicular stomatitis virus is labeled with Cy5 at the 5 'end and BHQ2 at the 3' end; the probe for detecting the Indiana type vesicular stomatitis virus is marked with HEX at the 5 'end and BHQ1 at the 3' end.
3. A kit for simultaneously detecting and identifying foot-and-mouth disease virus, new jersey type vesicular stomatitis virus and indiana type vesicular stomatitis virus, comprising the one-step multiplex fluorescent RT-PCR primer and probe of claim 1 or 2.
4. The kit of claim 3, further comprising RT-PCR reaction buffer, enzyme, positive control, and negative control.
5. The kit according to claim 4, wherein the positive control is in vitro reverse transcription cRNA of foot-and-mouth disease virus 3D gene, New Jersey type vesicular stomatitis virus L gene, Indiana type vesicular stomatitis virus L gene, respectively.
6. The kit according to claim 4, wherein the RT-PCR reaction buffer is a Multiplex RT-PCR reaction buffer and the enzyme is a Multiplex enzyme.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710241986.5A CN106929605B (en) | 2017-04-14 | 2017-04-14 | Detection kit, primer and probe capable of simultaneously detecting and identifying foot-and-mouth disease and vesicular stomatitis |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710241986.5A CN106929605B (en) | 2017-04-14 | 2017-04-14 | Detection kit, primer and probe capable of simultaneously detecting and identifying foot-and-mouth disease and vesicular stomatitis |
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| CN106929605B true CN106929605B (en) | 2020-12-25 |
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| CN1840703A (en) * | 2006-01-06 | 2006-10-04 | 云南出入境检验检疫局检验检疫技术中心 | Multiple RT-PCR identification and detection reagent for animal vesicular disease and preparation method and use thereof |
| CN103602757A (en) * | 2013-08-30 | 2014-02-26 | 天津出入境检验检疫局动植物与食品检测中心 | Development and application of multiple fluorescence RT-PCR detection method for foot-and-mouth disease, vesicular stomatitis and swine vesicular disease |
| CN104450966A (en) * | 2014-12-09 | 2015-03-25 | 艾军 | Multiplex PCR kit for simultaneously detecting four viruses carried by ruminants |
| CN104789694A (en) * | 2014-09-15 | 2015-07-22 | 中国农业科学院兰州兽医研究所 | Vesicular disease detection kit |
| CN105671201A (en) * | 2016-03-03 | 2016-06-15 | 广西壮族自治区兽医研究所 | Primer combination for identifying foot-and-mouth disease virus and vesicular stomatitis virus and application thereof |
| CN106350609A (en) * | 2016-10-25 | 2017-01-25 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Reagent and detection method for detecting vesicular stomatitis virus, and applications |
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| CN106929605A (en) | 2017-07-07 |
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