CN116769972A - RAA primer pair, composition, detection method and application for detecting TGEV - Google Patents
RAA primer pair, composition, detection method and application for detecting TGEV Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
The invention belongs to the technical field of virus detection, and particularly relates to a RAA primer pair, a composition, a detection method and application for detecting TGEV. The primer pair comprises a forward primer and a reverse primer, the nucleotide sequences of which are respectively shown as SEQ ID NO.3 and SEQ ID NO. 5. The primer probe composition comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.3, and a reverse probe primer and a forward probe primer with nucleotide sequences shown as SEQ ID NO.5 and 7 respectively. Meanwhile, the invention also constructs two methods for detecting TGEV by using a basic RAA amplification technology and a fluorescence type RAA amplification technology based on the primer pair and the primer probe composition. The two methods have better detection sensitivity, repeatability and specificity, and can realize the specific detection of the TGEV when the pig infected with the TGEV contains PRCV or other pathogens for mixed infection.
Description
Technical Field
The invention belongs to the technical field of virus detection, and particularly relates to a RAA primer pair, a composition, a detection method and application for detecting TGEV.
Background
Transmissible gastroenteritis (Porcine transmissible gastroenteritis, TGE) is an acute, high-contact gastrointestinal infectious disease of pigs caused by transmissible gastroenteritis virus (Porcine transmissible gastroenteritis virus, TGEV), and the main clinical symptoms include anorexia, sustained vomiting, watery diarrhea, severe dehydration and even death. Pigs at different stages and breeds are susceptible, and the death rate of piglets within 2 weeks of age is up to 100%. As the age of day increases, the mortality rate of infected pigs gradually decreases, and pigs over 5 weeks of age become more viable after infection with virus, but their subsequent growth and development is severely affected and carries the virus for a long period of time. The disease is widely distributed, seriously harms, and seriously affects the health level of pigs and the development of pig industry.
Due to the similarity of TGEV to other porcine diarrhea viruses in clinical manifestations, pathological changes, and common mixed and recessive infections, diagnosis of TGEVs often requires the aid of laboratory detection diagnostic techniques. The traditional TGEV detection and diagnosis method mainly comprises virus separation and identification, ELISA, electron microscope observation, virus neutralization test, nucleic acid probe hybridization technology, RT-PCR and the like, but has the defects of complicated operation, high requirement on instruments and equipment, long period and the like in practical application, so that a detection method which is simple, sensitive, quick and free of precise instruments is urgently needed to be established.
The recombinase-mediated isothermal amplification (RAA) technology is a novel nucleic acid detection method, can rapidly complete amplification under the constant temperature condition (37-42 ℃), and is successfully applied to detection of pathogens such as viruses, bacteria and the like. 2022, lv Qiao et al reported that specific primers and probes were designed for TGEV N gene, recombinant plasmid standard TGEV-N was constructed, and TGEV fluorescence RT-RAA detection method was established after screening and optimization. However, TGEV cannot be distinguished from porcine respiratory coronavirus by detection against the N gene (Porcine Respiratory Coronavirus, PRCV). Because the amino terminal of the coding region of the TGEVS protein gene forms 621-681 nucleotide deletion, PRCV with homology of 96% with TGEV sequence is formed, so that toxicity is reduced, and infected tissues are transferred from small intestine to respiratory tract. Therefore, if pigs only have PRCV or mixed infection of multiple pathogens containing PRCV, the detection method established by using the TGEV N gene can not realize detection and diagnosis of TGEV specificity, and diagnosis errors are easy to occur.
In the prior art, CN115927745A discloses an RT-PCR detection method for detecting Porcine Epidemic Diarrhea Virus (PEDV), TGEV, PRCV and porcine acute diarrhea syndrome coronavirus (Swine acute diarrhea syndrome coronavirus, SADS-CoV), porcine hemagglutinating encephalomyelitis virus ((Porcine hemagglutinating encephalomyelitis virus, PHEV) and/or porcine delta coronavirus (Porcine deltacoronavirus, PDCoV) in total, CN103275194A discloses an antigen of porcine transmissible gastroenteritis virus or porcine respiratory coronavirus antibody in porcine serum and an ELISA detection method thereof, but the RT-PCR detection method and the ELISA detection method have the defects of complex operation, high requirement on instrument and equipment, long period and the like in practical application.
Disclosure of Invention
In view of the above, in order to overcome the defects and shortcomings of the existing detection technology, the invention provides a TGEV detection diagnosis method based on the recombinase-mediated isothermal amplification technology, which is accurate, rapid and easy to operate and detect.
The invention aims to provide a primer pair for detecting the TGEV in the pig body with the mixed infection of the TGEV and the PRCV and/or other pathogens, which is designed according to a DNA sequence of 361bp which is unique to the TGEV and deleted by the PCRV, and can realize the specific detection of the TGEV by the PRCV or other pathogens in the pig body with the TGEV infection, and particularly eliminate the interference of the PCRV. The primer pair can also be used for constructing a recombinase-mediated isothermal amplification detection kit for specifically detecting the transmissible gastroenteritis virus of pigs, and provides technical support for the specific detection of the transmissible gastroenteritis virus of pigs.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
primer pairs for specifically detecting TGEV in pig bodies infected with PRCV and/or other pathogens, wherein the primer pairs comprise a forward primer with a nucleotide sequence shown as SEQ ID NO.3 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 5.
Further, the primer pair is designed according to a TGEV detection target molecule with a nucleotide sequence shown as SEQ ID NO. 1.
The second object of the invention is to provide a primer probe composition for specifically detecting TGEV in pig body infected by mixed TGEV, PRCV and/or other pathogens, the composition can be used for constructing a detection kit for specifically detecting transmissible gastroenteritis virus of pigs, and the visual detection of transmissible gastroenteritis virus of pigs is realized.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the primer probe composition for specifically detecting the TGEV in the pig body infected by the mixed infection of the TGEV, the PRCV and/or other pathogens comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.3, a reverse probe primer with a nucleotide sequence shown as SEQ ID NO.5 and a forward probe primer with a nucleotide sequence shown as SEQ ID NO. 7.
Further, the forward probe is modified by FITC at the 5 '-end, C3 Spacer at the 3' -end, intermediate/THF/interval and BHQ; the reverse primer is modified at the 5' -end Biotin.
It is a further object of the present invention to provide a kit comprising the primer set for specifically detecting TGEV in swine bodies infected with a mixture of TGEV and PRCV and/or other pathogens as described above.
Further, the kit further comprises at least one of a RAA amplification premix and magnesium acetate for basic RAA amplification, and water for RAA amplification.
The fourth object of the present invention is to provide a method for specifically detecting TGEV in swine infected with PRCV and/or other pathogen by using a basic RAA amplification technique, which has excellent detection sensitivity, repeatability and specificity, and can realize specific detection of TGEV in swine infected with TGEV accompanied with PRCV or other pathogen mixed infection.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for specifically detecting TGEV in swine infected with a combination of TGEV and PRCV and/or other pathogens using a basal RAA amplification technique, comprising the steps of:
(1) Extracting DNA or cDNA of a sample to be detected;
(2) Preparing a basic RAA amplification reaction system by using the kit containing the TGEV primer pair for specifically detecting the mixed infection of TGEV and PRCV and/or other pathogens in pigs, and performing amplification by using the DNA or cDNA extracted in the step (1) as a template to obtain an amplification product; the temperature of the amplification is 40 ℃ and the time is 30min;
(3) And (3) analyzing the amplified product obtained in the step (2) by agarose gel electrophoresis to judge whether the swine transmissible gastroenteritis virus is infected.
Further, the sample to be tested includes stool, anal swab, pharyngeal swab, cerebrospinal fluid and/or nasal swab specimens.
Further, the basic RAA amplification reaction system is a 50 μl system, comprising: 2 XBuffer V25. Mu.L, sterile water 14. Mu.L, forward primer 2. Mu.L with nucleotide sequence shown as SEQ ID NO.3, reverse primer 2. Mu.L with nucleotide sequence shown as SEQ ID NO.5, magnesium acetate I5. Mu.L, and DNA or cDNA 2. Mu.L of the sample to be tested.
Further, the concentration of the forward primer and the reverse primer was 10. Mu.M, and the concentration of the magnesium acetate I was 280mM.
Further, the positive control was at a concentration of 1.34×10 12 Copy number plasmid standard.
Further, negative controlIs ddH 2 O。
Further, the detection limit of the detection method based on the basic RAA amplification technology is as low as 2.68X10 1 Number of copies.
Further, if 196bp bands appear in the electrophoresis chart, the sample to be tested is infected with the transmissible gastroenteritis virus of the pig; otherwise, the swine transmissible gastroenteritis virus is not infected.
It is a fifth object of the present invention to provide a kit comprising the aforementioned primer probe composition for specifically detecting TGEV in swine body infected with a mixture of TGEV and PRCV and/or other pathogens.
Further, the kit further comprises at least one of RAA amplification premix and magnesium acetate for fluorescence RAA amplification, and at least one of water, a test strip and a test strip detection buffer for RAA amplification.
The sixth object of the present invention is to provide a method for visualizing TGEV in swine infected with mixed PRCV and/or other pathogens by using fluorescence-type RAA amplification technology, which also has excellent detection speed, sensitivity, reproducibility and specificity, and can realize specific detection of TGEV in swine infected with TGEV accompanied with mixed PRCV or other pathogens.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for visually detecting TGEV in swine infected with a mixture of TGEV and PRCV and/or other pathogens using fluorescence RAA amplification techniques, comprising the steps of:
(1) Extracting DNA or cDNA of a sample to be detected;
(2) Preparing an amplification reaction system by using the kit containing the primer probe composition for specifically detecting the TGEV, the PRCV and/or the TGEV in the pig body infected by other pathogens, and amplifying by using the DNA or cDNA extracted in the step (1) as a template to obtain an amplification product; the temperature of the amplification is 40 ℃ and the time is 30min;
(3) And (3) detecting the amplification product obtained in the step (2) by using a detection test paper strip, and judging whether the swine transmissible gastroenteritis virus is infected.
Further, when a detection strip appears on the detection test strip, the sample to be detected is indicated to be infected with the transmissible gastroenteritis virus of the swine; otherwise, the swine transmissible gastroenteritis virus is not infected.
Further, the sample to be tested includes stool, anal swab, pharyngeal swab, cerebrospinal fluid and/or nasal swab specimens.
Further, the fluorescence type RAA amplification reaction system is a 50. Mu.L system, comprising: 25 mu L of A Buffer, 1 mu L of forward probe with a nucleotide sequence shown as SEQ ID NO.7, 12.5 mu L of sterile and asepsis water, 2 mu L of forward primer with a nucleotide sequence shown as SEQ ID NO.3, 2 mu L of reverse primer with a nucleotide sequence shown as SEQ ID NO.5, 5 mu L of DNA or cDNA of a sample to be detected and 2.5 mu L of B Buffer; the forward probe is modified by FITC at the 5 '-end, C3 Spacer at the 3' -end, intermediate/THF/interval and BHQ; the reverse primer is modified at the 5' -end Biotin.
Further, the concentration of the forward primer and the forward probe was 10. Mu.M,
further, the positive control was at a concentration of 1.34×10 12 Copy number plasmid standard.
Further, the negative control was ddH 2 O。
Further, the detection limit of the detection method based on the fluorescence RAA amplification technology is as low as 1.34×10 1 copies/μL。
The seventh object of the present invention is to provide the use of the aforementioned primer pair, the aforementioned primer probe composition, the aforementioned kit comprising the aforementioned primer pair for specifically detecting TGEV in swine mixed with PRCV and/or other pathogens and/or the aforementioned kit comprising the aforementioned primer probe composition for specifically detecting TGEV in swine mixed with PRCV and/or other pathogens for preparing a product for specifically detecting TGEV in swine mixed with PRCV and/or other pathogens.
By adopting the two detection methods (the detection method based on the basic RAA amplification technology and the visual detection method based on the fluorescence RAA amplification technology) provided by the invention, only TGEV can be detected, and other strains or other non-viral strains are used as templates, including viruses such as PEDV, porcine coronavirus (PDCoV), porcine kukov virus (Porcine Kobuvirus, PKoV), porcine circovirus (porcine circovirus, PCV 2), blue-ear virus (Porcine Reproductive and Respiratory Syndrome Viruse, PRRSV), classical swine fever virus (Classical swine fever virus, CSFV), porcine rotavirus (Porcine rotavirus, poRV), african swine fever virus (Africa swine fever virus, ASFV) and the like; and pathogenic bacteria such as Escherichia coli (CVCC 1524), salmonella choleraesuis (CVCC 2139), atrophic rhinitis (CVCC 4083), staphylococcus aureus and Clostridium welchii were not detected. Both detection methods are shown to have good specificity.
The invention has the beneficial effects that:
1. according to the invention, a 361bp cDNA sequence which is unique to TGEV and is deleted by PCRV is selected as a detection target molecule according to a region which is different from PRCV in a TGEV genome, so that a detection primer and a probe are designed, and a kit and a detection method based on a basic RAA amplification reaction and a fluorescent RAA amplification reaction combined side-flow gold chromatography test strip are further constructed. The two methods have good detection sensitivity, repeatability and specificity, and can realize the specific detection of the TGEV when the pig body infected by the TGEV contains PRCV or other pathogenic mixed infection.
2. The kit and the detection method based on the fluorescence RAA amplification technology combined with the lateral flow gold chromatography test strip provided by the invention are rapid, sensitive and specific in detection, and the whole experimental process can be completed within 1 h. The visual detection method provided by the invention has fewer operation steps, no special instrument or equipment is needed, the result is directly judged by naked eyes, and the experimental operation is simple.
3. The visual specificity detection method based on the RAA technology is carried out under isothermal conditions, does not need to use complex thermal cycling equipment, omits complex temperature change steps in PCR, and reduces experimental complexity. The detection method can be carried out under the common environmental conditions without being limited to a laboratory, and has flexible detection field, convenient detection method and higher detection speed.
Drawings
FIG. 1 is a graph of the results of a BLAST alignment of 10 TGEV whole-genome DNA sequences with 4 PRCV whole-genome DNA sequences, cut out portions with significant differences;
FIG. 2 is an electrophoresis chart of a basic RAA amplification reaction product using a plasmid as a positive standard in example 2, wherein M represents a DNA molecule Marker DL500, C represents a blank, 1 represents F1/R1,2 represents F1/R2,3 represents F2/R1,4 represents F2/R2,5 represents F3/R1, and 6 represents F3/R2;
FIG. 3 is an amplification graph of fluorescence type RAA amplification in example 2, wherein 1 represents PF2/PR1,2 represents PF1/PR1, and 3 represents a positive control (kit self-contained);
FIG. 4 is an electrophoresis chart of a plasmid standard basic RAA amplification product with optimized amplification temperature in example 3, wherein 1,2, 3, 4, 5, 6 respectively represent 37 ℃,38 ℃,39 ℃,40 ℃,41 ℃,42 ℃, N represents a negative control, P represents a positive control (kit is self-contained), and M represents a DNA molecule Marker DL500;
FIG. 5 is a graph showing the detection results of the plasmid label fluorescence-type RAA amplification product test strips with optimized amplification temperature in example 3, wherein 1,2, 3, 4, 5 and 6 respectively represent 37 ℃,38 ℃,39 ℃,40 ℃,41 ℃ and 42 ℃;
FIG. 6 is an electrophoresis chart of the plasmid standard in example 3 at different time bases of RAA amplification products, wherein M represents a DNA molecule Marker DL500 and N represents a negative control;
FIG. 7 is a graph showing the detection of the fluorescence-type RAA amplification product test strip of the plasmid standard in example 3 at different times, wherein 1,2, 3, 4 and 5 respectively represent 30min, 35min, 40min, 45min and 50min;
FIG. 8 is a graph showing the detection results of the fluorescent RAA amplification product test strips of different probes for the plasmid markers in example 3, wherein N represents a negative control, and 1,2 and 3 represent the addition amounts of the probe primers of 1.0. Mu.L, 1.5. Mu.L and 2.0. Mu.L, respectively;
FIG. 9 is an electrophoresis chart of the reaction products of the basic RAA at different concentrations of the plasmid standard in example 3, wherein M represents the DNA standard molecule Marker DL500, N represents the negative control, and 1-9 represent the template plasmid standard concentrations 1.34×10, respectively 0 copies/μL、1.34×10 1 copies/μL、1.34×10 2 copies/μL、1.34×10 3 copies/μL、1.34×10 4 copies/μL、1.34×10 5 copies/μL、1.34×10 6 copies/μL、1.34×10 7 copies/μL、1.34×10 8 copies/μL；
FIG. 10 is a graph showing the detection results of the plasmid label fluorescence RAA amplification product test strips at different concentrations in example 3, wherein 1,2 and 3 respectively represent the concentration of the template plasmid label of 1.34×10 0 copies/μL、1.34×10 1 copies/μL、1.34×10 2 copies/μL；
FIG. 11 is an electrophoresis chart showing the results of the repeated tests of the basic RAA of the positive standard plasmid of example 4, wherein M represents the molecular Marker DL500, N represents the negative control, and 1,2 and 3 are the positive standard plasmid concentrations 1.34×10, respectively 1 COPIES/. Mu.L, P represents the positive control
FIG. 12 is a graph showing the reproducibility of the detection of the fluorescence amplification of RAA in example 4, wherein N represents a negative control and 1,2, 3 represent concentrations of 1.34×10 1 Positive standard plasmid of cobies/. Mu.L;
FIG. 13 is an electrophoresis chart of the results of the basic RAA-specific assay of example 5, wherein M represents molecular Marker DL500,1 represents ASFV,2 represents PCV2,3 represents Cookra virus, 4 represents PEDV,5 represents PRRSV,6 represents PDCoV,7 represents a negative control, and 8 represents TGEV;
FIG. 14 is a visual inspection chart of a fluorescent RAA product test strip of example 5, wherein 1 represents ASFV,2 represents PCV2,3 represents kubuvirus, 4 represents PEDV,5 represents PRRSV,6 represents PDCoV,7 represents TGEV, and 8 represents negative control.
Detailed Description
The technical scheme of the present invention will be further clearly and completely described in connection with specific embodiments. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. Therefore, all other embodiments obtained by those skilled in the art without undue burden are within the scope of the invention based on the embodiments of the present invention.
In the embodiment of the invention, the target molecule DNA for detection is synthesized by the division of engineering (Shanghai) and all probe primers are synthesized by the division of engineering (Shanghai).
In the embodiment of the invention, the lateral flow gold detection test strip/hybrid detection test strip is Universal lateral flow dipstick for detection of biotin-and FITC-labeled assays, and the manufacturer is Milenia Biotec GmbH company.
Example 1
1. Screening detection target sequences
The TGEV and PRCV whole genome sequences are downloaded from a GenBank database, and the most obvious difference in the genome level of the TGEV and the PRCV is found through the whole genome BLAST comparison and analysis, and the difference is mainly reflected on the encoding genes of the virus S protein, which accords with the previous report. Among them, the results of cutting out portions having significant differences with BLAST alignment of 10 TGEV whole genome DNA sequences and 4 PRCV whole genome DNA sequences are shown in fig. 1. According to the difference region between the TGEV genome and the PRCV, a 361bp DNA sequence which is unique to TGEV and is deleted by PCRV is selected as a detection target molecule for subsequent research. The nucleotide sequence of the detection target molecule is shown as SEQ ID NO. 1.
2. Preparation of positive standard quality granules
The target molecule DNA for detection was synthesized and cloned into pUC57-vecter vector to obtain a recombinant plasmid containing the target fragment. The recombinant plasmid is transformed into E.coli DH5 alpha for culture and amplified. The recombinant plasmid was extracted as a positive standard, and the plasmid concentration was measured (289.+ -. 0.2 ng/. Mu.L) with an ultra-micro ultraviolet spectrophotometer after the obtained plasmid was used, and the copy number was calculated. The original concentration is diluted according to a 10 multiplied gradient to prepare 1.34 multiplied by 10 12 ～1.34×10 0 copy/. Mu.L, stored at-20deg.C for further use.
3. Preparation of negative control product
The kit also comprises a negative control product, wherein the negative control product is preferably ddH 2 O。
EXAMPLE 2 RAA primer design and preference
1. Common primer
(1) Primer design
By taking a DNA sequence inserted in a positive plasmid as a template, adopting primer design software to carry out RAA primer design, combining the TGEV detection target molecule sequence through primer length, primer sequence, GC content analysis and the like, 5 conserved TGEV RAA primers are respectively designed, wherein 3 primers are forward primers (F) and 2 primers are reverse primers (R). All designed primer sequences and their numbering are shown in Table 1.
TABLE 1 RAA primers and numbering thereof
Each of the above primers was prepared at 10. Mu.M for use.
(2) Primer screening
The designed and synthesized primers are combined into primer pairs to form 6 pairs of primers of F1/R1, F1/R2, F2/R1, F2/R2, F3/R1 and F3/R2, and the amplification reaction is carried out by adopting a RAA kit, wherein the specific operation is as follows:
basic RAA amplification reaction: the invention selects RAA nucleic acid amplification kit with the batch of BF9MB0, which comprises 48 tubes of dry powder reagent, buffer solution V, magnesium acetate I, sterile non-enzymatic water and the like. The main components, concentration, dosage and the like of the basic RAA amplification reaction system are shown in Table 2, and RAA amplification is immediately carried out after uniform mixing; the reaction conditions were 39℃for 50min, and the amplified products were analyzed by 2% agarose gel electrophoresis, and the results of the gel electrophoresis image are shown in FIG. 2.
TABLE 2 basic RAA amplification reaction System
Results: by observing and analyzing the RAA amplification product electrophoretogram, the target band of the RAA amplification product electrophoretogram of the primer combination of the No.3 electrophoresis band (F2/R1) can be found to be bright and single, and no specific band is generated. However, the RAA electrophoresis bands of other primer pairs have problems such as excessive bands, diffuse bands, or dark bands in the RAA product electrophoresis pattern due to the generation of primer dimers.
2. Probe primer
(1) Primer design
Combining the optimal primer pair F2/R1 selected by the basic RAA with the DNA fragment expected to be amplified by the optimal primer pair; 3 Probe (P) primers were designed, and the sequence, the sequence length and the labeling conditions of the Probe primers are shown in Table 3.
TABLE 3 RAA probe primers (both 5 '-3')
In the tables, tgev-PF1 and tgev-PF2 represent FAM, FITC-labeled upstream probe primers, tgev-PR1 represents downstream probe primers, and each primer was prepared at 10. Mu.M for use.
(2) Probe primer screening
Fluorescence-type RAA amplification system: the invention adopts RAA nucleic acid amplification reagent (fluorescence type) with a batch of 2302101 by Nanning Zhuangbo biotechnology Co., ltd, and comprises 48 tubes of dry powder reagent and 1 tube of each of A Buffer, B Buffer, sterile and asepsis water. The main components, concentration, dosage and the like of the fluorescence RAA amplification reaction system are shown in Table 4, after the reaction mixture is fully and uniformly mixed, the obtained reaction system is put into a fluorescence PCR instrument, RAA amplification is immediately carried out, the reaction condition is that the reaction time is 21min (1 min;30s,40 cycles) at 39 ℃,1 fluorescence is collected every 30s through the fluorescence quantitative PCR instrument, and an amplification curve is generated to analyze the product, and the result is shown in FIG. 3.
TABLE 4 fluorescence RAA amplification System
Results: the fluorescent RAA amplification with FITC and FAM labeled probe primers all produced typical amplification curves, consistent with the expected results, indicating that the synthesized probe primers were all available, but the probe primers labeled with FITC were selected according to the subsequent visual detection requirements.
EXAMPLE 3 RAA amplification reaction System Condition optimization
1. Optimization of amplification temperature
The basic RAA amplification reaction system and the fluorescent RAA amplification reaction system are respectively prepared according to the above systems. The amplification reaction was carried out at 37℃at 38℃at 39℃at 40℃at 41℃at 42℃for 50min.
The basic RAA amplification product was analyzed by 2% agarose gel electrophoresis, and the results are shown in FIG. 4. The product electrophoretogram shows that DNA bands of the expected molecular weight appear under each isothermal amplification condition of 37 ℃,38 ℃,39 ℃,40 ℃,41 ℃ and 42 ℃, but the bands shown under the reaction condition of 40 ℃ are brightest.
For fluorescence RAA amplification products, 10-20 mu L of amplification products are added into 100 mu L of buffer solution of a lateral flow gold detection test strip, the test strip is inserted into the buffer solution after uniform mixing, and the reaction is carried out for 10-20min, and then the analysis is interpreted. The results are shown in FIG. 5. The expected bands were seen in the Test lines at 37℃at 38℃at 39℃at 40℃at 41℃at 42℃with the Test lines at 40℃at 41℃being most evident.
After comprehensive analysis and comparison, the RAA amplification temperature was determined to be 40 ℃.
2. Optimization of amplification time
The basic RAA amplification reaction system and the fluorescent RAA amplification reaction system are respectively prepared according to the above systems. The amplification reaction is carried out at 40 ℃ for 30min, 35min, 40min, 45min and 50min respectively. Wherein, for the basic RAA amplification product, the amplification product is analyzed by 2% agarose gel electrophoresis. As a result, as shown in FIG. 6, for 30min and 50min of the amplified products, the electrophoresis pattern showed clear DNA bands with expected molecular weights, and the difference was not significant.
For the fluorescent RAA amplification product, 10-20 mu L of the amplification product is added into a buffer solution of a lateral flow gold detection test strip 100, the test strip is inserted into the buffer solution after uniform mixing, and the reaction is carried out for 10-20min, and then the interpretation and analysis are carried out. As a result, as shown in FIG. 7, the amplification was performed at each of 30min, 35min, 40min, 45min and 50min, the expected bands were observed on the Test lines, and the difference between the Test lines was not significant at each time point.
Comprehensively considering, the RAA amplification time is determined to be 30min.
3. Fluorescent RAA primer condition exploration
The fluorescence RAA amplification reaction system is prepared according to the above system. When the probe primer is added to react at 0.6 mu L, the visual detection effect of the test strip is poor. Therefore, the quantitative condition exploration of RAA probe primer is carried out, namely, the fluorescent RAA nucleic acid amplification reaction system is prepared, the amplification temperature is 40 ℃, and the amplification time is 30min (1 min;30s,60 cycles). The amounts of primers and probes added were prepared as shown in Table 5, and RAA amplification was performed.
TABLE 5 RAA reactions with addition of different primers
After the reaction is finished, 10-20 mu L of amplification product is added into the buffer solution of the detection test strip 100 for the fluorescent RAA amplification product, the test strip is inserted into the buffer solution after the reaction is uniformly mixed, the reaction is carried out for 10-20min, and the analysis is interpreted, and the result is shown in figure 8. As can be seen from FIG. 8, the primer for fluorescence type RAA amplification, the reaction system of adding the primer probe under 3 different addition schemes, the detection test strip has the expected and visible detection band, and the reaction system of the tgev-PF2 in the use amount is 1.0 mu L comprehensively considering the cost of the FITC-labeled probe primer.
4. Detection sensitivity
The basic RAA amplification reaction system and the fluorescent RAA amplification reaction system are respectively prepared according to the above systems. For the basic RAA amplification product, the reaction temperature was 40℃and the amplification time was 30min, and positive standard plasmid (original concentration 1.34X 10) 12 cobies/. Mu.L) was diluted at a 10-fold ratio. However, the method is thatThereafter, 1.34×10 8 copies/μL～1.34×10 0 The concentration gradient of copies/. Mu.L 9 was used as a template with ddH 2 O is a negative control, and after the reaction is finished, the amplified product is analyzed by 2% agarose gel electrophoresis. As a result, as shown in FIG. 9, it can be seen from the graph that the detection technique established using the basic RAA amplification kit, in theory, has a detection limit as low as 2.68X10 in a 50. Mu.L reaction system 1 Genomic DNA of each copy of TGEV can be detected.
Thus, based on the detection result of the sensitivity of the basic RAA in gel electrophoresis, the visualized detection of fluorescent amplification was performed at a reaction temperature of 40℃and a template of 1.34X 10, respectively 0 copies/μL，1.34×10 1 copies/μL～1.34×10 8 The positive standard plasmid was diluted with a gradient of copies/. Mu.L and the amplification time was 30min (1 min;30s 60 cycles), and 1 fluorescence signal was collected every 30 s. After the reaction is finished, 10-20 mu L of the fluorescence RAA amplification product is added into a buffer solution of a lateral flow gold detection test strip 100, the test strip is inserted into the buffer solution after uniform mixing, and the reaction is carried out for 10-20min, and then the interpretation and analysis are carried out.
As a result, as shown in FIG. 10, the detection technique established by the fluorescence RAA amplification method was used, and the fluorescence detection value was about 1.34X 10 in a 50. Mu.L reaction system 1 The genome DNA of the copies/. Mu.L TGEV is detected, and the fluorescence detection product can be rapidly judged by a detection test strip.
In summary, the optimized fluorescence-type RAA amplification reaction system is shown in Table 6.
TABLE 6 fluorescence RAA amplification System after optimization
| Constituent components of the reaction System | Volume (mu L) |
| A Buffer | 25.0μL |
| Forward probe (tgev-PF 2, 10. Mu.M) | 1.0μL |
| Sterile water | 12.5μL |
| Forward primer (tgev-F2, 10. Mu.M) | 2.0μL |
| Reverse marker primer (tgev-PR 1) | 2.0μL |
| Plasmid (1.34×10) 12 Copy number | 5.0μL |
| B Buffer | 2.5μL |
| Total volume of | 50.0μL |
Example 4 method for detecting transmissible gastroenteritis Virus based on basic RAA amplification technique
(1) Extracting DNA or cDNA of a sample to be detected by adopting a conventional method in the field;
(2) Preparing a basic RAA amplification reaction system according to Table 2;
(3) Adding the DNA or cDNA extracted in the step (1) into the amplification reaction system prepared in the step (2) by taking the DNA or cDNA as a template, and amplifying for 30 minutes at 40 ℃; after purification of the amplified product, analysis was performed by agarose electrophoresis.
And (3) judging results: if 196bp bands appear in the electrophoresis diagram, the sample to be tested is indicated to be infected with the transmissible gastroenteritis virus of the pig; otherwise, the swine transmissible gastroenteritis virus is not infected.
Example 5 method for detecting transmissible gastroenteritis Virus based on fluorescence RAA amplification technique
(1) Extracting DNA or cDNA of a sample to be detected by adopting a conventional method in the field;
(2) Preparing a fluorescence RAA amplification reaction system according to Table 6;
(3) Adding the DNA or cDNA extracted in the step (1) into the amplification reaction system prepared in the step (2) to amplify for 30 minutes at 40 ℃ to obtain an amplified product;
(4) And adding 10-20 mu L of amplification product into 100 mu L of buffer solution of the lateral flow gold detection test strip, uniformly mixing, inserting the test strip into the buffer solution, reacting for 10-20min, and judging and analyzing.
And (3) judging results: if the test strip appears in the test strip, the test sample is infected with the transmissible gastroenteritis virus of the pig; otherwise, the swine transmissible gastroenteritis virus is not infected.
Example 6 repeatability test
The basic RAA amplification reaction system and the fluorescent RAA amplification reaction system were prepared in accordance with the above tables 2 and 6.
(1) Basic RAA amplification: the reaction temperature was 40℃and the template positive standard plasmid concentration was 1.34X 10 1 The amplification time was 30min for 3 replicates, and after the reaction was completed, the amplified product was analyzed by 2% agarose gel electrophoresis.
Results: as shown in fig. 11, the detection technology system established using the basic RAA amplification kit has good reproducibility of the amplification reaction.
(2) Fluorescence RAA amplification: based on the result of the reproducibility test of the basic RAA in gel electrophoresis, a visual detection reproducibility test of RAA fluorescence amplification was performed at a reaction temperature of 40℃with a template positive for the standard plasmid (concentration 1.34X 10) 1 The copies/. Mu.L), the amplification time was 30min, and the fluorescent signal was recorded every 30s for 3 replicates. After the reaction is finished, 20 mu L of fluorescence RAA amplification product is taken and added into 100 mu L of buffer solution of a hybrid detection test strip to be uniformly mixed, the test strip is inserted into the buffer solution, and the reaction is carried out for 10-20minAnd (5) interpretation analysis.
Results: as shown in fig. 12, the basic detection pattern is consistent with the detection result of the fluorescence detection product by the lateral flow gold detection test strip using the visualization test strip detection technology established by the fluorescence-type RAA amplification method. The fluorescence detection and the visualization of the test strip have good repeatability, the test line is clear, and the rapid judgment can be realized.
EXAMPLE 7 specificity test
The basic RAA amplification reaction system and the fluorescent RAA amplification reaction system were prepared in accordance with the above tables 2 and 6. In order to analyze the detection specificity of this method, in this example, genomic DNA or RNA of a pathogen is extracted, and if the viral nucleic acid is RNA, further reverse transcription is required to obtain cDNA.
(1) RNA extraction
The Trizol method is adopted, and the method is carried out according to the operation instruction provided by manufacturers, and comprises the following specific steps: taking 250 mu L of supernatant as a sample to be extracted, adding 750 mu L of Trizol, shaking and mixing uniformly, and standing at room temperature for 5min. Adding 250 mu L of chloroform, shaking and mixing, standing at room temperature for 5-15 min, and centrifuging at 4 ℃ for 15min at 12000 r/min. Taking 400-500 mu L of upper water phase, adding isopropyl alcohol with the same volume, uniformly mixing, standing at-20 ℃ for more than 2 hours or at-80 ℃ for 0.5 hour, and centrifuging at 4 ℃ for 10min at 12000 r/min. Removing supernatant, slowly adding 1.0ml of washing 12000r/min for 5min after 75% of alcohol (prepared by DEPC water). The supernatant was removed, dried at room temperature for 20min, and then 20. Mu.L of human DEPC water was added to dissolve it sufficiently.
(2) Preparation of cDNA by reverse transcription
The reverse transcription was performed according to the TaKaRa reverse transcription kit (Code No. RR014A), specifically: reverse transcription premix A (random primer 50. Mu.M, 1. Mu.L, dNTP mix 10mM 1. Mu.L); reverse transcription premix B (reverse transcription buffer 2. Mu.L, reverse transcriptase 0.5. Mu.L, RNase inhibitor 0.5. Mu.L, water for reverse transcription amplification make up to 10. Mu.L). After adding 8. Mu.L of RNA to reverse transcription premix A, the mixture was first incubated at 65℃for 5min and then rapidly cooled on ice. Transferring 10 mu L of the product of the last step into the reverse transcription premix B, slowly and uniformly mixing, reacting for 10min at 30 ℃ and reacting for 30min at 42 ℃; and then preserving the temperature at 95 ℃ for 5min to inactivate the enzyme, and finally placing the enzyme on ice for standby.
(3) Specific detection
The DNA obtained as described above was used as a template, and the method was subjected to detection-specific analysis. The sources of the templates mainly comprise: in terms of viruses, TGEV (SCJY-1 strain) and PEDV (SCSZ-1 strain) are given to Chongqing Australian biological products limited company; porcine coronavirus (PDCoV), porcine kubuvirus (Porcine Kobuvirus, PKoV), porcine circovirus (porcine circovirus, PCV 2), blue ear virus (Porcine Reproductive and Respiratory Syndrome Viruse, PRRSV); hog cholera virus (Classical swine fever virus, CSFV) and porcine rotavirus (Porcine rotavirus, poRV) are stored in this laboratory; other viruses such as African swine fever virus (Africa swine fever virus, ASFV) are a benefit of the agricultural university of south China. Bacterial aspects: pathogenic bacteria such as Escherichia coli (CVCC 1524), salmonella choleraesuis (CVCC 2139), atrophic rhinitis (CVCC 4083), staphylococcus aureus and Clostridium welchii (stored in the laboratory). The genomic DNA or cDNA obtained by reverse transcription is used as a template, and added into an amplification reaction system prepared by the system, and amplified for 30 minutes at 40 ℃, and after the basic amplification product is purified, agarose electrophoresis observation and analysis are performed. Wherein, TGEV (SCJY-1 strain), PEDV (SCSZ-1 strain), porcine coronavirus, porcine kubuvirus, porcine circovirus, blue ear virus, african swine fever virus and porcine circovirus are used as templates for the specific detection of the basic RAA. The results are shown in FIG. 13.
Similarly, the fluorescent RAA amplification products of the corresponding templates are used for carrying out visual detection on the amplification products by adopting an immunochromatography test strip: and adding 100 mu L of test strip detection buffer solution into 10-20 mu L of amplification product, uniformly mixing, inserting the test strip, reacting for 10-20min, and finally judging the result. The results are shown in FIG. 14.
All the results showed that only TGEV could be detected and none with other strains or other non-viral strains as templates.
Example 8 clinical sample testing
107 clinical samples (including faeces, anal swab, pharyngeal swab, cerebrospinal fluid and nasal swab specimens) are selected, and the detection is carried out simultaneously with the method by using a swine transmissible gastroenteritis virus RT-nPCR detection method (national agricultural industry standard NY/T2841-2015 of the people's republic of China). The detection method specifically comprises the following steps:
1. specimen processing
Sample RNA was rapidly extracted using a QIAamp viral RNA mini kit kit (purchased from Kaijer Corp., germany):
(1) Respectively adding 1mL of sterilized normal saline into samples such as faeces, anal swab, pharyngeal swab and the like, and fully shaking and shaking uniformly;
(2) The sample obtained in step (1) was centrifuged at 5000rpm for 1min, and 140. Mu.L of the supernatant was taken.
RNA extraction
Extracting by Trizol method, and storing for use, wherein the Trizol method has the specific steps as described in example 5.
3. Reverse transcription
The cDNA product was collected and stored for use as described in TaKaRa reverse transcription kit (Code No. RR014A) of example 5.
4. Fluorescence RAA reaction
The procedure was followed for the fluorescent RAA amplification kit set forth in table 6.
5. Test strip detection
Taking 10-20 mu L of RAA amplification product, adding 100 mu L of test strip detection buffer solution, mixing, inserting the test strip for reaction for 10-20min, and finally judging the result. The detection results of 107 samples are completely consistent with the fluorescent quantitative results, wherein 6 samples are positive samples and 101 samples are negative samples.
From the detection results of the embodiment, the invention can accurately detect the virus in the clinical specimen, the detection is quick, sensitive and specific, and the whole experimental process can be completed within 1 h. The operation steps are fewer, special instruments and equipment are not needed in the whole process, the result can be judged by direct naked eye observation, and the experimental operation is simple.
Claims (10)
1. Primer pair for the specific detection of TGEV in swine infected with PRCV and/or other pathogens, characterized in that the primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID No.3 and a reverse primer with a nucleotide sequence shown as SEQ ID No. 5.
2. The primer probe composition for specifically detecting the TGEV in the pig body infected by the mixed infection of the TGEV, the PRCV and/or other pathogens is characterized by comprising a forward primer with a nucleotide sequence shown as SEQ ID NO.3, a reverse probe primer with a nucleotide sequence shown as SEQ ID NO.5 and a forward probe primer with a nucleotide sequence shown as SEQ ID NO. 7.
3. The primer probe composition according to claim 2, wherein the forward probe is modified at the 5' -end by FITC,
3' -terminal C3 Spacer modification, mid/THF/Spacer modification, BHQ modification; the reverse probe primer is modified by Biotin at the 5' -end.
4. A kit comprising a primer pair for specifically detecting TGEV in swine mixed with PRCV and/or other pathogens according to claim 1.
5. A method for specifically detecting TGEV in swine infected with a mixture of TGEV and PRCV and/or other pathogens using a basic RAA amplification technique, comprising the steps of:
(1) Extracting DNA or cDNA of a sample to be detected;
(2) Preparing a basic RAA amplification reaction system by using the kit of claim 4, and performing amplification by using the DNA or cDNA extracted in the step (1) as a template to obtain an amplification product; the temperature of the amplification is 40 ℃ and the time is 30min;
(3) And (3) analyzing the amplified product obtained in the step (2) by agarose gel electrophoresis to judge whether the swine transmissible gastroenteritis virus is infected.
6. The method of claim 5, wherein the base RAA amplification reaction system is a 50 μl system comprising: 2 XBuffer V25. Mu.L, sterile water 14. Mu.L, forward primer 2. Mu.L with nucleotide sequence shown as SEQ ID NO.3, reverse primer 2. Mu.L with nucleotide sequence shown as SEQ ID NO.5, magnesium acetate I5. Mu.L, and DNA or cDNA 2. Mu.L of the sample to be tested.
7. A kit comprising a primer probe composition according to any one of claims 2-3 for the specific detection of TGEV in swine bodies infected with PRCV and/or other pathogens.
8. A method for visually detecting TGEV in swine infected with a mixture of TGEV and PRCV and/or other pathogens using fluorescence RAA amplification technology, comprising the steps of:
(1) Extracting DNA or cDNA of a sample to be detected;
(2) Preparing a fluorescence RAA amplification reaction system by using the kit of claim 7, and amplifying by using the DNA or cDNA extracted in the step (1) as a template to obtain an amplified product; the temperature of the amplification is 40 ℃ and the time is 30min;
(3) And (3) detecting the amplification product obtained in the step (2) by using a lateral flow chromatography detection test strip, and judging whether the swine transmissible gastroenteritis virus is infected.
9. The method of claim 8, wherein the fluorescent RAA amplification reaction system is a 50 μl system comprising: 25 mu L of A Buffer, 1 mu L of forward probe with a nucleotide sequence shown as SEQ ID NO.7, 12.5 mu L of sterile and asepsis water, 2 mu L of forward primer with a nucleotide sequence shown as SEQ ID NO.3, 2 mu L of reverse primer with a nucleotide sequence shown as SEQ ID NO.5, 5 mu L of DNA or cDNA of a sample to be detected and 2.5 mu L of B Buffer; the forward probe is 5'
-terminal FITC modification, 3' -terminal C3 Spacer modification, mid/THF/Spacer and BHQ modification; the reverse primer 5'
-terminal Biotin modification.
10. Use of the primer pair of claim 1, the primer probe composition of any one of claims 2-3, the kit of claim 4 and/or the kit of claim 7 for the preparation of a product for specifically detecting TGEV in a pig body infected with a mixture of TGEV and PRCV and/or other pathogens.
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