CN113046489A - Multiple RT-PCR primer group for detecting porcine astrovirus, kit and application thereof - Google Patents
Multiple RT-PCR primer group for detecting porcine astrovirus, kit and application thereof Download PDFInfo
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Abstract
The invention discloses a multiplex RT-PCR primer group for detecting porcine astrovirus, which comprises five pairs of specific primers, namely primers PAStV1-F and PAStV1-R, primers PAStV2-F and PAStV2-R, primers PAStV3-F and PAStV3-R, primers PAStV4-F and PAStV4-R, and primers PAStV5-F and PAStV5-R, wherein the primers have base sequences of sequence tables SEQ ID No.1 to SEQ ID No.10 respectively. Accordingly, the inventor also prepares a corresponding RT-PCR kit and establishes a corresponding RT-PCR detection method. The method is simple and convenient in actual operation, reduces the operation times, greatly avoids cross contamination, and saves the detection time and the reagent consumption. The established detection method aims at the gene amplification of the virus conserved region, and has strong pertinence and high sensitivity. Can meet the requirements of simple, convenient, economic, convenient and accurate inspection in clinical experiments.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a multiple RT-PCR primer group for detecting porcine astrovirus, a kit and application thereof.
Background
Porcine astrovirus was first discovered in pig manure by electron microscopy in 1980. Astrovirus is associated with diarrhea in the gut of a variety of animals, ranging from birds to mammals to humans to infections. Porcine astrovirus often infects young, old and immunocompromised animals. Meanwhile, rotavirus, coronavirus, calicivirus and other enterovirus mixed infection are often detected in positive samples of the porcine astrovirus. The porcine astrovirus has various genotypes, and at present, 5 genotypes (PAStV1-PAStV5) are found, and the sequence homology of each genotype is not high, which indicates that the genotypes are derived from different clades. Although the genetic background of each genotype is obviously different, the porcine astrovirus of different genotypes can be detected in the same area and even the same pig body, and the phenomenon of mixed infection of PAStVs is common. In addition, co-infection with different genotypes of PAstV often leads to genetic recombination phenomena with the potential risk of cross-species transmission and even zoonotic co-morbidity. The mixed infection of multiple genotypes is frequently generated among the genomes of the PAStV, and the infection of two or more genotypes is generated, so that the genetic variation of the virus is further accelerated, and the monitoring of the porcine astrovirus is challenged. It is worth mentioning that the porcine astrovirus interspecies barrier may not be strict, and genetic evolution analysis results suggest that the porcine astrovirus may cross the interspecies barrier of humans and other animals. Researchers find that recombination phenomena exist between human astrovirus and porcine astrovirus ORF2 genes, and indicate that the porcine astrovirus has close relation with the human astrovirus in the evolution process. Therefore, the establishment of an efficient and rapid detection method can monitor the infection condition of the porcine astrovirus in a swinery and analyze the recombination and variation rules among strains, which has important public health significance for the biological characteristics and pathogenic mechanism of the astrovirus and the prevention of the infection of the human and animal co-suffering astrovirus.
In the current intensive management production, the condition of polygenic mixed infection of the porcine astrovirus is common, the epidemiological monitoring of the porcine astrovirus is a long-term and very necessary work, and the establishment of a rapid and accurate diagnostic method becomes a powerful tool for monitoring the PAStVs. In contrast, the establishment of multiplex PCR detection technology can amplify multiple gene fragments simultaneously, and is low in cost, rapid, cheap and undoubtedly the best detection means at present.
Disclosure of Invention
The invention provides a multiplex RT-PCR primer group, a kit and application thereof for detecting porcine astrovirus, which are simple and convenient to operate, avoid cross contamination, save detection time, consume reagents, have strong pertinence and high sensitivity, and solve the problems of complicated operation and high cross contamination risk of single RT-PCR, and the specific scheme is as follows:
a multiplex RT-PCR primer group for detecting porcine astrovirus comprises five pairs of specific primers, namely primers PAStV1-F and PAStV1-R, primers PAStV2-F and PAStV2-R, primers PAStV3-F and PAStV3-R, primers PAStV4-F and PAStV4-R and primers PAStV5-F and PAStV5-R, which respectively have base sequences from a sequence table SEQ ID No.1 to a sequence table SEQ ID No. 10.
A multiplex RT-PCR kit for detecting porcine astrovirus, the kit comprising a multiplex RT-PCR primer set.
The kit also comprises 5 xbuffer, dNTPmix, M-MLVReverserTranscriptae, RNase inhibitor, RNA, TarakaEx xTaq, 10 xTaqXExTaqBuffer, dNTPmix, TemplatecDNA and ddH2O。
The application of the multiplex RT-PCR primer group for detecting the porcine astrovirus in RT-PCR amplification.
The RT-PCR amplification reaction system comprises multiple RT-PCR primer groups of 0.8 mu mol/L, TarakaEx XTaq 0.25 mu L and 10 XExTaqBuffer 5 mu L, dNTPMix4 mu L, cDNA template of 1 mu L respectively, and ddH after sterilization2The content of O is filled to 50 mu L.
The RT-PCR amplification reaction conditions are denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min and 30 cycles.
THE ADVANTAGES OF THE PRESENT INVENTION
1. Aiming at the lack of an effective and reliable technology for simultaneously detecting and diagnosing multi-genotype porcine astrovirus, the inventor researches and designs five pairs of specific primers, namely primers PAStV1-F and PAStV1-R, primers PAStV2-F and PAStV2-R, primers PAStV3-F and PAStV3-R, primers PAStV4-F and PAStV4-R and primers PAStV5-F and PAStV5-R, establishes a corresponding RT-PCR kit and a corresponding RT-PCR detection method. The RT-PCR detection method can be used for carrying out PCR amplification on cDNA (complementary deoxyribonucleic acid) obtained from the total RNA of the same detection sample as a template, judging the genotype of the sample to be detected infected with the virus through agarose gel electrophoresis according to the size of an amplification product, identifying five genotypes of the virus amplified simultaneously in the same reaction system, and having important practical effects on early detection and effective prevention and control of the occurrence and spread of the porcine astrovirus.
2. The method is simple and convenient in actual operation, reduces the operation times, greatly avoids cross contamination, and saves the detection time and the reagent consumption. The established detection method aims at the gene amplification of the virus conserved region, and has strong pertinence and high sensitivity. Can meet the requirements of simple, convenient, economic, convenient and accurate inspection in clinical experiments. Provides technical support for understanding the prevalence of the porcine astrovirus and preventing and controlling diseases. The method lays a foundation for monitoring the prevalence and mixed infection conditions of the porcine astrovirus types 1-5 and rapid diagnosis of molecular biology.
Drawings
FIG. 1 is a diagram showing the detection of specific primers corresponding to PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5, wherein the lanes are M from left to right: m is DL2000 DNA Marker; A. b, C, D, E are sequentially PAStV1, PAStV2, PAStV3, PAStV4 and PAStV 5;
FIG. 2 is a graph of the optimization of primer concentrations for multiplex RT-PCR, where M is DL2000 DNA Marker; the primer concentrations of 1, 2, 3, 4, and 5 were 0.2. mu. mol/L, 0.4. mu. mol/L, 0.6. mu. mol/L, 0.8. mu. mol/L, and 1.0. mu. mol/L, respectively.
FIG. 3 is a graph of the optimization of the annealing temperature for multiplex RT-PCR, wherein M is DL2000 DNA Marker; the annealing temperatures of 1, 2, 3, 4 and 5 are respectively 53 ℃; at 54 ℃; 55 ℃; 56 ℃; and 57 ℃.
FIG. 4 is a diagram showing the detection results of the primers of the present invention for simultaneously detecting the specificity of the primers corresponding to PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5, wherein the lanes in the diagram are, from left to right: m is DL2000 DNA Marker; 1: PAStV 1; 2: PAStV 2; 3: PAStV 3; 4: PAStV 4; 5: PAStV 5; 6: PAStV1, PAStV2, PAStV3, PAStV4, and PAStV 5; 7-13 non-target genes; 14: and (5) negative control.
FIG. 5 is a graph showing the results of single sensitivity experiments for simultaneously detecting PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5 according to the present invention, wherein the lanes are, from left to right: 1:10 ng; 2:1 ng; 3:100 pg; 4:10 pg; 5:1 pg; 6:0.1 pg; 7: negative control positive plasmid.
FIG. 6 is a graph showing the results of detection of multiple RT-PCR sensitivities simultaneously, wherein the lanes from left to right are: 1:10 ng; 2:1 ng; 3:100 pg; 4:10 pg; 5:1 pg; 6:0.1 pg; 7: negative control positive plasmid.
And (3) positive plasmids.
Detailed Description
The invention will be further explained and illustrated with reference to the drawings and the following specific examples, which do not limit the scope of the invention. Unless otherwise specified, the technical means in the examples are conventional means well known to those skilled in the art.
Example 1
1.1 Strain for test and clinical specimens
Positive nucleic acid samples of porcine astrovirus type 1, porcine astrovirus type 2, porcine astrovirus type 3, porcine astrovirus type 4, porcine astrovirus type 5, porcine enterovirus, porcine senecio virus, porcine pseudorabies virus, classical swine fever virus and porcine reproductive and respiratory syndrome virus are identified by the laboratory and stored at-80 ℃. Porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus triple live vaccine (Harbin Virginiaceae biotechnology development company), and porcine Japanese encephalitis B virus (Wuhanke pre-biological shares, Inc.). The clinical stool samples are all from a certain pig farm in Guangxi of 2020, and the total amount is 275.
1.2 primer design and Synthesis
According to the reported whole genome sequences of PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5 on GenBank, the genomes of different strains are compared and analyzed, conservative nucleic acid fragments of the strains are screened, five pairs of specific primers aiming at the five viruses are designed by using Pirmer 5.0 primer design software, and the five pairs of primers are analyzed on line through NCBI. Five pairs of primers (Table 1) synthesized by Biotechnology, Shanghai, Inc. were finally screened. The five pairs of primers can specifically amplify specific fragments of 125(PAStV1), 573(PAStV2), 175(PAStV3), 485(PAStV4) and 305(PAStV 5).
TABLE 1 multiplex RT-PCR primer sequence Listing
EXAMPLE 2cDNA template preparation
2.1 extraction of RNA of sample to be detected: collecting pig feces, diluting the feces sample with PBS by vortex oscillation, centrifuging at 8000r/min4 deg.C, collecting supernatant, and storing at-80 deg.C.
2.2 reverse transcription reaction conditions: the reaction was carried out at 42 ℃ for 1h, and the resulting cDNA was stored at-20 ℃ for PCR amplification.
2.3 TABLE 2 reverse transcription PCR System
2.4 extraction of cDNA: DNAs extracted from PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5 were stored at-20 ℃ according to the conventional method.
Example 3 Single PCR amplification sequence validation
The cDNAs of PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5 stored in example 2 were used as templates and subjected to single PCR amplification using five pairs of specific primers designed in example 1. The single PCR amplification system is shown in Table 3. The reaction condition is denaturation at 98 ℃ for 10 s; annealing at 55 ℃ for 30 s; extending for 1min at 72 ℃; 30 cycles. RT-PCR products were detected by 1.5% agarose gel electrophoresis. As shown in FIG. 1, the PCR product was purified to obtain the target gene by a purification kit and sequenced by Biotechnology engineering (Shanghai) Co., Ltd. The sequencing result shows that the nucleotide sequence of the strain is consistent with that of the corresponding strain.
TABLE 3RT-PCR reaction System
| Reagent | 25 μ L reaction System |
| Taraka Ex×Taq | 0.25μL |
| 10×Ex Taq Buffer | 5μL |
| dNTP Mix | 4μL |
| Upstream primer | 0.8μmol/L |
| Downstream primer | 0.8μmol/L |
| Template cDNA | Each 1 mu L |
| ddH2O | Make up to 50 μ L |
EXAMPLE 4 multiplex RT-PCR reaction
4.1 multiple RT-PCR reaction system and optimized condition;
under the condition that the reaction conditions are not changed (denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min and 30 cycles), mixing the PAStV1, the PAStV2, the PAStV3, the PAStV4 and the PAStV5 with the same concentration to be used as a multiple RT-PCR template, and optimizing the multiple RT-PCR reaction conditions including primer concentration and a reaction system.
And (3) primer concentration screening: the reaction system used was: 1.0 mu L of each upstream primer and downstream primer; TarakaEx × Taq0.25 μ L; 10 XExTaqBuffer 5 μ L; dNTPmix4 μ L; 2.5. mu.L each of cDNA templates; sterilized ddH2Supplementing O to 50 μ L; 5 different concentration gradients of 0.2. mu. mol/L, 0.4. mu. mol/L, 0.6. mu. mol/L, 0.8. mu. mol/L and 1.0. mu. mol/L were selected for PCR amplification, and the results of primer concentration screening were shown in FIG. 2.
Annealing stability optimization: the annealing temperature is selected to be 53 ℃; at 54 ℃; 55 ℃; 56 ℃; 5: PCR amplification was performed at 57 ℃ with 5 different gradients and the optimal annealing temperature was chosen, the results are shown in FIG. 3.
4.2 multiplex RT-PCR specificity;
the amplification results of the mixed cDNA template of PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5, the cDNA of positive sample of PEV, PRRSV, SVV, CSFV, JEV and porcine triple live vaccine (PEDV, RV and TGEV) and the DNA template of positive sample of PRV are shown (as shown in figure 4), and only the single cDNA of PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5 and the mixed cDNA of PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5 amplify the band which is in accordance with the size of the target fragment.
4.3 Single/multiplex RT-PCR sensitivity assay
Respectively measuring the cDNA concentrations of the PAStV1, the PAStV2, the PAStV3, the PAStV4 and the PAStV5 by using a spectrophotometer, and respectively adjusting the cDNA concentrations to 1:10 ng; 2:1 ng; 3:100 pg; 4:10pg, 5: 0.1 pg; 6:1 pg; 7:0.1pg the cDNA of each concentration was used as a template for PCR amplification, and the minimum amount detected was detected.
As shown in FIG. 5, 5 viral genotype plasmids were individually subjected to sensitive amplification with a minimum detection amount of cDNA of PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5 of 0.1pg, 1pg and 0.1pg, respectively, as shown in FIG. 6, and 5 viral genotype plasmids were mixed and subjected to sensitive amplification with a minimum detection amount of cDNA of PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5 of 10 pg.
EXAMPLE 5 preliminary application of clinical samples
The multiplex RT-PCR primer of the invention is used for RT-PCR detection of the following samples, 275 dejecta collected in each pig farm in Guangxi in 2020 is detected.
TABLE 4 clinical sample test results
Astrovirus has been prevalent in many countries around the world since its first discovery, and for a considerable period of time, its clinical symptoms have been considered to be only sporadic, mild diarrhoea, and the pathogenicity of astrovirus has not been appreciated. With the development of detection means, astrovirus is deeply understood, and astrovirus infection is accompanied by symptoms such as nephritis, hepatitis, neurological symptoms and the like. Further, it has been found that the astrovirus has a cross-species gene recombination phenomenon.
Pork is one of the main meat foods in China, and the price of the pork continuously rises along with the rapid development of intensification. The distribution range of the breeding industry is wide, the breeding scale is large, and the like, and the astrovirus infectivity is high due to the large-scale breeding mode. Although the cause of porcine diarrhea virus is not only astrovirus, astrovirus is often co-infected with other diarrhea virus and often plays a "culprit" role in diarrhea virus. Moreover, the astrovirus has polygenic mixed infection, so that diarrhea symptoms are aggravated, or nervous or encephalitis symptoms are caused, and economic losses are often caused to farmers. At present, diagnosis and detection aiming at astrovirus mostly stay in the detection level of single RT-PCR or double RT-PCR, and other detection methods such as: direct immunofluorescence tests, electron microscopy, agar immunodiffusion tests are expensive and not suitable for large-scale clinical testing. Therefore, the method for establishing the multiple RT-PCR detection aiming at different genotypes has high efficiency and saves cost.
The invention aims at the mixed infection condition of distinguishing all genotypes of the porcine astrovirus and establishes a multiple RT-PCR detection kit. The test subjects involved AstV1, PAstV2, PAstV3, PAstV4 and PAstV 5. The partial genotypes of the genes have great harm in swinery, the potential public health significance is particularly important, and the genes have important values for detecting epidemic situations and preventing epidemic diseases.
The multiple RT-PCR established by the invention has high sensitivity, and the minimum detection amount reaches 10 pg; and can specifically amplify the target gene; the requirement on the content of the extracted RNA is low, and the reaction system is simple and easy to operate.
In conclusion, the invention provides a detection and identification method which can specifically and sensitively detect the genotypes of the PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5 and the mixed infection condition, and the invention has important application value in public epidemiological investigation and the like.
The above-described embodiments are merely illustrative of the present invention, and not restrictive, and it should be understood that various changes and modifications in the spirit and scope of the invention may be made by those skilled in the art from the disclosure herein without departing from the spirit and scope of the invention.
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Claims (6)
1. The multiplex RT-PCR primer group for detecting the porcine astrovirus is characterized by comprising five pairs of specific primers, namely primers PAStV1-F and PAStV1-R, primers PAStV2-F and PAStV2-R, primers PAStV3-F and PAStV3-R, primers PAStV4-F and PAStV4-R, and primers PAStV5-F and PAStV5-R, wherein the primers have base sequences of sequence tables SEQ ID No.1 to SEQ ID No.10 respectively.
2. A multiplex RT-PCR kit for detecting porcine astrovirus, the kit comprising the multiplex RT-PCR primer set of claim 1.
3. The method of claim 2 for detecting porcine astrovirusThe multiplex RT-PCR kit of (1), wherein the kit further comprises 5 XBuffer, dNTPmix, M-MLV Reverse transcriptase, RNase inhibitor, RNA, Taraka Ex XTaq, 10 XExTaq Buffer, dNTPmix, Template cDNA and ddH2O。
4. The use of the multiplex RT-PCR primer set for detecting porcine astrovirus according to claim 1 in RT-PCR amplification.
5. The use of claim 4, wherein the RT-PCR amplification reaction system comprises multiple RT-PCR primer sets of 0.8 μmol/L each, 0.25 μ L of Taraka Ex xTaq, 5 μ L of 10 xEx Taq Buffer, 1 μ L of dNTP Mix4 μ L, cDNA template each, and ddH after sterilization2The content of O is filled to 50 mu L.
6. The use of claim 4, wherein the RT-PCR amplification reaction conditions are denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 1min for 30 cycles.
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