CN110592278A - Multiplex RT-PCR kit for PRoV, PoSaV and PAStV - Google Patents
Multiplex RT-PCR kit for PRoV, PoSaV and PAStV Download PDFInfo
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Abstract
The invention discloses a primer group for detecting multiple RT-PCR of PRoV, PoSaV and PAStV, which comprises three pairs of specific primers, namely primers PRoV-F and PRoV-R, primers PoSaV-F and PoSaV-R and primers PAStV-F and PAStV-R. Accordingly, the inventors have also developed a corresponding diagnostic kit. By applying the invention, single or mixed infection viruses of PRoV, PoSaV and PAStV can be simultaneously detected from a sample through one RT-PCR reaction, and the invention has the characteristics of high specificity, good sensitivity, short time consumption, low cost and the like. In conclusion, the invention provides a quick, simple and convenient tool for detecting the mixed infection of the swine herd of PRoV, PoSaV and PAStV, lays a solid foundation for clinical quick identification and detection and laboratory epidemiological investigation, is beneficial to the pig industry to establish a treatment scheme in time, and reduces the death rate and economic loss of the swine herd.
Description
Technical Field
The invention belongs to the field of virus nucleic acid detection, and particularly relates to a PRoV, PoSaV and PAStV multiplex RT-PCR kit.
Background
In recent years, with the intensive and large-scale development of pig raising industry, the situation that a swinery is infected by a plurality of pathogens in a mixed way is more common. Porcine rotavirus (PRoV), Sapovirus (PoSaV), and porcine astrovirus (PASTv), which are found in healthy suckling piglets and diarrhea piglets, are pathogens of viral intestinal diseases, and it is difficult to distinguish these diseases due to the similarity of their pathological changes in clinical symptoms and epidemiology. Meanwhile, the three viruses are reported to infect not only suckling piglets, but also main viruses causing diarrhea in infants. The mixed infection of the above diseases in swinery not only can bring huge challenges to laboratory diagnosis and clinical treatment, but also can cause significant economic loss to the pig industry and human health.
Porcine Rotavirus (PRoV) belongs to Rotavirus (Reoviridae) Rotavirus genus of Reoviridae family, is widely existed in infants and other mammals, is the most common factor causing acute diarrhea of newborn piglets and weaned piglets, and is easy to be secondarily infected by bacteria. Rotavirus (RV) is the major cause of viral diarrhea in infants and young children worldwide, and in developed and developing countries, RV infection in infants and young children is common, the virus is mainly transmitted through the fecal-oral route, usually the virus is developed 1-2 days after infection, almost every child <5 years old is infected with RV, and infants from 6 months to 2 years old are the major population of RV infection. The young piglets are most susceptible, the morbidity of the piglets at 1-4 weeks is over 80 percent, the mortality rate reaches 7-20 percent, and the piglets are mainly marked by severe diarrhea, vomiting, dehydration, emaciation, slow growth and the like.
Saporoviruses (PoSaV) is a member of the genus Saporovirus, the family Caliciviridae, and can be transmitted via the fecal-oral route to cause acute gastroenteritis in pigs, and is a potential zoonosis. Studies have shown that some PoSaV has high homology with the human SaV nucleotide sequence and more SaV recombinant new strains derived from human and pig are discovered, suggesting that PoSaV has the potential risk of cross-species infection and transmission to humans. The human sapporo virus genes have high variability, and the recombination sites of the porcine recombinant sapporo virus strains are located in the overlapping regions of the polymerase and the capsid protein. Although recombination of caliciviruses between different species has not been found so far, this phenomenon exists with many other RNA viruses, and thus, potential recombination of caliciviruses between different species may also lead to the emergence of new strains with different pathogenicity and virulence.
Porcine astrovirus (PAstV) is a member of the astroviridae family. Astrovirus (AstV) is a zoonotic pathogen widely present in humans and in a variety of mammals and birds, and is widely distributed throughout the world. The PAStV has large damage to piglets in the lactation period, and often causes clinical symptoms such as diarrhea, dehydration, vomiting and the like, so that the piglets can grow slowly, and can die in severe cases, thereby bringing certain economic loss to the pig raising industry. The literature reports that the detection rate of astrovirus tends to increase with age. Although the astrovirus infection rates reported at present are all low at home, foreign studies indicate that astrovirus should not be considered as the only pathogen of common intestinal infections in children, but there may also be severe central nervous system infections caused by astrovirus and other unrecognized diseases.
Research shows that diarrhea of pigs caused by mixed infection of various viruses seriously harms the production work of the pigs, and the diarrhea is difficult to distinguish according to clinical symptoms and epidemiology, and must be distinguished and diagnosed by means of laboratory detection technology. The common diagnostic techniques such as electron microscope observation, virus separation, immunohistochemistry, fluorescent antibody technique and the like are complex to operate and have high difficulty; the ELISA pathogen rapid detection kit and the conventional RT-PCR method are widely applied, but the serology method has lower accuracy and the common single PCR method consumes long time. Therefore, there is a need to establish a rapid early diagnosis method capable of detecting these various viruses simultaneously, so as to take effective preventive and control measures as soon as possible and reduce the harm of these diseases to the pig industry and human health.
Disclosure of Invention
The invention aims to solve the technical problem of providing a multiple RT-PCR kit for PRoV, PoSaV and PAStV, can simultaneously detect the three viruses in the same reaction tube, and is suitable for rapid detection and epidemiological investigation of the PRoV, PoSaV and PAStV in clinic and scientific research.
In order to solve the technical problems, the invention adopts the following technical scheme:
the primer group for detecting the multiple RT-PCR of the ProV, the PoSaV and the PAStV comprises three pairs of specific primers, namely primers PRoV-F and PRoV-R, primers PoSaV-F and PoSaV-R and primers PAStV-F and PAStV-R, which have base sequences of sequence tables SEQ ID No.1 to SEQ ID No.6 respectively.
The application of the PRoV, PoSaV and PAStV multiplex RT-PCR detection primer group in PCR amplification.
In the PCR amplification, PRoV-F and PRoV-R were designed at position 359-19 of VP6 for the gene of PRoV, primers PoSaV-F and PoSaV-R were designed at position 4363-4661 of RdRp for PoSaV, and primers PAStV-F and PAStV-R were designed at position 3332-3797 of ORF1 for the gene of PAStV.
The annealing temperature for PCR amplification was 50 ℃.
The multiple RT-PCR kit of PRoV, PoSaV and PAStV comprises the following reagents: PCR standard, positive reference, negative reference, and primer, all reagents are stored at-20 deg.C; the primers comprise three pairs of specific primers, namely primers PRoV-F and PRoV-R, primers PoSaV-F and PoSaV-R and primers PAStV-F and PAStV-R, which respectively have base sequences of sequence tables SEQ.ID.NO.1 to SEQ.ID.NO. 6.
The PCR standard comprises a PRoV standard, a PoSaV standard and a PAStV standard; the positive control contains positive plasmid mixture of three viruses including PRoV, PoSaV and PAStV (the concentration of the plasmid mixture is about 4.58X 10)8copies/. mu.L); the negative control is sterilized ultrapure water.
The PRoV standard, the PoSaV standard and the PAStV standard respectively have base sequences of sequence tables SEQ.ID.NO.7 to SEQ.ID.NO. 9.
In the primers, the upstream primers PRoV-F, PoSaV-F and PAStV-F of the three pairs of specific primers are packaged in the same tube, and the downstream primers PRoV-R, PoSaV-R and PAStV-R of the three pairs of specific primers are packaged in the same tube.
Aiming at the problems of the identification and detection of the porcine rotavirus, Sapovirus and porcine astrovirus, by applying a multiple RT-PCR technology, the inventor designs a primer group for the multiple RT-PCR detection of PRoV, PoSaV and PAStV respectively aiming at the gene VP6 of the PRoV, the gene of the PoSaV and the gene ORF1 of the PAStV, and the primer group comprises three pairs of specific primers, namely primers PRoV-F and PRoV-R, primers PoSaV-F and PoSaV-R, primers PAStV-F and PAStV-R, which respectively have base sequences from SEQ.ID.NO.1 to SEQ.ID.NO.6. Accordingly, the inventors have also developed a corresponding diagnostic kit comprising the following reagents: PCR standard substance, positive control substance, negative control substance and primer. By applying the invention, single or mixed infection viruses of PRoV, PoSaV and PAStV can be simultaneously detected from a sample through one RT-PCR reaction, and the invention has the characteristics of high specificity, good sensitivity, short time consumption, low cost and the like. In conclusion, the invention provides a quick, simple and convenient tool for detecting the mixed infection of the swine herd of PRoV, PoSaV and PAStV, lays a solid foundation for clinical quick identification and detection and laboratory epidemiological investigation, is beneficial to the pig industry to establish a treatment scheme in time, and reduces the death rate and economic loss of the swine herd.
Drawings
FIG. 1 is a diagram showing the results of specific detection of primers for simultaneous detection of porcine rotavirus, Saporo virus and porcine astrovirus in accordance with the present invention, in which: the lanes are from left to right: m is DL 1000 DNA Marker; 1: PRoV; 2, PoSaV; 3, PAStV; 4: PRoV, PoSaV and PAStV; and 5, negative control.
FIG. 2 is a diagram showing the results of specific detection of primers corresponding to the present invention for simultaneous detection of porcine rotavirus, Saporo virus and porcine astrovirus, in which: the lanes are from left to right: m is DL 1000 DNA Marker; 1: PRoV and PoSaV; 2: PRoV and PAStV; PoSaV and PAStV; 4: a non-target template; 5: and (5) negative control.
FIG. 3 is a graph showing the results of sensitivity detection of the present invention for simultaneous detection of porcine rotavirus, Saporo virus and porcine astrovirus, in which: the lanes are from left to right: m is DL 1000 DNA Marker; 1: 4.58X 109；2:4.58×108；3:4.58×107；4:4.58×106；5:4.58×105；6:4.58×104；7:4.58×103；8:4.58×102copies/. mu.L positive plasmid mixture; 9 negative control.
FIG. 4 is a graph showing the results of sensitivity tests for detecting porcine rotavirus alone in accordance with the present invention, in which: the lanes are from left to right: m is DL 1000 DNA Marker; 1: 1.38X 109；2:1.38×108；3:1.38×107；4:1.38×106；5:1.38×105；6:1.38×104；7:1.38×103；8:1.38×102；9:1.38×101；10:1.38×100copies/. mu.L positive plasmid; 11, negative control.
FIG. 5 is a graph showing the results of susceptibility assays for detecting Saporoviruses alone according to the present invention, in which: the lanes are from left to right: m is DL 1000 DNA Marker; 1: 8.33X 109；2:8.33×108；3:8.33×107；4:8.33×106；5:8.33×105；6:8.33×104；7:8.33×103；8:8.33×102；7:8.33×101；7:8.33×100copies/. mu.L positive plasmid; 11, negative control.
FIG. 6 is a graph showing the results of susceptibility testing of the porcine astrovirus alone of the invention, wherein: the lanes are from left to right: m is DL 1000 DNA Marker; 1: 4.40X 109；2:4.40×108；3:4.40×107；4:4.40×106；5:4.40×105；6:4.40×104；7:4.40×103；8:4.40×102；9:4.40×101；10:4.40×100copies/. mu.L positive plasmid; 11, negative control.
Detailed Description
First, primer design
Aiming at three viruses of porcine rotavirus, sapovirus and porcine astrovirus, the inventors designed primers shown in Table 1, and synthesized the primers by Shanghai Jieli biotechnology Limited, with the synthesis amount of 1OD per tube of primers.
TABLE 1 multiplex PCR primers designed according to the present invention
In the article of the Yang-Wen teacher, namely the establishment and preliminary application of the multiplex RT-PCR detection method for the porcine circovirus A, the porcine circovirus C and the porcine astrovirus, VP4, VP7 and VP6 genes of the porcine circovirus A (GARV) and the porcine circovirus C (GCRV) and ORF1a, ORF1b and ORF2 genes of PAStV are selected for conservative comparison, a reference gene region used for detecting the porcine circovirus A is 251bp-479bp of VP6, a reference gene region used for detecting the porcine circovirus C is 1bp-166bp, and a reference gene region used for detecting the porcine astrovirus is 7bp-416bp of ORF 2.
Different from the reference gene region selected by the Yangyu teacher, the detection primer of the porcine rotavirus (PRoV) is 160bp correspondingly and is positioned at target genes VP6, 359bp-518 bp; the detection primer of the Saporo virus (PoSaV) is 299bp in length, and the target gene is RdRp and is positioned in 4363bp-4661bp in full length; the corresponding length of the detection primer of the porcine astrovirus (PAStV) is 466bp, and the detection primer is located in the target gene ORF1, 3332bp-3797 bp. Moreover, researches show that the primer group has high specificity and stability, and can simultaneously and effectively amplify target fragments of three viruses in the same PCR reaction system, so that the detection process is simpler, more convenient, and saves time and labor.
Second, kit assembly and use
1. Kit assembly
The multiple RT-PCR kit of PRoV, PoSaV and PAStV comprises the following reagents: the kit comprises a PCR standard substance, a positive control substance, a negative control substance, primers and a kit body, wherein corresponding container holes are formed in the kit body and used for storing reagents, and all the reagents are stored at-20 ℃. Wherein the content of the first and second substances,
the PCR standard comprises a PRoV standard, a PoSaV standard and a PAStV standard; the positive control contains positive plasmid mixture of three viruses including PRoV, PoSaV and PAStV (the concentration of the plasmid mixture is about 4.58X 10)8copies/. mu.L); the negative control is sterilized ultrapure water.
The primers comprise three pairs of specific primers, namely primers PRoV-F and PRoV-R, primers PoSaV-F and PoSaV-R and primers PAStV-F and PAStV-R, which respectively have base sequences of sequence tables SEQ.ID.NO.1 to SEQ.ID.NO. 6. The upstream primers PRoV-F, PoSaV-F and PAStV-F of the three pairs of specific primers are packed in the same tube, and the downstream primers PRoV-R, PoSaV-R and PAStV-R of the three pairs of specific primers are packed in the same tube.
2. Use of the kit
Setting negative control and positive control according to specific conditions in each detection; diluting the standard product with sterile ultrapure water to 10%9～102copies/μL。
RNA virus sample extraction: viral nucleic acids were extracted from cell lines, fresh or frozen samples using the viral RNA extraction kit rnaasso Plus according to the product instructions.
Reverse transcription reaction: 8 muL of total RNA template prepared in the previous step, 2.5 muL of 5 Xreversatase M-MLV Buffer, 1 muL of dNTP mix, 0.5 muL of downstream primer, 0.25 muL of reversatrastase M-MLV, 0.25 muL of Ribolock RNase Inhibitor and 12.5 muL of total volume are sequentially added into an autoclaved 0.2mL PCR tube, the reaction condition is 42 ℃ for reaction for 1h, and after the reverse transcription reaction is finished, the obtained reaction solution is the cDNA template.
Detection of nucleic acids: mu.L of the prepared cDNA template was subjected to multiplex PCR reaction using 12.5. mu.L of 2 XTAQ Plus Master Mix II (Dye Plus), 0.5. mu.L of the forward primer Mix, and 0.5. mu. L, ddH of the reverse primer Mix2O8.5 mu L, cDNA 3 mu L, and the total volume is 25 mu L; the reaction program is pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extending for 5min at 72 ℃; the PCR product was detected by 2% agarose gel electrophoresis and analyzed after imaging by a gel imager.
And (3) reporting a result: and under the condition that the negative control and the positive control are established, identifying whether the porcine rotavirus, the Sa-SaV and the porcine astrovirus exist or not according to the existence of PRoV, PoSaV and PAStV amplified gene segments.
Third, specific experiment
According to the multiplex PCR reaction system, 12.5. mu.L of 2 XTAQ Plus Master Mix II (Dye Plus), 0.5. mu.L of upstream primer mixture, and 0.5. mu. L, ddH of downstream primer mixture2O8.5 mu L, respectively adding 3 mu L of positive sample cDNA template 1: PRoV; 2, PoSaV; 3, PAStV; 4: PRoV and PoSaV; 5, PRoV and PAStV; PoSaV and PAStV; PRoV, PoSaV and PAStV; 8, non-targeting template; 9: ultrapure water negative control. The reaction is carried out in a life ProFlex PCR amplification instrument, and the reaction program is pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extension at 72 ℃ for 5 min. And (3) taking 10 mu L of PCR product, spotting the product in a hole of a 2% agar gel electrophoresis plate, carrying out electrophoresis at 140V for about 20min, and carrying out ultraviolet imaging on the product by a gel imager and then photographing for judgment.
The results are shown in FIG. 1 and FIG. 2, and the sizes of the corresponding PCR products are respectively 160bp, 299bp and 466bp completely consistent with those of PRoV, PoSaV and PAStV in Table 1, which shows that the multiplex RT-PCR detection kit for simultaneously detecting porcine rotavirus, SapoV and porcine astrovirus constructed by the invention has good specificity.
Fourth, sensitivity test
Multiplex PCR sensitivity detection reaction system: 2 XTaq Plus Master Mix II (Dye Plus) 12.5. mu.L, forward primer Mix 0.5. mu.L, reverse primer Mix 0.5. mu.L, ddH2O8.5. mu.L, 3. mu.L of 10-fold serial dilutions of a mixture of PRoV, PoSaV and PAStV positive plasmids (4.58X 10, respectively)9～4.58×102copies/. mu.L) template. The reaction is carried out in a life ProFlex PCR amplification instrument, and the reaction program is as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extension at 72 ℃ for 5 min. 10 μ L of PCR product was taken and detected by electrophoresis on 2% agar gel.
Conventional single-plex PCR sensitivity detection reaction system: 2 XTaq Plus Master Mix II (Dye Plus) 12.5. mu.L, forward primer Mix 0.5. mu.L, reverse primer Mix 0.5. mu.L, ddH2O8.5. mu.L, 3. mu.L of 10-fold serial dilutions of PRoV, PoSaV and PAStV positive plasmid standards (10. mu.L) were added9～102copies/. mu.L) template. The reaction is carried out in a life ProFlex PCR amplification instrument, and the reaction program is as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extension at 72 ℃ for 5 min. 10 μ L of PCR product was taken and detected by electrophoresis on 2% agar gel.
ResultsAs shown in FIG. 3, the lower limit of the detection of the porcine rotavirus, Sapovirus and porcine astrovirus by the multiplex PCR of the present invention is 4.58X 104copies/. mu.L, has better sensitivity to three viruses. The lower limit of the detection of the conventional single PCR sensitive porcine rotavirus is 1.38 multiplied by 103The lower limit of the detection of copies/mu L and Saporo virus is 8.33X 102copies/. mu.L, the lower limit of detection of porcine astrovirus is 4.40X 102copies/μL。
Fifth, application example
The multiplex RT-PCR kit is applied to detect 18 clinical pig disease materials, and detects 1 part of wheel positive, 2 parts of Sappora positive and 2 parts of star positive.
Sequence listing
<110> Guangxi university
<120> multiplex RT-PCR kit for PRoV, PoSaV and PAStV
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atctgaagcg ttgagaaagt 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
agtaaatgaa gctgagtatg 20
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gtgacgagcg ccagtgttga c 21
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aggccgtaca cacaatcatc c 21
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
aacaacgaac ccgcacatct 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ggaggaacat acggtgtcgt 20
<210> 7
<211> 160
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atctgaagcg ttgagaaagt tgtcgggtat taagtttagg agaattaatt ttgacaattc 60
atctgattac attgaaaatt ggaatttgca aaatagacga cagcgtactg gattcgtgtt 120
ccacaaacca aatatacttc catactcagc ttcatttact 160
<210> 8
<211> 299
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gtgacgagcg ccagtgttga catcttggcc tcttacgctg aggacacgcc actcacttca 60
gcagctatag ccacactgtg ctcaccggcc gtcggccggc ttgatgacat tggtttgact 120
gtcacaactg ggctcccctc tggcatgcca ttcaccagtg tcataaactc tgtcaaccat 180
atgatatact ttgccatggc cgtgcttgaa gcttatgagg agttcaaggt gccctacatg 240
ggaaacatct ttgacaatga gactgtttac acgtatgggg atgattgtgt gtacggcct 299
<210> 9
<211> 466
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
aacaacgaac ccgcacatct gtgggccagt gtgggtggac gccaatgcaa ggtgggttta 60
aagccataat ggagaggctt gtttcaaaag gtaatagtta ctttgtagaa atggactgga 120
ctcgttacgg cggaaccatt ccaacagaac ttttcaggca cattaaagaa ctacgatgga 180
agctgatcaa caaagaacag cgtgagaaat acgcagaagt ccacaagtgg tatgttgaca 240
acctattaaa taggtatgtc ctcctgccgt ctggggaagt aaccctgcag acacggggca 300
atccatctgg acaattctca actaccatgg acaataacat gatcaacttc tggctccagg 360
catttgagtt tgcttggatt aatggcccaa acaaggaact ctggaagagc tatgacacca 420
ttgtatacgg tgatgatcgt ttaaacacga caccgtatgt tcctcc 466
Claims (8)
- The primer group for detecting the multiple RT-PCR of the PRoV, the PoSaV and the PAStV is characterized by comprising three pairs of specific primers, namely primers PRoV-F and PRoV-R, primers PoSaV-F and PoSaV-R and primers PAStV-F and PAStV-R, which have base sequences of sequence tables SEQ ID No.1 to SEQ ID No.6 respectively.
- 2. The use of the primer set for multiplex RT-PCR amplification of PRoV, PoSaV and PAStV according to claim 1.
- 3. Use according to claim 2, characterized in that: in the PCR amplification, PRoV-F and PRoV-R are designed against 359-19 th site of VP6 of the gene of PRoV, primers PoSaV-F and PoSaV-R are designed against 4363-4661 th site of RdRp of PoSaV, and primers PAStV-F and PAStV-R are designed against 3332-3797 th site of ORF1 of the gene of PAStV.
- 4. Use according to claim 3, characterized in that: the annealing temperature of the PCR amplification is 50 ℃.
- 5. A multiplex RT-PCR kit of PRoV, PoSaV and PAStV, characterized by comprising the following reagents: PCR standard substance, positive reference substance, negative reference substance and primer; the primers comprise three pairs of specific primers, namely primers PRoV-F and PRoV-R, primers PoSaV-F and PoSaV-R and primers PAStV-F and PAStV-R, and the primers respectively have base sequences of sequence tables SEQ ID No.1 to SEQ ID No. 6.
- 6. The multiplex RT-PCR kit of PRoV, PoSaV and PAstV according to claim 5, wherein: the PCR standard comprises a PRoV standard, a PoSaV standard and a PAStV standard; the positive control substance contains a positive plasmid mixed product of three viruses of PRoV, PoSaV and PAStV; the negative control product is ultrapure water after sterilization.
- 7. The multiplex RT-PCR kit of PRoV, PoSaV and PAstV according to claim 6, wherein: the PRoV standard, the PoSaV standard and the PAStV standard respectively have base sequences of sequence tables from SEQ.ID.NO.7 to SEQ.ID.NO. 9.
- 8. The multiplex RT-PCR kit of PRoV, PoSaV and PAstV according to claim 5, wherein: in the primers, the upstream primers PRoV-F, PoSaV-F and PAStV-F of the three pairs of specific primers are packaged in the same tube, and the downstream primers PRoV-R, PoSaV-R and PAStV-R of the three pairs of specific primers are packaged in the same tube.
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| CN113046489A (en) * | 2021-03-03 | 2021-06-29 | 广西大学 | Multiple RT-PCR primer group for detecting porcine astrovirus, kit and application thereof |
| CN113186359A (en) * | 2021-06-21 | 2021-07-30 | 浙江理工大学 | Multiplex PCR rapid diagnosis kit for detecting and parting pig astrovirus |
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Application publication date: 20191220 |