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Novel swine orthopneumovirus (SOV) infections have been identified in pigs in the USA and some European countries but not in Asian countries, including South Korea, to date. The current study reports the first SOV infections in four domestic pig farms located in four provinces across South Korea. The detection rate of SOV in oral fluid samples using qRT-PCR was 4.4% (14/389), indicating the presence of the virus in pigs at commercial farms in Korea. Two complete genome sequences and one glycoprotein (G) gene sequence were obtained from SOV-positive samples. The complete genome analysis of KSOV-2201 and KSOV-2202 strains showed 98.2 and 95.4% homologies with a previously reported SOV, and the phylogenetic tree exhibited a high correlation with a previously reported SOV strain from the US and a canine pneumovirus (CPnV) strain from China. Based on the genetic analysis of the viral G gene, the murine pneumonia virus (MPV)-like orthopneumoviruses (MLOVs) were divided into two genogroups (G1 and G2). Seventeen CPnVs and two feline pneumoviruses were grouped into G1, while the Korean SOV strains identified in this study were grouped into G2 along with one SOV and two CPnVs. These results will contribute to expanding our understanding of the geographical distribution and genetic characteristics of the novel SOV in the global pig population.
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Pneumovirus , Doenças dos Suínos , Camundongos , Suínos , Animais , Gatos , Cães , Sus scrofa , Vírus Sinciciais Respiratórios , Fazendas , Filogenia , Doenças dos Suínos/epidemiologia , República da Coreia/epidemiologiaRESUMO
For rapid and reliable detection of porcine epidemic diarrhea virus (PEDV) from pig clinical samples, a multiplex, real-time, reverse transcription loop-mediated isothermal amplification (mqRT-LAMP) was developed using two sets of primers and assimilating probes specific to the PEDV N gene and the Sus scrofa ß-actin gene, which was used as an endogenous internal positive control (EIPC) to avoid false-negative results. The assay specifically amplified both target genes of PEDV and EIPC in a single reaction without any interference but did not amplify other porcine viral nucleic acids. The limit of detection was 10 copies/µL, 100-fold lower than that of a reverse transcription-polymerase chain reaction (RT-PCR) and equivalent to that of quantitative/real-time RT-PCR (qRT-PCR). This assay has high repeatability and reproducibility with coefficients of variation < 4.0%. The positive signal of the mqRT-LAMP assay was generated within 25 min, demonstrating advantages in rapid detection of PEDV over RT-PCR or qRT-PCR assay, which require at least 2 h turnaround times. In clinical evaluation, the detection rate of PEDV by mqRT-LAMP assay (77.3%) was higher than that of RT-PCR assay (69.7%), and comparable to qRT-PCR (76.8%) with almost 100% concordance (kappa value 0.98). The developed mqRT-LAMP assay can serve as an advanced alternative method for PEDV diagnosis because it has high sensitivity and specificity, rapidity, and reliability even in resource-limited laboratories.
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Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Animais , Suínos , Vírus da Diarreia Epidêmica Suína/genética , Transcrição Reversa , Reprodutibilidade dos Testes , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/veterinária , Sensibilidade e Especificidade , Doenças dos Suínos/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodosRESUMO
Feline panleukopenia virus (FPV), a member of the species Protoparvovirus carnivoran1, is one of the most fatal pathogens of domestic and wild carnivores. The virus endemically infects domestic carnivores worldwide and its cross-species transmission threatens endangered wild carnivores, including Siberian tigers. In this study, a fatal FPV infection in endangered Siberian tigers was investigated to trace the origin of the virus and elucidate the reason behind FPV's infection of the vaccinated tigers. Our genetic characterization and phylogenetic analysis revealed that the virus detected in the infected tigers, designated as the KTPV-2305 strain, was closely related to FPV strains circulating in Korean cats, suggesting that it might have been transmitted from stray cats wandering around the zoo. Compared with the prototype FPV reference strains, the KTPV-2305 strain carried three distinct amino acid (aa) mutations in the VP2 protein sequence (I101T, I232V, and L562V) in this study. These three mutations are commonly found in most global FPV strains, including Korean strains, indicating that these mutations are common evolutionary characteristics of currently circulating global FPVs. The reason why the vaccinated tigers were infected with FPV was most likely the insufficient protective immunity of the affected tigress or vaccine failure triggered by the interference of maternal-derived antibodies in the affected tiger cubs. These findings suggest that improved vaccination guidelines are urgently needed to save the lives of wild carnivores from this fatal virus.
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Despite its many advantages, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay has yet to be developed for canine parainfluenza virus 5 (CPIV5). In this study, a visual RT-LAMP (vRT-LAMP) assay was developed for the rapid detection of CPIV5 in clinical samples. At a constant reaction temperature of 62 °C, the assay was completed within 40 min, and the results could be directly detected with the naked eye using a hydroxynaphthol blue (HNB) metal indicator without any additional detection apparatuses. The assay specifically amplified CPIV5 RNA with a limit of detection of 10 RNA copies/reaction, which was 10-fold more sensitive than the previously reported conventional reverse-transcription polymerase chain reaction (cRT-PCR) assay and was comparable to the previously reported real-time RT-PCR (qRT-PCR) assay. In a clinical evaluation using 267 nasopharyngeal swab samples collected from hospitalized dogs with respiratory symptoms, the CPIV5 detection rate using the vRT-LAMP assay was 5.24% (14/267), which was higher than that of the cRT-PCR assay (4.49%, 12/267) and consistent with that of the qRT-PCR assay, demonstrating 100% concordance with a kappa coefficient value (95% confidence interval) of 1 (1.00-1.00). The discrepancies in the results of the assays were confirmed to be attributed to the low sensitivity of the cRT-PCR assay. Owing to the advantages of a high specificity, rapidity, and simplicity, the developed vRT-LAMP assay using an HNB metal indicator will be a valuable diagnostic tool for the detection of CPIV5 in canine clinical samples, even in resource-limited laboratories.
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Porcine deltacoronavirus (PDCoV) is an emerging coronavirus that causes diarrhea in nursing piglets. Since its first outbreak in the United States in 2014, this novel porcine coronavirus has been detected worldwide, including in Korea. However, no PDCoV case has been reported since the last report in 2016 in Korea. In June 2022, the Korean PDCoV strain KPDCoV-2201 was detected on a farm where sows and piglets had black tarry and watery diarrhea, respectively. We isolated the KPDCoV-2201 strain from the intestinal samples of piglets and sequenced the viral genome. Genetically, the full-length genome and spike gene of KPDCoV-2201 shared 96.9-99.2% and 95.8-98.8% nucleotide identity with other global PDCoV strains, respectively. Phylogenetic analysis suggested that KPDCoV-2201 belongs to G1b. Notably, the molecular evolutionary analysis indicated that KPDCoV-2201 evolved from a clade different from that of previously reported Korean PDCoV strains and is closely related to the emergent Peruvian and Taiwanese PDCoV strains. Furthermore, KPDCoV-2201 had one unique and two Taiwanese strain-like amino acid substitutions in the receptor-binding domain of the S1 region. Our findings suggest the possibility of transboundary transmission of the virus and expand our knowledge about the genetic diversity and evolution of PDCoV in Korea.
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A duplex real-time quantitative reverse transcription-polymerase chain reaction (dqRT-PCR) assay was successfully developed to simultaneously detect canine parainfluenza virus 5 (CPIV5) and a canine endogenous internal positive control (EIPC) in canine clinical samples. Two sets of primers and probes for the CPIV5 L and canine 16S rRNA genes were included in the dqRT-PCR assay to detect CPIV and monitor invalid results throughout the qRT-PCR process. The developed dqRT-PCR assay specifically detected CPIV5 but no other canine pathogens. Furthermore, 16S rRNA was stably amplified by dqRT-PCR assay in all samples containing canine cellular materials. The assay's sensitivity was determined as below ten RNA copies per reaction, with CPIV5 L gene standard RNA and 1 TCID50/mL with the CPIV5 D008 vaccine strain, which was 10-fold higher than that of the previous HN gene-specific qRT-PCR (HN-qRT-PCR) assays and was equivalent to that of the previous N gene-specific qRT-PCR (N-qRT-PCR) assays, respectively. Moreover, the Ct values of the CPIV5-positive samples obtained using the dqRT-PCR assay were lower than those obtained using the previous HN- and N-qRT-PCR assays, indicating that the diagnostic performance of the dqRT-PCR assay was superior to those of previous HN- and N-qRT-PCR assays. The calculated Cohen's kappa coefficient values (95% confidence interval) between dqRT-PCR and the HN- or N-specific qRT-PCR assays were 0.97 (0.90-1.03) or 1.00 (1.00-1.00), respectively. In conclusion, the newly developed dqRT-PCR assay with high sensitivity, specificity, and reliability will be a promising diagnostic tool for the detection of CPIV5 in clinical samples and useful for etiological and epidemiological studies of CPIV5 infection in dogs.
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Two species of porcine parainfluenza viruses (PPIV), PPIV1 and PPIV5, are globally distributed in pig herds and associated with porcine respiratory diseases, and a diagnostic tool for the simultaneous detection of the two viruses is required. In this study, a TaqMan probe-based duplex real-time reverse transcription polymerase chain reaction (dqRT-PCR) assay was first developed for the differential detection of PPIV1 and PPIV5 nucleocapsid protein (NP) genes in porcine clinical samples. The dqRT-PCR assay was highly sensitive, its limit of detection was approximately 10 RNA copies/reaction, it specifically amplified the targeted NP genes of PPIV1 and PPIV5 without cross-reacting with other porcine pathogens, and their clinical detection rates were 15.2% and 0.7%, respectively. The results from 441 clinical samples taken from 278 Korean domestic pig farms showed that the prevalence of PPIV1 and PPIV5 was 11.2% and 1.1%, respectively, and co-infection of both viruses was confirmed in a farm, suggesting that PPIV1 and PPIV5 are co-circulating in current Korean pig herds. Phylogenetic analysis based on the partial NP genes suggested that genetically diverse PPIV1 strains are circulating in Korean pig herds. The developed dqRT-PCR assay was found to be an accurate, reliable, and quantitative detection tool for PPIV1 and PPIV5 RNA in clinical pig samples and will be useful for etiological and epidemiological studies and the control of viral infections in the field.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been frequently reported in companion dogs and cats worldwide during the ongoing coronavirus disease. However, RT-qPCR methods developed for humans have been used for the diagnosis of SARS-CoV-2 infections in suspected companion dogs and cats owing to the lack of the companion animal-tailored methods. Therefore, we developed a multiplex RT-qPCR (mRT-qPCR) using newly designed primers and probes targeting RdRp and N genes of all currently circulating SARS-CoV-2 variants as well as the canine or feline 16S rRNA gene as an endogenous internal positive control (EIPC) for reliable diagnosis of SARS-CoV-2 infection from suspected dogs and cats. The developed mRT-qPCR assay specifically detected the target genes of SARS-CoV-2 but no other canine or feline pathogens. Furthermore, canine and feline EIPCs were stably amplified by mRT-qPCR in samples containing canine- or feline-origin cellular materials. This assay has high repeatability and reproducibility, with an optimal limit of detection (<10 RNA copies per reaction) and coefficients of variation (<1.0%). The detection rate of SARS-CoV-2 of the developed mRT-qPCR was 6.6% for canine and feline nasopharyngeal samples, which was consistent with that of a commercial mRT-qPCR kit for humans. Collectively, the newly developed mRT-qPCR with canine and feline EIPC can efficiently diagnose and evaluate the viral load in field specimens and will be a valuable tool for etiological diagnosis, epidemiological study, and controlling SARS-CoV-2 infections in canine and feline populations.
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Porcine respirovirus 1 (PRV1) is a recently emerging porcine respiratory virus that belongs to the genus Respirovirus of the Paramyxoviridae family. Since its first detection in Hong Kong, China in 2009, PRV1 has been subsequently identified in several American and European countries, suggesting that the emerging virus may have been globally distributed. However, in Asia, the virus has been reported only in China. Here, we report that PRV1 was first detected in pigs from 16 farms located in seven provinces across Korea, with a prevalence of 71.4% based on the tested oral fluid samples, suggesting that the virus is already widespread in Korean pig herds. For further genetic characterization of the Korean PRV1 strains, a complete genome and two F gene sequences were obtained from PRV1-positive samples collected from three different pig farms. Phylogenetic analysis based on the complete genome and F gene sequences showed that all three Korean PRV1 strains were grouped into European lineage 1 and were closely related to strains from Hong Kong (China), Germany and Poland. We could not obtain evidence for the origin of Korean PRV1 because of the limited availability of PRV1 sequences. In conclusion, PRV1 was first identified in Korean pig herds and genetically characterized in the present study. These results contribute to a better understanding of the global geographical distribution and genetic characteristics of PRV1.
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Doenças dos Suínos , Animais , Suínos , Filogenia , Respirovirus/genética , China/epidemiologia , República da Coreia/epidemiologiaRESUMO
Human infection by avian-origin subtype H10 influenza viruses has raised concerns about the pandemic potential of these microbes. H10 subtype low pathogenic avian influenza viruses (LPAIVs) have been isolated from wild birds and poultry worldwide. Here, we isolated 36 H10 LPAIVs from wild bird habitats (a mean annual rate of 3.8% of all avian influenza virus isolations) from January 2010 to April 2019 through a nationwide active surveillance program for avian influenza viruses (AIVs). Phylogenetic analysis revealed that the haemagglutinin (HA) gene of H10 isolates formed eight distinct genetic subgroups (HA-A-H). Unlike other Eurasian-origin subgroups, the HA-H subgroup belonged to the North American lineage. Gene-constellation analysis revealed that 24 H10 LPAIVs constituted ≥18 distinct genotypes, representing high levels of genetic diversity. An intravenous pathogenicity index (IVPI) experiment showed that the pathogenicity of representative strains of the HA-B, E and G subgroups possessing an IVPI score >1.2 was associated with replication capacity in the chicken kidney in the absence of trypsin. Intranasal inoculation experiments showed that a representative strain of the HA-D subgroup replicated and transmitted in chickens without clinical signs. Subclinical virus shedding in chickens may contribute to its silent spread among the poultry population. Moreover, six representative viruses replicated in the lungs of mice without prior adaptation and a representative strain of the HA-C subgroup caused 40% mortality, with severe body weight loss. These findings highlight the importance of intensive surveillance of wild bird habitats, poultry farms and the animal-human interface, along with appropriate risk assessment of isolated viruses.
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Vírus da Influenza A , Influenza Aviária , Doenças dos Roedores , Animais , Animais Selvagens , Galinhas , Hemaglutininas , Humanos , Influenza Aviária/epidemiologia , Camundongos , Filogenia , Aves Domésticas , Tripsina/genéticaRESUMO
A novel porcine circovirus 4 has been recently identified in China and Korea. A sensitive and specific diagnostic method is urgently required to detect the virus in field samples. We developed a loop-mediated isothermal amplification (LAMP) the assay for the visual detection of PCV4 and evaluated its sensitivity, specificity, and applicability in clinical samples. This assay's results can be directly visualized by the naked eye using hydroxynaphthol blue after incubation for 40 min at 64 °C. The assay specifically amplified PCV4 DNA and no other viral nucleic acids. The sensitivity of the assay was <50 DNA copies/reaction, which was 10 times more sensitive than conventional polymerase chain reaction (cPCR) and comparable to real-time PCR (qPCR). Clinical evaluation revealed that the PCV4 detection rate in individual pig samples and at the farm level was 39.3 % (57/145) and 45.7 % (32/70), respectively, which were higher than cPCR (46 samples, 24 farms) and qPCR (52 samples, 29 farms) results. Cumulatively, owing to the advantages of high sensitivity and specificity, direct visual monitoring of the results, no possibility for cross-contamination, and being a low-cost equipment, the developed LAMP assay will be a valuable tool for the detection of the novel PCV4 in clinical samples, even in resource-limited laboratories.
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Infecções por Circoviridae , Circovirus , Doenças dos Suínos , Animais , Infecções por Circoviridae/diagnóstico , Infecções por Circoviridae/veterinária , Circovirus/genética , Circovirus/isolamento & purificação , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase em Tempo Real , República da Coreia , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologiaRESUMO
A simple reverse transcription loop-mediated isothermal amplification combined with visual detection method (vRT-LAMP) assay was developed for rapid and specific detection of porcine epidemic diarrhea virus (PEDV) in this study, which overcomes the shortcomings of previously described RT-LAMP assays that require additional detection steps or pose a risk of cross-contamination. The assay results can be directly detected by the naked eye using hydroxynaphthol blue after incubating for 40 min at 62 °C. The assay specifically amplified PEDV RNA and no other viral nucleic acids. The limit of detection of the assay was less than 50 RNA copies per reaction, which was 100 times more sensitive than conventional reverse transcription polymerase chain reaction (RT-PCR) and comparable to real-time RT-PCR (RRT-PCR). In the clinical evaluation, the PEDV detection rate of vRT-LAMP was higher than that of RRT-PCR, showing 99 % concordance, with a kappa value (95 % confidence interval) of 0.97 (0.93-1.01). Considering the advantages of high sensitivity and specificity, simple and direct visual monitoring of the results, no possibility for cross-contamination, and being able to be used as low-cost equipment, the developed vRT-LAMP assay will be a valuable tool for detecting PEDV from clinical samples, even in resource-limited laboratories.
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Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Animais , Colorimetria , Técnicas de Diagnóstico Molecular , Naftalenossulfonatos , Técnicas de Amplificação de Ácido Nucleico/métodos , Vírus da Diarreia Epidêmica Suína/genética , Transcrição Reversa , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/diagnósticoRESUMO
Porcine circovirus type 3 (PCV3) is an emerging viral pathogen that has been identified in pigs with various clinical signs. For rapid and specific detection of PCV3, an advanced real-time loop-mediated isothermal amplification (rLAMP) assay that uses both assimilating probes and swarm primers were developed and evaluated in this study. The assay specifically amplified PCV3 DNA, but it did not amplify other porcine viral nucleic acids. The limit of detection of rLAMP with swarm primers was 50 PCV3 DNA copies/reaction, which was comparable to that of the real-time quantitative polymerase chain reaction (qPCR) and 10 times more sensitive than rLAMP without swarm primers. In an evaluation of clinical samples, the rLAMP assay was able to detect PCV3 DNA within 17.34 ± 4.45 min, which is more rapid than what has been previously reported for the standard qPCR assay (31.78 ± 4.60 min). Detection with rLAMP was largely in agreement with that of the qPCR with a kappa value (95% confidence interval) of 0.98 (0.95-1.00). Taken together, these results suggest that the rLAMP assay presented will be a valuable tool for rapid, specific and reliable diagnosis of PCV3 in clinical samples.
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Infecções por Circoviridae/veterinária , Circovirus/isolamento & purificação , Doenças Transmissíveis Emergentes/veterinária , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Doenças dos Suínos/diagnóstico , Animais , Infecções por Circoviridae/diagnóstico , Infecções por Circoviridae/virologia , Circovirus/genética , Doenças Transmissíveis Emergentes/diagnóstico , Doenças Transmissíveis Emergentes/virologia , Primers do DNA , Sondas de DNA , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/virologiaRESUMO
Rapid and specific detection of foot-and-mouth disease virus (FMDV) is a key factor for promoting prompt control of FMD outbreaks. In this study, a real-time reverse transcription loop-mediated isothermal amplification (RRT-LAMP) assay with high sensitivity, rapidity and reliability was developed using a targeted gene-specific assimilating probe for real-time detection of seven FMDV serotypes. Positive assay signals were generated within 15 min for the lowest concentration of a standard RNA sample at 62°C; this was substantially faster than that achieved by the OIE (World Organisation for Animal Health)-recommended real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. The new assay specifically amplified the 3D gene of all seven FMDV serotypes and did not amplify other viral nucleic acids. The detection limit of the assay was 102 copies/µl which is comparable to that achieved by qRT-PCR. Furthermore, using clinical samples, the results of the RRT-LAMP assay were largely in agreement with those from the qRT-PCR assay with a kappa value (95% confidence interval [CI]) of 0.94 (0.86-1.02). The established RRT-LAMP assay that features assimilating probes is an advanced molecular diagnostic tool that is easily applicable to a wide range of circumstances and has high potential for use as an on-site diagnostic assay for rapid, specific, and reliable detection of FMDVs in clinical samples.
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Vírus da Febre Aftosa/isolamento & purificação , Febre Aftosa/diagnóstico , Técnicas de Diagnóstico Molecular/veterinária , Técnicas de Amplificação de Ácido Nucleico/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Animais , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Swarm primer-applied loop-mediated isothermal amplification (sLAMP) assay was developed for the rapid and specific detection of the ORF V1 gene of beak and feather disease virus (BFDV). The amplification can be completed in 40â¯min at 62⯰C, and the results can be visually detected by the naked eye. The assay specifically amplified BFDV DNA and not amplified other viral nucleic acids. The limit of detection of the assay was 5â¯×â¯102 DNA copies/reaction, which was lower than that of the previously described LAMP (preLAMP) assay and comparable to that of a previously reported real time quantitative PCR (qPCR) assay. The detection rates of BFDV from psittacine clinical samples by the sLAMP, qPCR and preLAMP assays were 36.0 %, 36.0 % and 25.6 %, respectively, and the sLAMP results showed 100.0 % concordance with the qPCR results with a kappa value of 1.0. On the other hand, the preLAMP assay did not detect nine out of the 31 samples that were positive by sLAMP and qPCR assays, probably due to low sensitivity of the assay. These data suggest that the newly developed sLAMP assay will be a valuable tool for the rapid, sensitive, specific and reliable detection of BFDV in suspected psittacine birds, even in resource-limited laboratories.
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Doenças das Aves/diagnóstico , Infecções por Circoviridae/diagnóstico , Infecções por Circoviridae/veterinária , Circovirus/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Papagaios/virologia , Animais , Doenças das Aves/virologia , Circovirus/genética , Primers do DNA/genética , Sensibilidade e EspecificidadeRESUMO
A colorimetric loop-mediated isothermal amplification (LAMP) assay was developed for the rapid and specific detection of the T gene of Aves polyomavirus 1 (APyV), a causative agent of budgerigar fledgling disease (BFD) in psittacine birds. The amplification can be completed in 40 min at 60 °C, and the results can be visually detected by the naked eye using hydroxyl naphthol blue as a colorimetric indicator. The assay specifically amplified APyV DNA but not other viral and bacterial nucleic acids. The limit of detection of the assay was 5 × 102 DNA copies/reaction, which was comparable to those of previously reported conventional polymerase chain reaction assays. In the clinical evaluation, the LAMP results showed 100% concordance with those of the previously reported PCR assays with regard to specificity, sensitivity, and percentage of overall agreement, with a kappa value of 1.0. These results indicate that the developed LAMP assay will be a valuable tool for the rapid, sensitive and specific detection of APyV from BFD-suspected psittacine bird samples even in resource-limited laboratories.
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Doenças das Aves/diagnóstico , Colorimetria/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Papagaios/virologia , Infecções por Polyomavirus/veterinária , Polyomavirus/isolamento & purificação , Infecções Tumorais por Vírus/veterinária , Animais , Compostos Azo/química , Doenças das Aves/virologia , Primers do DNA/genética , Sensibilidade e Especificidade , TemperaturaRESUMO
Porcine parvovirus 7 (PPV7) was first detected in Korean pig farms in 2017. The detection rate of PPV7 DNA was 24.0% (30/125) in aborted pig fetuses and 74.9% (262/350) in finishing pigs, suggesting that PPV7 has circulated among Korean domestic pig farms. Phylogenetic analysis based on capsid protein amino acid sequences demonstrated that the nine isolated Korean strains (PPV-KA1-3 and PPV-KF1-6) were closely related to the previously reported USA and Chinese PPV7 strains. In addition, the Korean strains exhibit genetic diversity with both insertion and deletion mutations. This study contributes to the understanding of the molecular epidemiology of PPV7 in Korea.
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Infecções por Parvoviridae/veterinária , Parvovirus Suíno/genética , Doenças dos Suínos/virologia , Feto Abortado/virologia , Sequência de Aminoácidos , Animais , Proteínas do Capsídeo/genética , DNA Viral/genética , Fazendas , Infecções por Parvoviridae/epidemiologia , Infecções por Parvoviridae/virologia , Filogenia , República da Coreia/epidemiologia , Suínos/virologia , Doenças dos Suínos/epidemiologiaRESUMO
Rapid and accurate diagnosis of foot-and-mouth disease viruses (FMDV) is essential for the prompt control of FMD outbreaks. Reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR (qRT-PCR) are used for routine FMDV diagnosis as World Organisation for Animal Health-recommended diagnostic assays. However, these PCR-based assays require sophisticated equipment, specialized labour, and complicated procedures for the detection of amplified products, making them unsuitable for under-equipped laboratories in developing countries. In this study, to overcome these shortcomings, a simple, rapid, and cost-effective reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the sensitive and specific detection of serotype A FMDV circulating in the pool 1 region. The amplification could be completed in 40 min at 62°C, and the results could be visually detected by the naked eye without any additional detection systems. The assay specifically amplified the VP1 gene of the Sea-97 genotype of serotype A FMDV, but it did not amplify other viral nucleic acids. The limit of detection of the assay was 102 TCID50 /ml, which is 10 times more sensitive than RT-PCR and is comparable to the sensitivity of qRT-PCR. Evaluation of the assay using different FMDV strain serotypes showed 100% agreement with the results of RT-PCR. Surprisingly, the previously reported RT-LAMP assay did not detect all eight tested strains of serotype A FMDVs, due to multiple mismatches between primer and template sequences, demonstrating that it is not suitable for detecting serotype A FMDVs circulating in pool 1-region countries. Conversely, the newly developed RT-LAMP assay using improved primers can rapidly and accurately diagnose the genotype of Sea-97 strains of serotype A FMDVs from the pool 1 region. The established RT-LAMP assay in this study is a simple, rapid, specific, sensitive, and cost-effective tool for the detection of serotype A FMDV in the resource-limited pool 1-region countries.
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Vírus da Febre Aftosa/isolamento & purificação , Febre Aftosa/virologia , Técnicas de Amplificação de Ácido Nucleico/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Animais , Proteínas do Capsídeo/genética , Linhagem Celular , Primers do DNA , Surtos de Doenças/veterinária , Febre Aftosa/diagnóstico , Febre Aftosa/epidemiologia , Vírus da Febre Aftosa/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , SorogrupoRESUMO
A sensitive and specific swarm primer-based reverse transcription loop-mediated isothermal amplification (sRT-LAMP) assay for the detection of serotype O foot-and-mouth disease virus (FMDV) was developed and evaluated. The assay specifically amplified the VP3 gene of serotype O FMDV, but did not amplify the VP3 gene of other serotype FMDVs or any other viruses. The limit of detection of the assay was 102 TCID50/mL or 103 RNA copies/µL, which is 100 times lower than that of the RT-LAMP assay without swarm primers. The new assay is 10 times more sensitive than reverse transcription-polymerase chain reaction (RT-PCR) and is comparable to the sensitivity of real time RT-PCR (qRT-PCR). Evaluation of the assay using different serotypes of FMDV strains showed 100% agreement with the RT-PCR results. The previously reported serotype O FMDV-specific RT-LAMP assay did not detect 20 out of 22 strains of serotype O FMDVs, probably due to multiple mismatches between the primer and template sequences, showing that it is not suitable for detecting the serotype O FMDVs circulating in Pool 1 region countries, including Korea. In contrast, the developed sRT-LAMP assay with improved primers can rapidly and accurately diagnose serotype O FMDVs circulating in Pool 1 region countries and will be a useful alternative to RT-PCR and qRT-PCR.
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Proteínas do Capsídeo/genética , Vírus da Febre Aftosa/isolamento & purificação , Febre Aftosa/virologia , Técnicas de Amplificação de Ácido Nucleico/veterinária , Animais , Pareamento Incorreto de Bases , Primers do DNA , Febre Aftosa/diagnóstico , Vírus da Febre Aftosa/classificação , Vírus da Febre Aftosa/genética , Limite de Detecção , República da Coreia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Reversa , Sensibilidade e Especificidade , SorogrupoRESUMO
A loop-mediated isothermal amplification (LAMP) assay using hydroxynaphthol blue was developed for the rapid and visual detection of the capsid gene of porcine circovirus 3 (PCV3). The amplification could be completed in 40â¯min at 62°C, and the results could be visually detected by the naked eye. The assay specifically amplified PCV3 DNA and not other porcine viral nucleic acids. The limit of detection of the assay was 50 PCV3 DNA copies, which was comparable to that of the real-time polymerase chain reaction (qPCR) and lower than that of conventional PCR. In the clinical evaluation, the PCV3 detection rate of the LAMP assay was higher than that of PCR and agreed 100% with that of qPCR. These results indicate that the LAMP assay will be a valuable tool for the rapid, sensitive, and specific detection of PCV3 in clinical samples.