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Establishment of a Direct PCR Assay for Simultaneous Differential Diagnosis of African Swine Fever and Classical Swine Fever Using Crude Tissue Samples
African swine fever (ASF) and classical swine fever (CSF) are contagious swine diseases that are clinically indistinguishable from each other; hence, reliable test methods for accurate diagnosis and differentiation are highly demanded. By employing a buffer system suitable for crude extraction of nu...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8948687/ https://www.ncbi.nlm.nih.gov/pubmed/35336904 http://dx.doi.org/10.3390/v14030498 |
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author | Nishi, Tatsuya Okadera, Kota Fukai, Katsuhiko Yoshizaki, Miwa Nakasuji, Ai Yoneyama, Syuji Kokuho, Takehiro |
author_facet | Nishi, Tatsuya Okadera, Kota Fukai, Katsuhiko Yoshizaki, Miwa Nakasuji, Ai Yoneyama, Syuji Kokuho, Takehiro |
author_sort | Nishi, Tatsuya |
collection | PubMed |
description | African swine fever (ASF) and classical swine fever (CSF) are contagious swine diseases that are clinically indistinguishable from each other; hence, reliable test methods for accurate diagnosis and differentiation are highly demanded. By employing a buffer system suitable for crude extraction of nucleic acids together with an impurity-tolerant enzyme, we established a multiplex assay of real-time reverse-transcription polymerase chain reaction (rRT-PCR) for simultaneous detection of ASF virus (ASFV), CSF virus (CSFV) and swine internal control derived genes in a sample without the need for prior purification of viral nucleic acids. We applied this method to test serum and tissue samples of infected pigs and wild boars and compared the statistical sensitivities and specificities with those of standard molecular diagnostic methods. When a serum was used as a test material, the newly established assay showed 94.4% sensitivity for both and 97.9 and 91.9% specificity for ASFV and CSFV detection, respectively. In contrast, the results were 100% identical with those obtained by the standard methods when a crude tissue homogenate was used as a test material. The present data indicate that this new assay offers a practical, quick, and reliable technique for differential diagnosis of ASF and CSF where geographical occurrences are increasingly overlapping. |
format | Online Article Text |
id | pubmed-8948687 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-89486872022-03-26 Establishment of a Direct PCR Assay for Simultaneous Differential Diagnosis of African Swine Fever and Classical Swine Fever Using Crude Tissue Samples Nishi, Tatsuya Okadera, Kota Fukai, Katsuhiko Yoshizaki, Miwa Nakasuji, Ai Yoneyama, Syuji Kokuho, Takehiro Viruses Article African swine fever (ASF) and classical swine fever (CSF) are contagious swine diseases that are clinically indistinguishable from each other; hence, reliable test methods for accurate diagnosis and differentiation are highly demanded. By employing a buffer system suitable for crude extraction of nucleic acids together with an impurity-tolerant enzyme, we established a multiplex assay of real-time reverse-transcription polymerase chain reaction (rRT-PCR) for simultaneous detection of ASF virus (ASFV), CSF virus (CSFV) and swine internal control derived genes in a sample without the need for prior purification of viral nucleic acids. We applied this method to test serum and tissue samples of infected pigs and wild boars and compared the statistical sensitivities and specificities with those of standard molecular diagnostic methods. When a serum was used as a test material, the newly established assay showed 94.4% sensitivity for both and 97.9 and 91.9% specificity for ASFV and CSFV detection, respectively. In contrast, the results were 100% identical with those obtained by the standard methods when a crude tissue homogenate was used as a test material. The present data indicate that this new assay offers a practical, quick, and reliable technique for differential diagnosis of ASF and CSF where geographical occurrences are increasingly overlapping. MDPI 2022-02-28 /pmc/articles/PMC8948687/ /pubmed/35336904 http://dx.doi.org/10.3390/v14030498 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Nishi, Tatsuya Okadera, Kota Fukai, Katsuhiko Yoshizaki, Miwa Nakasuji, Ai Yoneyama, Syuji Kokuho, Takehiro Establishment of a Direct PCR Assay for Simultaneous Differential Diagnosis of African Swine Fever and Classical Swine Fever Using Crude Tissue Samples |
title | Establishment of a Direct PCR Assay for Simultaneous Differential Diagnosis of African Swine Fever and Classical Swine Fever Using Crude Tissue Samples |
title_full | Establishment of a Direct PCR Assay for Simultaneous Differential Diagnosis of African Swine Fever and Classical Swine Fever Using Crude Tissue Samples |
title_fullStr | Establishment of a Direct PCR Assay for Simultaneous Differential Diagnosis of African Swine Fever and Classical Swine Fever Using Crude Tissue Samples |
title_full_unstemmed | Establishment of a Direct PCR Assay for Simultaneous Differential Diagnosis of African Swine Fever and Classical Swine Fever Using Crude Tissue Samples |
title_short | Establishment of a Direct PCR Assay for Simultaneous Differential Diagnosis of African Swine Fever and Classical Swine Fever Using Crude Tissue Samples |
title_sort | establishment of a direct pcr assay for simultaneous differential diagnosis of african swine fever and classical swine fever using crude tissue samples |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8948687/ https://www.ncbi.nlm.nih.gov/pubmed/35336904 http://dx.doi.org/10.3390/v14030498 |
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