68fc9046835f50c97003971e27eafc87 PAT_a2025v14p473.pdf 766d6ad0e5d39c67d3687cb8b5c21a0b9e4fe600 PAT_a2025v14p473.pdf aa6d883301039caf39d2121d5bfbf884d71e7a790059299842424e41eacb8c75 PAT_a2025v14p473.pdf Title: Simultaneous Detection of Classical and African Swine Fever Viruses by Duplex Taqman Real-Time PCR Assay in Pigs Infected with Both Diseases Subject: The increasing spread of African swine fever (ASF) in recent years and the presence of classical swine fever (CSF) subclinical forms in endemic countries suggests that the possibility of coinfection with ASF virus (ASFV) and CSF virus (CSFV) in pigs cannot be ruled out in areas where both diseases are prevalent. Thus, rapid and reliable diagnosis through molecular testing is essential for the timely implementation of control measures to prevent the spread of these devastating swine diseases. Here, we have coupled two of the most validated PCR assays for the detection of CSFV and ASFV in a single reaction tube. The combination of the two tests for the detection of two target nucleic acids did not affect the analytical sensitivity, and the duplex RT-qPCR assay was comparable with the standard molecular techniques. The detection limits for CSFV RNA and ASFV DNA were 0.12 TCID50/reaction and 0.25 TCID50/reaction, respectively. The test showed high repeatability and reproducibility, the coefficient of variation was below 2%, and excellent performance was demonstrated in clinical samples. The duplex assay shows great potential to become a robust diagnostic tool for the rapid and reliable detection and differentiation of CSFV and ASFV in areas where both viruses may be circulating. Keywords: early diagnosis; differential detection; duplex qPCR; CSFV; ASFV; doubly infected pigs; surveillance Author: Liani Coronado, Adriana Muñoz-Aguilera, Miaomiao Wang, Iván Muñoz, Cristina Riquelme, Saray Heredia, Katarzyna Stępniewska, Carmina Gallardo and Llilianne Ganges Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Wed May 21 09:55:54 2025 CEST ModDate: Wed May 21 10:00:40 2025 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 12 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1772142 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- KDNOIK+URWPalladioL-Roma Type 1 Custom yes yes yes 10 0 QUPTWG+URWPalladioL-Bold Type 1 Custom yes yes yes 16 0 HUWBVZ+URWPalladioL-Ital Type 1 Custom yes yes yes 21 0 OVNEXW+EURM10 Type 1 Builtin yes yes yes 47 0 IXLVLQ+CMSY10 Type 1 Builtin yes yes yes 52 0 EDKNAH+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 72 0 EDKMKJ+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 78 0 EDKMJI+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 84 0 EDKNAK+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 90 0 EDKNAH+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 159 0 EDKMKJ+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 165 0 EDKMJI+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 171 0 EDKNAK+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 177 0 EDKNAH+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 188 0 EDKMKJ+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 194 0 EDKMJI+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 200 0 EDKNAK+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 206 0 EDKNAH+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 217 0 EDKMKJ+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 223 0 EDKMJI+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 229 0 EDKNAI+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 235 0 EDKNAK+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 238 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-06-12 02:41:13 CEST RepresentationInformation: PAT_a2025v14p473.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-06-11 11:24:32 CEST Size: 1772142 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 378 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Cell and Viruses Destination: subsection.2.1 Item: Title: Nucleic Acid Extraction and CSFV and ASFV Molecular Detection Destination: subsection.2.2 Item: Title: CSF-ASF Duplex RT-qPCR Analytical Sensitivity Destination: subsection.2.3 Item: Title: Analytic Specificity Determination of the Duplex RT-qPCR Assay Destination: subsection.2.4 Item: Title: Validation of the Duplex RT-qPCR Using Samples from Inter-Laboratory Comparison Test (ILCT) Panels Destination: subsection.2.5 Item: Title: Duplex RT-qPCR Validation in Samples from Experimentally Infected Pigs Destination: subsection.2.6 Item: Title: Reproducibility of the Duplex RT-qPCR Destination: subsection.2.7 Item: Title: Results Destination: section.3 Children: Item: Title: Analytical Sensitivity of the Duplex RT-qPCR Assay Destination: subsection.3.1 Item: Title: Analytical Specificity of the Duplex RT-qPCR Assay Destination: subsection.3.2 Item: Title: Validation of the Duplex RT-qPCR Assay Destination: subsection.3.3 Item: Title: Intra- and Inter-Assay Variability Destination: subsection.3.4 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.6 Info: Title: Simultaneous Detection of Classical and African Swine Fever Viruses by Duplex Taqman Real-Time PCR Assay in Pigs Infected with Both Diseases Author: がどちのど〠ぃはひはのちつは〬〠ぁつひどちのち〠きふヱはぺ〭ぁでふどぬづひち〬〠きどちはねどちは〠しちので〬〠ぉぶメの〠きふヱはぺ〬〠ぃひどびぴどのち〠げどぱふづぬねづ〬〠こちひちべ〠えづひづつどち〬〠かちぴちひぺべのち〠こぴㄙばのどづぷびにち〬〠ぃちひねどのち〠ぇちぬぬちひつは〠ちのつ〠がぬどぬどちののづ〠ぇちのでづび Subject: The increasing spread of African swine fever (ASF) in recent years and the presence of classical swine fever (CSF) subclinical forms in endemic countries suggests that the possibility of coinfection with ASF virus (ASFV) and CSF virus (CSFV) in pigs cannot be ruled out in areas where both diseases are prevalent. Thus, rapid and reliable diagnosis through molecular testing is essential for the timely implementation of control measures to prevent the spread of these devastating swine diseases. Here, we have coupled two of the most validated PCR assays for the detection of CSFV and ASFV in a single reaction tube. The combination of the two tests for the detection of two target nucleic acids did not affect the analytical sensitivity, and the duplex RT-qPCR assay was comparable with the standard molecular techniques. The detection limits for CSFV RNA and ASFV DNA were 0.12 TCID50/reaction and 0.25 TCID50/reaction, respectively. The test showed high repeatability and reproducibility, the coefficient of variation was below 2%, and excellent performance was demonstrated in clinical samples. The duplex assay shows great potential to become a robust diagnostic tool for the rapid and reliable detection and differentiation of CSFV and ASFV in areas where both viruses may be circulating. 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