Journal: Frontiers in Veterinary Science

Article Title: One-Tube Nested Real-Time PCR Assay for Rapid Screening of Porcine Cytomegalovirus in Clinical Samples

doi: 10.3389/fvets.2020.586045

Figure Lengend Snippet: Schematic diagram of the DNA polymerase gene to distinguish PCMV from positions of primers and probes. A positive control was synthesized to verify the efficiency of the developed primers and probes. Primer sequences for conventional PCR and nested PCR were selected from the PCMV DNA polymerase gene determined by Hamel and Fryer , respectively. Primers and probe sequences for one-tube nested real-time PCR used in this study were selected at differed locations from the human and mouse cytomegalovirus DNA polymerase gene.

Article Snippet: Detection of PCMV in clinical samples was performed with Opti PCMV real-time PCR (Optipharm), a quantitative one-tube nested real-time PCR-based assay, using a CFX-96 real-time PCR system (Bio-Rad, Hercules, CA, USA) for thermal cycling and fluorescence detection.

Techniques: Positive Control, Synthesized, Nested PCR, Real-time Polymerase Chain Reaction

Journal: Frontiers in Veterinary Science

Article Title: One-Tube Nested Real-Time PCR Assay for Rapid Screening of Porcine Cytomegalovirus in Clinical Samples

doi: 10.3389/fvets.2020.586045

Figure Lengend Snippet: The detection limit of the one-tube nested real-time PCR assay was evaluated using 10-fold serial diluted samples. Serially diluted PCMV control DNA ranging from 10 6 copies to 1 copy per reaction were used to determine the detection limit of the one-tube nested real-time PCR assay. In the one-tube nested real-time PCR assay, the amplification curve of the specific probe (A) for detecting PCMV is shown. The overall detection limit of this assay for the PCMV ranged from approximately 1 copy DNA per reaction. C T was plotted against the input of the quantity of PCMV DNA (repeated 20 times). The linearity (B) was generated by plotting the log quantity of PCMV DNA vs. the corresponding C T value, and the coefficients of determination of the linear regression were 0.997 with a slope of −4.082. The fluorescence intensity is displayed on the Y-axis ( R 2 = reporter signal/passive reference signal). RFU, relative fluorescence unit; R 2 , fluorescence units.

Article Snippet: Detection of PCMV in clinical samples was performed with Opti PCMV real-time PCR (Optipharm), a quantitative one-tube nested real-time PCR-based assay, using a CFX-96 real-time PCR system (Bio-Rad, Hercules, CA, USA) for thermal cycling and fluorescence detection.

Techniques: Real-time Polymerase Chain Reaction, Control, Amplification, Generated, Fluorescence

Journal: Frontiers in Veterinary Science

Article Title: One-Tube Nested Real-Time PCR Assay for Rapid Screening of Porcine Cytomegalovirus in Clinical Samples

doi: 10.3389/fvets.2020.586045

Figure Lengend Snippet: Detection of PCMV DNA in 127 clinical samples suspected of PCMV infection using the conventional PCR, nested PCR, and one-tube nested real-time PCR assay.

Article Snippet: Detection of PCMV in clinical samples was performed with Opti PCMV real-time PCR (Optipharm), a quantitative one-tube nested real-time PCR-based assay, using a CFX-96 real-time PCR system (Bio-Rad, Hercules, CA, USA) for thermal cycling and fluorescence detection.

Techniques: Infection, Nested PCR, Real-time Polymerase Chain Reaction

Journal: Frontiers in Veterinary Science

Article Title: One-Tube Nested Real-Time PCR Assay for Rapid Screening of Porcine Cytomegalovirus in Clinical Samples

doi: 10.3389/fvets.2020.586045

Figure Lengend Snippet: Clinical sensitivity and specificity between the one-tube nested real-time PCR assay and conventional PCR, nested PCR, and sequence analysis methods stratified by PCMV suspected samples.

Article Snippet: Detection of PCMV in clinical samples was performed with Opti PCMV real-time PCR (Optipharm), a quantitative one-tube nested real-time PCR-based assay, using a CFX-96 real-time PCR system (Bio-Rad, Hercules, CA, USA) for thermal cycling and fluorescence detection.

Techniques: Real-time Polymerase Chain Reaction, Nested PCR, Sequencing

Journal: Frontiers in Veterinary Science

Article Title: One-Tube Nested Real-Time PCR Assay for Rapid Screening of Porcine Cytomegalovirus in Clinical Samples

doi: 10.3389/fvets.2020.586045

Figure Lengend Snippet: Phylogenetic analysis of 49 PCMV isolates. The phylogenetic tree was constructed using Phylogeny.fr software after the alignment of the 49 sequenced results. The Phylogeny.fr software offers MUSCLE for multiple sequence alignments, Gblocks for alignment curation, PhyML for phylogenetic reconstruction, and TreeDyn for graphical representation of trees. Analysis of phylogenetic tree showed that the sequences obtained from the one-tube nested real-time PCR assay were similar to those of the reference strains FJ01 and B6, with two divided groups (Groups A and B) accounting for 69.4% ( n = 34) and 30.6% ( n = 15), respectively.

Article Snippet: Detection of PCMV in clinical samples was performed with Opti PCMV real-time PCR (Optipharm), a quantitative one-tube nested real-time PCR-based assay, using a CFX-96 real-time PCR system (Bio-Rad, Hercules, CA, USA) for thermal cycling and fluorescence detection.

Techniques: Construct, Software, Sequencing, Real-time Polymerase Chain Reaction