quantitative real time pcr qrt pcr primescript rt master mix  (TaKaRa)

Journal: Journal of Virology

Article Title: Comprehensive Molecular Detection of Tick-Borne Phleboviruses Leads to the Retrospective Identification of Taxonomically Unassigned Bunyaviruses and the Discovery of a Novel Member of the Genus Phlebovirus

doi: 10.1128/JVI.02704-14

Figure Lengend Snippet: Sensitivity of RT-PCR using newly designed primers. (A) Conserved regions of tick-borne phlebovirus (TBPV) proteins were manually selected based on the multiple-sequence alignments. Two sets of primers (ppL1 and ppL2) were designed from the same conserved regions in the L protein ORF (indicated by black arrowheads) to minimize the degeneracy of nucleotide sequences. (B and C) The sensitivities of conventional one-step RT-PCR using these two primer sets were tested with serial dilutions of in vitro-synthesized RNAs (B) and viral RNAs (C) as the templates. The sensitivity of one-step quantitative RT-PCR (qRT-PCR) was tested using serial dilutions of in vitro-synthesized RNAs. (D) Average Cq values (five replicates) are plotted against the number of RNA copies, and trend lines calculated by semilog-line nonlinear regression analysis are also shown). Abbreviations: L, RNA-dependent RNA polymerase; NSs, nonstructural protein; N, nucleocapsid protein; SFTSV, severe fever with thrombocytopenia syndrome virus; BHAV, Bhanja virus; TCID50, 50% tissue culture infectious dose; Cq, quantification cycle.

Article Snippet: Quantitative real-time RT-PCR (qRT-PCR) was performed using the PrimeScript RT Master Mix Perfect Real Time (TaKaRa) for reverse transcription following the manufacturer's protocol and using SYBR Premix Ex Taq (Tli RNase H Plus; TaKaRa) for quantitative PCR.

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, In Vitro, Synthesized, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Comprehensive Molecular Detection of Tick-Borne Phleboviruses Leads to the Retrospective Identification of Taxonomically Unassigned Bunyaviruses and the Discovery of a Novel Member of the Genus Phlebovirus

doi: 10.1128/JVI.02704-14

Figure Lengend Snippet: Cross-reactivity of RT-PCR using newly designed primers with known TBPVs. Cross-reactivity of the TBPV RT-PCR was tested using viral RNAs of known TBPVs. Since viral RNA concentrations were not standardized, the band intensity does not reflect the sensitivity of the system. The phylogenetic tree, which was constructed by the neighbor-joining (NJ) algorithm, at the bottom indicates the genetic relationship among the TBPVs tested. Abbreviations: SFTSV, severe fever with thrombocytopenia syndrome virus; BHAV, Bhanja virus; PALV, Palma virus.

Article Snippet: Quantitative real-time RT-PCR (qRT-PCR) was performed using the PrimeScript RT Master Mix Perfect Real Time (TaKaRa) for reverse transcription following the manufacturer's protocol and using SYBR Premix Ex Taq (Tli RNase H Plus; TaKaRa) for quantitative PCR.

Techniques: Reverse Transcription Polymerase Chain Reaction, Construct

Journal: Journal of Virology

Article Title: Comprehensive Molecular Detection of Tick-Borne Phleboviruses Leads to the Retrospective Identification of Taxonomically Unassigned Bunyaviruses and the Discovery of a Novel Member of the Genus Phlebovirus

doi: 10.1128/JVI.02704-14

Figure Lengend Snippet: Retrospective identification of TBPVs. (A) Conventional RT-PCR was performed using RNA from uncharacterized bunyaviruses obtained from multiple sources. (B) Each amplified RT-PCR fragment (around 500 bp) was sequenced, and a phylogenetic tree was constructed based on the fragment sequences and reference sequences using the maximum likelihood (ML) method with 1,000 bootstrap replicates. Bootstrap probabilities above 70% are shown near the branches. The viruses that were newly sequenced in the present study are shown in boldface type and underlined. (C) The sequence identity of the fragment between the newly identified TBPVs (i.e., Kismayo virus, Silverwater virus, Kaisodi virus, and Lanjan virus) and known TBPVs. Each symbol shows the identity value for an individual tick, and the median value for the genetic group is shown as a black bar. Abbreviations: UUKV, Uukuniemi virus; SFTSV, severe fever with thrombocytopenia syndrome virus.

Article Snippet: Quantitative real-time RT-PCR (qRT-PCR) was performed using the PrimeScript RT Master Mix Perfect Real Time (TaKaRa) for reverse transcription following the manufacturer's protocol and using SYBR Premix Ex Taq (Tli RNase H Plus; TaKaRa) for quantitative PCR.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Construct, Sequencing

Journal: Journal of Virology

Article Title: Comprehensive Molecular Detection of Tick-Borne Phleboviruses Leads to the Retrospective Identification of Taxonomically Unassigned Bunyaviruses and the Discovery of a Novel Member of the Genus Phlebovirus

doi: 10.1128/JVI.02704-14

Figure Lengend Snippet: Identification of Shibuyunji virus (SHBV) from a field tick sample in Zambia. (A) The Shibuyunji district in Zambia is indicated by the black star. The Zambia location map by NordNordWest was obtained from Wikimedia Commons and modified by the authors. (B) RT-PCR was performed with field tick samples corresponding to homogenized single tick samples (CZCZT13-1 to CZCZT13-25). (C) Four out of eight RT-PCR fragments were sequenced (no sequencing reads was obtained with the other four fragments), and a phylogenetic tree was constructed based on these sequences, as well as reference sequences, using the maximum likelihood (ML) method with 1,000 bootstraps. Bootstrap probabilities above 70% are indicated near the branches. SFTSV, severe fever with thrombocytopenia syndrome virus.

Article Snippet: Quantitative real-time RT-PCR (qRT-PCR) was performed using the PrimeScript RT Master Mix Perfect Real Time (TaKaRa) for reverse transcription following the manufacturer's protocol and using SYBR Premix Ex Taq (Tli RNase H Plus; TaKaRa) for quantitative PCR.

Techniques: Modification, Reverse Transcription Polymerase Chain Reaction, Sequencing, Construct