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One-Step Reverse-Transcription FRET-PCR for Differential Detection of Five Ebolavirus Species.

Lu G, Zhang J, Zhang C, Li X, Shi D, Yang Z, Wang C - PLoS ONE (2015)

Bottom Line: It is important to be able to differentiate the Ebolavirus species as they significantly differ in pathogenicity and more than one species can be present in an area.The primers and FRET-probes we designed enabled us to differentiate five Ebolavirus species by distinct Tm (Zaire: flat peaks between 53.0°C and 56.9°C; Sudan: 51.6°C; Reston: flat peaks between 47.5°C and 54.9°C; Tai Forest: 52.8°C; Bundibugyo: dual peaks at 48.9°C and 53.5°C), and by different amplicon sizes (Zaire 255 bp, Sudan 211 bp, Reston 192 bp, Taï Forest 166 bp, Bundibugyo 146 bp).This one-size-fit-all assay enables the rapid detection and discrimination of the five Ebolavirus species in a single reaction.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University College of Veterinary Medicine, Yangzhou, Jiangsu, 225009, P. R. China.

ABSTRACT
Ebola is an emerging infectious disease caused by a deadly virus belonging to the family Filoviridae, genus Ebolavirus. Based on their geographical distribution, Ebolavirus has been classified into total five species so far, mainly Zaire, Sudan, Taï Forest, Bundibugyo and Reston. It is important to be able to differentiate the Ebolavirus species as they significantly differ in pathogenicity and more than one species can be present in an area. We have developed a one-step step-down RT-PCR detecting all five Ebolavirus species with high sensitivity (1 copy of Ebolavirus DNA, 10 copies of RNA and 320 copies of RNA spiked in 1 ml whole blood). The primers and FRET-probes we designed enabled us to differentiate five Ebolavirus species by distinct Tm (Zaire: flat peaks between 53.0°C and 56.9°C; Sudan: 51.6°C; Reston: flat peaks between 47.5°C and 54.9°C; Tai Forest: 52.8°C; Bundibugyo: dual peaks at 48.9°C and 53.5°C), and by different amplicon sizes (Zaire 255 bp, Sudan 211 bp, Reston 192 bp, Taï Forest 166 bp, Bundibugyo 146 bp). This one-size-fit-all assay enables the rapid detection and discrimination of the five Ebolavirus species in a single reaction.

No MeSH data available.


Related in: MedlinePlus

Amplification curves of reverse-transcription PCR on transcribed Ebolavirus RNAs and blood samples spiked with transcribed RNA.A: The different concentrations of transcribed Zaire ebolavirus RNA (105, 104, 103, 102, 101, 100 /10μl) were detected with the one-step reverse transcription FRET-PCR described in this study, and the detection limitation was 10 copies per reaction system. B: Serial concentrations of transcribed Zaire ebolavirus RNAs (106, 105, 104, 103, 102, 101/10μl) were spiked into human whole blood and were extracted with RNA purification kit, followed by the one-step reverse transcription FRET-PCR established in this study. The detection limitation was 100 RNA copies per reaction, equivalent to 320 copies of Zaire Ebolavirus in 1 ml whole blood. Triplicates (A) or duplicates (B) were performed for each concentration of tested Ebolavirus RNA.
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pone.0126281.g005: Amplification curves of reverse-transcription PCR on transcribed Ebolavirus RNAs and blood samples spiked with transcribed RNA.A: The different concentrations of transcribed Zaire ebolavirus RNA (105, 104, 103, 102, 101, 100 /10μl) were detected with the one-step reverse transcription FRET-PCR described in this study, and the detection limitation was 10 copies per reaction system. B: Serial concentrations of transcribed Zaire ebolavirus RNAs (106, 105, 104, 103, 102, 101/10μl) were spiked into human whole blood and were extracted with RNA purification kit, followed by the one-step reverse transcription FRET-PCR established in this study. The detection limitation was 100 RNA copies per reaction, equivalent to 320 copies of Zaire Ebolavirus in 1 ml whole blood. Triplicates (A) or duplicates (B) were performed for each concentration of tested Ebolavirus RNA.

Mentions: Our one-step RT FRET-PCR was capable of detecting a single DNA copy and 10 RNA copies of all five Ebolavirus species (Figs 3 and 5). When whole blood spiked with transcribed Ebolavirus RNA and VLPs were tested, the detection limit was 100 copies of RNA per PCR reaction (Fig 5), equivalent to 320 copies of Ebolavirus in 1 ml whole blood. Triplicates were performed for each assay. The BLASTN results for each of the 8 oligonucleotides we used in the one-step RT FRET-PCR showed they were specific for the Ebolavirus species and did not cross-react with partial amplicons or other viruses in the family Filoviridae. No PCR amplification was observed with nucleic acids of eight types of pathogens which induce hemorrhagic fever or similar clinical signs as Ebolavirus.


One-Step Reverse-Transcription FRET-PCR for Differential Detection of Five Ebolavirus Species.

Lu G, Zhang J, Zhang C, Li X, Shi D, Yang Z, Wang C - PLoS ONE (2015)

Amplification curves of reverse-transcription PCR on transcribed Ebolavirus RNAs and blood samples spiked with transcribed RNA.A: The different concentrations of transcribed Zaire ebolavirus RNA (105, 104, 103, 102, 101, 100 /10μl) were detected with the one-step reverse transcription FRET-PCR described in this study, and the detection limitation was 10 copies per reaction system. B: Serial concentrations of transcribed Zaire ebolavirus RNAs (106, 105, 104, 103, 102, 101/10μl) were spiked into human whole blood and were extracted with RNA purification kit, followed by the one-step reverse transcription FRET-PCR established in this study. The detection limitation was 100 RNA copies per reaction, equivalent to 320 copies of Zaire Ebolavirus in 1 ml whole blood. Triplicates (A) or duplicates (B) were performed for each concentration of tested Ebolavirus RNA.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4446292&req=5

pone.0126281.g005: Amplification curves of reverse-transcription PCR on transcribed Ebolavirus RNAs and blood samples spiked with transcribed RNA.A: The different concentrations of transcribed Zaire ebolavirus RNA (105, 104, 103, 102, 101, 100 /10μl) were detected with the one-step reverse transcription FRET-PCR described in this study, and the detection limitation was 10 copies per reaction system. B: Serial concentrations of transcribed Zaire ebolavirus RNAs (106, 105, 104, 103, 102, 101/10μl) were spiked into human whole blood and were extracted with RNA purification kit, followed by the one-step reverse transcription FRET-PCR established in this study. The detection limitation was 100 RNA copies per reaction, equivalent to 320 copies of Zaire Ebolavirus in 1 ml whole blood. Triplicates (A) or duplicates (B) were performed for each concentration of tested Ebolavirus RNA.
Mentions: Our one-step RT FRET-PCR was capable of detecting a single DNA copy and 10 RNA copies of all five Ebolavirus species (Figs 3 and 5). When whole blood spiked with transcribed Ebolavirus RNA and VLPs were tested, the detection limit was 100 copies of RNA per PCR reaction (Fig 5), equivalent to 320 copies of Ebolavirus in 1 ml whole blood. Triplicates were performed for each assay. The BLASTN results for each of the 8 oligonucleotides we used in the one-step RT FRET-PCR showed they were specific for the Ebolavirus species and did not cross-react with partial amplicons or other viruses in the family Filoviridae. No PCR amplification was observed with nucleic acids of eight types of pathogens which induce hemorrhagic fever or similar clinical signs as Ebolavirus.

Bottom Line: It is important to be able to differentiate the Ebolavirus species as they significantly differ in pathogenicity and more than one species can be present in an area.The primers and FRET-probes we designed enabled us to differentiate five Ebolavirus species by distinct Tm (Zaire: flat peaks between 53.0°C and 56.9°C; Sudan: 51.6°C; Reston: flat peaks between 47.5°C and 54.9°C; Tai Forest: 52.8°C; Bundibugyo: dual peaks at 48.9°C and 53.5°C), and by different amplicon sizes (Zaire 255 bp, Sudan 211 bp, Reston 192 bp, Taï Forest 166 bp, Bundibugyo 146 bp).This one-size-fit-all assay enables the rapid detection and discrimination of the five Ebolavirus species in a single reaction.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University College of Veterinary Medicine, Yangzhou, Jiangsu, 225009, P. R. China.

ABSTRACT
Ebola is an emerging infectious disease caused by a deadly virus belonging to the family Filoviridae, genus Ebolavirus. Based on their geographical distribution, Ebolavirus has been classified into total five species so far, mainly Zaire, Sudan, Taï Forest, Bundibugyo and Reston. It is important to be able to differentiate the Ebolavirus species as they significantly differ in pathogenicity and more than one species can be present in an area. We have developed a one-step step-down RT-PCR detecting all five Ebolavirus species with high sensitivity (1 copy of Ebolavirus DNA, 10 copies of RNA and 320 copies of RNA spiked in 1 ml whole blood). The primers and FRET-probes we designed enabled us to differentiate five Ebolavirus species by distinct Tm (Zaire: flat peaks between 53.0°C and 56.9°C; Sudan: 51.6°C; Reston: flat peaks between 47.5°C and 54.9°C; Tai Forest: 52.8°C; Bundibugyo: dual peaks at 48.9°C and 53.5°C), and by different amplicon sizes (Zaire 255 bp, Sudan 211 bp, Reston 192 bp, Taï Forest 166 bp, Bundibugyo 146 bp). This one-size-fit-all assay enables the rapid detection and discrimination of the five Ebolavirus species in a single reaction.

No MeSH data available.


Related in: MedlinePlus