BACKGROUND: Detection of nucleic acids of Rift Valley fever virus (RVFV) has been shown to be useful in field diagnostics. OBJECTIVES: To develop an isothermal 'recombinase polymerase amplification (RPA)' assay on an ESEquant tubescanner device. STUDY DESIGN: RPA was adapted for RNA amplification by first developing a two-step and then a one-step-RT-RPA protocol. Several RT enzymes were tested and the best sensitivity was achieved using Transcriptor (Roche). Finally an RT-RPA pellet containing a recombinant MuLV was tested in RVFV one-step-RT-RPA. RESULTS: The one-step-RT-RPA assay showed a sensitivity of 19 molecules detected as determined by probit analysis of eight runs using a RVFV S-segment based quantitative RNA standard and detected 20 different RVFV strains. The assays showed no cross detection of the human genome and several agents of a typical biothreat panel. It performed almost as good as the assay using glycerol buffer based Transcriptor albeit at a cost of 1-log(10) step in sensitivity. The presented combination of one-step-RT-RPA and portable fluorescence reading device could be a useful tool for field or point of care diagnostics.
BACKGROUND: Detection of nucleic acids of Rift Valley fever virus (RVFV) has been shown to be useful in field diagnostics. OBJECTIVES: To develop an isothermal 'recombinase polymerase amplification (RPA)' assay on an ESEquant tubescanner device. STUDY DESIGN: RPA was adapted for RNA amplification by first developing a two-step and then a one-step-RT-RPA protocol. Several RT enzymes were tested and the best sensitivity was achieved using Transcriptor (Roche). Finally an RT-RPA pellet containing a recombinant MuLV was tested in RVFV one-step-RT-RPA. RESULTS: The one-step-RT-RPA assay showed a sensitivity of 19 molecules detected as determined by probit analysis of eight runs using a RVFV S-segment based quantitative RNA standard and detected 20 different RVFV strains. The assays showed no cross detection of the human genome and several agents of a typical biothreat panel. It performed almost as good as the assay using glycerol buffer based Transcriptor albeit at a cost of 1-log(10) step in sensitivity. The presented combination of one-step-RT-RPA and portable fluorescence reading device could be a useful tool for field or point of care diagnostics.
Authors: Sebastian Kersting; Valentina Rausch; Frank F Bier; Markus von Nickisch-Rosenegk Journal: Mikrochim Acta Date: 2014-02-18 Impact factor: 5.833
Authors: Michael Mauk; Jinzhao Song; Haim H Bau; Robert Gross; Frederic D Bushman; Ronald G Collman; Changchun Liu Journal: Lab Chip Date: 2017-01-31 Impact factor: 6.799