Defining the relative performance of isothermal assays that can be used for rapid and sensitive detection of foot-and-mouth disease virus

Fig. 1. Analytical sensitivity of seven isothermal assay formats using a RNA standard. Results were compared to the diagnostic laboratory-based real-time reverse transcription (rRT)-PCR (▼). ● rRT-loop-mediated isothermal amplification using Dukes et al. (2006) primers (rRT-LAMP-D)-wet; ○rRT-LAMP-d-dry; ■ rRT-recombinase polymerase amplification (RPA)-exoRT; □rRT-RPA-nfo; ▲ rRT-LAMP using Toshiba primers (rRT-LAMP-T)-wet. Half shaded points represent samples where one duplicate was positive and the other negative; grey shaded points (for rRT-PCR) represent CT values that were over the diagnostic cut-off value of CT <32 (Shaw et al., 2007). For the molecular lateral-flow devices (LFD), the presence of a test (T) and control (C) line signifies a positive result; the presence of a single control band signifies a negative result (C). For all assays, both duplicates had to be positive for the sample to be regarded as a positive.

Fig. 2. Concordance between the diagnostic laboratory-based real-time reverse transcription (rRT)-PCR and isothermal assays using 43 clinical samples: rRT-loop-mediated isothermal amplification using Dukes et al. (2006) primes (rRT-LAMP-D)-wet (A), rRT-LAMP using Toshiba primers (rRT-LAMP-T)-wet (B), rRT-recombinase polymerase amplification (RPA)-exoRT (C) and rRT-RPA-nfo (D). Grey box highlights rRT-PCR CT values over the diagnostic threshold of CT < 32 (Shaw et al., 2007). Lyophilisation of rRT-LAMP-D did not impact assay performance.

Fig. 3. Comparison of sample preparation methods for real-time reverse transcription loop-mediated isothermal amplification using Dukes et al. (2006) primers (rRT-LAMP-d-wet). The top row (A-C) presents results for epithelial suspensions; the middle for serum samples (D-F); the bottom for oesophageal-pharyngeal (OP) fluid (G-I). A total of 11 sample preparation methods were trialled: (A,D,G): RNA extraction methods; (B,E,H): use of neat samples or dilution in nuclease-free water; (C,F,I): 1 in 5 dilutions subjected to further treatment using Chelex® or syringe filtering. (Extraction from antigen-detection lateral-flow devices [Ag-LFD] was trialled for neat epithelial suspensions only) Half shaded points represent samples where one duplicate was positive and the other negative; both duplicates had to be positive for the sample to be regarded as a positive.

Fig. 4. Comparison of sample preparation methods for real-time reverse transcription recombinase polymerase amplification using TwistAmp® exo RT kit (rRT-RPA-exoRT). Sample preparation methods were trialled across three sample types: epithelial suspensions (A-C), serum samples (D-F) and oesophageal-pharyngeal (OP) fluid (G-I). Eleven sample preparation methods were tested: (A,D,G): RNA extraction methods; (B,E,H): use of neat samples or dilution in nuclease-free water; (C,F,I): 1 in 5 dilutions subjected to Chelex® or syringe filtering (antigen-detection lateral-flow devices [Ag-LFD] extraction was trialled for neat epithelial suspensions only). NS: non-specific amplification detected (signified by amplification present in known negative samples); AI: assay inhibition. Half shaded points represent samples where one duplicate was positive and the other negative; both duplicates had to be positive for the sample to be regarded as a positive.