Journal: Archives of Virology

Article Title: Optimization of a magnetic capture RT-LAMP assay for fast and real-time detection of potato virus Y and differentiation of N and O serotypes

doi: 10.1007/s00705-017-3635-3

Figure Lengend Snippet: Performance of the Y4-set-primed RT-LAMP for PVY detection. (a) Comparison of the amplification speeds of the Y4, Y5 and N primer sets in the RT LAMP assay for PVY detection from total RNA isolated from plants infected with PVY NTN (isolate 12/94). We used total RNA from virus-free plants (nc) and a no-template control (ntc) as negative controls. The time-to-positive values are shown above the plot. (b) Comparison of the sensitivity of different RT-LAMP assays by serial dilution of total RNA. The RT-LAMP results are shown as time-to-positive (Tp) values as a function of the logarithm of the total RNA concentration. The total RNA (100 pg/µl) was serially diluted down to 0.001 pg/µl. The amounts of RNA [pg] detected by the specific RT-LAMP assays are indicated above the plot. (c) Specificity of the Y4 RT-LAMP for PVY detection expressed as amplification plots for reactions performed using total RNA isolated from plants infected with PVY NTN , PVA, PVM, PVS, PVX, or PLRV. (d) Second derivatives of annealing curves recorded for amplicons in the reactions described in panel c. The peak of the plot indicates the annealing temperature (T a ) of the amplicon produced by Y4 primers for a reaction containing PVY NTN RNA. The data are averages from at least two independent experiments performed in triplicate. Error bars indicate standard deviation

Article Snippet: The amplification was performed either with a Genie II Ultra rapid amplification instrument (OptiGene Ltd.) or with a CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad Ltd).

Techniques: Comparison, Amplification, RT Lamp Assay, Isolation, Infection, Virus, Control, Serial Dilution, Concentration Assay, Produced, Standard Deviation

Journal: Archives of Virology

Article Title: Optimization of a magnetic capture RT-LAMP assay for fast and real-time detection of potato virus Y and differentiation of N and O serotypes

doi: 10.1007/s00705-017-3635-3

Figure Lengend Snippet: Statistical analysis of differences between mean annealing (T a ) and melting temperatures (T m ) recorded for O- and N-type PVY isolates by Y5 and Y4 RT-LAMP assays. T a values were determined using a Genie II rapid amplification system (OptiGene Ltd.), and T m values were determined using a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.). EU, European PVY isolates; USA, USA PVY isolates

Article Snippet: The amplification was performed either with a Genie II Ultra rapid amplification instrument (OptiGene Ltd.) or with a CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad Ltd).

Techniques: Amplification, Real-time Polymerase Chain Reaction

Journal: Archives of Virology

Article Title: Optimization of a magnetic capture RT-LAMP assay for fast and real-time detection of potato virus Y and differentiation of N and O serotypes

doi: 10.1007/s00705-017-3635-3

Figure Lengend Snippet: Differentiation of PVY types N and O by the Y4-primer-set-based RT-LAMP. Amplification plots recorded for RT-LAMP reactions with the Y4 (a) and Y5 (b) primer sets and total RNA isolated from plants infected with the indicated European PVY isolates, with total RNA from a virus-free plant (nc), or with water instead of RNA (ntc). Second derivatives of annealing curves recorded for amplicons amplified using the Y4 (c) and Y5 (d) primer sets in reactions described in panels a and b, respectively. Annealing temperature (T a ) ranges of the RT-LAMP products obtained on European PVY strains by amplification with the Y4 (e) and Y5 (f) primer sets. The annealing temperatures were recorded using a Genie II apparatus (g and h). Melting temperature (T m ) ranges of the RT-LAMP products obtained using European (g) or North American (h) PVY strains by amplification with the Y4 primer set. The melting temperatures were measured using the Bio-Rad apparatus. PVY strains with coat protein O or N types are indicated by symbols with a black or white background, respectively. The data are averages from at least two independent experiments performed in triplicate. Error bars indicate standard deviation

Article Snippet: The amplification was performed either with a Genie II Ultra rapid amplification instrument (OptiGene Ltd.) or with a CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad Ltd).

Techniques: Amplification, Isolation, Infection, Virus, Standard Deviation

Journal: Archives of Virology

Article Title: Optimization of a magnetic capture RT-LAMP assay for fast and real-time detection of potato virus Y and differentiation of N and O serotypes

doi: 10.1007/s00705-017-3635-3

Figure Lengend Snippet: Effect of the method of RNA isolation on the performance (a) and sensitivity (b) of detection of PVY by Y4 RT-LAMP. (a) Amplification plots recorded for RNA purified from plants infected with PVY by magnetic capture (i) according to the manufacturer (ii) by a modified magnetic procedure and (iii) by a shortened magnetic procedure. (b) Comparison of the effect of the different RNA isolation methods on sensitivity. Sap from plants infected with PVY was serially diluted up to 2 × 10 6 times, and total RNA was purified from each dilution by the magnetic capture procedure recommended by the manufacturer, by the shortened magnetic capture procedure, and by silica capture. Time-to-positive results are shown as a function of the logarithm of the dilution factor. The data are averages from at least two independent experiments performed in triplicate. Error bars indicate standard deviation

Article Snippet: The amplification was performed either with a Genie II Ultra rapid amplification instrument (OptiGene Ltd.) or with a CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad Ltd).

Techniques: Isolation, Amplification, Purification, Infection, Modification, Comparison, Standard Deviation