A plot of the threshold time versus the

log of the initial template copy number showed a linear regression, with statistically this website significant regression coefficients (R2 = 0.9725 for pCS20 and 0.9473 for sodB LAMP). The detection limits for these assays, using a positive turbidity signal as the indicator, were 10 copies for pCS20 and 5 copies for sodB LAMP. Alternative detection methods included agarose gel electrophoresis of the LAMP products, which displayed the typical ladder-like pattern (Figure 1C and 1D, upper panels), as well as the detection of double stranded LAMP products APR-246 nmr using Gel-Red (Figure 1C and 1D, lower panels).

With smaller amounts of DNA in triplicate assays, 5 copies of pCS20 was amplified once, with a threshold time of 48.3 min, and 1 copy of sodB was amplified twice with threshold times of 45.7 and 49.4 min. Figure 1 Sensitivities of E. HKI-272 manufacturer ruminantium LAMP assays. The assays were performed with serial dilutions of plasmid DNA (104, 103, 102, 10, 5, and 1 copies per reaction) containing the pCS20 or sodB genes. (A and B) Real-time monitoring of pCS20 (A) and sodB (B) LAMP assays using the Loopamp real-time turbidimeter. Plots represent the mean threshold time (Turbidity of 0.1). The error bars represent the standard errors of the mean from three replicates. The plot of the mean

threshold time versus the log of the input DNA fit a linear function (R2 = 0.9725 for pCS20 LAMP and 0.9437 for sodB LAMP). (C and D) Visual detection of pCS20 (C) and sodB (D) LAMP products. LAMP products were visualized with Gel-Red TM under UV (lower panel) or electrophoresed RAS p21 protein activator 1 in a 2.0% agarose gel stained with Gel-Red TM (upper panel). Lanes: M, 100-bp molecular weight marker; 1 to 6, from left to right, 104 to 1 gene copy per reaction, as above; N, negative control. Specificity of LAMP assays The specificity of pCS20 and sodB LAMP assays was evaluated by using the genomic DNA of 18 known E. ruminantium isolates and five closely related species of Anaplasmataceae: E. canis, E. chaffeensis, Anaplasma centrale, A. marginale, and A. phagocytophilum. All isolates of E. ruminantium were positive in both LAMP assays, the pCS20 real-time PCR and the pCS20 PCR; whereas the pCS20 PCR was cross-reactive with both E. canis and E. chaffeensis (Table 1).