Figure 1.
Flow diagram describing organization of samples (circles) and compared factors (rectangles) used for improvement of mycobacterial DNA detection by real-time PCR in tissues.
Figure 2.
DNA amount measured at 260 nm after DNA extractions (a: van Soolingen procedure, b: Invisorb Spin Tissue Mini Kit from Invitek, c: PowerSoil DNA isolation Kit from MoBio) from big (+++: 10 log10 cells), medium (++: 9 log10 cells), and small (+: 8 log10 cells) pellets of MAP K-10 (A, n=54), and M. tuberculosis H37Ra (B, n=54) cells.
Error bars are standard deviation. LOQ are limit of quantification of the spectrophotometer.
Figure 3.
Comparison of MAP K-10 quantification methods in culture samples (A, n=28), or in liver (B, n=45) and spleen (C, n=45) samples from C57BL/6 mice: culture on 7H10-mycobactin versus TaqMan real-time PCR targeting MAP0865 gene, including 1 h, 24 h, or 48 h of lysozyme digestion (Ly), 1 or 2 phenol:chloroform:isoamyl purifications (PCI), no or 1 grinding step (G), and/or 0, 1, 2 or 3 steps of bead beating (BB) during DNA extraction procedure.
Error bars are standard deviation.
Figure 4.
in vitro limit of quantification (on the left) estimated by reproducible TaqMan real-time PCR amplifications of MAP0865 (n=63) and ext-RD9 (n=21) for MAP K-10 (A) and M. tuberculosis H37Rv quantification (B), and limits of quantification in tissues (on the right) estimated by distribution analyses of sample number according to MAP K-10 (A) and M. tuberculosis H37Rv (B) densities, estimated by TaqMan real-time PCR targeting MAP0865 (n=40 spleen, n=40 liver) or ext-RD9 (n=7 spleen, n=9 lung), respectively.
All these accurate real-time PCR results are observed for a host DNA amount < 3 µg in a final volume of 25 µl, and DNA purity at 260/280 nm ratio = 1.89±0.08.
Figure 5.
Accurate (green), inaccurate (red), and inhibited (blue) quantification of MAP-K10 in liver (A, n=36) and spleen (B, n=36) by TaqMan real-time PCR (MAP0865 gene), of M. tuberculosis H37Rv in lung (C, n=36) by TaqMan real-time PCR (ext-RD9 element), and of C57BL/6J mouse cells (D, n=36) by SyberGreen real-time PCR (PTGR2 gene), according to host DNA purity at 260/280 nm (on the left), host DNA quantity at 260 nm (on the right).