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BackgroundTuberculosis (TB) is difficult to diagnose in children using molecular tests, because children have difficulty providing respiratory samples. Stool could replace sputum for diagnostic TB testing if adequate sample processing techniques were available.MethodsWe developed a rapid method to process large volumes of stool for downstream testing by the Xpert MTB/RIF (Xpert) TB-detection assay. The method was tested and optimized on stool samples spiked with known numbers of M. tuberculosis colony forming units (CFU), and stools from M. tuberculosis-infected cynomolgus macaques (Macaca fascicularis). Performance was scored on number of positive Xpert tests, the cycle thresholds (Cts) of the Xpert sample-processing control (SPC), and the Cts of the M. tuberculosis-specific rpoB probes. The method was then validated on 20 confirmed TB cases and 20 controls in Durban, South Africa.ResultsThe assay’s analytical limit of detection was 1,000 CFU/g of stool. As much as one gram of spiked stool could be tested without showing increased PCR inhibition. In analytical spiking experiments using human stool, 1g samples provided the best sensitivity compared to smaller amounts of sample. However, in Macaques with TB, 0.6g stool samples performed better than either 0.2g or 1.2g samples. Testing the stool of pediatric TB suspects and controls suggested an assay sensitivity of 85% (95% CI 0.6–0.9) and 84% (95% CI 0.6–0.96) for 0.6g and 1.2g stool samples, respectively, and a specificity of 100% (95% CI 0.77–1) and 94% (95% CI 0.7–0.99), respectively.ConclusionThis novel approach may permit simple and rapid detection of TB using pediatric stool samples.

The noninvasive pH sensor facilitated the detection of most strains of bacterial contaminants within 3 days with no potential for sampling error.

It is well known that DNA strands bind to silica surfaces in the presence of high concentrations of chaotropic salts. We developed simple methods to evaluate binding and recovery of DNA on flat glass microscope slides and compared their properties to commercially available silica "spin columns". Surprisingly, genomic DNA was recovered efficiently from untreated glass slides. Binding and elution times from glass slides were optimized in experiments with DNA samples of various sizes and defined buffers. Average DNA recovery from 500 ng of input genomic DNA varied from 25-53 % for the glass slide protocol. Yields were comparable to spin columns. Human serum albumin and plasma components decreased DNA binding to glass several-fold in a concentration dependent manner. These results support the concept of using flat glass slides as DNA purification surfaces in microfluidics devices for PCR sample preparation. Keywords DNA extraction; silica surfaces; PCR; microfluidicsThe use of powdered glass or other high surface area silica materials has been widely adopted as a basic technique for purifying DNA from biological samples [1]. DNA strands bind efficiently to silica particles in the presence of high concentrations of chaotropic salts such as guanidinium thiocyanate or sodium perchlorate. After washing away contaminating substances with high salt buffers, the bound DNA is released from the silica surface in the presence of water or low salt buffer. The recovered DNA is suitable for use in PCR or other amplification methods. These methods are used extensively in medical diagnostics and forensics, in recombinant technology and for molecular genetics.Silica-based DNA purification is compatible with microfluidic diagnostic devices. Such "lab on a chip" devices may help bring the benefits of PCR-based molecular diagnosis to users and point-of-care settings that lack the capacity for standard laboratory-based DNA purification [2] [3]. In such applications there are significant pressures to keep costs low. Therefore, DNA purification devices must be designed to use the least expensive materials possible, and without unnecessary engineering. Early researchers showed that the curved inner surfaces of glass test tubes could be used to purify DNA from agarose gels, but their binding capacity was deemed too small for most preparative experiments [4]. Currently, it is standard practice to maximize DNA binding surface area, for example by using silica glass bead matrices. However, such engineering measures may not be necessary to achieve satisfactory DNA purification yields.