16S rRNA Next Generation Sequencing Analysis Shows Bacteria in Alzheimer's Post-Mortem Brain.

The neurological deterioration associated with Alzheimer's disease (AD), involving accumulation of amyloid-beta peptides and neurofibrillary tangles, is associated with evident neuroinflammation. This is now seen to be a significant contributor to pathology. Recently the tenet of the privileged status of the brain, regarding microbial compromise, has been questioned, particularly in terms of neurodegenerative diseases. It is now being considered that microbiological incursion into the central nervous system could be either an initiator or significant contributor to these. This is a novel study using 16S ribosomal gene-specific Next generation sequencing (NGS) of extracted brain tissue. A comparison was made of the bacterial species content of both frozen and formaldehyde fixed sections of a small cohort of Alzheimer-affected cases with those of cognitively unimpaired (normal). Our findings suggest an increase in bacterial populations in Alzheimer brain tissue compared with normal.

Kinetic characterisation of primer mismatches in allele-specific PCR: a quantitative assessment.

A novel method of estimating the kinetic parameters of Taq DNA polymerase during rapid cycle PCR is presented. A model was constructed using a simplified sigmoid function to represent substrate accumulation during PCR in combination with the general equation describing high substrate inhibition for Michaelis-Menten enzymes. The PCR progress curve was viewed as a series of independent reactions where initial rates were accurately measured for each cycle. Kinetic parameters were obtained for allele-specific PCR (AS-PCR) amplification to examine the effect of mismatches on amplification. A high degree of correlation was obtained providing evidence of substrate inhibition as a major cause of the plateau phase that occurs in the later cycles of PCR.

SNP genotyping using single-tube fluorescent bidirectional PCR.

SNP genotyping is a well-populatedfield with a large number of assay formats offering accurate allelic discrimination. However, there remains a discord between the ultimate goal of rapid, inexpensive assays that do not require complex design considerations and involved optimization strategies. We describe the first integration of bidirectional allele-specific amplification, SYBR Green I, and rapid-cycle PCR to provide a homogeneous SNP-typing assay. Wild-type, mutant, and heterozygous alleles were easily discriminated in a single tube using melt curve profiling of PCR products alone. We demonstrate the effectiveness and reliability of this assay with a blinded trial using clinical samples from individuals with sickle cell anemia, sickle cell trait, or unaffected individuals. The tests were completed in less than 30 min without expensive fluorogenic probes, prohibiting design rules, or lengthy downstream processing for product analysis.