The evolution of Pyrosequencing® for microbiology: From genes to genomes.

Pyrosequencing is a unique sequencing method that was developed as an alternative to classical DNA sequencing for short- to medium-read applications. Compared to other methods, it is highly quantitative, fast and inexpensive. Additional advantages include high accuracy, flexibility and ability to automate sample preparation. This article presents a historical overview of pyrosequencing, improvements that have been made through the years and its evolution into a platform that vastly expanded the scope of genetic analysis that could be performed outside of a big sequencing center. In addition, we describe numerous applications in microbiology that have benefited from the pyrosequencing method.

Toxin-co-regulated pilus cluster in non-O1, non-toxigenic Vibrio cholerae: evidence of a third allele of pilin gene.

Polymerase chain reaction has been used to detect the presence of the virulence associated gene, tcpA and part of the promoter distal region of the toxin-co-regulated pilus cluster in non-O1, non-toxigenic, Vibrio cholerae. The amplified regions were characterised by restriction fragment length polymorphism and heteroduplex motility assay. We describe the nucleotide sequence of the tcpA gene fragment from non-toxigenic vibrios from clinical and environmental sources. The present study shows that there are at least three types of the tcpA gene among V. cholerae and the primers specific for the classical tcpA gene, amplify all biotypes. A sequence similarity in other regions of the toxin-co-regulated pilus cluster is suggested. The evidences for the presence of this cluster among non-toxigenic vibrios is, to our knowledge, reported for the first time. The use of restriction fragment length polymorphism for typing the tcpA and studying the alleles distribution is proposed.