Successful pyrosequencing of GC-rich DNA sequences by partial substitution of deoxyguanosine with deoxyinosine.

We experienced significant problems while developing a PyrosequencingTM (Biotage, Uppsala, Sweden) assay to characterize the rifampin resistance-determining region of Mycobacteriumtuberculosis, a target with high guanosine-cytosine content. This paper describes the successful use of a modified pyrosequencing protocol through partial substitution of deoxyguanosine triphosphate with deoxyinosine triphosphate in the polymerase chain reaction.

Pyrosequencing for rapid detection of Mycobacterium tuberculosis resistance to rifampin, isoniazid, and fluoroquinolones.

After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively.

Recent advances in diagnostic microbiology.

The past decade has seen a surge in the development of a variety of molecular diagnostics designed to rapidly identify or characterize medically important microorganisms. We briefly review important advances in molecular microbiology, and then discuss specific assays that have been implemented in clinical microbiology laboratories throughout the country. We also discuss emerging methods and technologies that will soon be more widely used for the prompt and accurate detection of the agents of infectious diseases.