Molecular-based diagnostic assays are the gold standard for infectious diseases today, since they allow a rapid and sensitive identification and typing of various pathogens. While PCR can be designed to be specific for a certain pathogen, a subsequent sequence analysis is frequently required for confirmation or typing. The design of appropriate PCR-based assays is a complex task, especially whe...

Multi-tag pyrosequencing has become a key method in the analysis of microbial community composition. However, it is well known that kinetic bias during the initial PCR amplification of such microbial communities can dramatically distort amplicon abundance prior to downstream emulsion PCR and pyrosequencing. Here we present a simple protocol combining length-heterogeneity PCR fingerprinting with...

BACKGROUND Surveillance for HIV transmitted drug resistance (TDR) is performed using HIV genotype results from individual specimens. Pyrosequencing, through its massive parallel sequencing ability, can analyze large numbers of specimens simultaneously. Instead of using pyrosequencing conventionally, to sequence a population of viruses within an individual, we interrogated a single combined pool...

To identify the bacterial diversity (microbiota) in expectorated sputum, a pyrosequencing method that investigates complex microbial communities of expectorated sputum was done in 19 stable chronic obstructive pulmonary disease patients (mean (SD) FEV1: 47 (18%) of predicted value). Using conventional culture, 3 phyla and 20 bacterial genera were identified, whereas the pyrosequencing approach ...

Conventional phenotypic drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are laborious and very time-consuming. Early detection of drug-resistant tuberculosis (TB) is essential for prevention and control of TB transmission. We have developed a pyrosequencing method for simultaneous detection of mutations associated with resistance to rifampin, isoniazid, ethambutol, amik...

Pyrosequencing technology is a sequencing method that screens DNA nucleotide incorporation in real time. A set of coupled enzymatic reactions, together with bioluminescence, detects incorporated nucleotides in the form of light pulses, which produces a profile of characteristic peaks in a pyrogram. We used this technology to identify the warfare agent Bacillus anthracis by sequencing 4 single n...

Abstract Background Reverse transcription of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (+)RNA genome and subgenomic RNAs (sgRNAs) subsequent quantitative polymerase chain reaction (RT-qPCR) is reliable diagnostic gold standard for COVID-19 diagnosis identification potential spreaders. Apart from clinical relevance containment, specific questions, it might be interest to (...

DNA sequencing is one of the most important platforms for the study of biological systems today. Sequence determination is most commonly performed using dideoxy chain termination technology. Recently, pyrosequencing has emerged as a new sequencing methodology. This technique is a widely applicable, alternative technology for the detailed characterization of nucleic acids. Pyrosequencing has the...