RESUMO
The Hologic Panther Fusion® Open Access™ functionality allows implementation of laboratory-developed tests (LDTs), with fully automated sample extraction, real-time PCR, and result interpretation. We report the development and validation of a multiplex LDT for norovirus G1, norovirus G2, and rotavirus from stool samples on this system. The LDT was optimized for primer and probe sequences, salt concentration, and PCR annealing temperature. Reproducibility of the PCR and extraction process was assessed. Performance of the multiplex LDT assay was evaluated with external quality assessment (EQA) samples and compared to a commercial multiplex assay (Allplex™ GI-Virus Assay, Seegene) in clinical samples. Salt concentrations and annealing/extension temperature were optimized to 4 mM MgCl2, 70 mM KCl, 20 mM Tris, and 60 °C, respectively. The user-prepared part of the LDT PCR mix (containing salts, probes, and primers) was stable for ≥ 11 days onboard the instrument. We observed reproducible results of PCR and the extraction process. The LDT had a sensitivity comparable to or greater than the commercial Allplex™ assay and showed excellent linearity. Forty-five EQA samples yielded the expected result with the LDT. There was 100% concordance between LDT and Allplex™ results in 160 clinical samples. Results from the suspension and direct swab stool sample preparation methods were highly concordant in the LDT. We report the successful development and validation of a multiplex PCR LDT for detection of norovirus G1, norovirus G2, and rotavirus from stool samples on the Panther Fusion® system.
Assuntos
Infecções por Caliciviridae/diagnóstico , Gastroenterite/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Norovirus/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções por Rotavirus/diagnóstico , Rotavirus/isolamento & purificação , Automação Laboratorial/normas , Primers do DNA , Fezes/virologia , Gastroenterite/virologia , Genótipo , Humanos , RNA Viral/genética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
L-Histidine biosynthesis in Corynebacterium glutamicum is mainly regulated by L-histidine feedback inhibition of the ATP-phosphoribosyltransferase HisG that catalyzes the first step of the pathway. The elimination of this feedback inhibition is the first and most important step in the development of an L-histidine production strain. For this purpose, a combined approach of random mutagenesis and rational enzyme redesign was performed. Mutants spontaneously resistant to the toxic L-histidine analog ß-(2-thiazolyl)-DL-alanine (2-TA) revealed novel and unpredicted mutations in the C-terminal regulatory domain of HisG resulting in increased feedback resistance. Moreover, deletion of the entire C-terminal regulatory domain in combination with the gain of function mutation S143F in the catalytic domain resulted in a HisG variant that is still highly active even at L-histidine concentrations close to the solubility limit. Notably, the S143F mutation on its own provokes feedback deregulation, revealing for the first time an amino acid residue in the catalytic domain of HisG that is involved in the feedback regulatory mechanism. In addition, we investigated the effect of hisG mutations for L-histidine production on different levels. This comprised the analysis of different expression systems, including plasmid- and chromosome-based overexpression, as well as the importance of codon choice for HisG mutations. The combination of domain deletions, single amino acid exchanges, codon choice, and chromosome-based overexpression resulted in production strains accumulating around 0.5 g l(-1) L-histidine, demonstrating the added value of the different approaches.
Assuntos
ATP Fosforribosiltransferase/metabolismo , Corynebacterium glutamicum/genética , Retroalimentação Fisiológica/fisiologia , Histidina/metabolismo , Engenharia Metabólica/métodos , ATP Fosforribosiltransferase/química , ATP Fosforribosiltransferase/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/enzimologia , Escherichia coli/genética , Redes e Vias Metabólicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Mutação Silenciosa/genética , Mutação Silenciosa/fisiologiaRESUMO
l-Histidine biosynthesis is an ancient metabolic pathway present in bacteria, archaea, lower eukaryotes, and plants. For decades l-histidine biosynthesis has been studied mainly in Escherichia coli and Salmonella typhimurium, revealing fundamental regulatory processes in bacteria. Furthermore, in the last 15 years this pathway has been also investigated intensively in the industrial amino acid-producing bacterium Corynebacterium glutamicum, revealing similarities to E. coli and S. typhimurium, as well as differences. This review summarizes the current knowledge of l-histidine biosynthesis in C. glutamicum. The genes involved and corresponding enzymes are described, in particular focusing on the imidazoleglycerol-phosphate synthase (HisFH) and the histidinol-phosphate phosphatase (HisN). The transcriptional organization of his genes in C. glutamicum is also reported, including the four histidine operons and their promoters. Knowledge of transcriptional regulation during stringent response and by histidine itself is summarized and a translational regulation mechanism is discussed, as well as clues about a histidine transport system. Finally, we discuss the potential of using this knowledge to create or improve C. glutamicum strains for the industrial l-histidine production.