Background: The detection of SARS-CoV-2 RNA by real-time reverse transcription-polymerase chain reaction (rRT-PCR) is used to confirm the clinical diagnosis of COVID-19 by molecular diagnostic laboratories. We developed a multiplex rRT-PCR methodology for the detection of SARS-CoV-2 RNA. Methods: Three genes were used for multiplex rRT-PCR: the Sarbecovirus specific E gene, the SARS-CoV-2 specific N gene, and the human ABL1 gene as an internal control. Results: Good correlation of C q values was observed between the simplex and multiplex rRT-PCR methodologies. Low copies (< 25 copies/reaction) of SARS-CoV-2 RNA were detected by the novel multiplex rRT-PCR method. Conclusion:The proposed multiplex rRT-PCR methodology will enable highly sensitive detection of SARS-CoV-2 RNA, reducing reagent use and cost, and time required by clinical laboratory technicians.

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The taxonomic positions of two clinically isolated actinomycetes were established using a polyphasic approach. The two strains, IFM 10032 T , isolated from ear discharge of a 28-year-old Japanese female patient with external otitis, and IFM 10148, isolated from pleural fluid of a 60-year-old Japanese male patient with bronchitis, possessed meso-diaminopimelic acid as the diagnostic amino acid, MK-9(H 2 ) as the predominant menaquinone and mycolic acids ranging from 58 to 64 carbons. The 16S rRNA gene sequences of the two strains were most closely related to those of Gordonia aichiensis, Gordonia sputi and 'Gordonia jacobaea'. Differences in several phenotypic characteristics together with genotypic distinctiveness distinguish strains IFM 10032T and IFM 10148 from these three species. DNA-DNA hybridization results and the combination of genotypic and phenotypic data showed that the two strains belong to a single species, and merit recognition of a novel species within the genus Gordonia.

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BackgroundThe MALDI (matrix-assisted laser desorption/ionization) Biotyper system for bacterial identification has already been utilized in clinical microbiology laboratories as a successful clinical application of protoemics. However, in cases of Nocardia, mass spectra suitable for MALDI Biotyper identification are often not obtained if such specimens are processed like general bacteria. This problem is related to the insufficiencies in bacterial spectrum databases that preclude accurate specimen identification. Here, we developed a bacterial processing method to improve mass spectra from specimens of the genus Nocardia. In addition, with the new processing method, we constructed a novel in-house bacterial database that combines a commercial database and mass spectra of Nocardia strains from the Department of Clinical Laboratory at Chiba University Hospital (DCLC) and the Medical Mycology Research Center at Chiba University (MMRC).ResultsThe newly developed method (Nocardia Extraction Method at DCLC [NECLC]) based on ethanol-formic acid extraction (EFAE) improved mass spectra obtained from Nocardia specimens. The Nocardia in-house database at Chiba University Hospital (NDCUH) was then successfully validated. In brief, prior to introduction of the NECLC and NDCUH, 10 of 64 (15.6%) clinical isolates were identified at the species level and 16 isolates (25.0%) could only be identified at the genus level. In contrast, after the introduction, 58 isolates (90.6%) were identified at the species level and 6 isolates (9.4%) were identified at the genus level.ConclusionsThe results of this study suggest that MALDI-TOF (time-of-flight) Biotyper system can identify Nocardia accurately in a short time in combination with a simple processing method and an in-house database.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-015-9078-5) contains supplementary material, which is available to authorized users.