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The general methods to detect the RNA of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in clinical diagnostic testing involve reverse transcriptases and thermostable DNA polymerases. In this study, we compared the detection of SARS-CoV-2 by a one-step real-time RT-PCR method using a heat-resistant reverse transcriptase variant MM4 from Moloney murine leukemia virus, two thermostable DNA polymerase variants with reverse transcriptase activity from Thermotoga petrophila K4 and Thermococcus kodakarensis KOD1, or a wild-type DNA polymerase from Thermus thermophilus M1. The highest performance was achieved by combining MM4 with the thermostable DNA polymerase from T. thermophilus M1. These enzymes efficiently amplified specific RNA using uracil-DNA glycosylase (UNG) to remove contamination and human RNase P RNA amplification as an internal control. The standard curve was obtained from 5 to 10⁵ copies of synthetic RNA. The one-step real-time RT-PCR method’s sensitivity and specificity were 99.44% and 100%, respectively (n = 213), compared to those of a commercially available diagnostic kit. Therefore, our method will be useful for the accurate detection and quantification of SARS-CoV-2., 新型コロナウイルス検出酵素試薬の開発 --独自酵素開発酵素によるOne-Step Real-Time PCR法--. 京都大学プレスリリース. 2021-06-07.

Background46,XY disorders of sex development (46,XY DSD) are genetically heterogeneous conditions. Recently, a few submicroscopic genomic rearrangements have been reported as novel genetic causes of 46,XY DSD.Methodology/principal findingsTo clarify the role of cryptic rearrangements in the development of 46,XY DSD, we performed array-based comparative genomic hybridization analysis for 24 genetic males with genital abnormalities. Heterozygous submicroscopic deletions were identified in three cases (cases 1-3). A ∼8.5 Mb terminal deletion at 9p24.1-24.3 was detected in case 1 that presented with complete female-type external genitalia and mental retardation; a ∼2.0 Mb interstitial deletion at 20p13 was identified in case 2 with ambiguous external genitalia and short stature; and a ∼18.0 Mb interstitial deletion at 2q31.1-32 was found in case 3 with ambiguous external genitalia, mental retardation and multiple anomalies. The genital abnormalities of case 1 could be ascribed to gonadal dysgenesis caused by haploinsufficiency of DMRT1, while those of case 3 were possibly associated with perturbed organogenesis due to a deletion of the HOXD cluster. The deletion in case 2 affected 36 genes, none of which have been previously implicated in sex development.Conclusions/significanceThe results indicate that cryptic genomic rearrangements constitute an important part of the molecular bases of 46,XY DSD and that submicroscopic deletions can lead to various types of 46,XY DSD that occur as components of contiguous gene deletion syndromes. Most importantly, our data provide a novel candidate locus for 46,XY DSD at 20p13.