Simple and rapid detection method for qepA1 by loop-mediated isothermal amplification

Although fluoroquinolone (FQ) has been used for the treatment of various bacterial infectious diseases, its continued use has been problematic given the appearance of FQ-resistant bacteria. However, the recent discovery of four plasmid-mediated quinolone resistance (PMQR) genes comprising qnr, aac(6\u27)Ib-cr, qepA and OqxAB since 1998 has provided insights in the area of FQ-resistance. For practical detection of qepA in microbiology laboratory, a specific, simple, rapid and cost-effective isothermal amplification method designated as LAMP is the good candidate to use. In this study, the development of a new detection method using LAMP to identify qepA1, one variant of the qepA gene, was tried. As the results, the LAMP method using a qepA1-specific LAMP primer set comprising five primerscould detect all four qepA1-positive strains in addition to 17 qepA1-negative strains. The LAMP method is clearly much more advantageous for use in clinical laboratories. Furthermore, the time and accuracy benefits allow for the selection of antibiotics in a clinical setting

A Radioimmunoassay for Rat Serum Corticosterone

A reliable radioimmunoassay for rat serum corticosterone has been developed. 25 μ1 of diluted serum (1:100) was assayed with a specific antiserum raised against corticosterone-21-hemisuccinate conjugated to bovine serum albumin. The within-assay and between-assay coefficients of variation were 7.1% and 13.9%, respectively. The mean serum corticosterone concentration was 65.3±6.0 ng/ml (n=10). The corticosterone level increased to 208.4 ±23.7 ng/ml after ACTH administration, and was suppressed to the limit of assay sensitivity after dexamethasone administration

Steroidogenesis in Isolated Adrenal Cells of Rat

A reliable and reproducible system for the isolation of rat adrenal cells was developed, using 0.25% trypsin for cell dispersion. The suspending cell in Krebs-Ringer bicarbonate buffer containing 0.2% glucose and 0.5% bovine serum albumin was incubated for 120 minutes at 37℃ under 95% 02 and 5% C02. Corticosterone production induced by synthetic 1-24ACTH showed a dose-related increase in decapsular cells. The precision of the inter-experiment of corticosterone production was 5.0% (average coefficients of variation)

Analysis of Human Insulin Analogues in Vitro, Using Gel Chromatographic Method

The incubation medium and incubated human pancreas were gel chromatographed on the Bio-Gel P-30 column after extraction with acid ethanol. The extracted immunoreactive insulin (IRI) was successfully separated into two peaks at the position of 6000 molecular weight region. These two peaks corresponded to those which were detected in human serum. These findings suggest that the two groups of insulin are directly secreted from human pancreatic tissue. But the incorporated [3H] leucine peak into acid-ethanol extractable protein did not elute out at the same position as each insulin peak. Therefore, the measurement of [3H] leucine incorporation into acid-ethanol extractable protein is not a good indicator to evaluate insulin biosynthesis

Distribution and Elimination of Insulin and C-peptide in a Benign Insulinoma Patient

The distribution and elimination of insulin and C-peptide was evaluated in a case of benign insulinoma, using the method of gel chromatography. The significant differences between total Immunoreactive insulin (IRI) level and the level of Peak I plus Peak II of IRI was noticed in splenic vein. This fact suggested that intermediate and/or abnormal IRI could be released from the tumor. In order to diagnose a hypoglycemic patient with completely normal IRI and CPR level in peripheral blood, the gel chromatographic separation of IRI from splenic and/or portal blood could be useful

Genotyping of Mycoplasma pneumoniae strains isolated in Japan during 2019 and 2020: spread of p1 gene type 2c and 2j variant strains

We characterized 118 Mycoplasma pneumoniae strains isolated from three areas of Japan (Saitama, Kanagawa, and Osaka) during the period of 2019 and 2020. Genotyping of the p1 gene in these strains revealed that 29 of them were type 1 lineage (29/118, 24.6%), while 89 were type 2 lineage (89/118, 75.4%), thereby indicating that type 2 lineage was dominant in this period. The most prevalent variant of type 2 lineage was type 2c (57/89, 64%), while the second-most was type 2j, a novel variant identified in this study (30/89, 33.7%). Type 2j p1 is similar to type 2 g p1, but cannot be distinguished from reference type 2 (classical type 2) using the standard polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) with HaeIII digestion. Thus, we used MboI digestion in the PCR-RFLP analysis and re-examined the data from previous genotyping studies as well. This revealed that most strains reported as classical type 2 after 2010 in our studies were actually type 2j. The revised genotyping data showed that the type 2c and 2j strains have been spreading in recent years and were the most prevalent variants in Japan during the time-period of 2019 and 2020. We also analyzed the macrolide-resistance (MR) mutations in the 118 strains. MR mutations in the 23S rRNA gene were detected in 29 of these strains (29/118, 24.6%). The MR rate of type 1 lineage (14/29, 48.3%) was still higher than that of type 2 lineage (15/89, 16.9%); however, the MR rate of type 1 lineage was lower than that found in previous reports published in the 2010s, while that of type 2 lineage strains was slightly higher. Thus, there is a need for continuous surveillance of the p1 genotype and MR rate of M. pneumoniae clinical strains, to better understand the epidemiology and variant evolution of this pathogen, although M. pneumoniae pneumonia cases have decreased significantly since the COVID-19 pandemic