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E Hidaka

Publications and source records attributed to E Hidaka.

45 records · Page 3Linked to original sources
PubMed1998

[Clinical evaluation of RT-PCR method for detection of HCV-RNA: second report availability of RT-PCR system using PCR internal control for detection of HCV-RNA and the influence of interfering substances on the detection].

The COBAS Amplicor system is an automated diagnostic PCR system which contains a PCR internal control (P. I. C.) template to monitor the amplification. The applicability of COBAS Amplicor HCV was examined using sera of patients with hepatitis. Furthermore, the effects of possible interfering substance (total protein, triglyceride, hemoglobin, glucose, total bilirubin, heparin, lysis reagent including guanidium) on HCV-RNA detection were investigated. The sensitivity of COBAS Amplicor HCV was equivalent to the manual method of Amplicor HCV, moreover all of the results in 54 clinical samples analyzed on both COBAS Amplicor HCV and Amplicor HCV were in agreement. Detection sensitivity of HCV-RNA decreased in the presence of total bilirubin and heparin. Ten and 25mg/dl of total bilirubin affected HCV-RNA detection but did not affect P. I. C. This result suggested that total bilirubin interfered with the protein denature caused by the lysis reagent. Fifteen U/ml of heparin in the sample completely inhibited amplification both of the HCV-RNA and P. I. C. One U/ml of heparin did not affect amplification, but heparinized blood samples should not be used for the detection of HCV-RNA. To examine the effect of possible carry over contamination on the lysis reagent which contains guanidium, various concentrations of lysis reagent in P. I. C. were tested. RT-PCR was inhibited by 1/500 volume contamination of lysis reagent in specimen diluent. Other substances did not affect the sensitivity. Our results indicate that the carryover contamination of lysis reagent cause more "false negative" results than interfering substances in sera. In conclusion, HCV-RNA detection system containing P. I. C., such as COBAS Amplicor HCV, will become a very useful to differentiate "false negative" and "true negative" result.

Bilirubin↗
PubMed1997

[Clinical evaluation of RT-PCR method for detection of HCV-RNA--with special reference to Amplicor HCV].

Detection of the viral genome in serum is the most reliable way to analyze HCV viremia. In the present study, we evaluated the availability of a new detection kit for HCV-RNA, Amplicor HCV, which is based on the RT-PCR microplate hybridization protocol. The procedure of Amplicor HCV is simple, unlike the conventional RT-nested-PCR method. Although Amplicor HCV assay exhibited the lower sensitivity than the the conventional RT-nested-PCR method (10 x for HCV type 1a, 1b and 2b; 10(3) x for type 2a), Amplicor HCV assay could detect the HCV-RNA in all HCV-RNA positive cases by conventional RT-nested PCR method, except for one case who contained low concentration of HCV-RNA (10 copies/ml). The coincidence rate was 99.2% in 120 clinical samples between two assays. Amplicor HCV assay, moreover, could efficiently evaluate the viremia regardless of anti-HCV-2 antibody titer. This assay was useful for monitoring the effect of interferon therapy during and after administration. These results suggest that Amplicor HCV has an excellent availability for the clinical laboratories.

Hepacivirus↗
PubMed1997

[Differentiation of selected abnormal hemoglobin by PCR-RFLP method].

Abnormal hemoglobin is one of the most frequent genetic disorder, of which about 700 variants have been reported in the world. Some abnormal hemoglobins such as HbS and HbC are endemic among limited human populations. We have found five subjects with abnormal hemoglobin in routine hematology analysis using an automated system Sysmex NE-7000. We isolated genomic DNA from the peripheral blood leukocyte of each subject, and amplified a 558 bp fragment of DNA including the entire exons 1 and 2 as well as intron 1 of the beta-globin gene. The amplified DNA fragments were subjected to direct sequencing by the dideoxy termination method on an automated DNA sequencer. The sequence analysis showed the abnormalities including HbA/C, HbS/C, HbE/E, and HbA/G-Szuhu. In the present study, we report a simple method for the mutual differentiation of these abnormal hemoglobins using restriction fragment length polymorphism (PCR-RFLP) method. Digestion of the amplified fragments described above with a restriction enzyme MnlI gave different RFLP patterns for HbA, HbC or S, HbE, and for HbG-Szuhu. RFLP using another enzyme, DdeI, could distinguish for HbS from HbC.

Hemoglobins, Abnormal↗
PubMed1995

[Detection and identification of mycobacteria by PCR-RFLP method].

The frequency of isolation of Mycobacterium tuberculosis has reported to be decreased, however, Mycobacterium species including MOTT (Mycobacteria other than M. tuberculosis complex) still remain to be the important bacteria causing opportunistic infection. Two clinical procedures have long been adopted to date for the detection of acid fast bacteria belonging to the family Mycobacteriaceae. One is the direct microscopic examination of the specimen stained by Ziehl-Neelsen method, which is rapid, but is relatively insensitive and less specific. And another is a time- consuming culturing technique on slants of Ogawa's media, which takes 4 to 8 weeks of incubation. Recently, various methods to detect mycobacterial DNA have been reported. However, they require a complicated and tedious processes. We represent here a simple method for the detection and identification of Mycobacteria (M. tuberculosis, Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium chelonae) using PCR-RFLP method for the 65kDa antigen with high sensitivity and specificity. This includes the amplification of Mycobacterial gene encoding a part of the 65kDa antigen. Subsequently, we sequenced the region amplified by PCR from the seven standard bacterial strains. As the results, we found that HaeIII restriction enzyme is suitable for the prompt and easy discrimination among the seven strains examined. These findings led us to conclude that this method is time-saving and possibly applicable for the rapid detection of Mycobacterial species including MOTT from clinical specimens in routine clinical laboratories.

Base Sequence↗
PubMedDate not supplied

Losses of heterozygosity on chromosomes 17p and 9p/18q may play important roles in early and advanced phases of gallbladder carcinogenesis.

PURPOSE: This present study aimed to investigate the genetic changes in gallbladder carcinogenesis. METHODS: Eleven intramucosal gallbladder carcinomas were compared with 31 invasive lesions for loss of heterozygosity (LOH) on chromosomes 5q, 9p, 17p and 18q, frame-shift mutations in a ten-adenine repeat site within the gene encoding the transforming growth factor beta type II receptor (TGFbetaRII) and an eight-guanine repeat site within BAX, and point mutations in codon 12 of Ki-ras. RESULTS: The incidences of LOH in intramucosal and invasive carcinomas were 14% and 17% on 5q, 9% and 52% on 9p, 64% and 65% on 17p, and 13% and 32% on 18q. No frame-shift mutations were found at TGFbetaRII or BAX, and point mutations in codon 12 of Ki-ras were present in only 8% of the samples. Thus, LOH on 17p was by far the most frequent lesion with similar results in both intramucosal and invasive carcinomas. In contrast, the frequency of LOH on 9p and 18q was distinctly higher in invasive lesions. CONCLUSION: The present data suggest that LOH on 17p may play an important role in the evolution of gallbladder carcinoma from a relatively early phase, while LOH on 9p and 18q may play roles in progression.

Adenine↗
PubMedDate not supplied

Ultrastructural features of adenoma malignum of the uterine cervix: demonstration of gastric phenotypes.

Histochemical staining has shown that so-called adenoma malignum (the mucinous type of minimal deviation adenocarcinoma [mucinous MDA]) of the uterine cervix expresses gastric phenotypes. The present ultrastructural study was undertaken to explore the fine structure and phenotypic expression of this tumor, and to make comparisons with normal cervical glands and gastric pyloric mucosa. Post-embedding, double-immunogold staining for gastric gland mucous cell mucin (HIK1083-reactive mucin) and lysozyme revealed localization exclusively to the matrix and to the core of the mucin granules, respectively, both in mucinous MDA and gastric pyloric mucosa. Mucin granules of normal cervical gland cells lacked core structures and showed no immunoreactivity with HIK1083 or lysozyme. Thus, mucinous MDA was confirmed to be a tumor expressing gastric phenotypes ultrastructurally. Both markers should be useful for the identification of tumor cells.

Adenocarcinoma, Mucinous↗