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S Tamatsukuri

Publications and source records attributed to S Tamatsukuri.

18 recordsLinked to original sources
PubMed2001

Automated multiplex assay system for simultaneous detection of hepatitis B virus DNA, hepatitis C virus RNA, and human immunodeficiency virus type 1 RNA.

We have developed an automated multiplex system for simultaneously screening hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) in blood donations. The assay, designated AMPLINAT MPX HBV/HCV/HIV-1 Test (AMPLINAT MPX), consists of virus extraction and target sequence-specific probe capture on specimen preparation workstation GT-X (Roche Diagnostics K.K., Tokyo, Japan) and amplification and detection by TaqMan PCR on the ABI PRISM 7700 Analyzer (Perkin-Elmer Applied Biosystems, Foster City, Calif.). An internal control (IC) is incorporated in the assay to monitor the extraction, target amplification, and detection processes. The assay yields qualitative results without discrimination of the three targets. Detection limits (95% confidence interval) are 22 to 60 copies/ml for HBV, 61 to 112 IU/ml for HCV, and 33 to 66 copies/ml for HIV-1, using a specimen input volume of 0.2 ml. The AMPLINAT MPX assay detects a broad range of genotypes or subtypes for all three viruses and has a specificity of 99.6% for all three viruses with seronegative specimens. In an evaluation of seroconversion panels, the AMPLINAT MPX assay detects HBV infection an average of 24 days before the detection of HBsAg by enzyme immunoassay. HCV RNA was detected an average of 31 days before HCV antibody. HIV-1 RNA was detected an average of 14 days before HIV-1 antibody and an average of 9 days before p24 antigen. The Japanese Red Cross has been evaluating the AMPLINAT MPX system since October 1999. The clinical performance indicates that the AMPLINAT MPX system is robust, sensitive, and reproducible, with a high percentage of valid assay runs (96.8%), a low false-positive rate (0.34%), and a low IC failure rate (0.24%).

Blood Donors↗
PubMed2000

Detection of hepatitis C virus in saliva before and after scaling of dental calculus.

Dentists and dental health care workers are at risk of contracting hepatitis C virus (HCV) through dental treatment, since HCV RNA was reported to be easily detectable in the saliva of patients with chronic HCV liver disease. We tested for the presence of HCV RNA in saliva before and after removal of dental calculus, and in splashes on the chin-length face shields of dentists following treatment of six patients with HCV chronic liver diseases. We used a sensitive reverse transcription polymerase chain reaction (RT-PCR) method, to estimate exposure to HCV. All patients were anti-HCV and HCV RNA seropositive. HCV RNAs in saliva before or after scaling treatment were detected in three of the six, but none of the face shields showed positive samples. In conclusion, dentists and dental health care workers should be aware of the possibility of HCV infection via contact with serum and saliva during dental practice.

Dental Calculus↗
PubMed1999

Spontaneous negativation of serum hepatitis C virus RNA is a rare event in type C chronic liver diseases: analysis of HCV RNA in 320 patients who were followed for more than 3 years.

BACKGROUND/AIMS: The natural course of hepatitis C virus (HCV) replication in type C liver diseases has not yet been elucidated. The aim of the study was to investigate the spontaneous outcome of the viremia by examining the changes in HCV RNA in patients with chronic type C liver diseases. METHODS: Among patients who visited our liver clinic between June 1981 and December 1993, 320 patients with chronic type C liver diseases were followed for at least 3 years and had no history of interferon treatment. HCV RNA was examined by a highly specific reverse transcription-polymerase chain reaction method in paired serum samples obtained from these patients at the beginning and end of follow-up. RESULTS: Among the 320 cases, HCV RNA was seropositive in 310 (97%) cases at the beginning of follow-up. Of these 310, HCV RNA remained seropositive in 304 (98%) and became seronegative in six (2%) cases by the end of follow-up. All of these six patients had liver cancer. HCV RNA became seronegative after the patients entered the state of liver failure because of the development of tumors or portal thrombosis. The remaining 10 cases who were seronegative for HCV RNA at the beginning were seropositive at the end of follow-up. Among the 320 cases, serum alanine aminotransferase normalized and remained normal for more than 12 months until the end of follow-up in 11 cases (0.6%/year/case), but none became negative for HCV RNA. CONCLUSIONS: Thus, during the natural course of chronic HCV infection, spontaneous negativation of serum HCV RNA seems extremely rare, at least in patients with chronic active hepatitis or cirrhosis of the liver, and may occur primarily at the terminal stage when tumors cause liver failure.

Aged↗
PubMed1996

Real-time monitoring of HCV-RNA by single tube assay kit and potential importance for predicting virological sustained response in patients with chronic hepatitis C.

In an attempt to predict virological sustained responders among patients with chronic hepatitis C after interferon therapy, HCV-RNA in serum was measured by a one tube RT-PCR assay kit using the RNA corresponding to 5 microL serum (standard assay) or 300 microL serum (enhanced-sensitivity assay). Dilution analysis revealed that sensitivity of the 'enhanced-sensitivity assay' increased by 10-100-fold when compared with a 'standard assay'. Using these assays, prospective study of interferon therapy on 38 HCV-RNA seropositive cases with chronic hepatitis (total amount 702 MU; duration of treatment 5-6 months) was performed. At the end of treatment, six were still positive and 32 became negative by the "standard assay', whereas an additional eight cases became positive (total 14 cases positive; the remaining 24 cases negative) by the 'enhanced-sensitivity assay'. Hepatitis C viral RNA state at the end of treatment remained the same 6 months later in 23 cases (61%) by a 'standard assay' and in 31 (82%) by the 'enhanced-sensitivity assay'. Of importance was that all patients (14 cases) demonstrating HCV-RNA in serum at the end of therapy, even by the "enhanced-sensitivity assay', did not show the disappearance of HCV-RNA in serum despite the long follow up. From these results, in order to improve our treatment efficacy, we should try to modify our treatment protocol to the extent that at least HCV-RNA becomes undetectable. That can be only feasible during treatment by real-time monitoring of HCV-RNA.

Adult↗
PubMed1996

[A novel RNA capture method for the extraction of HCV-RNA].

A novel method for the extraction of HCV-RNA by using DNA probe coated-latex microparticle (AmpliTex) was developed. This high density particle, with a large surface area, could be dispersed uniformly in a guanidium lysate of the serum. The DNA probe on the particles could hybridize with a few copies of HCV-RNA in the lysate. The RNA thus extracted on the particle can be subjected to the reverse-transcription and PCR reaction. Analytical sensitivity to detect HCV in serum was approximately 20 copies per ml. AmpliTex and the conventional organic extraction gave the same sensitivity and specificity (both 100%) to detect the HCV-RNA from fifty clinical specimens. It was also possible to extract the HCV-RNA from heparinated plasma.

DNA Probes↗
PubMed1996

[Development of a detection system for Helicobacter pylori DNA in gastric juice].

A rapid and sensitive PCR-based microwell plate assay (PCR-MWP) system to detect the 16 S ribosome RNA gene of Helicobacter pylori was developed. Analytical sensitivity, evaluated with purified recombinant plasmid DNA and genomic DNA of H. pylori, was one copy of DNA per PCR. Specificity was validated with a panel of DNA from 75 kinds of microorganisms including Helicobacter showed weak positives, when 1 pg of DNA was input. Other microorganisms gave negative signals even when 100 pg of DNA was used for PCR. When compared with a Nested-PCR system to detect the urease A-subunit gene performed by a commercial reference laboratory, the results obtained (sensitivity 93.3% and specificity 73.3%) was almost equivalent. The PCR-MWP was rapid and easy for the detection of H. pylori DNA in gastric juice specimen.

DNA, Bacterial↗
PubMed1995

[Prior enrichment of HIV-DNA with probe-DNA particles for an efficient PCR diagnosis].

PCR mediated detection of HIV-DNA has been widely used. However, compared with traditional immunological diagnoses, the extraction of DNA is a laborious and time consuming step. We have developed a procedure for the efficient isolation and concentration of HIV-DNA from cell lysates. We report here a novel method by one can recover HIV-DNA in a small volume (approximately 50 microliters) of solution from a large volume of crude cell lysate which contains as few as several copies. The method uses the specific hybridization of HIV-DNA to HIV probe-DNA particles. This prior enrichment augmented the sensitivity in the detection of HIV-DNA by PCR, and allows us to make a diagnosis even if the specimen contained an extremely low copy number of HIV-DNA molecules in a large volume, which would have otherwise resulted in false-negative data with the conventional extraction method. The method also enables the examination of 100 individual blood specimens in a combined form. Thus, the application of the present enrichment procedure with HIV probe-DNA particles should reduce the labor and cost of HIV diagnosis, since the HIV positive samples represent a very minor group of people among specimens subjected to clinical laboratory tests, and particularly, among blood samples voluntarily donated to be used for transfusions.

Base Sequence↗
PubMed1995

Prior enrichment of human immunodeficiency virus DNA with probe DNA particles for efficient PCR diagnosis.

We report here a novel method by which one can recover human immunodeficiency virus (HIV) DNA in a small volume (approximately 50 microliters) of solution from 8 ml of crude cell lysate which contains as few as several HIV DNA copies. The method uses the specific binding of HIV DNA to HIV probe DNA particles. The HIV DNA thus concentrated on the particles can be subjected to the PCR assay. The method also enables the examination of 100 individual blood specimens in a combined form.

Base Sequence↗
PubMed1993

Application of oligo(dT)30-latex for rapid purification of poly(A)+ mRNA and for hybrid subtraction with the in situ reverse transcribed cDNA.

The carboxyl groups on the surface of latex beads were linked to amino moiety of cytidine residue of oligo(dC)10(dT)30. The resultant latex beads-(dC)10(dT)30 showed a very stable suspension and yet is precipitable to a small pellet by centrifugation. These properties merits the oligomer-linked beads to be applied for experiments in which poly(A)+ mRNAs are involved. An efficient (> 95%) hybridization to poly(A)+ mRNA occurred in a short reaction period (10 min), and more than 95% of bound mRNAs were recovered from the beads by heating (65 degrees C, 5 min) followed by centrifugation. Interestingly, the poly(A)+ mRNAs could be transcribed to cDNAs in situ by reverse transcriptase, with the covalently linked oligo(dT)30 as primers. These properties allowed the oligo(dT)30-latex to prepare the cDNA covalently bound to latex which was used for mRNA hybrid subtraction. In a model experiment with the mixture of vaccinia virus and HeLa mRNAs, about 200-fold enrichment of vaccinia mRNA species was obtained after four cycles of hybrid subtraction with HeLa cDNA-latex.

DNA↗
PubMed1992

A new method for constructing NotI linking and boundary libraries using a restriction trapper.

We have developed a novel method for constructing NotI linking and boundary libraries using a modified "solid-supported ligation primer" (restriction trapper). The restriction trapper could be used to purify the DNA fragments with a specific restriction enzyme cutting site(s) at their ends. The method uses a ligation and recutting reaction with double-stranded DNA ends of a hairpin-shaped oligolinker which is connected covalently to the surface of the latex beads. Selectivity is based on the specificity of the restriction enzyme for its recognition site, resulting in efficient purification. We applied this technique to the construction of high-quality NotI linking and NotI boundary libraries, which contain almost all the NotI sites of the genome and, in addition, are free of illegitimately ligated clones.

Base Sequence↗
PubMed1992

Enzyme-labeled oligonucleotide probes for detection of the genes for thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus.

Alkaline phosphatase conjugated oligonucleotide probes were developed to detect the genes (tdh and trh) coding for the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus. Using dot blot hybridization, probes were tested with 94 clinical isolates of V. parahaemolyticus. Results agreed well with those obtained using radio-labeled recombinant DNA probes for the genes tdh and trh. Specificity and sensitivity of enzyme tdh probes for detection of the trh gene were 100 and 93%, respectively, and those of the trh probes for trh gene detection were 93 and 86%, respectively. The tdh probes also hybridized with tdh-like genes processed by all strains of V. hollisae, and some strains of V. mimicus and V. cholerae non-O1, but neither tdh nor trh probes reacted with other bacterial species isolated from diarrheal stools. However, some V. parahaemolyticus strains that were negative with the enzyme trh probe hybridized weakly with a radio-labeled trh DNA fragment probe at medium stringency, and a few strains that were negative in high stringency conditions with a radio-labeled trh DNA fragment probe hybridized with the enzyme trh probe. This suggests that some strains of V. parahaemolyticus may carry another gene resembling trh.

Alkaline Phosphatase↗
PubMed1991

Detection of a heat-labile enterotoxin gene in enterotoxigenic Escherichia coli by densitometric evaluation using highly specific enzyme-linked oligonucleotide probes.

Two alkaline phosphatase-conjugated 24-mer oligonucleotide probes were developed to detect the heat-labile enterotoxin gene in enterotoxigenic Escherichia coli. Probes were antisense codon sequences, which are transcribed into mRNA, of the heat-labile enterotoxin gene of enterotoxigenic Escherichia coli of human origin. Using dot-blot hybridization, probes were tested with 100 clinical isolates and evaluated by a reflectance-type densitometer. Results agreed very well with those of an immunological test, the Biken test, and a 32P-labelled recombinant DNA probe. The oligonucleotide probes did not react with nucleic acids prepared from other diarrhoeagenic bacterial pathogens. Thus, the alkaline phosphatase-conjugated oligonucleotide probes seem to be highly sensitive and specific for detection of heat-labile enterotoxin-producing enterotoxigenic Escherichia coli. Moreover, the results indicate a potential usefulness for densitometric evaluation of DNA hybridization.

Alkaline Phosphatase↗
PubMed1987

Solid phase synthesis of oligoribonucleotides using the o-nitrobenzyl group for 2'-hydroxyl protection and H-phosphonate chemistry.

Oligoribonucleotides with chain length of 7, 11, 15, 17, 24 and 34 were synthesized on long chain alkylamine controlled pore glass beads (LCA-CPG) using o-nitrobenzyl protection of 2'-hydroxyls via a H-phosphonate approach either manually or by using an automatic synthesizer. The oligoribonucleotides were obtained in yields of 0.6 0.6-20%, based on initial nucleoside bound to the LCA-CPG support.

Automation↗
PubMed1986

Solid phase synthesis of oligoribonucleotides using o-nitrobenzyl protection of 2'-hydroxyl via a phosphite triester approach.

The hepta and undecaribonucleotide were synthesized on a controlled pore glass beads using o-nitrobenzyl protection of 2'-hydroxyls via a phosphite approach. By using 5-p-nitrophenyltetrazole for the activation of nucleoside-phosphoramidite, the condensation reaction was carried out very rapidly (2.5 min). The time required for one cycle was only 16 min. The hepta-(UACUAAC) and undecaribonucleotides (GUAUGUUAAUA) were obtained in yields of 28 and 17% respectively from the original resin.

Methods↗
PubMed1986

Synthesis of 5'-phosphorylated oligonucleotides on a polymer support.

Highly lipophilic trityloxyethylamine (TEAm) or trityloxyethylaniline (TEAn) were prepared and used for the protection of the 5'-terminal phosphate. We synthesized 5'-phosphorylated oligodeoxyribonucleotides by the solid phase synthesis. TEAm or TEAn groups enabled us to isolate the desired oligonucleotide easily by the reversed phase column due to their high lipophilicity and could be removed by the aq. AcOH treatment.

Base Sequence↗