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 (PerkinElmer 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%).
The amounts of volatile substances responsible for the malodor of human waste (feces and urine) obtained from the storage tank of a community waste-water treatment plant were determined. Thus far, there has been little systematic research on malodor-causing substances of human waste. These substances were collected using Tenax-TA, and their concentrations were determined by the usual thermal-desorption coldtrap injector/gas chromatography/mass spectrometry (TCT/GC/MS). About 90% of the malodor-causing substances were fatty acids: acetic acid, propionic acid and butyric acid. The proportion of ammonia was 6.5%. Other malodor-causing and minor substances detected were indole, skatole, pyridine, pyrrole, hydrogen sulfide, and methyl mercaptan. In addition, a small amount of paradichlorobenzene used as a deodorizer in household toilets was also recognized.
Abstract:The first nationwide nucleic acid amplification testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-l) of voluntarily donated blood after serological pre-screening and before release of cellular components and plasma for fractionation was implemented by the Japanese Red Cross Blood Transfusion Services. From February 1, 2000 to April 30, 2001, specimens from 6,805,010 units of serologically negative donation were screened in minipools of 50 samples within 24 hr after blood donation by NAT using multiplex HBV/HCV/HIV-l reagent for blood transfusion including short shelf-life platelets. Among them, 112 HBV DNA-positives, 25 HCV RNA positives and 4 HIV-l RNA positives were screened out and we could prevent transfusion of these NAT positive units. Subtypes/genotypes of HBV DNA, adr/C, adw/A, adwlB, adw/C, ayr/C and aywlD were found and adr/C was predominant. A total of 61.6 % of them (69/112) were negative by overnight EIA. Sixth three of HBV NATpositive samples carried virus loads less than 10 4 copies ImL and 92.1 % of them (58/63) were negative by overnight EIA. The virus growth curves of HBV in 6 cases obtained by retrospective and prospective follOW-Up study showed exponential straight lines in the early stage of serological window periods and the log times ofHBV growth (10 fold increase) in serological window period were between 4.6 and 7.6 days. NAT screening with highly sensitive reagents in pool of specimens is useful to exclude blood units with low level of HBV and HBV mutants from blood transfusion.
The conformational conversion of prion protein (PrP) from an alpha-helix-rich normal cellular isoform (PrPC) to a beta-sheet-rich pathogenic isoform (PrPSc) is a key event in the development of prion diseases, and it takes place in caveolae, cavelike invaginations of the plasma membrane. A peptide homologous to residues 106-126 of human PrP (PrP106-126) is known to share several properties with PrPSc, e.g., the capability to form a beta-sheet and toxicity against PrPC-expressing cells. PrP106-126 is thus expected to represent a segment of PrP that is involved in the formation of PrPSc. We have examined the effect of lipid membranes containing negatively charged ganglioside, an important component of caveolae, on the secondary structure of PrP106-126 by circular dichroism. The peptide forms an alpha-helical or a beta-sheet structure on the ganglioside-containing membranes. The beta-sheet content increases with an increase of the peptide:lipid ratio, indicating that the beta-sheet formation is linked with self-association of the positively charged peptide on the negatively charged membrane surface. Analogous beta-sheet formation is also induced by membranes composed of negatively charged and neutral glycerophospholipids with high and low melting temperatures, respectively, in which lateral phase separation and clustering of negatively charged lipids occur as shown by Raman spectroscopy. Since ganglioside-containing membranes also exhibit lateral phase separation, clustered negative charges are concluded to be responsible for the beta-sheet formation of PrP106-126. In caveolae, clustered ganglioside molecules are likely to interact with the residue 106-126 region of PrPC to promote the PrPC-to-PrPSc conversion.
Inhibitors of enzymatic amplification in serum may cause false-negative results for direct detection of hepatitis C virus (HCV) by polymerase chain reaction (PCR). This study was undertaken to demonstrate the importance of the internal control in a PCR assay for detection of HCV-RNA to monitor false-negatives due to inhibitors. HCV-RNA was extracted using RNA extraction kit (SepaGene RV-R, Sanko Junyaku) and a prototype instrument for automated specific capture of HCV-RNA with probes and magnetic bead/fluid separation (Roche Molecular Systems). The extracted HCV-RNA and internal control were detected by an automated PCR machine (Cobas Amplicor, Roche Diagnostic Systems). Addition of hemoglobin (up to 4.5 g/l) to the sera followed by RNA extraction with SepaGene RV-R had no inhibitory effect on the detection of either HCV-RNA or the internal control. In contrast, addition of heparin to the sera showed an inhibitory effect with a dose-dependent manner on the detection of both HCV-RNA and the internal control, with a greater effect at lower copy number of HCV. When HCV-RNA was extracted by the automated system, the inhibitory effect of heparin was successfully eliminated. In the assays of 65 serum samples positive for anti-HCV antibodies, positivity for the internal control indicated efficient amplification and validated 14 negative and two equivocal results for detection of HCV-RNA. Detection of the internal control was negatively correlated with viral copy number in sera suggesting competitive inhibition of high viral copy number on amplification of the internal control. Extraction, co-amplification and detection of the internal control appears useful for estimating effects of inhibitors on amplification in each assay for the detection of HCV-RNA, and for evaluating efficacy of RNA extraction methods.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.