Highly sensitive multiplex assay for detection of human immunodeficiency virus type 1 and hepatitis C virus RNA.

Various nucleic acid assays have been developed and implemented for diagnostics and therapeutic monitoring of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections. The high-throughput, semiautomated assays described here were developed to provide a method suitable for screening plasma specimens for the presence of HIV-1 and HCV RNAs. Three assays were developed: a multiplex HIV-1/HCV assay for simultaneous detection of HIV-1 and HCV, and discriminatory assays for specific detection of HIV-1 and HCV. The assay systems utilize three proprietary technologies: (i) target capture-based sample preparation, (ii) transcription-mediated amplification (TMA), and (iii) hybridization protection assay (HPA). An internal control is incorporated into each reaction to control for every step of the assay and identify random false-negative reactions. The assays demonstrated a sensitivity of at least 100 copies/ml for each target, and they detected with similar sensitivity all major variants of HCV and HIV-1, including HIV-1 group O strains. Assay sensitivity for one virus was not affected by the presence of the other. The specificity of these TMA-driven assays was >or=99.5% in both normal donor specimens and plasma containing potentially interfering substances or other blood-borne pathogens. Statistical receiver operating characteristic plots of 1 - specificity versus sensitivity data determined very wide analyte cutoff values for each assay at the point at which the assay specificity and sensitivity were both >or=99.5%. The sensitivity, specificity, and throughput capability predict that these assays will be valuable for large-volume plasma screening, either in a blood bank setting or in other diagnostic applications.

Is the learn unit a fundamental measure of pedagogy?

We propose a measure of teaching, the learn unit, that explicitly describes the interaction between teachers and their students. The theoretical, educational research, and applied behavior analysis literatures all converge on the learn unit as a fundamental measure of teaching. The theoretical literature proposes the construct of the interlocking operant and embraces verbal behavior, social interaction, and translations of psychological constructs into complex theoretical respondent-operant interactions and behavior-behavior relations. Research findings in education and applied behavior analysis on engaged academic time, opportunity to respond, active student responding, teacher-student responding, student-teacher responding, tutor-tutee responding, tutee-tutor responding, and verbal episodes between individuals all support a measure of interlocking responses. More recently, research analyzing the components of both the students' and teachers' behavior suggests that the learn unit is the strongest predictor of effective teaching. Finally, we propose applications of the learn unit to other issues in pedagogy not yet researched and the relation of learn units to the verbal behavior of students.

Construction of single amino acid substitution mutants of cloned Bacillus stearothermophilus DNA polymerase I which lack 5'-->3' exonuclease activity.

Two individual amino acid substitutions were engineered at a selected site in the 5' --> 3' exonuclease domain of the cloned Bacillus stearothermophilus DNA polymerase I gene. These mutations resulted in the expression of enzymes lacking the 5' --> 3' exonuclease activity while maintaining normal polymerizing activity. The mutated and non-mutated enzymes were each constitutively expressed in an Escherichia coli host without the use of an exogenous or inducible promoter, and the mutated enzymes were demonstrated to be equivalent to the subtilisin large fragment of the native holoenzyme in sequencing reactions.

Rapid and quantitative detection of enzymatically amplified HIV-1 DNA using chemiluminescent oligonucleotide probes.

A hybridization protection assay (HPA) that uses acridinium ester (AE) labeled oligonucleotide probes which are specific for a conserved gag gene region of human immunodeficiency virus type 1 (HIV-1) was developed to measure the amount of HIV-1 nucleic acid. Hybridization of the single-stranded probes with their target HIV-1 sequences protected the chemiluminescent AE group from subsequent alkaline hydrolysis. The chemiluminescence from the residual AE could be easily quantitated in a luminometer. The entire process comprising template dissociation, hybridization, alkaline hydrolysis, and chemiluminescence measurement can be completed in less than one hour and does not require the separation of hybridized probe from unhybridized probe. We demonstrated that HPA could quantitatively measure the amount of DNA amplified by polymerase chain reaction. A comparative study using amplified DNA from the peripheral blood mononuclear cells (PBMC) of HIV seropositive and seronegative persons showed that HPA was as sensitive as the previous methods using 32P-labeled DNA probes.

Cell-related sequences in the DNA genome of human cytomegalovirus strain AD169.

Human cytomegalovirus (HCMV) cloned EcoRI fragments R and b hybridized strongly, under standard high-stringency conditions, to uninfected cellular DNA of human, murine, or sea urchin origin. Less hybridization was detected with fragments, A, C, E, WL(F), WN(H), I, M, O, P, Q, V, c, d, and e. Southern blot analysis of the HCMV-related human DNA localized the major sites of hybridization of HCMV EcoRI fragments R, b, and d to defined regions of the 28S rRNA gene.

Analysis of the major transcripts encoded by the long repeat of human cytomegalovirus strain AD169.

In this report, we describe the size and kinetics of appearance of RNAs from the long repeat of human cytomegalovirus. The most abundant RNA from this region was a 2.7-kilobase (kb) species that was detected throughout the infection and was most abundant at 27 and 72 h after infection. The 2.7-kb RNA was the only major species detected with a probe that included the terminus of the long repeat and the heterogeneous L-S junction region. Other transcripts were detected with probes from the internal portion of the long repeat, including an immediate-early RNA of 1.3 kb, early and late RNAs of 1.2 kb, and minor late transcripts of 4.4, 3.6, 3.3, and 1.8 kb. S1 nuclease and exonuclease VII protection analyses of RNA from immediate-early, early, midpoint, and late times in the infection indicated that the major 2.7-kb RNA was not spliced and that the RNA mapped within the long repeat, 1.6 kb from the heterogeneous region. No evidence for temporally regulated changes in transcription initiation, splicing, or choice of 3' end of this RNA was observed. Nuclease protection analysis also demonstrated that the second most abundant late RNA from this region, the 1.2-kb species, was not spliced and had the same polarity as the 2.7-kb RNA. The 1.2-kb also mapped entirely within the long repeat, with its 3' terminus 1.7 kb upstream from the 5' terminus of the 2.7-kb RNA.

Transcription in human fibroblasts permissively infected by human cytomegalovirus strain AD169.

We used cloned subgenomic DNA fragments of human cytomegalovirus strain AD169 as hybridization reagents to analyze the sites of transcription and abundance of viral RNA in permissively infected human embryonic lung cells. RNA extracted from immediate early, early, middle, and late times in the infection was attached to filters and hybridized with excess cloned subgenomic fragments. Each hybridization was performed with an internal standard to allow quantitation of the RNA concentration and standardization for the variation in probe complexity and specific activity. We found immediate early transcription in the AD169 strain occurring primarily from three regions in the long unique segment at 0.061-0.110, 0.117-0.142, and 0.588-0.616 map units. A low level of transcription was also detected from the long unique segment at 0.230-0.372, 0.419-0.437 and 0.703-0.707 map units and the short unique segment and terminal repeats at 0.00-0.046, 0.776-0.854, and 0.892-1.00 map units. At 8 hr after infection in the presence of de novo protein synthesis, the transcription pattern was changed. Two of the major immediate early sites at 0.061-0.110 and 0.117-0.142 map units were represented less abundantly in the absence of cycloheximide while the site at 0.588-0.616 map units was represented at the same level. The most abundant early RNA was synthesized from the terminal repeat sequences and part of the short unique segment at 0.00-0.046, 0.776-0.822, and 0.892-1.00 map units. Steady-state RNA from the midpoint of the infection hybridized with most regions of the genome. Abundantly transcribed regions included the abundant early sites mentioned above (0.00-0.046, 0.588-0.616, 0.776-0.822, and 0.892-1.00 map units), several new sites in the long unique segment at 0.104-0.110, 0.576-0.588, 0.653-0.664, 0.703-0.707, and 0.766-0.776 map units and the L-S junction fragment at 0.804-0.854 map unit. RNA from late in the infection hybridized to all subgenomic fragments. The sites of de novo RNA synthesis along the genome were determined by hybridizing pulse-labeled RNA to individual cloned subgenomic fragments attached to filters. Transcription from the terminal repeat sequences and the long unique segment at 0.576-0.588 map unit accounted for 40% of the total viral RNA synthesized from 22 to 28 hr after infection. Of the total de novo RNA synthesis from 71 to 77 hr after infection, 4.3% was virus specific and 47% of the total de novo synthesized viral RNA hybridized to the terminal repeat sequences.