PubMed · 3187491
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T M Beardsley. 1988. Supertests.. https://doi.org/10.1038/scientificamerican0288-34
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Three highly sensitive reverse transcriptase (RT) assays were recently published that are at least one million times more sensitive than conventional RT assays. These assays derive their high sensitivities through the ability to amplify the complementary DNA (cDNA) product of the RT reaction by the polymerase chain reaction (PCR). We describe a modified PCR-based RT (PBRT) assay that retains the high sensitivities of the original assays while reducing their inherent background signals. The background signal of the PBRT assay was found to be due to an intrinsic RNA-dependent DNA polymerase activity of the Taq DNA polymerase, the enzyme used for the PCR. It could be eliminated by inserting a ribonuclease digestion step prior to amplifying the cDNA product of the RT reaction by PCR and by using a thermostable DNA polymerase identified as having reduced RNA-dependent DNA polymerase activity. Comparable results were obtained using three RNA templates with two purified RT enzymes. This modified assay is capable of detecting reliably between 10 and 100 molecules of RT, which is equivalent to between 1 and 10 retrovirus particles.
Infection with hepatitis B virus can result in asymptomatic seroconversion with viral clearance, fulminant hepatic failure and death, or chronic, typically lifelong, transmissible infection. The mechanism(s) of viral persistence are poorly understood but viral clearance and fulminant hepatic failure are generally thought to result from co-ordinated and effective and abnormally vigorous immune responses, respectively, whereas viral persistence results from immunological failure in addition to poorly characterized viral factors promoting persistence. This paper proposes (1) that the predominant viral factor(s) promoting persistence of hepatitis B virus are homeostatic mechanism(s) responsible for modulating its replication and mutation and (2) that chronic hepatitis B results when these mechanisms are successful and other outcomes occur when these homeostatic mechanism(s) fail. Furthermore, it is proposed that seroconversion (e.g. from HBsAg to anti HBsAg positivity), when it occurs, is a consequence facilitated by restricted viral antigenic diversity and reduced viral replication rather than a proximate cause of it. The specific homeostatic mechanisms proposed--negative feedback inhibition of hepatitis B virus DNA polymerase/reverse transcriptase mediated by HBs antigen and a hepatitis B virus DNA polymerase fidelity modulating function of HBeAg--are consistent with the available data and resolve many paradoxical clinical observations. But, more importantly, this model has clear implications for therapy, including the rational design of drugs and therapeutic vaccines.
We describe a method for multiplex detection of mutations in which the solid-phase minisequencing principle is applied to an oligonucleotide array format. The mutations are detected by extending immobilized primers that anneal to their template sequences immediately adjacent to the mutant nucleotide positions with single labeled dideoxynucleoside triphosphates using a DNA polymerase. The arrays were prepared by coupling one primer per mutation to be detected on a small glass area. Genomic fragments spanning nine disease mutations, which were selected as targets for the assay, were amplified in multiplex PCR reactions and used as templates for the minisequencing reactions on the primer array. The genotypes of homozygous and heterozygous genomic DNA samples were unequivocally defined at each analyzed nucleotide position by the highly specific primer extension reaction. In a comparison to hybridization with immobilized allele-specific probes in the same assay format, the power of discrimination between homozygous and heterozygous genotypes was one order of magnitude higher using the minisequencing method. Therefore, single-nucleotide primer extension is a promising principle for future high-throughput mutation detection and genotyping using high density DNA-chip technology.