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Biomedical subjects

M J Pallen

Publications and source records attributed to M J Pallen.

9 recordsLinked to original sources
PubMed1992

Degenerate PCR primers for the amplification of fragments from genes encoding response regulators from a range of pathogenic bacteria.

Many bacterial responses to environmental stimuli are mediated by response regulators which coordinately regulate genes involved in particular adaptive responses. Degenerate oligonucleotide primers were used to amplify by the polymerase chain reaction (PCR), fragments from genes encoding eleven novel response regulators. Sequence and phylogenetic analysis revealed that phoB, phoP and creB gene fragments had been amplified from Yersinia enterocolitica and Yersinia pseudotuberculosis, and that a creB sequence had been amplified from Campylobacter jejuni. Four amplified fragments from C. jejuni, Listeria monocytogenes, Mycobacterium tuberculosis and Escherichia coli clearly came from response regulator genes, but were not closely related to any of the known genes. Mutagenesis of the newly identified genes should allow us to determine their function and the genes under their control.

Amino Acid Sequence↗
PubMed1991

New strategies in microbiological diagnosis.

With the advent of the polymerase chain reaction (PCR), molecular biology is at last poised to enter the clinical microbiology laboratory. We describe this technique, and review its present and future applications in the diagnosis of infectious disease, with particular emphasis on its potential in diagnostic bacteriology. We discuss the suitability of different sequences as targets for DNA amplification. The disadvantages of PCR as a diagnostic strategy are covered, and current technical problems with the method are surveyed. We briefly mention two alternative strategies--the transcript-based amplification system and replicatable RNA reporter systems based on the Q beta replicase.

Gene Amplification↗
PubMed1991

Rapid screening for toxigenic Corynebacterium diphtheriae by the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to discriminate between toxigenic and non-toxigenic isolates of Corynebacterium diphtheriae. Primers specific to the diphtheria toxin gene were used to amplify a toxin gene fragment from simple boiled-cell preparations. Eight recent clinical isolates and four reference strains were tested. The result of the PCR agreed with the traditional toxigenicity assays (the Elek test and guinea pig inoculation) in all cases. PCR has several advantages over the Elek test: it gives a same-day result, it works on colonies taken from selective media, and it detects the toxin gene in mixed cultures. One potential drawback is that the PCR might give a false positive result with the occasional isolate carrying an inactive toxin gene. The good predictive value of a negative PCR result, however, should make it a valuable screening test.

Base Sequence↗
PubMed1989

The 45 kilodalton molecule of Mycobacterium tuberculosis identified by immunoblotting and monoclonal antibodies as antigenic in patients with tuberculosis.

The object of this study was to discover new M. tuberculosis antigens which are recognized by patients with tuberculosis, because effective serodiagnostic tests are likely to require combinations of different antigens. In our early experiments using immunoblotting, the findings suggested that human sera from smear-negative tuberculosis patients bound to an antigen in the 45 kDa region. Subsequently, estimates of molecular weight in the immunoblots confirmed that the murine monoclonal antibody (MAB) HGT-6 and sera from patients both recognized the same 45 kDa molecule. An antibody-antibody competition assay between MAB HGT-6 and sera from smear-positive tuberculosis patients yielded a positive result in 23 out of 43 sera from patients, but in only four out of 23 from controls. This is further evidence that the 45 kDa antigen is recognized by tuberculous patients. We analysed whether a combination of the 45 kDa antigen results and those of known antigens might better discriminate between minimal smear-negative disease and healthy controls than could test with single antigens. There is no clinically useful laboratory test for smear-negative tuberculosis. In immunoblotting, combining the results with the 65, 45, 38 and 10 kDa antigens gave the best discrimination. This suggests that future serodiagnostic tests for minimal disease, such as the antibody-antibody competition assay, should contain a MAB against the 45 kDa antigen and possibly also against the 10 kDa antigen.

Antibodies, Monoclonal↗