1887

Abstract

A universal stool extraction method for recovery of nucleic acids (NAs) from gastrointestinal pathogens was developed to support rapid diagnostics for the London 2012 Olympics. The method involved mechanical disruption (bead beating) of the stools, followed by automated extraction and detection using real-time PCR. This method had been used extensively in the Second Infectious Intestinal Disease Study (IID2) for the isolation of NA from bacteria and parasites (and was effective for the robust recovery of spp.) but had not been used for enteric viruses. To ensure this method was universally suitable, panels of samples known to contain target bacteria, viruses or parasites were processed in triplicate using the pre-treatment method routinely used for each target and the new extraction method (bead beating). The extracts were tested using real-time PCR and the cycle threshold values were compared. The results from this study showed that bead beating improved yields for the bacterial and parasitic targets and was suitable for the viral targets. The implementation of this universal method should confer cost- and time-saving benefits and streamline the processes required for the characterization of an array of pathogens from faecal samples.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.058743-0
2013-10-01
2020-01-19
Loading full text...

Full text loading...

/deliver/fulltext/jmm/62/10/1535.html?itemId=/content/journal/jmm/10.1099/jmm.0.058743-0&mimeType=html&fmt=ahah

References

  1. Boom R., Sol C. J. A., Salimans M. M. M., Jansen C. L., Wertheim-van Dillen P. M. E., van der Noordaa J.. ( 1990;). Rapid and simple method for purification of nucleic acids. . J Clin Microbiol 28:, 495–503.[PubMed]
    [Google Scholar]
  2. Chalmers R. M., Katzer F.. ( 2013;). Looking for Cryptosporidium: the application of advances in detection and diagnosis. . Trends Parasitol 29:, 237–251. [CrossRef][PubMed]
    [Google Scholar]
  3. Elwin K., Robinson G., Hadfield S. J., Fairclough H. V., Iturriza-Gómara M., Chalmers R. M.. ( 2012;). A comparison of two approaches to extracting Cryptosporidium DNA from human stools as measured by a real-time PCR assay. . J Microbiol Methods 89:, 38–40. [CrossRef][PubMed]
    [Google Scholar]
  4. Espy M. J., Uhl J. R., Sloan L. M., Buckwalter S. P., Jones M. F., Vetter E. A., Yao J. D., Wengenack N. L., Rosenblatt J. E.. & other authors ( 2006;). Real-time PCR in clinical microbiology: applications for routine laboratory testing. . Clin Microbiol Rev 19:, 165–256. [CrossRef][PubMed]
    [Google Scholar]
  5. Jorm L. R., Thackway S. V., Churches T. R., Hills M. W.. ( 2003;). Watching the Games: public health surveillance for the Sydney 2000 Olympic Games. . J Epidemiol Community Health 57:, 102–108. [CrossRef][PubMed]
    [Google Scholar]
  6. Lee A. V., Atkinson C., Manuel R. J., Clark D. A.. ( 2011;). Comparative evaluation of the QIAGEN QIAsymphony® SP system and bioMérieux NucliSens easyMAG automated extraction platforms in a clinical virology laboratory. . J Clin Virol 52:, 339–343. [CrossRef][PubMed]
    [Google Scholar]
  7. McLauchlin J., Pedraza-Díaz S., Amar-Hoetzeneder C., Nichols G. L.. ( 1999;). Genetic characterization of Cryptosporidium strains from 218 patients with diarrhea diagnosed as having sporadic cryptosporidiosis. . J Clin Microbiol 37:, 3153–3158.[PubMed]
    [Google Scholar]
  8. O’Brien S. J., Rait G., Hunter P. R., Gray J. J., Bolton F. J., Tompkins D. S., McLauchlin J., Letley L. H., Adak G. K.. & other authors ( 2010;). Methods for determining disease burden and calibrating national surveillance data in the United Kingdom: the second study of infectious intestinal disease in the community (IID2 study). . BMC Med Res Methodol 10:, 39. [CrossRef][PubMed]
    [Google Scholar]
  9. Tam C. C., Rodrigues L. C., Viviani L., Dodds J. P., Evans M. R., Hunter P. R., Gray J. J., Letley L. H., Rait G.. & other authors ( 2012;). Longitudinal study of infectious intestinal disease in the UK (IID2 study): incidence in the community and presenting to general practice. . Gut 61:, 69–77. [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.058743-0
Loading
/content/journal/jmm/10.1099/jmm.0.058743-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error