Detection of diarrhoeal pathogens in human faeces using an automated, robotic platform

• Published in: Molecular and cellular probes Vol. 26; no. 1; pp. 11 - 15
• Main Authors: Jex, Aaron R., Stanley, Keith K., Lo, William, Littman, Rachael, Verweij, Jaco J., Campbell, Bronwyn E., Nolan, Matthew J., Pangasa, Aradhana, Stevens, Melita A., Haydon, Shane, Gasser, Robin B.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.02.2012
• Subjects: Adenoviridae - isolation & purification; Adenovirus; Campylobacter; Clostridium difficile; Clostridium difficile - isolation & purification; Cryptosporidium; Cryptosporidium - isolation & purification; Diarrhea - microbiology; Diarrhea - virology; Diarrhoeal disease; Feces - microbiology; Feces - virology; Giardia; Giardia - isolation & purification; Humans; Norovirus; Pathogens; PCR; Polymerase Chain Reaction - methods; Polymorphism, Single-Stranded Conformational; Robotics; Salmonella; Sensitivity and Specificity; Shigella; Shigella - isolation & purification; Pathogens; PCR; Diarrhoeal disease
• ISSN: 0890-8508; 1096-1194; 1096-1194
• DOI: 10.1016/j.mcp.2011.10.004

Infectious diarrhoeal diseases represent a major socio-economic burden to humans, and are linked to a range of pathogens, including viruses, bacteria and protists. The accurate detection of such pathogens is central to control. However, detection often relies on methods that have limited diagnostic sensitivity and specificity. Here, we assessed an automated, robotic platform for the simultaneous detection of eight major pathogens associated with infectious diarrhoea. Genomic DNA samples ( n = 167) from faeces from humans with diarrhoea and diagnosed as cryptosporidiosis, and 100 uninfected control subjects, were tested for adenovirus 40/41, norovirus, Clostridium difficile, Campylobacter, Salmonella, Shigella, Cryptosporidium and Giardia by multiplexed-tandem PCR, and also characterized by single-strand conformation polymorphism analysis (SSCP) and selective sequencing. All 167 samples tested positive for Cryptosporidium, five for adenovirus 40/41, four for Campylobacter, three for C. difficile and seven for Shigella spp., with no false positive results for any assay. The automated PCR exhibited a high sensitivity, with <10 individual pathogens being readily detected. The robotic detection platform assessed here represents a sensitive, high-throughput tool for key pathogens linked to infectious diarrhoea in humans. This platform requires little molecular biological expertise and is well suited to various diagnostic facilities and settings.