Keywords
Lysis, Neonatal Sepsis, Point-of-care, LAMP
Presentation Type
Poster
Research Abstract
Neonatal sepsis, resulting from a bloodstream infection within the first few weeks of life, is the leading cause of newborn deaths worldwide. The gold standard of neonatal sepsis diagnosis requires a blood culture to identify the infecting bacteria, however require days of incubation, expensive equipment, and expertise. Any delay in diagnosis is critical, as the condition can be treated easily if appropriate antibiotics are administered promptly. A low-cost, rapid, and sensitive diagnostic test would enable more timely treatment and lead to better patient outcomes with fewer required resources. Point-of-care, nucleic acid amplification assays are a promising alternative to blood culture that quickly deliver sensitive results with minimal sample volume. However, these require isolated and purified template DNA from the pathogenic bacteria, a task that is difficult to achieve in a field setting. This study sought to develop a simple one-step lysis and amplification protocol for three common bacterial causes of neonatal sepsis, Streptococcus agalacitae, Klebsiella pnemoniae, and Staphylococcus aureus. The combined efficacy of enzymes, proteinase K and achromopeptidase (ACP), and heat to lyse each of these bacteria for direct DNA amplification was examined. Results showed that all three strains could be effectively lysed by applying 1 U of ACP and incubating for 10 minutes at 37° C. No adverse effects were seen in amplification reactions containing ACP if the compound was inactivated prior to amplification at 85° C for 2 minutes. The demonstrated effectiveness of ACP in rapid bacterial lysis validates its usefulness in a point-of-care device for neonatal sepsis.
Session Track
Biotechnology
Recommended Citation
Gregory Berglund, Elizabeth A. Phillips, and Jacqueline C. Linnes,
"Lysis and Amplifciation of Neonatal Sepsis Causing Pathogens"
(August 4, 2016).
The Summer Undergraduate Research Fellowship (SURF) Symposium.
Paper 17.
https://docs.lib.purdue.edu/surf/2016/presentations/17
Lysis and Amplifciation of Neonatal Sepsis Causing Pathogens
Neonatal sepsis, resulting from a bloodstream infection within the first few weeks of life, is the leading cause of newborn deaths worldwide. The gold standard of neonatal sepsis diagnosis requires a blood culture to identify the infecting bacteria, however require days of incubation, expensive equipment, and expertise. Any delay in diagnosis is critical, as the condition can be treated easily if appropriate antibiotics are administered promptly. A low-cost, rapid, and sensitive diagnostic test would enable more timely treatment and lead to better patient outcomes with fewer required resources. Point-of-care, nucleic acid amplification assays are a promising alternative to blood culture that quickly deliver sensitive results with minimal sample volume. However, these require isolated and purified template DNA from the pathogenic bacteria, a task that is difficult to achieve in a field setting. This study sought to develop a simple one-step lysis and amplification protocol for three common bacterial causes of neonatal sepsis, Streptococcus agalacitae, Klebsiella pnemoniae, and Staphylococcus aureus. The combined efficacy of enzymes, proteinase K and achromopeptidase (ACP), and heat to lyse each of these bacteria for direct DNA amplification was examined. Results showed that all three strains could be effectively lysed by applying 1 U of ACP and incubating for 10 minutes at 37° C. No adverse effects were seen in amplification reactions containing ACP if the compound was inactivated prior to amplification at 85° C for 2 minutes. The demonstrated effectiveness of ACP in rapid bacterial lysis validates its usefulness in a point-of-care device for neonatal sepsis.