Reverse Transcription-Multiplex PCR Assay for Simultaneous Detection of Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi

A sensitive method for specific detection of viable Escherichia coli O157:H7 cells, including viable but nonculturable (VBNC) cells, in water samples was developed. This method involved capture of the bacterial cells on a lowprotein-binding membrane and direct extraction and purification of RNA followed by reverse transcription-PCR and electronic microarray detection of the rfbE and fliC genes of E. coli O157:H7. It detected as few as 1 CFU of E. coli O157:H7 in diluted cultures, 3 to 4 CFU/liter in tap water, 7 CFU/liter in river water, and 50 VBNC cells in 1 liter of river water, demonstrating the best limit of detection reported to date for VBNC cells in environmental water samples.

show abstract

“…The RT-PCRs were performed under conditions that were previously described in detail (27). These procedures are briefly described in Fig.…”

Section: Methodsmentioning

confidence: 99%

Detection of Viable but Nonculturable Escherichia coli O157:H7 Bacteria in Drinking Water and River Water

Liu

Gilchrist

Zhang

et al. 2008

Appl Environ Microbiol

Self Cite

116

View full text Add to dashboard Cite

show abstract

“…Some studies have attempted to remedy this by utilizing the amplification of RNA instead of DNA, which degrades rapidly after cell death and in particular, using messenger RNA targets as this is a highly unstable molecule and is only produced by metabolically active cells (85–88). The major disadvantage to this approach, however, lies in the inherent difficulties associated with isolating RNA from samples, including the need for stringent sample storage conditions following sample collection, in addition to sample processing regimes to prevent RNA degradation (86).…”

Section: Microbial Cell Viability the Microbiome And Clinical Transmentioning

confidence: 99%

Exploring Preterm Birth as a Polymicrobial Disease: An Overview of the Uterine Microbiome

2014

View full text Add to dashboard Cite

Infection is a leading cause of preterm birth (PTB). A focus of many studies over the past decade has been to characterize microorganisms present in the uterine cavity and document any association with negative pregnancy outcome. A range of techniques have been used to achieve this, including microbiological culture and targeted polymerase chain reaction assays, and more recently, microbiome-level analyses involving either conserved, phylogenetically informative genes such as the bacterial 16S rRNA gene or whole shotgun metagenomic sequencing. These studies have contributed vast amounts of data toward characterization of the uterine microbiome, specifically that present in the amniotic fluid, fetal membranes, and placenta. However, an overwhelming emphasis has been placed on the bacterial microbiome, with far less data produced on the viral and fungal/yeast microbiomes. With numerous studies now referring to PTB as a polymicrobial condition, there is the need to investigate the role of viruses and fungi/yeasts in more detail and in particular, look for associations between colonization with these microorganisms and bacteria in the same samples. Although the major pathway by which microorganisms are believed to colonize the uterine cavity is vertical ascension from the vagina, numerous studies are now emerging suggesting hematogenous transfer of oral microbiota to the uterine cavity. Evidence of this has been produced in mouse models and although DNA-based evidence in humans appears convincing in some aspects, use of methodologies that only detect viable cells as opposed to lysed cells and extracellular DNA are needed to clarify this. Such techniques as RNA analyses and viability polymerase chain reaction are likely to play key roles in the clinical translation of future microbiome-based data, particularly in confined environments such as the uterus, as detection of viable cells plays a key role in diagnosis and treatment of infection.

show abstract

“…Detection of RNA and especially of the highly unstable mRNA thus tends to indicate the presence of live cells far better than the detection of DNA. In addition, messenger RNA (mRNA) is only produced by metabolically active cells, making mRNA suitable to specifically detect living microorganisms (Bleve et al, 2003;Morin et al, 2004). Nevertheless the same labile nature qualifying mRNA as a suitable target for detecting live cells at the same time makes working with it more challenging.…”

Section: Introductionmentioning

confidence: 99%

Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification

Fittipaldi

Nocker

Codony

2012

Journal of Microbiological Methods

350

283

View full text Add to dashboard Cite

Reverse Transcription-Multiplex PCR Assay for Simultaneous Detection of Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi

Cited by 57 publications

References 34 publications

Detection of Viable but Nonculturable Escherichia coli O157:H7 Bacteria in Drinking Water and River Water

Detection of Viable but Nonculturable Escherichia coli O157:H7 Bacteria in Drinking Water and River Water

Exploring Preterm Birth as a Polymicrobial Disease: An Overview of the Uterine Microbiome

Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification

Contact Info

Product

Resources

About