Detection of mRNA by Reverse Transcription-PCR as an Indicator of Viability in
            <i>Escherichia coli</i>
            Cells

Sheridan, G. E. C.; Masters, C. I.; Shallcross, Jamie; Mackey, B.M.

doi:10.1128/aem.64.4.1313-1318.1998

Cited by 337 publications

(170 citation statements)

References 34 publications

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“…The half-life of the S-layer gene mRNA has been reported as 15 min in L. acidophilus [16] and 14 min in L. brevis [17]. Detection of mRNA is an indicator of living cells or those only recently dead at the time of sampling [6]. Moreover, mRNA of L. helveticus GCL1001 was not detected in the feces of volunteers when heat-killed cells were ingested (data not shown).…”

Section: Viability Of L Helveticus Gcl1001 In Fecesmentioning

confidence: 93%

Monitoring the cell number and viability ofLactobacillus helveticusGCL1001 in human feces by PCR methods

Saito¹,

Sakamoto

Takizawa³

et al. 2004

FEMS Microbiology Letters

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Real-time polymerase chain reaction (PCR) and nested reverse transcription (RT) PCR were applied to demonstrate the viability of lactobacilli in the feces of volunteers fed fermented milk containing lactobacilli. Two sets of specific primers and a TaqMan probe for realtime PCR were constructed using the S-layer gene as a target. After fermented milk ingestion, Lactobacillus helveticus GCL1001 was detected in the feces of 12 volunteers over a few days, with the maximum number being between 10 4:5 and 10 7:8 cells g 31 of feces. Moreover, mRNA from this strain was detected in the feces of all volunteers by nested RT-PCR. The results show that these methods are applicable to the demonstration of bacterial viability in feces, and that ingested L. helveticus GCL1001 can survive through the gastrointestinal tract.

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Section: Viability Of L Helveticus Gcl1001 In Fecesmentioning

confidence: 93%

Monitoring the cell number and viability ofLactobacillus helveticusGCL1001 in human feces by PCR methods

Saito¹,

Sakamoto

Takizawa³

et al. 2004

FEMS Microbiology Letters

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“…It has been reported that prokaryotic mRNA has a much shorter half life compared with prokaryotic rRNA [4,13,14] and in dead bacterial cells degraded to undetectable levels after 2^16 h at room temperature. In contrast, 16S rRNA transcripts remained intact, and were detected by RT-PCR in dead cells kept at room temperature for 16 h [15], suggesting that 16S rRNA is more stable compared to mRNA inside bacterial cells. The persistence of RNA molecules in stored bacterial samples depends on the method of cell ¢xation and subsequent holding conditions [15].…”

Section: Introductionmentioning

confidence: 94%

RNA recovery and detection of mRNA by RT-PCR from preserved prokaryotic samples

Bachoon¹

2001

FEMS Microbiology Letters

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The effectiveness of maintaining prokaryotic RNA in Synechococcus and Pseudomonas cells, fixed in 96% ethanol, 4% paraformaldehyde, or suspended in RNAlater, and held in cold storage for 3 months was compared. Fluorometric determination of the RNA extracted from Synechococcus and Pseudomonas cells indicated that the cell storage treatments tested were equally effective at maintaining their total RNA content. There was not any detectable decrease in the quantity of RNA isolated from the preserved samples during storage. Intact mRNA transcripts of the RuBisCO (rbcL) and nir genes were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) from preserved bacterial cells throughout 3 months of storage. In contrast, RT-PCR failed to amplify the mRNA of the rbcL and nitrite reductase genes in unfixed and/or unpreserved bacterial samples, suggesting that bacterial mRNA can be well maintained during a prolonged storage when cells are preserved properly. In addition, RNAlater is a useful reagent for the storage and maintenance of high quality RNA in unfrozen samples. ß

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“…128,129 In some clinical situations, detection of RNA species by RT-PCR has been shown to correlate well with the presence of viable organisms and has been effectively used to monitor antibiotic therapy. [130][131][132][133][134] Clinical application of RNA-based approaches will need further improvement, however, because they have been hampered in development by difficulties in extracting detectable concentrations of intact RNA from small numbers of bacteria.…”

Section: Clinical Significance Of Positive Pcrmentioning

confidence: 99%

PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings

Yang

Rothman

2004

The Lancet Infectious Diseases

796

545

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Molecular diagnostics are revolutionising the clinical practice of infectious disease. Their effects will be significant in acute-care settings where timely and accurate diagnostic tools are critical for patient treatment decisions and outcomes. PCR is the most well-developed molecular technique up to now, and has a wide range of already fulfilled, and potential, clinical applications, including specific or broad-spectrum pathogen detection, evaluation of emerging novel infections, surveillance, early detection of biothreat agents, and antimicrobial resistance profiling. PCR-based methods may also be cost effective relative to traditional testing procedures. Further advancement of technology is needed to improve automation, optimise detection sensitivity and specificity, and expand the capacity to detect multiple targets simultaneously (multiplexing). This review provides an up-to-date look at the general principles, diagnostic value, and limitations of the most current PCR-based platforms as they evolve from bench to bedside.

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Detection of mRNA by Reverse Transcription-PCR as an Indicator of Viability in Escherichia coli Cells

Cited by 337 publications

References 34 publications

Monitoring the cell number and viability ofLactobacillus helveticusGCL1001 in human feces by PCR methods

Monitoring the cell number and viability ofLactobacillus helveticusGCL1001 in human feces by PCR methods

RNA recovery and detection of mRNA by RT-PCR from preserved prokaryotic samples

PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings

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