Determination of Mycobacterium leprae Viability by Polymerase Chain Reaction Amplification of 71-kDa Heat-Shock Protein mRNA

Patel, Bharvin K.R.; Banerjee, D.; Butcher, Philip D.

doi:10.1093/infdis/168.3.799

Cited by 53 publications

(31 citation statements)

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“…One disadvantage of DNA-based methods is that they do not distinguish between living and dead cells (17). With our viability test, we have shown that groEL mRNA was undetectable immediately after cell inactivation, as previously shown for other primer systems (2,28). The suitability of mRNAs as viability indicators needs to be proven before their use in a reliable technique for the sterility testing of PCs.…”

Section: Discussionsupporting

confidence: 64%

Two Novel Real-Time Reverse Transcriptase PCR Assays for Rapid Detection of Bacterial Contamination in Platelet Concentrates

2004

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The incidence of platelet bacterial contamination is approximately 1 per 2,000 units and has been acknowledged as the most frequent infectious risk from transfusion. In preliminary studies, the sterility of platelet concentrates (PCs) was tested with an automated bacterial blood culturing system and molecular genetic assays. Two real-time reverse transcriptase PCR (RT-PCR) assays performed in a LightCycler instrument were developed and compared regarding specificity and sensitivity by the use of different templates to detect the majority of the clinically important bacterial species in platelets. Primers and probes specific for the conserved regions of the eubacterial 23S rRNA gene or the groEL gene (encoding the 60-kDa heat shock protein Hsp60) were designed. During the development of the 23S rRNA RT-PCR, problems caused by the contamination of reagents with bacterial DNA were noted. Treatment with 8-methoxypsoralen and UV irradiation reduced the level of contaminating DNA. The sensitivity of the assays was greatly influenced by the enzyme system which was used. With rTth DNA polymerase in a one-enzyme system, we detected 500 CFU of Escherichia coli or Staphylococcus epidermidis/ml. With a two-enzyme system consisting of Moloney murine leukemia virus RT and Taq DNA polymerase, we detected 16 CFU/ml. With groEL mRNA as the target of RT-PCR under optimized conditions, we detected 125 CFU of E. coli/ml, and no problems with false-positive results caused by reagent contamination or a cross-reaction with human nucleic acids were found. Furthermore, the use of mRNA as an indicator of viability was demonstrated. Here we report the application of novel real-time RT-PCR assays for the detection of bacterial contamination of PCs that are appropriate for transfusion services.

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Section: Discussionsupporting

confidence: 64%

Two Novel Real-Time Reverse Transcriptase PCR Assays for Rapid Detection of Bacterial Contamination in Platelet Concentrates

2004

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“…Thus, the presence of mRNA is usually indicative of the continuous transcription of a gene. Patel et al (31) used reverse transcription (RT)-PCR to examine the viability of heat-killed M. leprae and detected dnaK mRNA only in living cells. Recently, Hellyer et al (15) found that exposure of log-phase M. tuberculosis to isoniazid and rifampin for 24 h reduced the levels of fbpB (85B, ␣-antigen) mRNA to Ͻ4 and Ͻ0.01%, respectively, of those in the drug-free control.…”

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confidence: 99%

Detection of mRNA Transcripts and Active Transcription in Persistent Mycobacterium tuberculosis Induced by Exposure to Rifampin or Pyrazinamide

et al. 2000

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Mycobacterium tuberculosis can persist in an altered physiological state for many years after initial infection, and it may reactivate to cause active disease. An analogous persistent state, possibly consisting of several different subpopulations of bacteria, may arise during chemotherapy; this state is thought to be responsible for the prolonged period required for effective chemotherapy. Using two models of drug-induced persistence, we show that both microaerophilic stationary-phase M. tuberculosis treated with a high dose of rifampin in vitro and pyrazinamide-induced persistent bacteria in mice are nonculturable yet still contain 16S rRNA and mRNA transcripts. Also, the in vitro persistent, plate culture-negative bacteria incorporate radioactive uridine into their RNA in the presence of rifampin and can rapidly up-regulate gene transcription after the replacement of the drug with fresh medium and in response to heat shock. Our results show that persistent M. tuberculosis has transcriptional activity. This finding provides a molecular basis for the rational design of drugs targeted at persistent bacteria.Tuberculosis is an important infectious disease, with over 1 billion people subclinically infected and the 3 million new cases each year resulting in 7% of the total number of deaths (20). Case finding and treatment are the main control measures, but chemotherapy takes 6 months to achieve a cure because bacteria persist during chemotherapy and cause relapse (2 to 5% in 5 years) after the end of treatment (14). These persistent bacteria are usually drug sensitive at relapse, and so their resistance to chemotherapy is phenotypic (17) (or tolerant) rather than genetic. It is suggested that an altered physiological state of persistent Mycobacterium tuberculosis accounts for its tolerance to drugs as well as the ability to survive in the host for many years. Persistence is likely to be a combined effect of both the immune system and bacterial physiology, resulting in what is generally referred to as a latent state (1). A key question is whether the metabolism of persistent bacteria is switched off with no cell division (spore-like) or, alternatively, is active (30).The first major advance in the detection of quiescent M. tuberculosis was the development of the Cornell mouse model (25). In this model, animals are infected with M. tuberculosis and treated for 3 months with chemotherapy which renders the tissues sterile; then steroids are administered, which leads to relapse in a high proportion of the mice. Immediately after the end of antibiotic therapy, the persistent bacteria are invisible by microscopy and are uncultivable, but about 10 5 genome equivalents of M. tuberculosis DNA per ml of tissue in the organs have been detected by DNA amplification (4). The presence of DNA could be indicative of dead bacilli or, possibly, of live bacilli which can multiply when the host immune system becomes weakened by steroids. Since DNA can be detected in dead bacteria (9,16,24), targeting genomic DNA is not suitable for the ...

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“…If such molecules have species-specific sequences, they might provide specific information on the growth activity of individual microbial species present in highly complex samples. For example, specific mRNA molecules were used to assess the viability of noncultivable Mycobacterium leprae cells in animal tissues (26). Since bacterial mRNA can be difficult to detect and of undefined phylogenetic specificity, assays targeting bacterial rRNA have also been developed.…”

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confidence: 99%

Depletion of pre-16S rRNA in starved Escherichia coli cells

Cangelosi

Brabant

1997

J Bacteriol

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Specific hybridization assays for intermediates in rRNA synthesis (pre-rRNA) may become useful for monitoring the growth activity of individual microbial species in complex natural systems. This possibility depends upon the assumption that rRNA processing in microbial cells continues after growth and pre-rRNA synthesis cease, resulting in drainage of the pre-rRNA pool. This is not the case in many eukaryotic cells, but less is known about the situation in bacteria. Therefore, we used DNA probes to measure steady-state cellular pre-16S rRNA pools during growth state transitions in Escherichia coli. Pre-16S rRNA became undetectable when cells entered the stationary phase on rich medium and was replenished upon restoration of favorable growth conditions. These fluctuations were of much greater magnitude than concurrent fluctuations in the mature 16S rRNA pool. The extent of pre-16S rRNA depletion depended upon the circumstances limiting growth. It was significantly more pronounced in carbon-energy-starved cells than in nitrogen-starved cells or in cells treated with energy uncouplers. In the presence of the transcriptional inhibitor rifampin, rates of pre-16S rRNA depletion in carbon-energy-starved cells and nitrogen-starved cells were similar, suggesting that the difference between these conditions resides primarily at the level of pre-rRNA synthesis. Chloramphenicol, which inhibits the final steps in rRNA maturation, halted pre-16S rRNA depletion under all conditions. The data show that E. coli cells continue to process pre-rRNA after growth and rrn operon transcription cease, leading to drainage of the pre-rRNA pool. This supports the feasibility of using pre-rRNA-targeted probes to monitor bacterial growth in natural systems, with the caveat that patterns of pre-rRNA depletion vary with the conditions limiting growth.

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Determination of Mycobacterium leprae Viability by Polymerase Chain Reaction Amplification of 71-kDa Heat-Shock Protein mRNA

Cited by 53 publications

References 0 publications

Two Novel Real-Time Reverse Transcriptase PCR Assays for Rapid Detection of Bacterial Contamination in Platelet Concentrates

Two Novel Real-Time Reverse Transcriptase PCR Assays for Rapid Detection of Bacterial Contamination in Platelet Concentrates

Detection of mRNA Transcripts and Active Transcription in Persistent Mycobacterium tuberculosis Induced by Exposure to Rifampin or Pyrazinamide

Depletion of pre-16S rRNA in starved Escherichia coli cells

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