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PubMed1997

An investigation of the physical characteristics of 66Ga as an isotope for PET imaging and quantification.

Isotopes commonly used for PET imaging and quantification have a straightforward decay scheme involving "pure" positron (beta +) emission, i.e., 95%-100% beta + abundance, with no additional gamma rays. 66Ga (Emax = 4.2 MeV, T1/2 = 9.5 h) is a member of a category of isotopes with a lower abundance of beta +'s (57%) and a more complicated spectrum involving combinations of gamma rays that are emitted in cascade. These additional gamma rays tend to cause a higher singles rate, resulting in more random coincidence events. The most abundant positron (51.5%) in the spectrum has one of the highest energies considered for PET imaging. For the purpose of monoclonal antibody dosimetry using 66Ga, it is important to verify the quantification in phantoms prior to initiating human studies. A series of quantitative phantom measurements were performed on the PC4600, a head-optimized BGO based scanner with multiple detector rings. Count rate linearity was verified over concentrations ranging from 4.0 kBq/cc to 37 kBq/cc (0.11-1.0 microCi/cc); resolution averaged 16 mm full width half-maximum in the x and y directions in both the direct and cross planes. Axial resolution was 14 mm. The range of the energetic positrons (up to 4.153 MeV, range 7.6 mm in tissue) was verified as a primary source of resolution degradation. Within the limits outlined above, 66Ga is a suitable isotope for use as 66Ga citrate or with monoclonal antibodies in the detection and staging of tumors and other lesions. In addition, the energetic positrons have possible therapeutic applications when used as a monoclonal antibody label.

Beta Particles↗
PubMed1998

Accumulation of amphotericin B in human macrophages enhances activity against Aspergillus fumigatus conidia: quantification of conidial kill at the single-cell level.

A cytofluorometric assay that allowed assessment of damage to phagocytosed Aspergillus fumigatus conidia at the single-cell level was developed. After ingestion by monocyte-derived macrophages (MDMs), conidia were reisolated by treatment of the cells with streptolysin O, a pore-forming toxin with lytic properties on mammalian cells but not on fungi. The counts obtained by staining of damaged conidia with propidium iodide and quantification by cytofluorometry correlated with colony counts. By the use of this method, we demonstrate that MDMs differentiated in vitro by low-dose granulocyte-macrophage colony-stimulating factor and gamma interferon have only a limited capacity to damage Aspergillus conidia in vitro. The killing rate 12 h after phagocytosis was found to be only 10 to 15%. However, intracellular loading of the phagocytes with amphotericin B (AmB) dose dependently enhanced the anticonidial activity. Preincubation of macrophages with only 1 microg of AmB per ml resulted in an uptake of 18 fg of AmB/cell, leading to killing rates of 50 to 60%. The experimental protocol provides a new tool for the rapid quantification of anticonidial activity against A. fumigatus in vitro. Intracellular accumulation of AmB may represent an important factor underlying the efficacy of this antifungal drug in the prophylaxis and treatment of Aspergillus infections.

Amphotericin B↗
PubMed2003

Ribavirin quantification in combination treatment of chronic hepatitis C.

Ribavirin in combination with alpha 2 interferon is the consensus treatment for chronic hepatitis C. However, recent preliminary pharmacological studies have suggested that the bioavailability of ribavirin displays great interindividual variability. In order to monitor serum ribavirin levels during combination treatment, we developed and validated a quantitative assay using an approach adaptable for routine hospital laboratories. The method involved solid-phase extraction on phenyl boronic acid cartridges followed by high-performance liquid chromatography with a C(18)-bonded silica column and a mobile phase containing 10 mM ammonium phosphate buffer (with the pH adjusted to 2.5) and UV detection (207 nm). The sensitivity, recovery, linearity of the calibration curve, intra- and interassay accuracies, precision, and stability at 4 degrees C were consistent with its use in the laboratory routine. In addition, other nucleoside analogues sometimes used with ribavirin in patients coinfected with hepatitis C virus (HCV) and human immunodeficiency virus did not interfere with the quantification of ribavirin levels. The ribavirin concentration was quantified in 24 serum samples from patients with chronic hepatitis C treated with a combination of ribavirin and alpha 2 interferon. The mean serum ribavirin concentration was 2.67 +/- 1.06 micro g/ml (n = 24) at week 12 of treatment (W12) and 3.24 +/- 1.35 micro g/ml (n = 24) at week 24 of treatment (W24). In addition, ribavirin concentrations displayed high interindividual variabilities: the coefficients of variation of the serum ribavirin concentrations adjusted to the administered dose were 44 and 48% at W12 and W24, respectively. Moreover, the ribavirin concentration was higher in patients with a sustained virological response (n = 11) than in patients with treatment failure (n = 13), with significant intergroup differences at W12 (P = 0.030) and W24 (P = 0.049). The present study describes a simple analytical method for the quantification of ribavirin in human serum that could be a useful tool for the monitoring of ribavirin concentrations in HCV-infected patients in order to improve the efficacy and safety of therapy with ribavirin plus interferon.

Adult↗
PubMed2006

Quantification of in vitro and in vivo Cryptosporidium parvum infection by using real-time PCR.

Established methods for quantifying experimental Cryptosporidium infection are highly variable and subjective. We describe a new technique using quantitative real-time PCR (qPCR) that can be used to measure in vitro and in vivo laboratory infections with Cryptosporidium. We show for the first time that qPCR permits absolute quantification of the parasite while simultaneously controlling for the amount of host tissue and correlates significantly with established methods of quantification in in vitro and in vivo laboratory models of infection.

Animals↗
PubMed2006

Quantification of the effects of salt stress and physiological state on thermotolerance of Bacillus cereus ATCC 10987 and ATCC 14579.

The food-borne pathogen Bacillus cereus can acquire enhanced thermal resistance through multiple mechanisms. Two Bacillus cereus strains, ATCC 10987 and ATCC 14579, were used to quantify the effects of salt stress and physiological state on thermotolerance. Cultures were exposed to increasing concentrations of sodium chloride for 30 min, after which their thermotolerance was assessed at 50 degrees C. Linear and nonlinear microbial survival models, which cover a wide range of known inactivation curvatures for vegetative cells, were fitted to the inactivation data and evaluated. Based on statistical indices and model characteristics, biphasic models with a shoulder were selected and used for quantification. Each model parameter reflected a survival characteristic, and both models were flexible, allowing a reduction of parameters when certain phenomena were not present. Both strains showed enhanced thermotolerance after preexposure to (non)lethal salt stress conditions in the exponential phase. The maximum adaptive stress response due to salt preexposure demonstrated for exponential-phase cells was comparable to the effect of physiological state on thermotolerance in both strains. However, the adaptive salt stress response was less pronounced for transition- and stationary-phase cells. The distinct tailing of strain ATCC 10987 was attributed to the presence of a subpopulation of spores. The existence of a stable heat-resistant subpopulation of vegetative cells could not be demonstrated for either of the strains. Quantification of the adaptive stress response might be instrumental in understanding adaptation mechanisms and will allow the food industry to develop more accurate and reliable stress-integrated predictive modeling to optimize minimal processing conditions.

Bacillus cereus↗
PubMed2006

Comparison of methods of extracting Salmonella enterica serovar Enteritidis DNA from environmental substrates and quantification of organisms by using a general internal procedural control.

This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general internal procedural control. Inclusion of this general internal procedural control permitted more accurate quantification of extraction and amplification of S. enterica serovar Enteritidis in these samples and reduced the possibility of false negatives. With this protocol it was found that the optimal extraction method differed for soil (Mobio soil DNA extraction kit), manure (Bio101 soil DNA extraction kit), and compost (Mobio fecal DNA extraction kit). With each method, as little as 1.2 x 10(3) to 1.8 x 10(3) CFU of added serovar Enteritidis per 100 mg of substrate could be detected by direct DNA extraction and subsequent S. enterica-specific TaqMan PCR. After bacterial enrichment, as little as 1 CFU/100 mg of original substrate was detected. Finally, the study presents a more accurate molecular analysis for quantification of serovar Enteritidis initially present in soil or manure using DNA extraction and TaqMan PCR.

Animals↗
PubMed1990

Enumeration of anaerobic chytridiomycetes as thallus-forming units: novel method for quantification of fibrolytic fungal populations from the digestive tract ecosystem.

An endpoint dilution procedure, based on the technique of most probable numbers, was developed to enumerate anaerobic chytridiomycetes as thallus-forming units. The method does not distinguish between zoospores and thalli, but does permit enumeration of fungal populations with respect to their ability to digest plant cell walls. Fibrolytic populations in batch culture, ruminal contents, and feces were compared by relating viable counts to the dry matter content of enumerated samples (i.e., thallus-forming units per gram of dry matter). Batch cultures of Neocallimastix sp. strain R1 grown on wheat straw were used to assess the enumeration procedure and to demonstrate the potential of the technique for quantification of anaerobic fungi in vivo. Determination of total ruminal contents from steers enabled the quantification of the entire population of fiber-degrading anaerobic fungi in the reticulorumen. The enumeration procedure revealed substantial populations of fibrolytic anaerobic fungi in fresh and air-dried feces. Populations in fresh feces were equivalent to those in ruminal contents, but declined exponentially with time in dry feces. Minimum values were obtained from dry feces 90 days after drying, and anaerobic fungi were detectable for up to 210 days thereafter.

Journal Article↗
PubMed1990

Characterization of Egg Yolk Antibodies for Detection and Quantification of Selenomonas ruminantium by Using an Enzyme-Linked Immunosorbent Assay.

The specificity of polyclonal antibodies prepared against strains of Selenomonas ruminantium, the effect of assay conditions, and quantification of individual strains in mixed-cell suspensions of selenomonad strains were examined in this study. Whole-cell suspensions were prepared with pure cultures of S. ruminantium PC18, HD(4), GA192, and D. Each cell suspension was injected into a Leghorn laying hen, and polyclonal antibodies were harvested from eggs laid in week 3 or 7 following initial immunization. Antibodies made to the S. ruminantium strains readily discerned the homologous strain from the heterologous strains. Cross-reactivity among antibodies and the heterologous S. ruminantium strains ranged from 5 to 26%. Among non-S. ruminantium species, cross-reactivity of S. ruminantium antibodies was greatest with Selenomonas sputigena (3 to 34%) and Succinivibrio dextrinosolvens (0 to 37%). Antibodies made to strains GA192 and D were used to quantify a mixture of the two strains. Both antibodies responded to graded concentrations of the homologous antigen in the biculture mixtures in accord with the change in the direct cell counts for each strain (strain D, R = 0.92; strain GA192, R = 0.90). This enzyme-linked immunosorbent assay enabled concurrent and accurate quantification of two strains of S. ruminantium subsp. ruminantium in a mixed-cell suspension with a precision of much less than 1 order of magnitude.

Journal Article↗
PubMed1994

Quantification of frankia strains and other root-associated bacteria in pure cultures and in the rhizosphere of axenic seedlings by high-performance liquid chromatography-based muramic Acid assay.

Application of a high-performance liquid chromatography-based muramic acid assay with precolumn fluorescence derivatization to quantification of root-associated bacteria was studied both in pure cultures and in the rhizosphere of axenic Festuca rubra seedlings. Quantities of muramic acid from acid-hydrolyzed cells of Frankia strains, Streptomyces griseoviridis, Enterobacter agglomerans, Klebsiella pneumoniae, Pseudomonas sp., and Bacillus polymyxa were mostly proportional to the respective cell protein and carbon quantities, but in some strains, culture age and particularly sporulation affected these ratios considerably. The muramic acid/cell protein ratio was generally 2 to 4 times higher in strains of the two actinomycete genera, Frankia and Streptomyces, than in the rest of the strains. Quantification of Frankia strains, S. griseoviridis, E. agglomerans, and Pseudomonas sp. was also attempted from the rhizosphere of F. rubra seedlings which had been inoculated with pure cultured bacteria and incubated briefly. It was possible to quantify Frankia cells by use of the muramic acid assay from both the root and the growth medium, whereas cells of the rest of the bacterial genera could only be detected in the medium. The detection limit for muramic acid was about 10 ng/ml hydrolysis volume, and from the Festuca rhizosphere, 28 to 63% of the muramic acid in the Frankia inoculum was recovered.

Journal Article↗
PubMed1997

A simple method for quantification of uncultured microorganisms in the environment based on in vitro transcription of 16S rRNA.

A simple method for the quantification of uncultured microorganisms in the environment was developed. In vitro-transcribed 16S rRNA is used as a template for midpoint dissociation temperature (Td) determinations of specific oligonucleotide probes and as a standard in quantitative probing. It replaces the need for total nucleic acids extracted from pure cultures of the organisms to be quantified. A sense RNA of a size almost identical to that of native 16S rRNA can be transcribed from ribosomal DNA clones recovered in studies of the phylogenetic diversity of microbial communities. This in vitro-transcribed rRNA yields dissociation curves typical of oligonucleotides. They parallel curves determined with total nucleic acids but yield slightly higher Td values. Neither unspecific sticking of the probe nor probe washing off the DNA template at low temperatures fully accounted for the discrepancy in probe release from the two templates. This suggests that the native rRNA itself has melting characteristics different from those of its in vitro-transcribed counterpart. However, this difference does not affect the performance of in vitro-transcribed rRNA compared with total nucleic acids as a standard in quantitative hybridizations. No difference was found between the estimates of the relative quantity of a single bacterial species in a mixed community obtained with the two standards, regardless of whether DNA was removed from the samples. This protocol will allow the large-scale quantification of the ecological importance of uncultured microorganisms in natural environments for the first time.

Bacteria↗
PubMed1997

PCR assay based on a microsatellite-containing locus for detection and quantification of Epichloë endophytes in grass tissue.

A PCR assay which allows detection and quantification of Epichloë endophytes in tissues of the grass Bromus erectus is described. PCR with specific primers flanking a microsatellite-containing locus (MS primers) amplified fragments 300 to 400 bp in length from as little as 1.0 pg of fungal genomic DNA in 100 ng of DNA from infected plant material. When annealing temperatures were optimized, all Epichloë and Acremonium strains tested, representing many of the known taxonomic groups, yielded an amplification product, indicating that the MS primers may be useful for in planta detection of a variety of related species, including agronomically important Acremonium coenophialum and Acremonium lolii. No fragments were generated from DNA isolates from uninfected plant material or from unrelated fungi isolated from B. erectus. For diagnostic applications, a B. erectus-specific primer pair was designed for use in multiplex PCR to allow simultaneous amplification of plant and fungal DNA sequences, providing an internal control for PCR failure caused by inhibitory plant compounds present in DNA extracts. For quantitative applications, a heterologous control template in primer binding sites complementary to the MS primers was constructed for use in competitive PCR, allowing direct quantification of Epichloë in plant DNA extracts. The fungal DNA present in infected leaves of B. erectus between 1 and 20 pg per 100 ng of leaf DNA, but the amounts of fungal DNA present in the sheath and blade of a given leaf were correlated, indicating that the degree of infection varied between plant individuals but that leaves were colonized in a uniform way.

Acremonium↗
PubMed1998

Nucleic acid (DNA, RNA) quantification and RNA/DNA ratio determination in marine sediments: comparison of spectrophotometric, fluorometric, and HighPerformance liquid chromatography methods and estimation of detrital DNA

In this study, we compared three methods for extraction and quantification of RNA and DNA from marine sediments: (i) a spectrophotometric method using the diphenylamine assay; (ii) a fluorometric method utilizing selective fluorochromes (thiazole orange for total nucleic acids and Hoechst 33258 for DNA); and (iii) a high-pressure liquid chromatography (HPLC) method which uses a specific column to separate RNA and DNA and UV absorption of the nucleic acids for quantification. Sediment samples were collected in the oligotrophic Cretan Sea (eastern Mediterranean, from 40 to 1,540 m in depth) and compared to the distribution and composition of the benthic microbial assemblages (i.e., bacteria and microprotozoa). DNA concentrations measured spectrophotometrically and by HPLC were not significantly different, while fluorometric yields were significantly lower. Such differences appear mainly due to fact that the stain-DNA complex is strongly dependent on the DNA composition and structure. RNA concentrations determined by the three methods displayed some differences; fluorometric and spectrophotometric methods obtain RNA concentration by difference and therefore may be biased by DNA estimates. By contrast, the HPLC method provides independent assessments of RNA and DNA concentrations. We tentatively estimated the contribution of the detrital DNA to the total DNA pools in two ways. The two calculations provided quite similar results indicating that the majority of the DNA pool in the deep-sea sediments was detrital. Microbial RNA generally accounted for almost the entire sedimentary RNA pools below 100-m depth. RNA concentrations were found to decrease along the Cretan shelf and slope. The RNA/DNA ratio calculated by using fluorometric DNA concentrations was significantly correlated with values of sediment community oxygen consumption only below 100-m depth (dominated by the microbial biomass). These data suggest that the RNA/DNA ratio, based on fluorometric estimates of DNA, can be used as an indicator of benthic metabolic activity, but only when metazoan contribution to the microbial DNA is negligible.

Journal Article↗
PubMed1999

Quantification of bias related to the extraction of DNA directly from soils.

In recent years, several protocols based on the extraction of nucleic acids directly from the soil matrix after lysis treatment have been developed for the detection of microorganisms in soil. Extraction efficiency has often been evaluated based on the recovery of a specific gene sequence from an organism inoculated into the soil. The aim of the present investigation was to improve the extraction, purification, and quantification of DNA derived from as large a portion of the soil microbial community as possible, with special emphasis placed on obtaining DNA from gram-positive bacteria, which form structures that are difficult to disrupt. Furthermore, we wanted to identify and minimize the biases related to each step in the procedure. Six soils, covering a range of pHs, clay contents, and organic matter contents, were studied. Lysis was carried out by soil grinding, sonication, thermal shocks, and chemical treatments. DNA was extracted from the indigenous microflora as well as from inoculated bacterial cells, spores, and hyphae, and the quality and quantity of the DNA were determined by gel electrophoresis and dot blot hybridization. Lysis efficiency was also estimated by microscopy and viable cell counts. Grinding increased the extracellular DNA yield compared with the yield obtained without any lysis treatment, but none of the subsequent treatments clearly increased the DNA yield. Phage lambda DNA was inoculated into the soils to mimic the fate of extracellular DNA. No more than 6% of this DNA could be recovered from the different soils. The clay content strongly influenced the recovery of DNA. The adsorption of DNA to clay particles decreased when the soil was pretreated with RNA in order to saturate the adsorption sites. We also investigated different purification techniques and optimized the PCR methods in order to develop a protocol based on hybridization of the PCR products and quantification by phosphorimaging.

Actinomycetales↗
PubMed2000

Rapid detection and quantification of members of the archaeal community by quantitative PCR using fluorogenic probes.

We describe a rapid, reproducible, and sensitive method for detection and quantification of archaea in naturally occurring microbial communities. A domain-specific PCR primer set and a domain-specific fluorogenic probe having strong and weak selectivity, respectively, for archaeal rRNA genes (rDNAs) were designed. A universal PCR primer set and a universal fluorogenic probe for both bacterial and archaeal rDNAs were also designed. Using these primers and probes, we demonstrated that detection and quantification of archaeal rDNAs in controlled microbial rDNA assemblages can be successfully achieved. The system which we designed was also able to detect and quantify archaeal rDNAs in DNA samples obtained not only from environments in which thermophilic archaea are abundant but also from environments in which methanogenic archaea are abundant. Our findings indicate that this method is applicable to culture-independent molecular analysis of microbial communities in various environments.

Archaea↗
PubMed2002

Multiplex fluorogenic real-time PCR for detection and quantification of Escherichia coli O157:H7 in dairy wastewater wetlands.

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C(T)) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the C(T) and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 x 10(-5) pg of E. coli O157:H7 DNA ml(-1) equivalent to approximately 6.4 x 10(3) CFU of E. coli O157:H7 ml(-1) based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were >/=3.5 x 10(4) CFU g(-1). E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g(-1) with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.

Bacteriological Techniques↗
PubMed2002

Real-time PCR quantification of rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase) mRNA in diatoms and pelagophytes.

Transcriptional activity is often used as a surrogate for gene expression in environmental microbial communities. We developed a real-time PCR assay in which the ABI-Prism (PE Applied Biosystems) detection system is used for quantification of large-subunit ribulose-1,5-bisphosphate caboxylase/oxygenase (rbcL) mRNA in diatoms and pelagophytes both in cultures and from natural phytoplankton communities. Plasmid DNA containing rbcL inserts, as well as in vitro transcribed mRNA of the plasmids, was used to generate standard curves with a dynamic range of more than 6 orders of magnitude with high accuracy and precision (R(2) = 0.998). Expression levels in a cultured diatom (Phaeodactylum tricornutum) were quantified through one light-dark cycle by using traditional 35S-labeled oligonucleotide hybridization and real-time PCR. The mRNA levels detected by the two techniques were similar and correlated well (R(2) = 0.95; slope = 1.2). The quantities obtained by hybridization were slightly, yet significantly, larger (t = 5.29; P = 0.0011) than the quantities obtained by real-time PCR. This was most likely because partially degraded transcripts were not detected by real-time PCR. rbcL mRNA detection by real-time PCR was 3 orders of magnitude more sensitive than rbcL mRNA detection by hybridization. Diatom and pelagophyte rbcL mRNAs were also quantified in a profile from an oligotrophic site in the Gulf of Mexico. We detected the smallest amount of diatom rbcL expression in the surface water and maximum expression at a depth that coincided with the depth of the subsurface chlorophyll maximum. These results indicate that real-time PCR may be utilized for quantification of microbial gene expression in the environment.

Animals↗
PubMed2003

Real-time PCR for simultaneous detection and quantification of Borrelia burgdorferi in field-collected Ixodes scapularis ticks from the Northeastern United States.

The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays based on microscopy. In this study, a real-time quantitative PCR (qPCR) protocol targeting the Borrelia burgdorferi-specific recA gene was adapted for use with a Lightcycler for rapid detection and quantification of the Lyme disease spirochete, B. burgdorferi, in field-collected Ixodes scapularis ticks. The sensitivity of qPCR for detection of B. burgdorferi DNA in infected ticks was comparable to that of a well-established nested PCR targeting the 16S-23S rRNA spacer. Of the 498 I. scapularis ticks collected from four northeastern states (Rhode Island, Connecticut, New York, and New Jersey), 91 of 438 (20.7%) nymphal ticks and 15 of 60 (25.0%) adult ticks were positive by qPCR assay. The number of spirochetes in individual ticks varied from 25 to 197,200 with a mean of 1,964 spirochetes per nymphal tick and a mean of 5,351 spirochetes per adult tick. No significant differences were found in the mean numbers of spirochetes counted either in nymphal ticks collected at different locations in these four states (P = 0.23 by one-way analysis of variance test) or in ticks infected with the three distinct ribosomal spacer restriction fragment length polymorphism types of B. burgdorferi (P = 0.39). A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment estimate of k = 0.279) was observed. From the frequency distribution data and previously published transmission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal tick to be able to transmit infection to mice while feeding on mice. These data indicate that real-time qPCR is a reliable approach for simultaneous detection and quantification of B. burgdorferi infection in field-collected ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes.

Animals↗
PubMed2005

Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples.

The distinction between viable and dead cells is a major issue in many aspects of biological research. The current technologies for determining viable versus dead cells cannot readily be used for quantitative differentiation of specific cells in mixed populations. This is a serious limitation. We have solved this problem by developing a new concept with the viable/dead stain ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR). A dynamic range of approximately 4 log(10) was obtained for the EMA-PCR viable/dead assay. Viable/dead differentiation is obtained by covalent binding of EMA to DNA in dead cells by photoactivation. EMA penetrates only dead cells with compromised membrane/cell wall systems. DNA covalently bound to EMA cannot be PCR amplified. Thus, only DNA from viable cells can be detected. We evaluated EMA-PCR with the major food-borne bacterium Campylobacter jejuni as an example. Traditional diagnosis of this bacterium is very difficult due to its specific growth requirements and because it may enter a state where it is viable but not cultivable. The conditions analyzed included detection in mixed and natural samples, survival in food, and survival after disinfection or antibiotic treatment. We obtained reliable viable/dead quantifications for all conditions tested. Comparison with standard fluorescence-based viable/dead techniques showed that the EMA-PCR has a broader dynamic range and enables quantification in mixed and complex samples. In conclusion, EMA-PCR offers a novel real-time PCR method for quantitative distinction between viable and dead cells with potentially very wide application.

Affinity Labels↗