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PubMed2005

SAGE reveals expression of Wnt signalling pathway members during mouse prostate development.

To identify genes and pathways not previously implicated in the mesenchymal-epithelial (M/E) interactions that are critical for normal mouse prostate development, we constructed six serial analysis of gene expression (SAGE) libraries. Bioinformatic analyses revealed expression of various members of numerous signalling pathways and the differential expression of several members of the wingless-related MMTV integration site (Wnt) signalling pathway. This pathway has not been previously implicated in prostate development thus expression of selected Wnt pathway members in the developing prostate was confirmed by RT-qPCR. Of particular interest, an antagonist of the Wnt pathway, secreted frizzled related protein 2 (Sfrp2), was highly expressed in the early prostate libraries and down regulated at later developmental stages. The expression levels of four Wnt ligands reported to interact with Sfrp2 were, therefore, examined by RT-qPCR. We found that only Wnt4 transcripts were detectable in the developing prostate. Expression of Sfrp2 was validated using RT-qPCR and localization of Sfrp2 transcripts and protein was carried out using in situ hybridization and immunofluorescence, respectively. These studies provide the first evidence that Wnt pathway members are expressed in the developing prostate. Functional analyses are now required to establish the biological significance of this observation.

Animals↗

PubMed2026

Human m6A demethylase FTO modulates the flowering time of tomato plants under low-temperature stress.

N6-methyladenosine (m6A) RNA modification plays an important role in plant development and environmental stress responses. However, whether m6A demethylation modulates flowering under low-temperature (LT) stress in tomatoes remains unclear. Here, we investigated whether ectopic expression of FTO, a well-characterized human m6A demethylase, influences flowering and post-transcriptional behaviour in tomato (Solanum lycopersicum) under LT conditions. Flowering of transgenic tomato plants expressing FTO was analyzed under LT and normal conditions (NC), and the impacts of FTO on transcripts-specific m6A level, mRNA stability and splicing efficiency of flowering-related genes were evaluated using RT-qPCR, LC-MS/MS, m6A-IP-qPCR, and RNA decay and splicing analyses. FTO-expressing plants exhibited accelerated flowering specifically under LT, whereas no significant differences were observed under normal growth conditions. This phenotype was accompanied by increased expression of positive floral regulators (SlMC, SlFCA, and SlJ2) and decreased expression of negative regulators (SlSVP, SlSP, and SlTMF) under LT conditions. Notably, these expression changes were associated with altered mRNA stability, with positive regulators showing increased stability and negative regulators showing reduced stability under LT conditions. m6A-IP-qPCR analysis indicated reduced m6A enrichment in these selected transcripts in FTO-expressing plants. In addition to effects on mRNA stability, FTO expression was associated with changes in the splicing efficiency of SlMC transcripts. Collectively, our findings indicate that human FTO functions as an mRNA m6A demethylase in tomatoes and is associated with altered RNA regulatory processes under LT conditions. These findings suggest that m6A-mediated post-transcriptional regulation contributes to stress-induced flowering plasticity under LT conditions, rather than direct activation of canonical flowering pathways.

Abiotic stress↗

PubMed2006

Reactivation and growth of non-culturable indicator bacteria in anaerobically digested biosolids after centrifuge dewatering.

Recent literature has reported that high concentrations of indicator bacteria such as fecal coliforms (FCs) were measured in anaerobically digested sludges immediately after dewatering even though low concentrations were measured prior to dewatering. This research hypothesized that the indicator bacteria can enter a non-culturable state during digestion, and are reactivated during centrifuge dewatering. Reactivation is defined as restoration of culturability. To examine this hypothesis, a quantitative polymerase chain reaction (qPCR) method was developed to enumerate Escherichia coli, a member of the FC group, during different phases of digestion and dewatering. For thermophilic digestion, the density of E. coli measured by qPCR could be five orders of magnitude greater than the density measured by standard culturing methods (SCMs), which is indicative of non-culturable bacteria. For mesophilic digestion, qPCR enumerated up to about one order of magnitude more E. coli than the SCMs. After centrifuge dewatering, the non-culturable organisms could be reactivated such that they are enumerated by SCMs, and the conditions in the cake allowed rapid growth of FCs and E. coli during cake storage.

Anaerobiosis↗

PubMed2002

Comparison of inhibition of ovalbumin-induced bronchoconstriction in guinea pigs and in vitro inhibition of tumor necrosis factor-alpha formation with phosphodiesterase 4 (PDE4) selective inhibitors.

Phosphodiesterase 4 (PDE4) inhibitors elevate cyclic adenosine 5'-monophosphate (cAMP), and this elevation has been shown to inhibit inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). Using TNF-alpha as a biomarker, we have developed transcription-based assays to examine inhibition of PDE4 activity in human and guinea pig whole blood. In vitro inhibition by PDE4 inhibitors was measured using quantitative PCR (qPCR) analysis of TNF-alpha mRNA levels in whole blood stimulated with lipopolysaccharide (LPS). The kinetics of human TNF-alpha mRNA production were analyzed and shown to be highest 4 hr following LPS stimulation. The guinea pig displayed kinetics of TNF-alpha transcription similar to those of the human. Analysis of inhibition of human TNF-alpha protein production was performed by immunoassay and shown to correlate with inhibition of transcription for three of the four compounds tested. Roflumilast was found to be 9-fold more potent for TNF-alpha inhibition in the qPCR assay than in the protein assay. The potencies of L-826,141 and roflumilast were determined in human and guinea pig whole blood by qPCR, with IC(50) values of 270 and 20 nM, respectively, in humans and 100 and 10 nM, respectively, in guinea pigs. These results show that the potency of PDE4 inhibitors can be monitored in whole blood using a transcription-based assay, and that this type of assay can be adapted to various species provided the TNF-alpha nucleotide sequence is known. The in vitro whole blood IC(50) for TNF-alpha inhibition was compared to inhibition in the ovalbumin-challenged guinea pig model of bronchoconstriction. Obtaining plasma levels at the IC(50) determined in vitro for L-826,141 and roflumilast provides significant inhibition of bronchoconstriction. This suggests that TNF-alpha can be used as a whole blood biomarker in the guinea pig for PDE4 inhibition in this inflammatory model.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1999

Quantitation and mapping of aflatoxin B1-induced DNA damage in genomic DNA using aflatoxin B1-8,9-epoxide and microsomal activation systems.

Aflatoxin B1 (AFB1) is a mutagenic and carcinogenic mycotoxin which may play a role in the etiology of human liver cancer. In vitro studies have shown that AFB1 adducts form primarily at the N7 position of guanine. Using quantitative PCR (QPCR) and ligation-mediated PCR (LMPCR), we have mapped total AFB1 adducts in genomic DNA treated with AFB1-8,9-epoxide and in hepatocytes exposed to AFB1 activated by rat liver microsomes or human liver and enterocyte microsomal preparations. The p53 gene-specific adduct frequencies in DNA, modified in cells with 40-400 microM AFB1, were 0.07-0.74 adducts per kilobase (kb). In vitro modification with 0. 1-4 ng AFB1-8,9-epoxide per microgram DNA produced 0.03-0.58 lesions per kb. The adduct patterns obtained with the epoxide and the different microsomal systems were virtually identical indicating that adducts form with a similar sequence-specificity in vitro and in vivo. The lesions were detected exclusively at guanines with a preference towards GpG and methylated CpG sequences. The methods utilizing QPCR and LMPCR thus provide means to assess gene-specific and sequence-specific AFB1 damage. The results also prove that microsomally-mediated damage is a suitable method for avoiding manipulations with very unstable DNA-reactive metabolites and that this damage can be detected by QPCR and LMPCR.

Aflatoxin B1↗

PubMed2003

Development and clinical application of a fully controlled quantitative PCR assay for cell-free cytomegalovirus in human plasma.

BACKGROUND: Cytomegalovirus (HCMV) disease continues to be a major problem in certain patient groups, including bone marrow transplant (BMT) recipients. The quantification of HCMV genome is clinically useful for the diagnosis of HCMV disease, for the virological surveillance of high-risk patients and for monitoring antiviral therapy. OBJECTIVES: To develop a novel, robust, and fully controlled PCR (qPCR) for the quantification of HCMV DNA in plasma samples and to demonstrate its clinical usefulness in the BMT setting. STUDY DESIGN: The newly developed HCMV qPCR employs cell culture-derived murine CMV as an internal control for both extraction and amplification. Following amplification using common primers, detection of both internal control and patient HCMV amplicons is by specific probes and a chemiluminescence microtitre plate system. Its performance was evaluated using the routine non-quantitative nested HCMV PCR on whole blood (NQPCR) and correlated with clinical events such as disease and antiviral therapy. RESULTS: A high level of concordance (85.1%) was found between the novel assay and the NQPCR, with the qPCR being slightly more sensitive. The samples giving discordant results generally had levels of HCMV DNA close to the limit of detectability or had been stored for prolonged periods. CONCLUSIONS: The use of plasma as an analyte by the newly developed assay avoids the detection of cell-associated virus. On the other hand, testing a comparatively large volume of plasma ensures that sensitivity is not compromised by not detecting cell-associated HCMV. In a small preliminary evaluation in BMT recipients, changes in HCMV 'viral load' correlated with initiation and discontinuation of antiviral therapy and were biologically plausible.

Base Sequence↗

PubMed2006

Quantitative PCR to detect, discriminate and quantify intracellular parasites in their host: an example from three microsporidians in Daphnia.

Reliable detection, discrimination and quantification of parasites are important for host-parasite studies and diagnostics. Microsporidial infections are problematic in this respect. Their discrimination and quantification using light microscopy is difficult because spores are the only light microscopically visible form of the parasite and they offer few distinct characters. We developed a quantitative PCR (qPCR) assay based on SYBR Green chemistry to quantify the microsporidia Glugoides intestinalis, Octosporea bayeri and Ordospora colligata in their host, the freshwater crustacean Daphnia magna. The assay allows the quantification of infection intensities in whole animals and is more than an order of magnitude more sensitive than light microscopy. Sampling and DNA extraction account for more than 90% of the residual variance in infection intensity data and this variance considerably impairs the resolution of qPCR. Where higher resolution is required, we propose using the ratio of parasite to host DNA as the measure of infection intensity. We show that this measure is robust and greatly improves resolution of qPCR. Additionally, this method can be applied to compare samples of unequal volume.

Animals↗

PubMed2001

Molecular monitoring of the tumor load predicts progressive disease in patients with multiple myeloma after high-dose therapy with autologous peripheral blood stem cell transplantation.

The clinical relevance of the assessment of minimal residual disease (MRD) in patients with multiple myeloma (MM) to predict disease recurrence has not been proven. In the present study, we retrospectively analyzed the tumor load in peripheral blood (PB) and bone marrow (BM) samples of 13 patients with MM both in remission after high-dose therapy (HDT) with autologous PBSC transplantation (PBSCT) and at the time of progressive disease (PD). For six patients, subsequent samples obtained in remission could be included in the study. Tumor cells were assessed by means of quantitative PCR with allele-specific oligonucleotides (ASO-qPCR) based on the method of limiting dilutions. PD was documented with ASO-qPCR in BM samples (median concentration of tumor cells in remission vs at PD: 0.18% vs 4.6%) representing a significant increase by a median factor of 8.7. In PB, the median tumor load was 799 cells/ml in remission and 23 400 cells/ml at PD. With a median factor of 45, the increase was much more pronounced. Comparing the results of the molecular monitoring in PB with those of the determination of the monoclonal protein, routinely applied as parameter for the course of the disease, revealed a superiority of the molecular monitoring because of the significantly higher increase in the tumor load. Analyzing the subsequent remission samples showed an increase of the malignant cells in four out of six PB samples and in all four BM samples available, indicating the potential of ASO-qPCR for an early PD recognition.

Adult↗

PubMed2006

Once daily ganciclovir as initial pre-emptive therapy delayed until threshold CMV load > or =10000 copies/ml: a safe and effective strategy for allogeneic stem cell transplant patients.

Quantitative polymerase chain reaction (QPCR) for cytomegalovirus (CMV) is emerging as the preferred screening method for detection of CMV viremia in patients following allogeneic bone marrow and peripheral blood stem cell transplant. However, there are currently no universally accepted QPCR treatment thresholds at which to start pre-emptive therapy. We report here results of a pre-emptive therapy strategy using ganciclovir (GCV) 5 mg/kg initiated once daily (ODG) delayed till a threshold CMV load of > or =10 000 copies/ml whole blood in clinically stable patients. Sixty-nine at risk patients underwent allogeneic stem cell transplant. 48/69 (70%) patients had an initial episode of CMV viremia. 5/48 (10%) cleared viremia without requiring treatment. 28/43 (65%) patients requiring treatment initiated treatment with ODG. 17/28 (61%) patients successfully cleared CMV viremia on ODG, 10/28 (36%) patients required dose escalation to twice daily GCV for increasing viral loads. There were two cases of CMV disease (colitis) and no deaths due to CMV disease in patients initiating treatment with ODG. We conclude delaying pre-emptive therapy with ODG until whole blood QPCR> or =10 000 copies/ml is a safe and effective strategy for CMV viremia after allogeneic stem cell transplant in clinically stable patients.

Adult↗

PubMed2004

Quantitative PCR analysis of house dust can reveal abnormal mold conditions.

Indoor mold concentrations were measured in the dust of moldy homes (MH) and reference homes (RH) by quantitative PCR (QPCR) assays for 82 species or related groups of species (assay groups). About 70% of the species and groups were never or only rarely detected. The ratios (MH geometric mean : RH geometric mean) for 6 commonly detected species (Aspergillus ochraceus, A. penicillioides, A. unguis, A. versicolor, Eurotium group, and Cladosporium sphaerospermum) were >1 (Group I). Logistic regression analysis of the sum of the logs of the concentrations of Group I species resulted in a 95% probability for separating MH from RH. These results suggest that it may be possible to evaluate whether a home has an abnormal mold condition by quantifying a limited number of mold species in a dust sample. Also, four common species of Aspergillus were quantified by standard culturing procedures and their concentrations compared to QPCR results. Culturing underestimated the concentrations of these four species by 2 to 3 orders of magnitude compared to QPCR.

Air Pollution, Indoor↗

PubMed2006

Prognostic significance of homozygous deletions and multiple duplications at the CDKN2A (p16INK4a)/ARF (p14ARF) locus in urinary bladder cancer.

OBJECTIVE: The 9p21 locus is a major target in the pathogenesis of human urinary bladder cancer. This locus harbours the CDKN2A/ARF tumour suppressor gene, which encodes two cell-cycle regulatory proteins: p16INK4a and p14ARF. We studied how homozygous deletions and multiple duplications at this locus affect prognosis and survival in patients with bladder cancer. MATERIAL AND METHODS: Real-time quantitative polymerase chain reaction (QPCR), based on simultaneous amplification of ARF and a reference gene, glyceraldehyde-3-phosphate dehydrogenase, was used to measure homozygous deletions and multiple duplications in a population-based material consisting of 478 patients with urinary bladder cancer. Results from real-time QPCR were compared with clinico-pathological parameters and survival curves were generated using the Kaplan-Meier method. RESULTS: Real-time QPCR analysis showed 71 (15%) homozygous deletions and 8 (2%) multiple duplications. We were unable to find any association between either stage or grade and urinary neoplasms with homozygous deletions. However, although there were only a limited number of patients with multiple duplications, 7/8 of them had highly malignant tumours (G2b-G4 or > or = T1; p = 0.02). CONCLUSIONS: Urinary bladder cancers constitute a spectrum of neoplasms with varying clinical manifestations. We were unable to establish a prognostic relevance for patients with tumours harbouring homozygous deletions at the CDKN2A/ARF locus. However, our data did indicate that patients with multiple duplications at the CDKN2A/ARF locus had poor survival. This suggests that multiple duplications, in combination with other genetic changes, have cooperative effects which have a negative outcome on urinary bladder cancer prognosis.

Carcinoma, Transitional Cell↗

PubMed2006

Mold infestation of wet spray-applied cellulose insulation.

Mold investigations were conducted in four buildings that had been insulated with wet spray-applied cellulose insulation (WSACI). Bulk WSACI samples were collected and analyzed by quantitative polymerase chain reaction (QPCR) methods. Airborne mold was evaluated using both Burkard total mold spore and Andersen culturable/viable sampling methods. Although reportedly treated with biocidal borates, QPCR analyses indicated that elevated concentrations of mold cells (reported as spore equivalents per gram) may be present in WSACI. QPCR analyses showed the following: (1) very high concentrations of Penicillium chrysogenum in samples from two of four buildings; (2) very high concentrations of Stachybotrys chartarum in samples from one building and a more moderate presence in a second; (3) moderately high concentrations of Aspergillus versicolor in samples from one building and more moderate concentrations in a second; (4) the presence of the opportunistic pathogen, Aspergillus fumigatus, in samples from three of the four buildings, and (5) the presence of 22 of 23 target mold species. Elevated airborne total mold spore concentrations were observed in all four of the buildings investigated. Culturable/viable airborne mold concentrations were moderately elevated in three of the four buildings. Mold genera/types present were relatively consistent among airborne mold samples collected by both methods and bulk sample analyses. Results of this study suggest that WSACI has the potential to cause elevated airborne mold levels in buildings where it has been applied and pose significant mold exposure and public health risks.

Air Microbiology↗

PubMed2006

Real-time PCR detection and speciation of Cryptosporidium infection using Scorpion probes.

At least eight species of Cryptosporidium can cause human infection and disease. A real-time PCR (qPCR) assay based on the 18S rRNA gene and utilizing a Scorpion probe was developed to detect all human-pathogenic Cryptosporidium without the usual need for nested amplification. Sensitivity of detection in stool samples was highest using a glass bead-based DNA extraction method (under 10(3) oocysts per stool sample). The assay was validated against 123 human stool specimens from Bangladesh and Tanzania, exhibited a sensitivity and specificity of >91% versus microscopy, and detected an additional eight microscopy-negative infections. Cryptosporidium parvum-specific and Cryptosporidium meleagridis-specific Scorpion qPCR assays that provided 100% accurate speciation compared with VspI RFLP analysis and sequencing were developed subsequently. These Scorpion probe qPCR assays are simpler to perform than existing nested PCR and RFLP methods for diagnosis and epidemiological investigation of cryptosporidiosis.

Animals↗

PubMed2005

Detection of Renibacterium salmoninarum in chinook salmon, Oncorhynchus tshawytscha (Walbaum), using quantitative PCR.

A quantitative polymerase chain reaction (QPCR) assay has been developed to detect varying levels of Renibacterium salmoninarum, the causative agent of bacterial kidney disease. This assay allows for the direct enumeration of bacterial DNA or RNA copy number within tissues and body fluids. The assay can be applied non-lethally and can be used to determine whether R. salmoninarum is transcriptionally active. The presence of R. salmoninarum in kidney tissues from 430 chinook salmon collected from five Idaho Fish and Game operated hatcheries was initially evaluated using the widely employed enzyme-linked immunosorbent assay (ELISA) with two sets of Kirkegaard and Perry Laboratories polyclonal antibodies, 'mother batches' 1 and 2. The same tissue samples were then analysed using the novel QPCR assay and the results compared. At moderate to high levels of infection [optical density (OD > 0.5)], ELISA values and estimated DNA copy number were highly correlated (r(2) > 0.80), although correlation to specific antibody batches varied. However, lower ELISA values (OD < 0.5) observed with either antibody batch did not correlate well with the QPCR assay (R(2) <or= 0.43). Negative controls, run concurrently with the PCR assay, did not indicate extraneous DNA contamination in the PCR samples.

Actinomycetales Infections↗

PubMed2005

Detection of hemizygosity in Hermansky-Pudlak syndrome by quantitative real-time PCR.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by oculocutaneous albinism, a bleeding diathesis and, in some patients, pulmonary fibrosis or granulomatous colitis. HPS is associated with biosynthesis defects of melanosomes, platelet-dense bodies, and lysosomes. There are seven genetic HPS subtypes; HPS-1 is the most common. We used a real-time quantitative PCR (qPCR) approach to investigate six HPS-1 patients, previously assigned as having homozygous mutations in the HPS1 gene. HPS1 gene copy numbers, calculated by use of a comparative Ct method, revealed that one patient was in fact hemizygous for her c.1189delC (S396delC) HPS1 mutation. The causative deletion/insertion was 13,966 bp in size, with defined breakpoints, and involved an adjacent gene (C10orf33). A mechanism of formation is proposed for the deletion/insertion, and both multiplex and qPCR indicated that the deletion/insertion was present in the patient, her brother, and her father. qPCR amplification is valuable for detecting deletions too small to be identified by fluorescence in situ hybridization. This demonstration of hemizygosity, performed using genomic DNA, can eliminate concerns about non-paternity and can verify the diagnosis of an autosomal recessive disorder when a DNA alteration appears to be homozygous by standard PCR and sequencing methods, and its pathogenicity is in doubt.

Adolescent↗

PubMed2005

Phenotypic characterization of Rice Cluster III archaea without prior isolation by applying quantitative polymerase chain reaction to an enrichment culture.

A so far uncultured member of the Euryarchaeota was enriched from an anoxic riparian soil and phenotypically characterized using quantitative polymerase chain reaction (qPCR; "real-time PCR"). The microorganism is related to the Thermoplasmatales and belongs to Rice Cluster III (RC-III). Enrichment cultures utilized yeast extract (YE) by transiently accumulating acetate as major fermentation product, which was subsequently converted to methane. The abundance of RC-III archaea within the enrichment cultures was quantified by analysis of the terminal restriction fragment length polymorphism (T-RFLP) and by qPCR. We developed qPCR assays targeting the 16S rRNA genes (16S rDNA) specific for RC-III as well as for the Archaea in general. The enrichment cultures consisted of a mixed methanogenic community of Bacteria and Archaea, the latter consisting of up to 60% of members of RC-III. The other archaea belonged to Methanosarcinaceae, Methanomicrobiaceae and Methanobacteriaceae. The enriched RC-III archaea were represented by two sequences (LL25A, LL37A) that were highly similar to each other and to those detected in the soil inoculum (>98% similarity). However, the 16S rDNA copy numbers of RC-III archaea were about 1000-fold lower than those of Bacteria. Nevertheless, we were able to estimate growth parameters and physiological properties of one of the enriched RC-III archaea (LL25A) by measuring the increase of 16S rDNA copy numbers specific for this group under different growth conditions. The enriched RC-III archaeon grew optimally at temperatures between 20 and 30 degrees C and neutral pH using YE, meat extract, peptone or tryptone under anoxic conditions. Doubling time was approximately 3 days. No proliferation was detected on carbohydrates, amino acids, fatty acids, glycerol, alcohols, aromatic compounds, purine and pyrimidine bases or pyruvate. Various exogenous electron acceptors (e.g. ferric iron, S(0)) did not support growth on YE. Proliferation of the enriched RC-III archaeon was hardly affected by the antibiotics ampicillin, kanamycin and streptomycin. These findings suggest that the enriched archaeon is a mesophilic anaerobe, which can grow heterotrophically on peptides. Further enrichment on peptone and kanamycin eventually allowed the microscopic detection of coccoid cells stained by fluorescence in situ hybridization (FISH).

Acetates↗

PubMed2006

Rapid separation and concentration of food-borne pathogens in food samples prior to quantification by viable-cell counting and real-time PCR.

Buoyant density gradient centrifugation has been used to separate bacteria from complex food matrices, as well as to remove compounds that inhibit rapid detection methods, such as PCR, and to prevent false-positive results due to DNA originating from dead cells. Applying a principle of buoyant density gradient centrifugation, we developed a method for rapid separation and concentration following filtration and low- and high-speed centrifugation, as well as flotation and sedimentation buoyant density centrifugation, for 12 food-borne pathogens (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, Campylobacter jejuni, Vibrio cholerae O139, Vibrio parahaemolyticus O3K6, Vibrio vulnificus, Providencia alcalifaciens, Aeromonas hydrophila, Bacillus cereus, Staphylococcus aureus, and Clostridium perfringens) in 13 different food homogenates. This method can be used prior to real-time quantitative PCR (RTi-qPCR) and viable-cell counting. Using this combined method, the target organisms in the food samples theoretically could be concentrated 250-fold and detected at cell concentrations as low as 10(1) to 10(3) CFU/g using the RTi-qPCR assay, and amounts as small as 10(0) to 10(1) CFU/g could be isolated using plate counting. The combined separation and concentration methods and RTi-qPCR confirmed within 3 h the presence of 10(1) to 10(2) CFU/g of Salmonella and C. jejuni directly in naturally contaminated chicken and the presence of S. aureus directly in remaining food items in a poisoning outbreak. These results illustrated the feasibility of using these assays for rapid inspection of bacterial food contamination during a real-world outbreak.

Animals↗

PubMed2006

A real-time PCR method for quantifying viable ascaris eggs using the first internally transcribed spacer region of ribosomal DNA.

Worldwide, 1.4 billion people are infected with the intestinal worm Ascaris lumbricoides. As a result, Ascaris eggs are commonly found in wastewater and sludges. The current microscopy method for detecting viable Ascaris eggs is time- and labor-intensive. The goal of this study was to develop a real-time quantitative PCR (qPCR) method to determine the levels of total and viable Ascaris eggs in laboratory solutions using the first internally transcribed spacer (ITS-1) region of ribosomal DNA (rDNA) and rRNA. ITS-1 rDNA levels were proportional to Ascaris egg cell numbers, increasing as eggs developed from single cells to mature larvae and ultimately reaching a constant level per egg. Treatments causing >99% inactivation (high heat, moderate heat, ammonia, and UV) eliminated this increase in ITS-1 rDNA levels and caused decreases that were dependent on the treatment type. By taking advantage of this difference in ITS-1 rDNA level between viable, larvated eggs and inactivated, single-celled eggs, qPCR results were used to develop inactivation profiles for the different treatments. No statistical difference from the standard microscopy method was found in 75% of the samples (12 of 16). ITS-1 rRNA was detected only in samples containing viable eggs, but the levels were more variable than rDNA levels and ITS-1 rRNA could not be used for quantification. The detection limit of the rDNA-based method was approximately one larvated egg or 90 single-celled eggs; the detection limit for the rRNA-based method was several orders of magnitude higher. The rDNA qPCR method is promising for both research and regulatory applications.

Animals↗