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PubMed2004

Convenient determination of DNA extraction efficiency using an external DNA recovery standard and quantitative-competitive PCR.

Molecular biology techniques have advanced the field of microbial ecology through the analysis of nucleic acids. Most techniques that use DNA or RNA require their extraction from environmental matrices, which can be tedious and inefficient. While a number of extraction methods, both laboratory-based and commercially available, have been developed, none of these include a convenient method to determine extraction efficiency. We have developed an external DNA recovery standard, Lambda DNA (target DNA) contained within pBR322, allowing routine determinations of DNA recovery efficiency. Target DNA was added to sediments as whole cells, total DNA extracted using commercial DNA extraction/purification kits and the amount of target DNA recovered quantified by quantitative-competitive PCR (QC-PCR). Three commercially available kits (UltraClean Soil DNA, FastDNA SPIN and Soil Master DNA Extraction) were evaluated for recovery efficiency. Recoveries for the three kits ranged from undetectable to 43.3% with average recoveries of 14.9+/-16.0%, 28.3+/-10.5% and 2.4+/-0.1% (UltraClean, FastDNA and Soil Master, respectively). Quantification of target DNA proved robust in sediments heavily polluted with polycyclic aromatic hydrocarbons and the external recovery standard could be detected following extraction and amplification from as few as 1 x 10(3) cells added to 0.5 g sediment (wet weight). The external DNA recovery standard was also added directly to the sediment as purified plasmid DNA prior to extraction. It was recovered with similar efficiency as when added as whole cells, suggesting its usefulness in estimating DNA recovery in ribosomal DNA studies. These results show that, while the commercial kits offer expedited sample processing, the extraction efficiencies vary on a sample-by-sample basis and were <100%. Therefore, quantitative DNA studies require an estimation of DNA recovery.

Bacteriophage lambda↗
PubMed1993

[The diagnosis of HIV infection by detecting specific nucleotide sequence].

There are several methods to detect human immunodeficiency virus (HIV) infection; the detection of antibodies against HIV, the detection of HIV particles, and the detection of HIV infected cells. The detection of anti-HIV antibodies is practical, but the antibodies are undetectable both on a seronegative person at the superacute phase, and the new-born baby from a seropositive mother. No other method is ideal for all purposes. Thus the detection of the HIV-specific nucleotide sequence especially using the PCR method has been expected. We developed a quantitational PCR method to assay HIV-infected PBMC cell number, and the result correlated with that of the quantitational culture method, and also correlated with the CD4 count, that is inversely related to the clinical stage of HIV infection. Our quantitational PCR method must be improved before application to a practical routine test. A new PCR kit to detect HIV infected cells qualitatively has been developed and purchased in Europe. This kit is especially workable to test specimens where the antibody detection method cannot be used. PCR can be used on other specimens and can be applied to HIV infection. HIV RNA (both genomic RNA and viral mRNA) can be detected by a combination with the reverse transcriptase reaction. This method (RT-PCR) will be ideal to detect HIV particles. With PCR, the sequence-differentiation between each clinical virus strain may be detected, thus the characterization of a virus strain such as drug resistance, latency, or the prognosis may be recognized. PCR is indeed a valuable method to detect HIV-infection, provided its limits are properly understood.

Adult↗
PubMed2005

Balb/c mouse model and real-time quantitative polymerase chain reaction for evaluation of the immunoprotectivity against Q fever.

The Balb/c mice were infected with Coxiella burnetii and samples of blood and major organs from the infected mice were collected on days 1 to 14 after infection. The DNAs extracted from the samples were detected by a developed real-time quantitative PCR specific for C. burnetii and high loads of C. burnetii were found in spleens, livers, and lungs, particularly in spleens. The Balb/c mice were immunized with whole cell antigen (WCA) of C. burnetii and coxiella loads in spleens of mice were assessed by the real-time quantitative PCR on day 7 after challenge with C. burnetii. The analysis suggested that phase I whole cells were excellent immunogen that elicited complete protection against coxiella infection by two-booster but not one-booster immunization. The results suggest that the combination of Balb/c model and the real-time quantitative PCR assay is a reliable and sensitive way to evaluate the efficiency of vaccines against Q fever.

Animals↗
PubMed2004

[Application of real-time fluorescence quantitative polymerase chain reaction techniques for prenatal diagnosis of Down syndrome].

OBJECTIVE: To investigate the value of real-time fluorescence quantitative PCR in diagnosis of Down syndrome with uncultured amniotic cells. METHODS: The uncultured amniocytes of 80 fetuses who were confirmed disomy 21 by chromosome analysis and 5 fetuses detected trisomy 21 and peripheral blood samples of 7 children diagnosed as Down syndrome were collected to extract gDNA and the real-time fluorescence quantitative PCR method was used to detect the original copies of Down syndrome critical region gene(3) (DSCR(3)) and GAPDH gene and then the ratio of DSCR(3)/GAPDH was calculated. RESULTS: The PCR product ratios of DSCR(3) to GAPDH in trisomy 21 from amniocytes and peripheral blood were ranged from 1.64 to 1.98 while in the normal control the ratio was only from 0.46 to 1.30. CONCLUSION: Real-time fluorescence quantitative PCR is a valuable method for rapid and accurate prenatal diagnosis of Down syndrome in uncultured amniotic cells.

Down Syndrome↗
PubMed1999

Presence of donor- and recipient-derived DNA in cell-free urine samples of renal transplantation recipients: urinary DNA chimerism.

BACKGROUND: Previous studies have indicated that microchimerism is present in body tissues, peripheral blood, and plasma of recipients after organ transplantation. We hypothesize that donor-derived DNA may also be present in cell-free urine of renal transplant recipients and that the concentrations of urine DNA may be correlated with graft rejection. METHODS: Thirty-one female patients who had renal transplantation were enrolled in the study. In women with male organ donors, the SRY gene on the Y chromosome was used as a marker for donor-derived DNA. Real-time quantitative PCR for the SRY and beta-globin genes was carried out on cell-free urinary DNA from these patients. Serial urine samples from a female renal transplant recipient undergoing an acute rejection episode were also collected and analyzed with the beta-globin quantitative PCR system. RESULTS: SRY sequences were detected in the urine of 14 of 17 female patients with male organ donors. None of the 14 patients with female organ donors had detectable SRY sequences in urinary DNA. The median fractional concentration of donor-derived DNA was 8.7% (interquartile range, 1.9-26.4%). During the acute rejection episode, urinary concentrations of the beta-globin gene were markedly increased, with the concentrations returning rapidly to normal following antirejection treatment. CONCLUSIONS: Our results demonstrate that urinary DNA chimerism is present following renal transplantation. The measurement of urinary DNA using quantitative PCR may be useful for the diagnosis and monitoring of graft rejection.

Biomarkers↗
PubMed2000

Quantitative and bioluminescent assay to measure efficacy of conventional and DNA vaccinations against Helicobacter pylori.

Vaccination against Helicobacter pylori using DNA sequences encoding Urease A and B subunits was compared to immunization with urease antigen and MTP-PE in a liposome formulation. To determine the effectiveness of a vaccine against H. pylori in a mouse model it is essential to quantify the number of H. pylori remaining in the stomachs following challenge with an inoculum of live bacteria. Culture assays and enzymatic assays produce inconsistent results often unsuitable to conclude if vaccine candidates are protective. To overcome this problem, we developed two assays: 1) a competitive quantitative PCR using a colorimetric readout and 2) a non-competitive direct quantitative PCR using a highly sensitive bioluminescent readout. The competitive PCR requires coamplification of a segment of the urease C sequence and an internal control standard in a competitive manner using a single set of primers. PCR products were quantified colorimetrically by an enzyme-linked immunosorbent assay and compared with known quantities of the internal control standard added to the PCR reaction. The highly sensitive, bioluminescent assay measures the amplified DNA directly using a flash-type luminescent tag and a specific probe. The Sydney strain of H. pylori was used for the mouse infection model. Quantification of H. pylori by either the bioluminescent assay or the competitive PCR was reliable, specific and sensitive compared to quantitative growth assays which often gave false results. The bioluminescent assay was much more sensitive and less labor/time intensive than the competitive PCR. The bioluminescent assay was able to quantitate as few as 100 bacteria, while the competitive assay could not detect less than 10(3) bacteria per mouse stomach. Quantification of H. pylori by bioluminescent assay was superior to the competitive assay and may be used for research applications, such as the development of vaccines, pathogenesis of gastric disease and monitoring of antibiotic treatment.

Animals↗
PubMed2005

The potential lymphangiogenic effects of hepatocyte growth factor/scatter factor in vitro and in vivo.

Lymphangiogenesis is key to the lymphatic spread of cancer cells. The current study examined the potential effect of hepatocyte growth factor (HGF), a factor known to have strong biological effects on endothelial cells, on the lymphangiogenic function of endothelial cells and the formation of lymphatic vessels using both in vitro and in vivo models. Human endothelial cells that have lymphatic characteristics, human prostate and breast cancer cells PC-3 and MDA MB 231, were used. Expression of lymphatic markers, podoplanin, Prox-1, vascular endothelial growth factor receptor 3 (VEGF-R3) and LYVE-1 was determined using reverse transcription polymerase reaction and quantitative PCR. In nude mice prostate and breast xenograft tumour models, either HGF or an HGF-producing fibroblast cell line MRC-5 was given with or without the HGF antagonist, NK4. The lymphangiogenic marker and lymphatic vessels in tumour tissues were also assessed using quantitative PCR and immunohistochemistry, respectively. In the mice tumour models, infusion of rhHGF significantly increased the levels of podoplanin and LYVE-1 in the tumour (p=0.05 for podoplanin and p<0.05 for LYVE-1 vs. without HGF in the prostate tumour model, p<0.05 for podoplanin and p<0.01 for LYVE-1 vs. without HGF for the breast tumour model; p<0.05 for podoplanin and p<0.01 for LYVE-1 vs. without HGF in the breast tumour model). The increased level of LYVE-1 transcript was supported by an increase in the number of LYVE-1-positive lymphatic vessels in tumours, using immunohistochemical analysis. Co-injection of MRC5 cells also increased the levels of LYVE-1 and number of LYVE-1-positive vessels in tumour tissues. The effects of HGF and MRC5 were significantly reduced by the HGF antagonist, NK4. In the in vitro models, rhHGF significantly increased the level of both podoplanin and LYVE-1, as shown by quantitative PCR analysis. Hepatocyte growth factor has potential lymphangiogenic activities, and this may have important implications in the nodal spread of cancer cells.

Animals↗
PubMed2003

Quantification of mitochondrial DNA deletion, depletion, and overreplication: application to diagnosis.

BACKGROUND: Many mitochondrial pathologies are quantitative disorders related to tissue-specific deletion, depletion, or overreplication of mitochondrial DNA (mtDNA). We developed an assay for the determination of mtDNA copy number by real-time quantitative PCR for the molecular diagnosis of such alterations. METHODS: To determine altered mtDNA copy number in muscle from nine patients with single or multiple mtDNA deletions, we generated calibration curves from serial dilutions of cloned mtDNA probes specific to four different mitochondrial genes encoding either ribosomal (16S) or messenger (ND2, ND5, and ATPase6) RNAs, localized in different regions of the mtDNA sequence. This method was compared with quantification of radioactive signals from Southern-blot analysis. We also determined the mitochondrial-to-nuclear DNA ratio in muscle, liver, and cultured fibroblasts from a patient with mtDNA depletion and in liver from two patients with mtDNA overreplication. RESULTS: Both methods quantified 5-76% of deleted mtDNA in muscle, 59-97% of mtDNA depletion in the tissues, and 1.7- to 4.1-fold mtDNA overreplication in liver. The data obtained were concordant, with a linear correlation coefficient (r(2)) between the two methods of 0.94, and indicated that quantitative PCR has a higher sensitivity than Southern-blot analysis. CONCLUSIONS: Real-time quantitative PCR can determine the copy number of either deleted or full-length mtDNA in patients with mitochondrial diseases and has advantages over classic Southern-blot analysis.

Adolescent↗
PubMed2001

Detection of minimal residual disease.

A high percentage of patients with leukemia, lymphoma, and solid tumors achieve a complete clinical remission after initial treatment, but the majority of these patients will finally relapse from residual tumor cells detectable in clinical remission only by the most sensitive methods. The in vitro amplification of tumor-specific DNA or RNA sequences by polymerase chain reaction (PCR) allows identification of a few neoplastic cells in 10(4) to 10(6) normal cells. Depending on the underlying malignant disease and therapeutic treatment, the presence of residual tumor cells in an individual patient may herald relapse, but a long-term stable situation or slowly vanishing tumor cells are also possible. Molecular monitoring of residual leukemia and lymphoma cells by quantitative PCR techniques has provided important information about the effectiveness of treatment and the risk of recurrent disease as shown by minimal residual disease (MRD) analysis in patients with various malignant diseases. Such diseases include childhood acute lymphoblastic leukemia, after induction therapy; acute promyelocytic leukemia, during and after chemotherapy; and chronic myelogenous leukemia, during treatment with alpha-interferon and after allogeneic bone marrow transplantation. Evaluation of the predictive value of the detection of MRD has to take into account its evolution and course, the pathogenesis, biology, and natural course of the underlying malignant disease, the molecular genetic lesion, and finally, the type of treatment. Quantification of minimal residual cells by the recently developed real-time quantitative PCR technique will surely have a major impact on our therapeutic strategies for patients with leukemia, lymphomas, and solid tumors. Based on quantitative PCR data, the terms molecular remission and molecular relapse have to be exactly defined and validated in prospective clinical trials to assess the biological and clinical significance of MRD in various types of malignancies.

Antigens, CD↗
PubMed2005

Quantitative studies on SMN1 gene and carrier testing of spinal muscular atrophy.

OBJECTIVE: To construct a method for detecting the copy number of survival of motor neuron 1 gene (SMN1) with single copy difference based on real-time fluorescence quantitative PCR, and to make practical use of the method for acquiring the data on SMN1 copy number in Chinese as well as for screening the carriers of spinal muscular atrophy (SMA) from healthy individuals and SMA families. METHODS: Exon 7 and flanking area of SMN1 gene were amplified by real-time fluorescence quantitative PCR in 264 healthy individuals, in 1 standard sample having 2 SMN1 but having no SMN2, and in 88 parents of SMA patients. The samples for detecting were diluted to 30 ng/microL and the standard sample was diluted to 15 ng/microL, 30 ng/microL, 45 ng/microL, 60 ng/microL; the unknown samples and 4 standard samples with different concentrations were amplified at the same time, a standard curve could be drawn out according to the results of the 4 standard samples, then the copy number of samples could be calculated. RESULTS: Of 88 parents' samples, 84 samples each had 1 copy of SMN1, and the rest 4 each had 2 copies of SMN1. Of 264 healthy individuals' samples, 5 samples each had only 1 copy of SMN1 (an indicator of definite gene carriers), 232 samples each had 2 copies of SMN1, 25 samples each had 3 copies of SMN1, and 2 samples each had 4 copies of SMN1. Of the samples of 32 members of SMA families, 2 samples each had only 1 copy of SMN1 indicating definite gene carriers, 25 samples each had 2 copies of SMN1, and 5 samples each had 3 copies of SMN1. CONCLUSION: SMN1 copy number could be detected precisely by real-time fluorescence quantitative PCR; the screening of gene carriers could provide essential data for genetic counseling.

Exons↗
PubMed2004

Clinical investigation of posttransfusion Kidd blood group typing using a rapid normalized quantitative polymerase chain reaction.

BACKGROUND: Accurate typing of a patient's RBCs in the setting of prior transfusion or a hemolytic transfusion reaction is crucial in the selection of compatible blood but is time consuming, technically difficult, and sometimes impossible. To address this problem, a simple, rapid, and inexpensive quantitative PCR method was developed to identify the single nucleotide polymorphism (SNP) of the Kidd blood group. We applied this method in a clinical investigation of 54 multiple-transfusion patients. STUDY DESIGN AND METHODS: Patients were eligible if they had received at least one RBC transfusion within 30 days and had a sample referred to our regional reference lab for assistance with compatibility testing requiring reticulocyte separation, hypotonic saline treatment, or chemical modification to remove IgG. We compared serologic result to the normalized quantitative PCR. For discrepants, or where no serologic type could be assigned, DNA sequencing characterized the patient's Kidd SNP. RESULTS: Of the 54 patients, the reference lab could assign a serologic Kidd type for 33. Quantitative PCR assigned a Kidd type for 53 of the 54. In three cases, where serology and PCR were discrepant, and for all cases where serology could not assign a Kidd type, DNA sequencing verified the Kidd typing assigned by PCR. CONCLUSION: A simple, rapid, and accurate technique has been developed. The assay performs well in the clinical setting. With further study, and inclusion of other blood group systems, this may become an important supplemental technique for selected patients in the immunohematology reference laboratory.

Calibration↗
PubMed1999

Clinical evaluation of an in-house reverse transcription-competitive PCR for quantitation of human immunodeficiency virus type 1 RNA in plasma.

An in-house reverse transcription (RT)-competitive PCR (RT-cPCR) for the quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma samples was developed and validated. The procedure involves (i) extraction of RNA with spin columns, (ii) ready-to-use bead-mediated RT, (iii) competitive PCR in a microtiter plate, (iv) agarose gel electrophoresis of the reaction products, and (v) densitometric analysis of the digitized image of the gel. Quadruplicate tests and dilution studies showed that the sensitivity and intertest coefficient of variability of the RT-cPCR are comparable to those of the reference AMPLICOR HIV-1 MONITOR test. The results obtained by the two assays with a panel of 45 clinical samples were in good agreement (mean difference, 0.36 +/- 0.25 log units). Analysis of 1,982 clinical samples by the in-house RT-cPCR yielded the typical range of plasma HIV-1 RNA levels with the expected inverse correlation between CD4 counts and HIV-1 RNA titers. In addition, testing of plasma from 36 subjects at weeks 0 and 4 with respect to the time of initiation of protease inhibitor therapy detected a significant decrease in HIV-1 viremia. The mean reduction in the HIV-1 RNA level was 0.914 log unit for those receiving saquinavir (P = 0.0210), 1.584 log units for those receiving indinavir (P = 0.0047), and 1.904 log units for those receiving ritonavir (P < 0.0001). The in-house RT-cPCR assay is simple to develop and perform and allows quantitation of HIV-1 RNA in 100 to 200 samples per operator per week. Since the cost is 1/8 to 1/10 of those of reference commercial assays, this procedure could be conveniently used in medium-scale laboratories.

Anti-HIV Agents↗
PubMed1998

Calibrated user-friendly reverse transcriptase-PCR assay: quantitation of epidermal growth factor receptor mRNA.

We report a competitive reverse transcriptase-PCR (RT-PCR) assay and a calibrated user-friendly RT-PCR assay (CURT-PCR) for epidermal growth factor receptor (EGFR) mRNA. A calibrator was prepared from isolated rat liver RNA, and the amount of EGFR mRNA was determined by competitive RT-PCR. In CURT-PCR, a calibration curve was developed by plotting the ratio between the amount of PCR product originating from the calibrator and the RNA internal standard vs the amount of EGFR mRNA present in the calibrator. A fixed amount of RNA internal standard was coamplified with the EGFR mRNA present in the calibrator or in the sample, using the same primer set. The primers were chosen in regions of the EGFR mRNA that show 100% homology between human, rat, and mouse. The amount of EGFR in the unknown samples was calculated from the calibration curve based on the ratio between PCR product originating from the sample and the corresponding RNA internal standard. Competitive RT-PCR and CURT-PCR were used for rat liver samples from 21 different animals. Comparable results were obtained by the two methods. The imprecision of the CURT-PCR method was 8% (n = 20), and the imprecision of the traditional competitive RT-PCR was 16% (n = 17). We conclude that the CURT-PCR method developed is suitable for routine applications such as quantitation of EGFR expression in tumor biopsies. The imprecision is relatively low. Furthermore, the use of a calibration curve makes it possible to analyze a large number of samples in one analytical run and to accept or reject the results according to existing rules for quality assurance.

Animals↗
PubMed2003

Application of real-time PCR for quantitative detection of Campylobacter jejuni in poultry, milk and environmental water.

Campylobacter jejuni is a leading human food-borne pathogen. The rapid and sensitive detection of C. jejuni is necessary for the maintenance of a safe food/water supply. In this article, we present a real-time polymerase chain reaction (PCR) assay for quantitative detection of C. jejuni in naturally contaminated poultry, milk and environmental samples without an enrichment step. The whole assay can be completed in 60 min with a detection limit of approximately 1 CFU. The standard curve correlation coefficient for the threshold cycle versus the copy number of initial C. jejuni cells was 0.988. To test the PCR system, a set of 300 frozen chicken meat samples, 300 milk samples and 300 water samples were screened for the presence of C. jejuni. 30.6% (92/300) of chicken meat samples, 27.3% (82/300) of milk samples, and 13.6% (41/300) of water samples tested positive for C. jejuni. This result indicated that the real-time PCR assay provides a specific, sensitive and rapid method for quantitative detection of C. jejuni. Moreover, it is concluded that retail chicken meat, raw milk and environmental water are commonly contaminated with C. jejuni and could serve as a potential risk for consumers in eastern China, especially if proper hygienic and cooking conditions are not maintained.

Animals↗
PubMed2003

Quantitative measurement of fungal DNA extracted by three different methods using real-time polymerase chain reaction.

Quantification of fungal populations in the environment is important for gaining a better understanding of various microbial processes. Recently, the development of real-time quantitative PCR (RTQ-PCR) has eliminated the variability associated with conventional quantitative PCR, thereby allowing the routine and reliable quantification of PCR products. Thus, in this present study, RTQ-PCR was used to quantify the fungal target DNA extracted by three commonly used DNA extraction protocols (bead mill homogenization, grinding in the presence of liquid nitrogen, and hot detergent SDS based enzymatic lysis combined with bead beating) to determine the suitability of the quantification of target DNA. For the purpose of this study, pure culture of Aspergillus fumigatus (model organism), sterilized soil seeded with a known amount ofA. fumigatus (model soil system), and woodland and grassland soil samples (environmental samples) were chosen to extract DNA by the above three different protocols. The extracted DNA was then quantified by spectroscopy and a RTQ-PCR system. 18S rDNA specific universal fungal primers were used to quantify the target part and then amplification products were verified by agarose gel electrophoresis. Standard curves used for the quantification by RTQ-PCR revealed strong linear relationships (R2=0.9994 for the primer pair NS1 and NS2 and 0.9938 for the primer pair nu-SSU-0817 and nu-SSU-1196) with a higher amplification efficiency, e=0.983 for the primer pair NS1 and NS2 and 0.956 for the primer pair nu-SSU-0817 and nu-SSU-1196. Although for pure culture the hot detergent SDS based enzymatic lysis combined with bead beating method showed the highest target DNA copy number (1.5 x 10(9) copies/microl), for the model soil system and both environmental samples the bead beating method was found to be suitable on the basis of the high target DNA copy numbers (6.16 x 10(8) and 2.7 x 10(8) copies/microl for woodland and grassland, respectively), high DNA yield (6.4 microg/g and 1.8 microg/g of soil for woodland and grassland, respectively), and high recovery on the basis of the target DNA copy number (39.2%), suggesting an overall high extraction efficiency.

Journal Article↗
PubMed2006

TMPRSS2:ETV4 gene fusions define a third molecular subtype of prostate cancer.

Although common in hematologic and mesenchymal malignancies, recurrent gene fusions have not been well characterized in epithelial carcinomas. Recently, using a novel bioinformatic approach, we identified recurrent gene fusions between TMPRSS2 and the ETS family members ERG or ETV1 in the majority of prostate cancers. Here, we interrogated the expression of all ETS family members in prostate cancer profiling studies and identified marked overexpression of ETV4 in 2 of 98 cases. In one such case, we confirmed the overexpression of ETV4 using quantitative PCR, and by rapid amplification of cDNA ends, quantitative PCR, and fluorescence in situ hybridization, we show that the TMPRSS2 (21q22) and ETV4 (17q21) loci are fused in this case. This result defines a third molecular subtype of prostate cancer and supports the hypothesis that dysregulation of ETS family members through fusions with TMRPSS2 may be an initiating event in prostate cancer development.

Adenovirus E1A Proteins↗
PubMed2003

Molecular characterization of Legionella populations present within slow sand filters used for fungal plant pathogen suppression in horticultural crops.

The total bacterial community of an experimental slow sand filter (SSF) was analyzed by denaturing gradient gel electrophoresis (DGGE) of partial 16S rRNA gene PCR products. One dominant band had sequence homology to Legionella species, indicating that these bacteria were a large component of the SSF bacterial community. Populations within experimental and commercial SSF units were studied by using Legionella-specific PCR primers, and products were studied by DGGE and quantitative PCR analyses. In the experimental SSF unit, the DGGE profiles for sand column, reservoir, storage tank, and headwater tank samples each contained at least one intense band, indicating that a single Legionella strain was predominant in each sample. Greater numbers of DGGE bands of equal intensity were detected in the outflow water sample. Sequence analysis of these PCR products showed that several Legionella species were present and that the organisms exhibited similarity to strains isolated from environmental and clinical samples. Quantitative PCR analysis of the SSF samples showed that from the headwater sample through the sand column, the number of Legionella cells decreased, resulting in a lower number of cells in the outflow water. In the commercial SSF, legionellae were also detected in the sand column samples. Storing prefilter water or locating SSF units within greenhouses, which are often maintained at temperatures that are higher than the ambient temperature, increases the risk of growth of Legionella and should be avoided. Care should also be taken when used filter sand is handled or replaced, and regular monitoring of outflow water would be useful, especially if the water is used for misting or overhead irrigation.

Crops, Agricultural↗
PubMed2002

Use of gDNA as internal standard for gene expression in staphylococci in vitro and in vivo.

An internal RNA standard proved less suitable in bacterial gene expression experiments. We therefore developed a method for simultaneous RNA and gDNA (genomic DNA) isolation from in vitro and in vivo samples containing staphylococci and combined it with quantitative PCR. The reliability of gDNA for bacterial quantification and for standardisation in gene expression experiments was evaluated. Quantitative PCR proves equivalent to quantitative culture for in vitro samples, and superior for in vivo samples. In gene expression experiments, gDNA permits a good standardisation for the initial amount of bacteria. The average interassay variability of the in vitro expression is 20.1%. The in vivo intersample variability was 73.3%. This higher variability can be attributed to the biological variation of gene expression in vivo. This method permits exact quantification of the number of bacteria and the expression of genes in staphylococci in vivo (e.g., in biofilms, evolution in time) and in vitro.

Colony Count, Microbial↗