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PubMed2025

A Quantitative Real-Time PCR Assay for Measuring Poxvirus Replication and Cell Binding.

Quantitative real-time PCR (qPCR) is a fast and reliable method to quantify viral genomes as a surrogate to titering on monolayers of cells for measuring virus replication. Whether it be for determining the number of virions released, the total number of genomes produced during infection, or the number of virions bound to a cell, qPCR assays can be adapted to quickly enumerate total viral genomes in a broad range of experiments comparing virus replication under different conditions. In addition, qPCR offers several advantages compared to plaque assays including time, linearity over 9 logs, and scalability from tens-to-hundreds of samples, depending on the qPCR machine. Here we describe a qPCR assay for quantifying vaccinia virus' dsDNA genome that can be used to determine the total number of virions produced. Furthermore, we describe a straightforward protocol for a cell-binding assay that is sensitive enough to use with small concentrations of inoculating virions. This protocol is suitable for measuring the cell-binding ability of mutations that affect virus production and infectivity.

Virus Replication↗

PubMed2007

Quantitative PCR based expression analysis on a nanoliter scale using polymer nano-well chips.

The analysis of gene expression is an essential element of functional genomics. Expression analysis is mainly based on DNA microarrays due to highly parallel readout and high throughput. Quantitative PCR (qPCR) based expression profiling is the gold standard for the precise monitoring of selected genes, and therefore used for validation of microarray data. Doing qPCR-based expression analysis in an array-like format can combine the higher sensitivity and accuracy of the qPCR methodology with a high data density at relatively low costs. This paper describes the development of an open-well based miniaturized platform for liquid PCR-based assays on the nanoliter scale using cost-effective polypropylene micro reactors (microPCR Chip). We show the quantification ability and reliability of qPCR in 200 nl with the microPCR chip down to 5 starting target molecules using TaqMan chemistry. An RNA expression analysis of four genes in mouse brain, liver and kidney tissues showed similar results in 200 nl as compared to standard 10 microl assays. The high sensitivity and quantification capability of the microPCR chip platform developed herein makes it a promising technology for performing high-throughput qPCR-based analysis in the nanoliter volume range.

Animals↗

PubMed2006

A real-time PCR assay for the enumeration and detection of Zygosaccharomyces bailii from wine and fruit juices.

Zygosaccharomyces bailii is a major food and beverage spoilage organism. Existing methods for its detection involve lengthy enrichment techniques and then the result does not always differentiate between Z. bailii and Saccharomyces cerevisiae. In this work, we developed a quantitative real-time PCR assay for the rapid detection of Z. bailii from fruit juices and wine even in the presence of non-target DNA. Primers were designed to the gene coding for the D1/D2 loop of the 26S ribosomal RNA subunit producing a single PCR product with a melting temperature of 83.5 degrees C. As few as 2 cells per ml could be detected by the assay in cranberry raspberry and apple juices and 22 cells per ml from grape juice. The assay was equally efficient in wine, detecting 6 cells per ml even in the presence of 10(7)S. cerevisiae cells. The CFU/ml as determined by plating on YM media showed excellent correlation with the CFU/ml established by the QPCR assay for all the beverages examined. Unknown samples of Z. bailii were prepared in the juices and wine and examined by QPCR. The QPCR estimated cell number was in good agreement with the cell counts obtained by plating, the exception being the cranberry raspberry juice sample. It was determined by live/dead cell counts that the Z. bailii cells were less viable in this juice thus leading to an overestimation of CFU/ml by QPCR. However, the correlation was high between QPCR and total cell count as determined by fluorescent microscopy. This assay provides a rapid and accurate method to establish the levels of the total Z. bailii population which consists of both viable and nonviable cells.

Beverages↗

PubMed2004

A sensitive and quantitative single-tube real-time reverse transcriptase-PCR for detection of enteroviral RNA.

BACKGROUND: Enteroviruses (EVs) are significant human pathogens. Rapid and sensitive diagnostic techniques are desirable. OBJECTIVES: To develop a quantitative single-tube real-time reverse transcription-polymerase chain reaction (RT-PCR) for human enterovirus ribonucleic acid (RNA) (QPCR), with protection against amplimer contamination. STUDY DESIGN: The method was evaluated with serial dilutions of EV, 62 cerebrospinal fluid (CSF) specimens from meningitis patients, and the third and fourth European Union Concerted Action Enterovirus Proficiency Panels. A commercial EV PCR test was run in parallel. RESULTS: Optimisation included RNA extraction procedure, design and concentrations of primers and probes from the 5' non-coding region as well as recombinant Thermus thermophilus polymerase (rTth), Mn(OAc)(2) and thermolabile UNG concentrations. Of 62 CSF samples from cases of meningitis submitted for QPCR testing, 34 (76%) and 21 (47%) were positive by QPCR and a commercial EV RNA detection kit, respectively. The detection limit of QPCR was 0.001 TCID(50)/ml (50% tissue culture-infective dose per millilitre) for a coxsackievirus B2 preparation and <10 copies of a plasmid containing coxsackievirus B2 complementary deoxyribonucleic acid (cDNA). The relation between threshold cycle (C(t)) and amount of virus was linear (r = 0.99) over a range of 10(-3) to 10(4) TCID(50)/ml of coxsackievirus B2. CONCLUSIONS: The QPCR method allows a large number of samples to be screened rapidly. Its sensitivity, simplicity, and reproducibility make it a suitable tool for the routine laboratory.

5' Untranslated Regions↗

PubMed2005

Detection of hemizygous deletions in genomic DNA from leukaemia specimens for the diagnosis of patients.

Hemizygous deletions in genomic DNA appear to play an important role in tumorigenesis. The loss or inactivation of tumour suppressor genes (TSGs) is of critical importance in most malignancies, and has been shown to affect response to therapy. Here, we report a quantitative real-time polymerase chain reaction (qPCR) designed to detect two TSGs at the CDKN2A locus, p16(INK4A) and p14(ARF) that allows the detection of hemizygous deletions. Testing by qPCR of 18 bone marrow specimens from paediatric acute lymphoblastic leukaemia (ALL) patients at diagnosis revealed nine to be GG, six to be GD and three to be DD for exon 2 of p14(ARF)/p16(INK4A), concordant with Southern blotting analysis. A panel of 13 ALL cell lines was investigated for deletions at the CDKN2A locus and one of the lines, typed as GD for all exons, was further assessed by fluorescence in situ hybridisation, confirming the qPCR findings. The expression levels of p16(INK4A) and p14(ARF) were measured in all cell lines and these quantitative reverse transcriptase PCR results also agreed with the typing by qPCR. The qPCR method described is suitable for detection of hemizygous loss in primary patient material and the accuracy of the method was verified by three independent techniques.

Alleles↗

PubMed2005

Quantitative real-time polymerase chain reaction for determination of plasmid copy number in bacteria.

A method for determination of plasmid copy number (PCN) in bacteria by real-time quantitative polymerase chain reaction (QPCR) was developed as an alternative to current PCN assays. Conventional methods for PCN estimation are generally not of high throughput, laborious, have low reproducibility, require large amounts of biological samples and are applicable only for a narrow dynamic range. Real-time QPCR, using the ABI Prism 7000, was able to sensitively detect the quantity of the pUC ori based plasmid, NS3, transformed into Escherichia coli host, DH5alpha, to be 411+/-6.1. The PCN of pBR322 plasmid DNA in DH5alpha was estimated to be 40+/-0.6 which is within its previously reported PCN range of approximately 30 to 70. QPCR was found to show good reproducibility and high sensitivity in detecting a two fold difference in template concentration, and a wide linear dynamic range covering 0.5 pg to 50 ng of DNA. PCNs of DH5alpha bearing plasmids pBR322 and NS3 computed from real-time QPCR assay were validated by that of agarose gel assay, and a marginal difference of only 13.0% and 10.7% was found for the two plasmids respectively. The QPCR assay was able to detect changes in PCN of plasmid producing DH5alpha during the course of a 2 l batch fermentation.

Culture Media↗

PubMed2006

Development and evaluation of a Taqman duplex real-time PCR quantification method for reliable enumeration of Legionella pneumophila in water samples.

This study describes the development and evaluation of a specific Legionella pneumophila Taqman duplex real-time PCR (qPCR) for fast and reliable quantification of this human pathogen in suspected man-made water systems. The qPCR assay was 100% specific for all L. pneumophila serogroups 1-15 with a sensitivity of 60 genome units/l and an amplification efficiency of 98%. Amplification inhibitors were detected via an exogenous internal positive control, which was amplified simultaneously with L. pneumophila DNA using its own primer and probe set. Mean recovery rates of the qPCR assay for tap water and cooling circuit water, spiked with a known number L. pneumophila bacteria, were 93.0% and 56.3%, respectively. Additionally, by using the Ultraclean Soil DNA isolation kit, we were able to remove amplification inhibitors ubiquitously present in cooling water. The practical value of our qPCR assay was evaluated through analysis of 30 water samples from showers, taps, eyewash stations, fire sprinklers and recirculation loops with qPCR and traditional culture. In conclusion, the described L. pneumophila Taqman duplex real-time assay proved to be specific, sensitive and reproducible. This makes it a promising method complementing the current time-consuming culture standard method.

DNA, Bacterial↗

PubMed2004

Comparison of Enterococcus measurements in freshwater at two recreational beaches by quantitative polymerase chain reaction and membrane filter culture analysis.

Cell densities of the fecal pollution indicator genus, Enterococcus, were determined by a rapid (3 h or less) quantitative polymerase chain reaction (QPCR) analysis method in 100 ml water samples collected from recreational beaches on Lake Michigan and Lake Erie during the summer of 2003. Measurements by this method were compared with counts of Enterococcus colony-forming units (CFU) determined by Method 1600 membrane filter (MF) analysis using mEI agar. The QPCR method had an estimated 95% confidence, minimum detection limit of 27 Enterococcus cells per sample in analyses of undiluted DNA extracts and quantitative analyses of multiple lake water samples, spiked with known numbers of these organisms, gave geometric mean results that were highly consistent with the spike levels. At both beaches, the geometric means of ambient Enterococcus concentrations in water samples, determined from multiple collection points during each sampling visit, showed approximately lognormal distributions over the study period using both QPCR and MF analyses. These geometric means ranged from 10 to 8548 cells by QPCR analysis and 1-2499 CFU by MF culture analysis in Lake Michigan (N=56) and from 8 to 8695 cells by QPCR and 3-1941 CFU by MF culture in Lake Erie (N=47). Regression analysis of these results showed a significant positive correlation between the two methods with an overall correlation coefficient (r) of 0.68.

Agar↗

PubMed1997

A quantitative PCR assay for the detection of low amounts of malignant cells in multiple myeloma.

BACKGROUND: High-dose chemotherapy (HDT) with autografting of hematopoietic stem cells induces up to 50% of complete remissions in patients with multiple myeloma. Cases of molecular remissions have been reported. However, qualitative assays determine only the absence or presence of a monoclonal population depending on their sensitivity. Therefore reliable and sensitive methods to quantitate tumor loads are necessary. MATERIALS AND METHODS: We have established a quantitative PCR assay (qPCR) with allele-specific primers complementary to hypervariable CDR3 regions. Sample DNA was serially diluted in 0.5 log steps and amplified in 10 replicates. PCR results were analysed by likelihood maximization and chi 2 minimization to calculate the tumor load. RESULTS: Three approaches were taken to validate the qPCR. 1) Single copies of the CDR3 region of U266 cells could be detected. 2) Analysis of a bone marrow sample by FACS for CD 38+2 and kappa/lambda restricted plasma cells and by qPCR yielded results of 1.4 and 2.5% respectively. 3) qPCR results with plasmids carrying CDR3 regions simulating different tumor loads diverged by no more than a factor of 1.6 from the expected values. CONCLUSION: We consider the qPCR to be an accurate method for assessing samples with low amounts of malignant cells.

Base Sequence↗

PubMed2002

Prospective validation of quantitative polymerase chain reaction for management of cytomegalovirus disease in solid-organ transplant patients.

This study evaluates the utility of quantitative polymerase chain reaction (QPCR) to determine duration of treatment of transplant patients with human cytomegalovirus (HCMV) disease. Eighteen patients with HCMV disease were prospectively evaluated and followed for recurrence using a QPCR assay. We used plasma samples from which nucleic acid was extracted. Quantification was determined by using an internal standard that contained the same primer sequences as for HCMV. During treatment, weekly QPCR assays were performed. Patients were treated with HCMV immunoglobulin-G for a finite period, but intravenous ganciclovir was continued until less than 100 viral copies (vc) per mL was detectable. After cessation of therapy, patients were followed for 6 months with monthly clinical assessment and QPCR. No patient developed recurrence of HCMV at a mean follow-up of 16 months. This preliminary study suggests that the use of QPCR to assess viral load is useful in deciding the length of HCMV treatment with ganciclovir but requires further randomized validation.

Adolescent↗

PubMed2006

Improved enumeration of lactic acid bacteria in mesophilic dairy starter cultures by using multiplex quantitative real-time PCR and flow cytometry-fluorescence in situ hybridization.

Nucleic acid-based assays were developed to enumerate members of the three taxa Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, and Leuconostoc spp. in mesophilic starter cultures. To our knowledge the present is the first study to present a multiplex quantitative PCR (qPCR) strategy for the relative enumeration of bacteria. The multiplex qPCR strategy was designed to quantify the target DNA simultaneously relative to total bacterial DNA. The assay has a high discriminatory power and resolves concentration changes as low as 1.3-fold. The methodology was compared with flow cytometric fluorescence in situ hybridization (FLOW-FISH) and 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-Gal)-calcium citrate agar-based plate counting. For enumeration by FLOW-FISH, three new probes having the same specificity as the qPCR assay were designed and established. A combination with flow cytometry greatly reduced the time consumed compared to manual enumeration. Both qPCR and FLOW-FISH yielded similar community compositions for 10 complex starter cultures, with all detected subpopulations being highly significantly correlated (P < 0.001). Correlations between X-Gal-calcium citrate agar-based CFU and qPCR-derived counts were highly significant (P < 0.01 and P < 0.001, respectively) for the number of acidifiers versus L. lactis subsp. cremoris and for Leuconostoc spp. as quantified by the two techniques, respectively. This confirmed that most acidifiers in the studied PROBAT cultures are members of L. lactis subsp. cremoris. Quantitative real-time PCR and FLOW-FISH were found to be effective and accurate tools for the bacterial community analysis of complex starter cultures.

Agar↗

PubMed2001

Combination of competitive quantitative PCR and constant-denaturant capillary electrophoresis for high-resolution detection and enumeration of microbial cells.

A novel quantitative PCR (QPCR) approach, which combines competitive PCR with constant-denaturant capillary electrophoresis (CDCE), was adapted for enumerating microbial cells in environmental samples using the marine nanoflagellate Cafeteria roenbergensis as a model organism. Competitive PCR has been used successfully for quantification of DNA in environmental samples. However, this technique is labor intensive, and its accuracy is dependent on an internal competitor, which must possess the same amplification efficiency as the target yet can be easily discriminated from the target DNA. The use of CDCE circumvented these problems, as its high resolution permitted the use of an internal competitor which differed from the target DNA fragment by a single base and thus ensured that both sequences could be amplified with equal efficiency. The sensitivity of CDCE also enabled specific and precise detection of sequences over a broad range of concentrations. The combined competitive QPCR and CDCE approach accurately enumerated C. roenbergensis cells in eutrophic, coastal seawater at abundances ranging from approximately 10 to 10(4) cells x ml(-1). The QPCR cell estimates were confirmed by fluorescent in situ hybridization counts, but estimates of samples with <50 cells x ml(-1) by QPCR were less variable. This novel approach extends the usefulness of competitive QPCR by demonstrating its ability to reliably enumerate microorganisms at a range of environmentally relevant cell concentrations in complex aquatic samples.

Animals↗

PubMed2006

Prochlorococcus ecotype abundances in the North Atlantic Ocean as revealed by an improved quantitative PCR method.

The cyanobacterium Prochlorococcus numerically dominates the photosynthetic community in the tropical and subtropical regions of the world's oceans. Six evolutionary lineages of Prochlorococcus have been described, and their distinctive physiologies and genomes indicate that these lineages are "ecotypes" and should have different oceanic distributions. Two methods recently developed to quantify these ecotypes in the field, probe hybridization and quantitative PCR (QPCR), have shown that this is indeed the case. To facilitate a global investigation of these ecotypes, we modified our QPCR protocol to significantly increase its speed, sensitivity, and accessibility and validated the method in the western and eastern North Atlantic Ocean. We showed that all six ecotypes had distinct distributions that varied with depth and location, and, with the exception of the deeper waters at the western North Atlantic site, the total Prochlorococcus counts determined by QPCR matched the total counts measured by flow cytometry. Clone library analyses of the deeper western North Atlantic waters revealed ecotypes that are not represented in the culture collections with which the QPCR primers were designed, explaining this discrepancy. Finally, similar patterns of relative ecotype abundance were obtained in QPCR and probe hybridization analyses of the same field samples, which could allow comparisons between studies.

Atlantic Ocean↗

PubMed2006

Quantitative PCR targeting 16S rRNA and reductive dehalogenase genes simultaneously monitors multiple Dehalococcoides strains.

The 16S rRNA gene provides insufficient information to infer the range of chloroorganic electron acceptors used by different Dehalococcoides organisms. To overcome this limitation and provide enhanced diagnostic tools for growth measurements, site assessment, and bioremediation monitoring, a quantitative real-time PCR (qPCR) approach targeting 16S rRNA genes and three Dehalococcoides reductive dehalogenase (RDase) genes with assigned function (i.e., tceA, bvcA, and vcrA) was designed and evaluated. qPCR standard curves generated for the RDase genes by use of genomic DNA from Dehalococcoides pure cultures correlated with standard curves obtained for both Bacteria- and Dehalococcoides-targeted 16S rRNA genes, suggesting that the RDase genes are useful targets for quantitative assessment of Dehalococcoides organisms. RDase gene probe/primer pairs were specific for the Dehalococcoides strains known to carry the diagnostic RDase gene sequences, and the qPCR method allowed the detection of as few as 1 to 20 and quantification of as few as 50 to 100 tceA, bvcA, or vcrA gene targets per PCR volume. The qPCR approach was applied to dechlorinating enrichment cultures, microcosms, and samples from a contaminated site. In characterized enrichment cultures where known Dehalococcoides strains were enumerated, the sum of the three RDase genes equaled the total Dehalococcoides cell numbers. In site samples and chloroethane-dechlorinating microcosms, the sum of the three RDase genes was much less than that predicted by Dehalococcoides-targeted qPCR, totaling 10 to 30% of the total Dehalococcoides cell numbers. Hence, a large number of Dehalococcoides spp. contain as-yet-unidentified RDase genes, indicating that our current understanding of the dechlorinating Dehalococcoides community is incomplete.

Bacterial Typing Techniques↗

PubMed2005

Potential application of ELAVL4 real-time quantitative reverse transcription-PCR for detection of disseminated neuroblastoma cells.

BACKGROUND: Reliable detection of neuroblastoma cells in bone marrow (BM) is critical because BM involvement influences staging, risk assessment, and evaluation of therapeutic response in neuroblastoma patients. Standard cytomorphologic examination of BM aspirates is sensitive enough to detect single tumor cells. Consequently, more sensitive and specific detection methods are indispensable. METHODS: We used real-time quantitative reverse transcription-PCR (QPCR) of the tyrosine hydroxylase (TH), GD2 synthetase (GALGT), and embryonic lethal, abnormal vision, Drosophila-like 4 (ELAVL4) genes to detect disseminated neuroblastoma cells. We assessed assay sensitivity by addition experiments and then analyzed 97 neuroblastic tumor, BM, peripheral blood (PB), or peripheral blood stem cell (PBSC) samples from 30 patients. The QPCR results were compared with those of a standardized immunocytochemical assay. RESULTS: The molecular markers were highly expressed in all evaluated tumor samples. In addition, 32%, 11%, and 38% of all BM, PB, and PBSC samples scored positive for TH, GALGT, or ELAVL4, respectively. The TH and ELAVL4 assays could detect 1 neuroblastoma cell in 10(6) mononuclear cells. By contrast, the GALGT QPCR assay could detect 1 neuroblastoma cell in 10(4) mononuclear cells. We assessed the potential prognostic value of TH, GALGT, and ELAVL4 QPCR by analyzing subsequent samples from 3 patients with stage 4 disease. Preliminary results indicated that persistence of high ELAVL4 expression has prognostic value. CONCLUSIONS: ELAVL4 QPCR can be used to detect residual neuroblastoma cells in clinical samples. However, combination of several molecular markers and screening techniques should be considered to ensure reliable detection of rare neuroblastoma cells.

Adolescent↗

PubMed2006

Increased integrity of free circulating DNA in sera of patients with colorectal or periampullary cancer: direct quantitative PCR for ALU repeats.

BACKGROUND: Cell-free DNA circulating in blood is a candidate biomarker for malignant tumors. Unlike uniformly truncated DNA released from apoptotic nondiseased cells, DNA released from dead cancer cells varies in size. We developed a novel method to measure the ratio of longer to shorter DNA fragments (DNA integrity) in serum as a potential biomarker for patients with colorectal cancer (CRC) or periampullary cancers (PACs). METHODS: Sera from 32 patients with CRC (3 stage I, 14 stage II, 6 stage III, and 9 stage IV patients), 19 patients with PACs (2 stage I, 9 stage II, 1 stage III, and 7 stage IV patients), and 51 healthy volunteers were assessed by quantitative real-time PCR of ALU repeats (ALU-qPCR) with 2 sets of primers (115 and 247 bp) amplifying different lengths of DNA. We used serum directly as a template for ALU-qPCR without DNA purification. DNA integrity was determined as ratio of qPCR results of 247-bp ALU over 115-bp ALU. RESULTS: ALU-qPCR had a detection limit of 0.01 pg of DNA. Eliminating DNA purification reduced technical artifacts and reagent/labor costs. Serum DNA integrity was significantly increased for stage I/II and III/IV CRC and stage I/II and III/IV PACs (P = 0.002, P = 0.006, P = 0.022, and P <0.0001, respectively). ROC curves for detecting CRC and PACs had areas under the curves of 0.78 and 0.80, respectively. CONCLUSIONS: Direct ALU-qPCR is a robust, highly sensitive, and high-throughput method to measure serum DNA integrity. DNA integrity is a potential serum biomarker for detection and evaluation of CRC and PACs.

Aged↗

PubMed2005

Neonatal cytomegalovirus blood load and risk of sequelae in symptomatic and asymptomatic congenitally infected newborns.

OBJECTIVE: Human cytomegalovirus (CMV) is a ubiquitous human-specific DNA virus and is the main cause of congenital virus infection in developed countries leading to psychomotor impairment and deafness. Diagnostic techniques for CMV detection have greatly improved during recent years with the advent of sophisticated serological and virological methods. The aim of the present study was to assess the diagnostic and prognostic value of detection and quantification of virus in neonatal blood samples of symptomatic and asymptomatic newborns with CMV congenital infection. METHODS: Between January 1997 and December 2003, we studied 99 newborns who were born to women with primary, recurrent, and undefined CMV infection during pregnancy. CMV congenital infection was identified by isolation of the virus in urine within the second week of life. Fifty-eight of 99 infants were infected and were assessed clinically for disease in the newborn period and classified as having symptomatic or asymptomatic infection on the basis of physical, instrumental, and laboratory findings. The infants were followed up from birth according to a protocol of the tertiary NICU at the University of Bologna in a prospective study of long-term sequelae of congenital infection. Forty-seven blood samples were obtained from 47 infants in the neonatal period: 34 were examined for pp65 antigenemia test and 44 for qualitative and quantitative polymerase chain reaction (PCR and qPCR). Sequelae at 12 months were evaluated in a group of 50 infants. RESULTS: Antigenemia was positive in only 10 of 34 samples of infected newborns (29.4% sensitivity). PCR was performed in 44 samples of infected newborns and was positive in all (100% sensitivity). qPCR showed a finding of > or =100 copies per 10(5) of polymorphonuclear leukocytes (PMNLs) in 39 of 44 samples; in the other 5 cases, the number of copies per 10(5) PMNLs was <100. Between symptomatic and asymptomatic newborns, the mean values of viral blood load determined by qPCR turned out to be significantly higher in symptomatic newborns. Mean values of neonatal blood viral load were statistically higher in newborns who developed sequelae than in those who did not. Of 20 children with a neonatal viral blood load of <1000 copies per 10(5) PMNLs, 19 did not develop sequelae (negative predictive value: 95%), whereas 2 of 3 with a viral blood load of >10,000 copies did develop sequelae. CONCLUSIONS: Different viremia value ranges are correlated to a different risk of sequelae: approximately 70% sequelae were found in newborns with a qPCR higher than 10,000 copies per 10(5) PMNLs. Low neonatal viral blood load detected by pp65 antigenemia test and qPCR was highly predictive of absence of sequelae: DNAemia <1000 copies per 10(5) PMNLs has a negative predictive value of 95%. As an independent predictive factor of outcome, neonatal viremia is another useful element for neonatal counseling and therapeutic choices in symptomatic and asymptomatic newborns.

Central Nervous System Diseases↗

PubMed2006

Application of a real-time PCR assay to detect and quantify the myxozoan parasite Ceratomyxa shasta in river water samples.

Ceratomyxa shasta is a virulent pathogen of salmonid fishes that is enzootic in the Pacific Northwest of North America. Current parasite detection methods involve sentinel fish exposures that are laborious and time-consuming. As a substitute, a filtering protocol and a quantitative real-time TaqMan polymerase chain reaction (QPCR) assay were developed to detect and enumerate parasite spores in river water. Fluorescence was detected from both the myxospore and actinospore stages of the parasite but not from the fish or polychaete hosts or from 9 other myxozoans tested. Less than 1/1000th of a spore was detected, indicating each had >1000 copies of the target 18S rRNA gene. The assay detected 1 spore in 1 l river water. Inhibition of the assay by some river samples was overcome by reducing the template volume and including bovine serum albumin in the reaction; occasionally a second purification step was required. The QPCR methodology was utilised to investigate the temporal and spatial distribution of C. shasta in the Klamath River, Oregon/ California. The parasite was detected throughout the river, and 2 of 5 tributaries tested contributed parasites to the mainstem. Correlation of QPCR cycle threshold values with a standard curve for known starting numbers of whole spores revealed several sites where parasite abundance was in excess of 20 spores l(-1). Although QPCR data corroborated results of sentinel fish exposures, spore numbers did not correlate consistently with mortality in the exposure groups. The water sampling and filtering protocol combined with the QPCR assay is a simple and relatively rapid method for detection and quantification of parasite levels in environmental water samples.

Animals↗