Quantitative PCR Assay For Detection Research Articles

Serial quantitative PCR assay for detection, species-discrimination and quantification of Leishmania spp. in human samples

Serial quantitative PCR assay for detection, species-discrimination and quantification of Leishmania spp. in human samples

Development of a real-time quantitative PCR assay for detection of a stable genomic region of BK virus

BackgroundBK virus infections can have clinically significant consequences in immunocompromised individuals. Detection and monitoring of active BK virus infections in certain situations is recommended and therefore PCR assays for detection of BK virus have been developed. The performance of current BK PCR detection assays is limited by the existence of viral polymorphisms, unknown at the time of assay development, resulting in inconsistent detection of BK virus. The objective of this study was to identify a stable region of the BK viral genome for detection by PCR that would be minimally affected by polymorphisms as more sequence data for BK virus becomes available.ResultsEmploying a combination of techniques, including amino acid and DNA sequence alignment and interspecies analysis, a conserved, stable PCR target region of the BK viral genomic region was identified within the VP2 gene. A real-time quantitative PCR assay was then developed that is specific for BK virus, has an analytical sensitivity of 15 copies/reaction (450 copies/ml) and is highly reproducible (CV ≤ 5.0%).ConclusionIdentifying stable PCR target regions when limited DNA sequence data is available may be possible by combining multiple analysis techniques to elucidate potential functional constraints on genomic regions. Applying this approach to the development of a real-time quantitative PCR assay for BK virus resulted in an accurate method with potential clinical applications and advantages over existing BK assays.

An integrated cell culture and reverse transcription quantitative PCR assay for detection of infectious rotaviruses in environmental waters

Rotaviruses exist widely in water environments and are the major cause to the gastroenteritis in children. To overcome the limitations associated with the current methods for detecting rotaviruses in environmental samples, such as long duration with the traditional cell culture-based plaque assay, inability to detect infectivity with RT-PCR-based molecular methods and lower sensitivity with ELISA tests, we developed an integrated cell culture and reverse transcription quantitative PCR (ICC-RT-qPCR) assay to detect infectious rotaviruses based on detection of viral RNA during replication in cells. The cell culturing step before qPCR allows the infectious rotaviruses to replicate and be detected because they are the only ones that can infect cells and produce RNA. The results showed that as low as 0.2 PFU/ml rotaviruses were detected by ICC-RT-qPCR after 2 days of incubation. With samples, the copy numbers of VP7 gene of rotaviruses linearly correlated (with a coefficient ( R 2) of 0.9575) with initial virus concentrations ranging from 0.2 to 200 PFU/ml. In parallel comparing tests, the ICC-RT-qPCR exhibited higher sensitivity than both the plaque assay and the RT-qPCR when applied to field samples. ICC-RT-qPCR detected infectious rotavirus in 42% (10/24) of secondary effluents, while only 21% (5/24) and 12% (3/24) of samples were positive with either the plaque counting or the RT-qPCR method, respectively. Concentrations of rotaviruses in secondary effluent samples were determined to be 1–30 PFU/l. The results demonstrated that the developed ICC-RT-qPCR method reduced test duration and improved sensitivity towards infectious rotavirus and therefore can be an effective and quantitative tool for detecting infectious rotaviruses in water environments.

Development and evaluation of a real-time quantitative PCR assay for detection and enumeration of yeasts of public health interest in dairy products

Yeast contamination is a problem in the food industry as a cause of spoilage. Moreover, various species of yeasts are known to be capable of causing opportunistic infections in humans. We have developed a real-time quantitative PCR (qPCR) assay to directly detect and quantify nine emerging opportunistic yeast species ( Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Clavispora lusitaniae, Filobasidiella neoformans, Issatchenkia orientalis, Trichosporon asahii, and Trichosporon jirovecii) in dairy product samples. We designed six primer pairs, conserved sequences of the variable D1/D2 domains of the 26S rRNA gene, to detect the yeasts and demonstrated their specificity. The qPCR assay could accurately quantify emerging opportunistic yeasts in an artificially contaminated dairy product. qPCR with the primer pairs we designed, was very sensitive and will allow producers to enumerate contaminating yeasts and identify whether they are opportunistic pathogens, in only 4 to 5 h. This assay can easily be extended to other food items and to a variety of food-monitoring initiatives.

Development and clinical application of a broad range 16S quantitative PCR assay for detection of bacteria in the uterus of the mare

Current treatments for Persistent mating-induced endometritis such as uterine lavage and oxytocin therapy focus on aiding the uterus in removal of inflammatory products, but these treatments do not modulate the inciting inflammatory response. Biological treatments, such as autologous conditioned serum (ACS) and mesenchymal stem cells (MSCs), have been used in human and veterinary medicine for immunomodulation for over 10 years. The objectives of this project were to evaluate the ability of ACS or MSCs to modulate the inflammatory response to spermatozoa after breeding. Two experiments were performed with six normal mares in each study to evaluate the effects of intrauterine administration of ACS, dexamethasone, or a placebo (experiment 1), or allogeneic MSCs or a placebo (experiment 2) on the inflammatory response to spermatozoa using clinical and biochemical endpoints. Treatment with ACS and MSCs significantly (P < 0.05) reduced the number of neutrophils in the uterine lumen 6 hours after the sperm challenge. An increase (P < 0.05) in the anti-inflammatory cytokine IL-1Ra was observed after treatment with MSCs before exposure to spermatozoa. There was no difference in IL-1Ra concentration in mares treated with ACS, dexamethasone, or a placebo. Mesenchymal stem cells and ACS were able to modulate the immune response to spermatozoa in normal mares. The effect may be due to an increase in IL-1Ra in MSCs-treated mares, but other bioactive molecules may be responsible for the decrease in neutrophils in ACS-treated mares. Autologous conditioned serum and bone-derived culture expanded MSCs were able to modulate the uterine inflammatory response to spermatozoa in normal mares. Treatment with allogeneic stem cells may be beneficial if a similar modulation in inflammatory cytokines occurs in mares affected by persistent mating–induced endometritis.

Establishment of Real-Time TaqMan-Fluorescence Quantitative RT-PCR Assay for Detection and Quantification of Porcine Lipoprotein Lipase mRNA

Porcine lipoprotein lipase (LPL) cDNA was cloned as the standard for real-time quantifying LPL mRNA and the TaqMan-fluorescence quantitative PCR assay for detection was established. The total RNA extracted from Longissimus dorsi of porcine was reverse-transcribed to cDNA. LPL cDNA was ligated with pGM-T vector and transformed into Escherichia coli TOP10. Plasmid DNA extracted from positive clones was verified by PCR amplification and sequenced. LPL was amplified by real-time fluorescence quantitative PCR from the plasmid DNA. The concentration of DNA template purified was detected by analyzing absorbance in 260 nm and then the combined plasmid was diluted to series as standard for fluorescence quantitative PCR (FQ-PCR). The method of LPL mRNA real-time PCR was well established, which detected as low as 10 3 with the linear range 10 3 to 10 10 copies. The standard curves showed high correlations ( R 2 = 0.9871). A series of standards for real-time PCR analysis have been constructed successfully, and real-time TaqMan-fluorescence quantitative RT-PCR is reliable to quantitatively evaluate FQ-PCR mRNA in L. dorsi of porcine.

Role of respiratory pathogens in infants hospitalized for a first episode of wheezing and their impact on recurrences

In order to evaluate the infectious agents associated with the first episode of severe acute wheezing in otherwise healthy infants and to define the role of each of them in recurrences, 85 patients in Italy, aged <12 months, hospitalized because of a first acute episode of wheezing, were prospectively enrolled between 1 October 2005 and 31 March 2006. Upon enrolment, nasopharyngeal swabs were collected for the real‐time PCR detection of respiratory syncytial virus (RSV) types A and B, influenza virus types A and B, adenovirus, parainfluenza viruses types 1, 2, 3 and 4, rhinovirus, human metapneumovirus, human coronavirus types 229E, OC43, NL63, and HKU1, bocavirus, enterovirus, and paraechovirus; nasopharyngeal aspirates were also obtained to detect atypical bacteria. At least one infectious agent was identified in 76 children (89.4%). RSV was the most frequently detected pathogen and its prevalence was significantly higher than that of the other pathogens in both age groups, and significantly higher in the children aged 3–12 months than in those aged <3 months. Only the children with RSV infection experienced recurrent wheezing. Viral load was significantly higher in children with than in those without recurrent wheezing. This study shows that RSV is the main reason for hospitalization during the first wheezing episode in infants, and that it appears to be the only pathogen associated with a high frequency of recurrences. A high viral load seems to be strictly related to the likelihood of recurrence.

Development and evaluation of a quantitative PCR assay for detection of Hepatozoon sp.

With the aim to improve current molecular diagnostic techniques of Hepatozoon sp. in carnivore mammals, we developed a quantitative PCR (qPCR) assay with SYBR Green I ®. The method, consisting of amplification of a 235 bp fragment of the 18S rRNA gene, is able to detect at least 0.1 fg of parasite DNA. Reproducible quantitative results were obtained over a range of 0.1 ng–0.1 fg of Hepatozoon sp. DNA. To assess the performance of the qPCR assay, DNA samples from dogs (140) and cats (50) were tested with either standard PCR or qPCR. Positive samples were always confirmed by partial sequencing of the 18S rRNA gene. Quantitative PCR was 15.8% more sensitive than standard PCR to detect H. canis in dogs. In cats, no infections were detected by standard PCR, compared to two positives by qPCR (which were infected by H. canis as shown by sequencing).

Development of an Internal Control for Evaluation and Standardization of a Quantitative PCR Assay for Detection of Helicobacter pylori in Drinking Water

Due to metabolic and morphological changes that can prevent Helicobacter pylori cells in water from growing on conventional media, an H. pylori-specific TaqMan quantitative PCR (qPCR) assay was developed that uses a 6-carboxyfluorescein-labeled probe (A. E. McDaniels, L. Wymer, C. Rankin, and R. Haugland, Water Res. 39:4808-4816, 2005). However, proper internal controls are needed to provide an accurate estimate of low numbers of H. pylori in drinking water. In this study, the 135-bp amplicon described by McDaniels et al. was modified at the probe binding region, using PCR mutagenesis. The fragment was incorporated into a single-copy plasmid to serve as a PCR-positive control and cloned into Escherichia coli to serve as a matrix spike. It was shown to have a detection limit of five copies, using a VIC dye-labeled probe. A DNA extraction kit was optimized that allowed sampling of an entire liter of water. Water samples spiked with the recombinant E. coli cells were shown to behave like H. pylori cells in the qPCR assay. The recombinant E. coli cells were optimized to be used at 10 cells/liter of water, where they were shown not to compete with 5 to 3,000 cells of H. pylori in a duplex qPCR assay. Four treated drinking water samples spiked with H. pylori (100 cells) demonstrated similar cycle threshold values if the chlorine disinfectant was first neutralized by sodium thiosulfate.

Evolution of Resistant M414T Mutants among Hepatitis C Virus Replicon Cells Treated with Polymerase Inhibitor A-782759

Treatment of hepatitis C virus (HCV) replicon cells with any single specific anti-HCV inhibitor in vitro leads to a rapid selection of resistant mutants. However, the source and the kinetic evolution of these resistant mutants during treatment are poorly understood. In this study we developed allele-specific real-time PCR assays for quantitative detection of the M414T mutant that was selected by a number of benzothiadiazine HCV polymerase inhibitors. Low levels of preexisting M414T mutants were detected in both 1b-con1 (0.22%) and 1b-N (0.18%) subgenomic replicon cell lines, as well as in 6 of 15 HCV RNA isolated from the sera of treatment-naive HCV-infected patients ranging from 0.11 to 0.60%. The proportion of M414T mutants in replicons rapidly increased in a dose-dependent manner upon treatment with benzothiadiazine inhibitor A-782759. After 4 days of treatment, 2.5, 26, or 60% of the replicon population contained M414T mutants with the use of A-782759 at 1x, 10x, or 100x its 50% effective concentration, respectively. In addition, the short 4-day treatment resulted in significant changes in inhibitor susceptibility in the replicon cells. Our results indicated that the resistant mutant preexisted as a minor population in replicon cells and that the mutant was selected within days of treatment with the inhibitor. The findings from this study suggested that early application of combination therapy of an HCV-specific inhibitor with interferon-based regimens or other classes of available inhibitors will be necessary to avoid quick viral rebound or treatment failure.

Detection of Borrelia burgdorferi DNA in tissues from dogs with presumptive Lyme borreliosis

To develop a quantitative PCR assay for detection of Borrelia burgdorferi DNA in formalin-fixed, paraffin-embedded tissues; compare results of this assay with results of immunohistochemical staining of tissues from seropositive dogs; and determine whether B burgdorferi DNA could be detected in renal tissues from dogs with presumptive Lyme nephritis. Cohort study. Archived tissue samples from 58 dogs. A quantitative PCR assay was performed on formalin-fixed, paraffin-embedded tissue sections from the dogs. Results were compared with results of immunohistochemical staining, B burgdorferi serostatus, clinical signs, and necropsy findings. 38 dogs were classified as having positive or equivocal results for Lyme borreliosis, and 20 were classified as having negative results on the basis of clinical signs, serologic findings, and pathologic abnormalities. Borrelia burgdorferi DNA was amplified from tissue samples from only 4 (7%) dogs, all of which had been classified as having positive or equivocal results for Lyme borreliosis and had signs of presumptive Lyme nephritis. Results of PCR assays of renal tissue were positive for only 1 dog, and there was no agreement between results of immunohistochemical staining (ie, detection of B burgdorferi antigen) and results of the PCR assay (ie, detection of B burgdorferi DNA) for renal tissues. Results indicated that detection of B burgdorferi DNA in formalin-fixed, paraffin-embedded tissues is feasible, but that intact B burgdorferi DNA is rarely found in tissues from naturally infected dogs, even tissues from dogs with presumptive Lyme borreliosis. Further, findings support the contention that Lyme nephritis may be a sterile, immune complex disease.

Real-time PCR-based methods for detection of Mycobacterium avium subsp. paratuberculosis in water and milk

A real-time PCR assay for quantitative detection of Mycobacterium avium paratuberculosis has been developed. It targets and amplifies sequences from the IS 900 insertion element which is specific for this bacterium, and includes an internal amplification control. The assay was tested against 18 isolates of M. avium paratuberculosis, 17 other mycobacterial strains, and 25 non-mycobacterial strains, and was fully selective. It is capable of detecting <3 genomic DNA copies with 99% probability or alternatively, using cells directly in the reaction, 12 cells can be detected with 99% probability. Using prior centrifugation, the assay was able to consistently and quantifiably detect 10 2 M. avium paratuberculosis cells in 20 ml artificially contaminated drinking water. With a simple detergent and enzymatic sample pretreatment before centrifugation and nucleic acid extraction, the assay was able to consistently detect 10 2 M. avium paratuberculosis in 20 ml artificially contaminated semi-skimmed milk. The assay will be a useful addition to the range of diagnostic tools available for the study of M. avium paratuberculosis.

Development of a real time quantitative PCR assay for detection of porcine endogenous retrovirus

Real time PCR technology was applied to the development of assays for detection and quantitation of porcine endogenous retrovirus (PERV) RNA and DNA sequences in tissues and cells of human or animal origin. A plasmid construct encoding the PERV- pol gene or the in vitro transcribed RNA derived from the plasmid (cRNA) serves as a standard template for amplification of a 178 bp fragment. This study showed that the detection of this target sequence was linear over a range from 20 copies to 2 million copies of the plasmid and from 100 copies to 1 million copies of the cRNA. In addition, amplification of the target sequence was not inhibited by the presence of exogenous genomic DNA. These results demonstrate that a real time (TaqMan-based) PCR or RT–PCR assay can provide a sensitive, reproducible, and robust method for detecting and quantifying PERV DNA or RNA sequences in samples of human or guinea pig origin.

Comparison of DNA and RNA based real-time PCR assays for quantitative detection of chromosomal translocations

Human malignant chromosomal translocation involved immunoglobulin gene (Ig) at chromosome 14 and bcl-2 protooncogene on chromosome 18 is frequently used as a molecular marker for lymphomas. We studied the application of real-time PCR (TaqMan PCR) for quantitative detection of the human lymphocyte cells bearing chromosomal translocation. Three approaches for real-time PCR standards were compared (1) mixing cells with and without translocation before DNA extraction, (2) dilution of plasmid DNA with translocation marker into background DNA, (3) dilution of plasmid RNA with translocation marker into background RNA with a following reverse-transcriptase reaction. This study was conducted to investigate potential differences in sensitivity, resolution and variability between different real-time assays. We found the dilution of plasmid DNA with translocation fragment at background DNA to be the most accurate and sensitive method for building real-time PCR standards.

A competitive PCR assay for quantitative detection of Neospora caninum

A quantitative-competitive PCR (QC-PCR) assay was developed for measurement of Neospora caninum levels in the tissues of infected animals. A molecule was synthesised for use in PCR as a competitor to the target Neospora-specific Nc5 genomic sequence. The assay was used to evaluate the relative level of parasites in the brain and lungs of mouse pups in a model of vertical transmission of N. caninum. Infection on day 11 of gestation resulted in similar levels of parasites in all offspring. The assay should be useful in evaluation of vaccines against Neospora infection. Incorporation of the competitor molecule in the detection assay also provides a control for PCR failure and facilitates identification of truly negative samples.

A novel real-time quantitative PCR assay for detection of Helicobacter pylori in the gastric mucosa

BACKGROUND: Although the assessment of the density of Helicobacter pylori (H. pylori) colonization may be important, precise quantitative evaluation of H. pylori in the gastric mucosa has been difficult. We have developed a novel real-time quantitative PCR technique based on 5' -3' exonuclease assay for detection of H. pylori. Using this system, we evaluated number of H. pylori organisms present in gastric biopsy specimens. METHODS: A primer pair for PCR amplification was made according to the reported sequence of ureA gene of H. pylori, and an oligonucleotide probe labeled with two fluorescent dyes (a reporter dye at the 5' end and a quencher dye at the 3' end) was also designed. While the probe is intact, the quencher dye greatly reduces the fluorescent signal by the reporter dye (Fluorescence resonance energy transfer phenomenon). During the PCR extension phase, the annealed probe is cleaved by 5' -3' exonuclease activity of Taq DNA polymerase, increasing the reporter dye signal. The reporter dye signal during a PCR assay was monitored with a ABI PRISM 7700 Sequence Detector (Perkin-Elmer). Purified PCR products of ureA gene were used as standard samples for making standard curves plotting log starting copy number and threshold cycle (Ct: PCR cycle number at which the reporter fluorescence signal reached above baseline). Biopsy specimens were collected from antral mucosa of consecutive 32 patients underwent gastroscopy (GU 9, DU 9, GC 1, NUD 13). Three biopsy specimens were obtained from each patient to avoid the effect of patchy distribution of H. pylori. DNA was extracted by conventional phenol-chloroform method. Since the size of each biopsy specimen was variable, quantitative PCR assay described above was performed using 0.5 ~tg of DNA extracted from each sample. !~_!LL_.T~: In each amplification reaction, a liner standard curve could be generated in a wide range of starting copy numbers (6xl07 to 6xl0 copies of the target sequences, R 2 > 0.99). Nine of 32 samples (DU 2, NUD 7) showed no increase in the fluorescence signal during PCR process, indicating the absence of H. pylori, which was also confirmed by histological examinations. In H. pylori positive patients, the detected ureA copy numbers (i.e. number of H. pylori organisms) were variable among patients (range 1.4x102 1.2x105 copies/0.5Mg DNA, mean 2.6x104 copies/0.51ag DNA). The average ureA copy numbers of samples from DU and GU patients were not significantly different from that of NUD patients [GU (2.1 _+ l.l)xl04, DU (1.8 _+ 0.6)xl04, NUD (2.6 + 1.1)xl04 copies/0.51ag DNA, respectively]. The sample of one GC patient showed high density of H. pylori colonization (1.2x105 copies/0.5Mg DNA). CONCLUSIONS: Accurate quantitation of H. pylori in biopsy specimens was possible with this PCR system, and it will be useful for 1-1. pylori research.

Knowledge analysis

Commensal rodent species are key reservoirs for Toxoplasma gondii in the domestic environment. In rodents, different T. gondii strains show variable patterns of virulence according to host species. Toxoplasma gondii strains causing non-lethal chronic infections in local hosts will be more likely to persist in a given environment, but few studies have addressed the possible role of these interactions in shaping the T. gondii population structure. In addition, the absence of validated techniques for upstream detection of T. gondii chronic infection in wild rodents hinders exploration of this issue under natural conditions. In this study, we took advantage of an extensive survey of commensal small mammals in three coastal localities of Senegal, with a species assemblage constituted of both native African species and invasive species. We tested 828 individuals for T. gondii chronic infection using the modified agglutination test for antibody detection in serum samples and a quantitative PCR assay for detection of T. gondii DNA in brain samples. The infecting T. gondii strains were genotyped whenever possible by the analysis of 15 microsatellite markers. We found (i) a very poor concordance between molecular detection and serology in the invasive house mouse, (ii) significantly different levels of prevalence by species and (iii) the autochthonous T. gondii Africa 1 lineage strains, which are lethal for laboratory mice, only in the native African species of commensal small mammals. Overall, this study highlights the need to reconsider the use of MAT serology in natural populations of house mice and provides the first known data about T. gondii genetic diversity in invasive and native species of small mammals from Africa. In light of these results, we discuss the role of invasive and native species, with their variable adaptations to different T. gondii strains, in shaping the spatial structure of T. gondii genetic diversity in Africa.