Rapid quantification of semen hepatitis B virus DNA by real-time polymerase chain reaction

Aim: To examine the sensitivity and accuracy of real-time polymerase chain reaction (PCR) for the quantification of hepatitis B virus (HBV) DNA in semen. Methods: Hepatitis B viral DNA was isolated from HBV carriers' semen and sera using phenol extraction method and QIAamp DNA blood mini kit (Qiagen, Germany). HBV DNA was detected by conventional PCR and quantified by TaqMan technology-based real-time PCR (quantitative polymerase chain reaction (qPCR)). The detection threshold was 200 copies of HBV DNA for conventional PCR and 10 copies of HBV DNA for real time PCR per reaction. Results: Both methods of phenol extraction and QIAamp DNA blood mini kit were suitable for isolating HBV DNA from semen. The value of the detection thresholds was 500 copies of HBV DNA per mL in the semen. The viral loads were 7.5×10 7 and 1.67×10 7 copies of HBV DNA per mL in two HBV infected patients' sera, while 2.14×10 5 and 3.02×10 5 copies of HBV DNA per mL in the semen. Conclusion: Real-time PCR is a more sensitive and accurate method to detect and quantify HBV DNA in the semen. © 2005 The WJG Press and Elsevier Inc. All rights reserved.published_or_final_versio

Sensitive PCR method for the detection and real-time quantification of human cells in xenotransplantation systems

The sensitive detection of human cells in immunodeficient rodents is a prerequisite for the monitoring of micrometastasis of solid tumours, dissemination of leukaemic cells, or engraftment of haematological cells. We developed a universally applicable polymerase chain reaction method for the detection of a human-specific 850-bp fragment of the α-satellite DNA on human chromosome 17. The method allows the detection of one human cell in 106 murine cells and could be established as both, a conventional DNA polymerase chain reaction-assay for routine screening, and a quantitative real-time polymerase chain reaction-assay using TaqMan-methodology. It was applied to the following xenotransplantation systems in SCID and NOD/SCID mice: (1) In a limiting dilution assay, cells of the MDA-MB 435 breast carcinoma were injected into the mammary fat pad of NOD/SCID mice. It could be shown that 10 cells mouse−1 were sufficient to induce a positive polymerase chain reaction signal in liver and lung tissue 30 days after transplantation as an indicator for micrometastasis. At this time a palpable tumour was not yet detectable in the mammary fat pad region. (2) Cells of a newly established human acute lymphatic leukaemia were administered intraperitoneally to SCID mice. These cells apparently disseminated and were detectable as early as day 50 in the peripheral blood of living mice, while the leukaemia manifestation was delayed by day 140. (3) In a transplantation experiment using mature human lymphocytes we wanted to standardise conditions for a successful survival of these cells in NOD/SCID mice. It was established that at least 5×107 cells given intravenously were necessary and that the mice had to be conditioned by 2 Gy body irradiation to get positive polymerase chain reaction bands in several organs. (4) Engraftment studies with blood stem cells originating from cytapheresis samples of tumour patients or from cord blood were undertaken in NOD/SCID mice in order to define conditions of successful engraftment and to use this model for further optimisation strategies. The polymerase chain reaction method presented allowed a reliable prediction of positive engraftment and agreed well with the results of immunohistochemical or FACS analysis. All together, the polymerase chain reaction method developed allows a sensitive and reliable detection of low numbers of human cells in immunodeficient hosts. In combination with real-time (TaqMan) technique it allows an exact quantification of human cells. As this method can be performed with accessible material of living animals, follow up studies for the monitoring of therapeutic interventions are possible in which the survival time of mice as evaluation criteria can be omitted

A sensitive detection method for MPLW515L or MPLW515K mutation in chronic myeloproliferative disorders with locked nucleic acid-modified probes and real-time polymerase chain reaction.

Acquired mutations in the juxtamembrane region of MPL (W515K or W515L), the receptor for thrombopoietin, have been described in patients with primary myelofibrosis or essential thrombocythemia, which are chronic myeloproliferative disorders. We have developed a real-time polymerase chain reaction assay for the detection and quantification of MPL mutations that is based on locked nucleic acid fluorescent probes. Mutational analysis was performed using DNA from granulocytes. Reference curves were obtained using cloned fragments of MPL containing either the wild-type or mutated sequence; the predicted sensitivity level was at least 0.1% mutant allele in a wild-type background. None of the 60 control subjects presented with a MPLW515L/K mutation. Of 217 patients with myelofibrosis, 19 (8.7%) harbored the MPLW515 mutation, 10 (52.6%) with the W515L allele. In one case, both the W515L and W515K alleles were detected by real-time polymerase chain reaction. By comparing results obtained with conventional sequencing, no erroneous genotype attribution using real-time polymerase chain reaction was found, whereas one patient considered wild type according to sequence analysis actually harbored a low W515L allele burden. This is a simple, sensitive, and cost-effective procedure for large-scale screening of the MPLW515L/K mutation in patients suspected to have a myeloproliferative disorder. It can also provide a quantitative estimate of mutant allele burden that might be useful for both patient prognosis and monitoring response to therapy

Genetic Amplification: Quantitative Polymerase Chain Reaction and Its Problems and Uses

Quantitative polymerase chain reaction (qPCR), also called real-time PCR, has become a cornerstone of DNA analysis, enabling detection of minute amounts of nucleic acids (Whittwer et. al, 1997). In 1983, Kary Mullis developed a new method of genetic amplification—the polymerase chain reaction [PCR] (Bartlett & Stirling, 2003). A little over 20 years later, PCR now is a common and often crucial technique used in medical and biological research laboratories for a variety of applications. Some of these applications include DNA cloning for sequencing, DNA-based phylogeny, the diagnosis of hereditary diseases, the identification of genetic fingerprints (used in forensic sciences and DNA paternity testing), and the detection and diagnosis of infectious diseases. qPCR is a modification of the classic PCR method which, due to the presence of a fluorescent-labeled probe, allows for the quantification of DNA. Precise DNA quantification is a valuable insight that qPCR provides over other diagnostic techniques and can affect treatment options or preventative measures. This is especially the case in the detection of infectious diseases, where pathogens may be harmless in insignificant amounts but cause disease once the infectious dose is reached. With the prevalence of this technique and its many uses, it is important to research qPCR and its successes as well as its potential issues

An overview of real-time quantitative PCR: Principles and formats for environmental microbiology studies

© Global Science Publications. A fluorescence-based real-time quantitative polymerase chain reaction (qPCR) is a powerful and commonly used molecular technique for quantifying the rRNA or DNA of targeted organisms in environmental samples. qPCR assays are easy to perform, making them capable of high throughput and can combine high sensitivity with reliable specificity. qPCR analysis is the combination of the traditional endpoint PCR attached with fluorescents to record the accumulation of the amplicons in real time during each cycle of the PCR. Detection of amplicons during the early exponential phase enables quantification of the gene numbers because they are proportional to the starting template. This review is focussed on currently used qPCR platforms, the chemistries involved in real-time PCR systems mainly applied for the environmental microbiology studies. The various factors affecting quantification of environmental microbial communities using qPCR have also been discussed

Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

Background: State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. Methodology and Principal Findings: The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. Conclusions and Significance: The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution fo

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included

Master of Science

thesisMolecular detection of Tropheryma whipplei is an important tool in the diagnosis of Whipple's Disease and other T. whipplei infections. This thesis describes the design and validation of a real-time polymerase chain reaction assay that detects T. whipplei in cerebrospinal fluid, whole blood, tissue, formalin-fixed paraffin-embedded tissue, serum, and plasma samples. The assay detects a repeated sequence present seven times in the T. whipplei genome and the rpoB gene in a multiplex format. The assay is highly specific for T. whipplei and is capable of detecting less than four copies per PCR reaction with certain sample types. The validated assay has been in use at ARUP Laboratories since April 2016 and has detected T. whipplei in three patient samples. This thesis also describes a method for manufacturing formalin-fixed paraffin- embedded blocks containing cultured bacteria as a means of simulating positive patient samples when genuine positive samples are not available. However, quantification of the number of bacteria in a sample using this method requires postextraction quantification of target copy number. Digital polymerase chain reaction is capable of absolute quantification of a target sequence without reference to a standard curve and was used to quantify target copy number in manufactured formalin-fixed paraffin-embedded samples. The World Health Organization produces International Standards which serve as a common calibration material for many types of diagnostic testing. This includes several quantification standards for various pathogens which are quantified by molecular methods. However, these standards are not well characterized in terms of true copy number and nucleic acid sequence, which can lead to discordant results between molecular assays targeting different regions of a given pathogen's genome. This thesis also describes the use of digital polymerase chain reaction to evaluate the World Health Organization 1st International Standard for Epstein-Barr virus and the World Health Organization 1st International Standard for Hepatitis D virus. This provides better characterization of each standard by estimating the viral copy number

Chip-Oriented Fluorimeter Design and Detection System Development for DNA Quantification in Nano-Liter Volumes

The chip-based polymerase chain reaction (PCR) system has been developed in recent years to achieve DNA quantification. Using a microstructure and miniature chip, the volume consumption for a PCR can be reduced to a nano-liter. With high speed cycling and a low reaction volume, the time consumption of one PCR cycle performed on a chip can be reduced. However, most of the presented prototypes employ commercial fluorimeters which are not optimized for fluorescence detection of such a small quantity sample. This limits the performance of DNA quantification, especially low experiment reproducibility. This study discusses the concept of a chip-oriented fluorimeter design. Using the analytical model, the current study analyzes the sensitivity and dynamic range of the fluorimeter to fit the requirements for detecting fluorescence in nano-liter volumes. Through the optimized processes, a real-time PCR on a chip system with only one nano-liter volume test sample is as sensitive as the commercial real-time PCR machine using the sample with twenty micro-liter volumes. The signal to noise (S/N) ratio of a chip system for DNA quantification with hepatitis B virus (HBV) plasmid samples is 3 dB higher. DNA quantification by the miniature chip shows higher reproducibility compared to the commercial machine with respect to samples of initial concentrations from 103 to 105 copies per reaction