A standard curve based method for relative real time PCR data processing

Background  

Currently real time PCR is the most precise method by which to measure gene expression. The method generates a large amount of raw numerical data and processing may notably influence final results. The data processing is based either on standard curves or on PCR efficiency assessment. At the moment, the PCR efficiency approach is preferred in relative PCR whilst the standard curve is often used for absolute PCR. However, there are no barriers to employ standard curves for relative PCR. This article provides an implementation of the standard curve method and discusses its advantages and limitations in relative real time PCR.     

Revealing biases inherent in recombination protocols

Background  

The recombination of homologous genes is an effective protein engineering tool to evolve proteins. DNA shuffling by gene fragmentation and reassembly has dominated the literature since its first publication, but this fragmentation-based method is labor intensive. Recently, a fragmentation-free PCR based protocol has been published, termed recombination-dependent PCR, which is easy to perform. However, a detailed comparison of both methods is still missing.     

Determination of PCR efficiency in chelex-100 purified clinical samples and comparison of real-time quantitative PCR and conventional PCR for detection of Chlamydia pneumoniae

Background  

Chlamydia pneumoniae infection has been detected by serological methods, but PCR is gaining more interest. A number of different PCR assays have been developed and some are used in combination with serology for diagnosis. Real-time PCR could be an attractive new PCR method; therefore it must be evaluated and compared to conventional PCR methods.     

Development and comparison of a real-time PCR assay for detection of <Emphasis Type="Italic">Dichelobacter nodosus</Emphasis> with culturing and conventional PCR: harmonisation between three laboratories

Background  

Ovine footrot is a contagious disease with worldwide occurrence in sheep. The main causative agent is the fastidious bacterium Dichelobacter nodosus. In Scandinavia, footrot was first diagnosed in Sweden in 2004 and later also in Norway and Denmark. Clinical examination of sheep feet is fundamental to diagnosis of footrot, but D. nodosus should also be detected to confirm the diagnosis. PCR-based detection using conventional PCR has been used at our institutes, but the method was laborious and there was a need for a faster, easier-to-interpret method. The aim of this study was to develop a TaqMan-based real-time PCR assay for detection of D. nodosus and to compare its performance with culturing and conventional PCR.     

A new approach to 'megaprimer' polymerase chain reaction mutagenesis without an intermediate gel purification step

Background  

Site-directed mutagenesis is an efficient method to alter the structure and function of genes. Here we report a rapid and efficient megaprimer-based polymerase chain reaction (PCR) mutagenesis strategy that by-passes any intermediate purification of DNA between two rounds of PCR.     

Effects of polymerase,template dilution and cycle number on PCR based 16 S rRNA diversity analysis using the deep sequencing method

Background  

The primer and amplicon length have been found to affect PCR based estimates of microbial diversity by pyrosequencing, while other PCR conditions have not been addressed using any deep sequencing method. The present study determined the effects of polymerase, template dilution and PCR cycle number using the Solexa platform.     

Shape based kinetic outlier detection in real-time PCR

Background  

Real-time PCR has recently become the technique of choice for absolute and relative nucleic acid quantification. The gold standard quantification method in real-time PCR assumes that the compared samples have similar PCR efficiency. However, many factors present in biological samples affect PCR kinetic, confounding quantification analysis. In this work we propose a new strategy to detect outlier samples, called SOD.     

Relative quantification of mRNA: comparison of methods currently used for real-time PCR data analysis

Background  

Fluorescent data obtained from real-time PCR must be processed by some method of data analysis to obtain the relative quantity of target mRNA. The method chosen for data analysis can strongly influence results of the quantification.     

Long‐range quantitative PCR for determining inactivation of adenovirus 2 by ultraviolet light

Aims

An extra‐long‐range quantitative PCR (LR‐qPCR) method was developed for estimating genome damage to adenovirus 2 caused by UV irradiation. The objective was to use LR‐qPCR as a rapid method to determine adenovirus UV inactivation.

Methods

The LR‐qPCR consisted of two steps: a long‐range PCR (up to 10 kb fragment) and a real‐time, quantitative (q) PCR for quantifying the products of the first PCR. We evaluated LR‐qPCR with adenovirus irradiated with medium‐pressure (MP, polychromatic emission) and low‐pressure (LP, 254 nm) mercury vapour lamps and compared results with cell culture infectivity.

Results

Using LR‐qPCR, a fragment of 6 kb estimated DNA damage in a linear relationship to doses between 0 and 20 mJ cm^?2, and a 1‐kb fragment related linearly to doses between 20 and 100 mJ cm^?2. The LR‐qPCR results for the 6‐kb fragment were similar to infectivity assays results for adenovirus exposed to MP UV. For adenovirus irradiated with LP lamps, LR‐qPCR results for the shorter fragment size (1 kb) were similar to reduction in viral infectivity. No difference was observed between 10 and 6 kb LR‐qPCR results.

Conclusion

The LR‐qPCR can be used as a tool for estimating DNA damage caused by UV in adenovirus. The LR‐qPCR results were related to reduction in viral infectivity.

Significance and Impact of the Study

The use of LR‐qPCR to determine DNA damage and estimate inactivation of adenovirus 2 from UV disinfection allows for same‐day results compared with >7 days required for cell culture. This accelerates adenovirus inactivation results for the water industry where adenovirus is used as a representative virus for crediting UV systems. This PCR approach provides a framework that can be used for other viral viability assays using the inhibition of amplification of viral nucleic acid after pretreatments, such as propidium monoazide, and for cellular biology studies of DNA damage.     

Statistical analysis of real-time PCR data

Background  

Even though real-time PCR has been broadly applied in biomedical sciences, data processing procedures for the analysis of quantitative real-time PCR are still lacking; specifically in the realm of appropriate statistical treatment. Confidence interval and statistical significance considerations are not explicit in many of the current data analysis approaches. Based on the standard curve method and other useful data analysis methods, we present and compare four statistical approaches and models for the analysis of real-time PCR data.     

Easy and rapid method for the determination of gene expression in cumulus cells incubated for oocyte maturation

Background  

The objectives of this study were to develop an easy and rapid method for measuring gene expression in a small number of cells by real-time PCR without RNA extraction and purification, and to use this method to determine more precisely IGF-I gene expression in the cumulus cells surrounding oocytes.     

BISMA - Fast and accurate bisulfite sequencing data analysis of individual clones from unique and repetitive sequences

Background  

Bisulfite sequencing is a popular method to analyze DNA methylation patterns at high resolution. A region of interest is targeted by PCR and about 20-50 subcloned DNA molecules are usually analyzed, to determine the methylation status at single CpG sites and molecule resolution.     

Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences

Background  

The most common method of GMO detection is based upon the amplification of GMO-specific DNA amplicons using the polymerase chain reaction (PCR). Here we have applied the loop-mediated isothermal amplification (LAMP) method to amplify GMO-related DNA sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene) junctions.     

Selection of housekeeping genes for gene expression studies in larvae from flatfish using real-time PCR

Background  

Flatfish metamorphosis involves major physiological and morphological changes. Due to its importance in aquaculture and as a model for developmental studies, some gene expression studies have focused on the understanding of this process using quantitative real-time PCR (qRT-PCR) technique. Therefore, adequate reference genes for accurate normalization are required.     

IS<Emphasis Type="Italic">1111</Emphasis> insertion sequences of <Emphasis Type="Italic">Coxiella burnetii</Emphasis>: characterization and use for repetitive element PCR-based differentiation of <Emphasis Type="Italic">Coxiella burnetii</Emphasis> isolates

Background  

Coxiella burnetii contains the IS1111 transposase which is present 20 times in the Nine Mile phase I (9Mi/I) genome. A single PCR primer that binds to each IS element, and primers specific to a region ~500-bp upstream of each of the 20 IS1111 elements were designed. The amplified products were characterized and used to develop a repetitive element PCR genotyping method.     

Development of a real-time multiplex PCR assay for the detection of multiple <Emphasis Type="Italic">Salmonella</Emphasis> serotypes in chicken samples

Background  

A real-time multiplex PCR assay was developed for the detection of multiple Salmonella serotypes in chicken samples. Poultry-associated serotypes detected in the assay include Enteritidis, Gallinarum, Typhimurium, Kentucky and Dublin. The traditional cultural method according to EN ISO 6579:2002 for the detection of Salmonella in food was performed in parallel. The real-time PCR based method comprised a pre-enrichment step in Buffered Peptone Water (BPW) overnight, followed by a shortened selective enrichment in Rappaport Vasilliadis Soya Broth (RVS) for 6 hours and subsequent DNA extraction.     

Product category rules alignment workshop, October 4, 2011 in Chicago, IL, USA

Purpose  

A workshop on Product Category Rule (PCR) alignment was organized by the American Center for LCA PCR Committee. PCR alignment refers to the process of assuring that PCRs (rules for developing LCA-based claims like EPDs) developed by different parties are consistent within product categories.     

Molecular characterization of Streptococcus agalactiae strains isolated from fishes in Malaysia

Aims

The aim of this study was to characterize Streptococcus agalactiae strains that were isolated from fishes in Malaysia using random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (REP‐PCR) techniques.

Methods and Results

A total of 181 strains of Strep. agalactiae isolated from red hybrid tilapia (Oreochromis sp.) and golden pompano (Trachinotus blochii) were characterized using RAPD and REP‐PCR techniques. Both the fingerprinting techniques generated reproducible band patterns, differing in the number and molecular mass amplicons. The RAPD technique displayed greater discriminatory power by its production of more complex binding pattern and divided all the strains into 13 groups, compared to 9 by REP‐PCR technique. Both techniques showed the availability to differentiate the genetic profiles of the strains according to their geographical location of origin. Three strains of Strep. agalactiae that were recovered from golden pompano showed a genetic dissimilarity from the strains isolated from red hybrid tilapia, while the strain of ATCC 27956 that recovered from bovine displayed a unique profile for both methods.

Conclusions

Both techniques possess excellent discriminative capabilities and can be used as a rapid means of comparing Strep. agalactiae strains for future epidemiological investigation.

Significance and Impact of the Study

Framework as the guideline in traceability of this disease and in the search for potential local vaccine candidates for streptococcosis in this country.     

Multiple source genes of HAmo SINE actively expanded and ongoing retroposition in cyprinid genomes relying on its partner LINE

Background  

We recently characterized HAmo SINE and its partner LINE in silver carp and bighead carp based on hybridization capture of repetitive elements from digested genomic DNA in solution using a bead-probe [1]. To reveal the distribution and evolutionary history of SINEs and LINEs in cyprinid genomes, we performed a multi-species search for HAmo SINE and its partner LINE using the bead-probe capture and internal-primer-SINE polymerase chain reaction (PCR) techniques.