Comparison of proteases in DNA extraction via quantitative polymerase chain reaction

We compared four proteases in the QIAamp DNA Investigator Kit (Qiagen) to extract DNA for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate alternate proteases for improved DNA recovery as compared with proteinase K for forensic, biochemical research, genetic paternity and immigration, and molecular diagnostic purposes. The Quantifiler Kit TaqMan quantitative PCR assay was used to measure the recovery of DNA from human blood, semen, buccal cells, breastmilk, and earwax in addition to low-template samples, including diluted samples, computer keyboard swabs, chewing gum, and cigarette butts. All methods yielded amplifiable DNA from all samples.     

Robust regression methods for real-time polymerase chain reaction

Current real-time polymerase chain reaction (PCR) data analysis methods implement linear least squares regression methods for primer efficiency estimation based on standard curve dilution series. This method is sensitive to outliers that distort the outcome and are often ignored or removed by the end user. Here, robust regression methods are shown to provide a reliable alternative because they are less affected by outliers and often result in more precise primer efficiency estimators than the linear least squares method.     

Urine as another potential source for template DNA in polymerase chain reaction (PCR)

This technical note examines the potential for preparing template DNA in polymerase chain reactions (PCR) from urine in Japanese macaques (Macaca fuscata). Microsatellite band patterns from urine samples showed close agreement with those of blood and fecal samples, and only a few hundred μl of urine yielded a template DNA for PCR. This research will increase the opportunity for scientists to examine the genetic backgrounds of their target animals by using non‐invasive sample collection in the wild. Am. J. Primatol. 48:299–304, 1999. © 1999 Wiley‐Liss, Inc.     

双重荧光定量PCR检测志贺毒素stx1和stx2基因

目的建立一种双重荧光定量PCR检测志贺毒素stx1和stx2基因的方法。方法根据不同细菌来源的stx1和stx2序列,设计PCR引物和TaqMan探针,建立双重实时荧光定量PCR检测体系,进行灵敏度、特异性和重复性评价,并对腹泻患者粪便样本进行检测分析。结果双重实时荧光定量PCR检测含志贺毒素基因重组质粒的最低检测下限为102copies/mL;该法对12种常见肠道病原菌均无特异性扩增,对不同浓度的标准质粒检测重复性高,Ct值变异系数均小于10%;对急性腹泻粪便标本的检测阳性率高于细菌分离培养。结论建立的双重实时荧光定量PCR可作为不同细菌来源的志贺毒素基因的快速鉴定方法,亦可用于人感染性腹泻标本的快速筛查。     

任意引物PCR及其应用研究进展

任意引物PCR技术又称为随机扩增多态性DNA技术,它是在PCR技术基础上发展起来的一项分子检测技术。它具有简便、快速,一套引物可用于多个物种的分析,不需预知分析对象的核酸序列,可以显示差异表达基因等特点,已广泛应用于病原微生物的分型鉴定、物种亲源关系分析、遗传育种研究和特异表达基因的克隆与鉴定等方面。     

Accurate quantitation of standard peptides used for quantitative proteomics

MS‐based proteomics has become an indispensable tool in system biology generating a need for accurate and precise quantitation of peptide standards. The presented method utilizes ultra performance LC‐MS/MS (UPLC‐MS/MS) to accurately quantify peptide standards at concentrations of 0.1–10 μM. The ability for accurate quantitation of micro‐molar concentrations has the advantages that quantitation can be performed routinely with high precision and the high sensitivity of the method minimizes the amounts required.     

Quantitative polymerase chain reaction used for the rapid detection of Carnobacterium species from French soft cheeses

An identification method by PCR, specific to the Carnobacterium genus, was optimised by testing it on 28 bacterial strains. Primers from the literature were tested to differentiate Carnobacterium strains present among various bacterial species. The DNA of Carnobacterium species (C. alterfunditum, C. divergens, C. funditum, C. gallinarum, C. inhibens, C. maltaromaticum, C. mobile, C. viridans), specifically amplified by the Cb1-Cb2R primer couple at a hybridization temperature of 69 degrees C, gave only one band of 340 bp. The validation of this technique was carried out on a French soft cheese made with pasteurised milk inoculated with C. maltaromaticum LMA 28. Using classical PCR, detection was not possible for decimal dilutions of the cheese above 1 g L(-1). With Sybr Green I real time PCR, the specificity of the reaction was confirmed by the T(m) value. The standard curve constructed using the main threshold cycle and various concentrations of C. maltaromaticum LMA 28 (ranging from 10(0) to 10(8) cfu mL(-1)) showed good linearity and a sensitivity limit of > or = 10(4) cfu g(-1) of cheese. This technique was applied on commercially available cheeses made from raw cow's milk. The Sybr Green I real time PCR method constitutes an effective and easy-to-perform method to quantify Carnobacterium sp. in cheese.     

Detecting and resolving position-dependent temperature effects in real-time quantitative polymerase chain reaction

Real-time quantitative polymerase chain reaction (qPCR) depends on precise temperature control of the sample during cycling. In the current study, we investigated how temperature variation in plate-based qPCR instruments influences qPCR results. Temperature variation was measured by amplicon melting analysis as a convenient means to assess well-to-well differences. Multiple technical replicates of several SYBR Green I-based qPCR assays allowed correlation of relative well temperature to quantification cycle. We found that inadequate template denaturation results in an inverse correlation and requires increasing the denaturation temperature, adding a DNA destabilizing agent, or pretreating with a restriction enzyme. In contrast, inadequate primer annealing results in a direct correlation and requires lowering the annealing temperature. Significant correlations were found in 18 of 25 assays. The critical nature of temperature-dependent effects was shown in a blinded study of 29 patients for the diagnosis of Prader–Willy and Angelman syndromes, where eight diagnoses were incorrect unless temperature-dependent effects were controlled. A method to detect temperature-dependent effects by pairwise comparisons of replicates in routine experiments is presented and applied. Systematic temperature errors in qPCR instruments can be recognized and their effects eliminated when high precision is required in quantitative genetic diagnostics and critical complementary DNA analyses.     

Selection of reference genes for quantitative real-time polymerase chain reaction studies in rat osteoblasts

Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to evaluate gene expression, but its accuracy depends on the choice and stability of the reference genes used for normalization. In this study, we aimed to identify reference genes for studies on osteoblasts derived from rat bone marrow mesenchymal stem cells (bone marrow osteoblasts), osteoblasts derived from newborn rat calvarial (calvarial osteoblasts), and rat osteosarcoma cell line UMR-106. The osteoblast phenotype was characterized by ALP activity and extracellular matrix mineralization. Thirty-one candidates for reference genes from a Taqman^® array were assessed by qRT-PCR, and their expressions were analyzed by five different approaches. The data showed that several of the most traditional reference genes, such as Actb and Gapdh, were inadequate for normalization and that the experimental conditions may affect gene stability. Eif2b1 was frequently identified among the best reference genes in bone marrow osteoblasts, calvarial osteoblasts, and UMR-106 osteoblasts. Selected stable and unstable reference genes were used to normalize the gene expression of Runx2, Alp, and Oc. The data showed statistically significant differences in the expression of these genes depending on the stability of the reference gene used for normalization, creating a bias that may induce incorrect assumptions in terms of osteoblast characterization of these cells. In conclusion, our study indicates that a rigorous selection of reference genes is a key step in qRT-PCR studies in osteoblasts to generate precise and reliable data.     

Chemically modified primers for improved multiplex polymerase chain reaction

Multiplex polymerase chain reaction (PCR), the amplification of multiple targets in a single reaction, presents a new set of challenges that further complicate more traditional PCR setups. These complications include a greater probability for nonspecific amplicon formation and for imbalanced amplification of different targets, each of which can compromise quantification and detection of multiple targets. Despite these difficulties, multiplex PCR is frequently used in applications such as pathogen detection, RNA quantification, mutation analysis, and (recently) next generation DNA sequencing. Here we investigated the utility of primers with one or two thermolabile 4-oxo-1-pentyl phosphotriester modifications in improving multiplex PCR performance. Initial endpoint and real-time analyses revealed a decrease in off-target amplification and a subsequent increase in amplicon yield. Furthermore, the use of modified primers in multiplex setups revealed a greater limit of detection and more uniform amplification of each target as compared with unmodified primers. Overall, the thermolabile modified primers present a novel and exciting avenue for improving multiplex PCR performance.     

Multiplex event-specific qualitative polymerase chain reaction for detecting three transgenic rice lines and application of a standard plasmid as a quantitative reference molecule

The three most well-known genetically modified (GM) rice lines in China are TT51-1, KMD1, and KF6. The purposes of this study were to establish a multiplex event-specific qualitative polymerase chain reaction (meqPCR) system for simultaneous detection of the three transgenic rice events and to construct a plasmid as the reference molecule for quantitative analysis. Event-specific primers for each event were selected or designed by focusing on the transgene borders between the inserted DNA and the flanking rice DNA. The developed meqPCR was anticipated to detect distinct amplicons as 454, 398, 301, and 250 bp from KF6, KMD1, TT51-1, and the rice endogenous reference gene, respectively. The robustness of the meqPCR was tested with different levels of the three transgenic rice genomic DNAs, and the sensitivity threshold of the meqPCR was at least 50 ng of 0.1% rice DNA for each event when the three transgenic rice events present and with other GM materials together. The constructed plasmid was evaluated using mixed samples with known GM contents in real-time quantitative PCR. The results indicated that the constructed plasmid was acceptable and suitable for GM rice quantitative analysis.     

A quantitative polymerase chain reaction assay for detecting and identifying fungal contamination in human allograft tissue

To complement donor selection and tissue processing, rapid and reliable detection, discrimination, and quantification of fungal pathogens are extremely important for tissues destined to be implanted into humans. The current detection method for fungal pathogens, in particular, is difficult and time-consuming. Quantitative polymerase chain reaction (qPCR) technology is considered one of the most sensitive methods to detect low levels of DNA. Here a qPCR method is described that can detect clinically relevant, pathogenic fungal organisms. The assay allowed the quantification of fungal organisms within a tissue implant and provides a means to identify the contaminating species. The primers for the qPCR assay were designed to amplify a conserved region of the L2 region of the large ribosomal subunit (LSU) gene. This set of primers was able to detect fewer than 10 colony forming units from Aspergillus and Candida species in spiked samples. Clinical samples were also evaluated using this method and the data compared positively to the existing accepted 28-day fungal culture method for fungal detection. The qPCR method described herein significantly reduced the time required to identify fungal contamination in tissue implants.     

Detection of nonauthorized genetically modified organisms using differential quantitative polymerase chain reaction: application to 35S in maize

Detection of nonauthorized genetically modified organisms (GMOs) has always presented an analytical challenge because the complete sequence data needed to detect them are generally unavailable although sequence similarity to known GMOs can be expected. A new approach, differential quantitative polymerase chain reaction (PCR), for detection of nonauthorized GMOs is presented here. This method is based on the presence of several common elements (e.g., promoter, genes of interest) in different GMOs. A statistical model was developed to study the difference between the number of molecules of such a common sequence and the number of molecules identifying the approved GMO (as determined by border-fragment-based PCR) and the donor organism of the common sequence. When this difference differs statistically from zero, the presence of a nonauthorized GMO can be inferred. The interest and scope of such an approach were tested on a case study of different proportions of genetically modified maize events, with the P35S promoter as the Cauliflower Mosaic Virus common sequence. The presence of a nonauthorized GMO was successfully detected in the mixtures analyzed and in the presence of (donor organism of P35S promoter). This method could be easily transposed to other common GMO sequences and other species and is applicable to other detection areas such as microbiology.     

Fluorescence-based temperature control for polymerase chain reaction

The ability to accurately monitor solution temperature is important for the polymerase chain reaction (PCR). Robust amplification during PCR is contingent on the solution reaching denaturation and annealing temperatures. By correlating temperature to the fluorescence of a passive dye, noninvasive monitoring of solution temperatures is possible. The temperature sensitivity of 22 fluorescent dyes was assessed. Emission spectra were monitored and the change in fluorescence between 45 and 95 °C was quantified. Seven dyes decreased in intensity as the temperature increased, and 15 were variable depending on the excitation wavelength. Sulforhodamine B (monosodium salt) exhibited a fold change in fluorescence of 2.85. Faster PCR minimizes cycling times and improves turnaround time, throughput, and specificity. If temperature measurements are accurate, no holding period is required even at rapid speeds. A custom instrument using fluorescence-based temperature monitoring with dynamic feedback control for temperature cycling amplified a fragment surrounding rs917118 from genomic DNA in 3 min and 45 s using 35 cycles, allowing subsequent genotyping by high-resolution melting analysis. Gold-standard thermocouple readings and fluorescence-based temperature differences were 0.29 ± 0.17 and 0.96 ± 0.26 °C at annealing and denaturation, respectively. This new method for temperature cycling may allow faster speeds for PCR than currently considered possible.