Biological Reagents Research Articles

Capillary electrophoresis with stationary nanogel zones of galactosidase and Erythrina cristagalli lectin for the determination of β(1–3)-linked galactose in glycans

A thermally responsive nanogel is used to create stationary zones of enzyme and lectin in a separation capillary. Once patterned in the capillary, analyte is driven through the zone, where it is converted to a specific product if an enzyme is used or captured if a lectin is used. These stationary zones are easily expelled after the analysis and then re-patterned in the capillary. The nanogel is compatible with enzymes and lectins and improves the stability of galactosidase, enabling more cost-effective use of biological reagents that provide insight into glycan structure. A feature of using stationary zones is that the reaction time can be controlled by the length of the zone, the applied field controlling the analyte mobility, or the use of electrophoretic mixing by switching the polarity of the applied voltage while the analyte is located in the zone. The temperature, applied voltage, and length of the stationary zone, which are factors that enhance the performance of the enzyme, are characterized. The combined use of enzymes and lectins in capillary electrophoresis is a new strategy to advance rapid and automated analyses of glycans using nanoliter volumes of enzymes and lectins. The applicability of this use of stationary zones of enzyme and lectin in capillary electrophoresis is demonstrated with the identification of β(1–3)-linked galactose in N-glycan.

Investigation of an N-Terminal Prohormone of Brain Natriuretic Peptide Point-of-Care ELISA in Clinically Normal Cats and Cats With Cardiac Disease.

BackgroundN‐terminal prohormone of brain natriuretic peptide (NT‐proBNP) concentrations may be increased in cats with various cardiac disorders. The point‐of‐care (POC) ELISA assay uses the same biologic reagents as the quantitative NT‐proBNP ELISA. Previous studies have evaluated the sensitivity and specificity of the POC ELISA in cats with cardiac disease.ObjectivesTo prospectively evaluate the diagnostic utility of the POC ELISA in a select population of cats.AnimalsThirty‐eight client‐owned cats presented to the University of Florida Cardiology Service for cardiac evaluation. Fifteen apparently healthy cats recruited as part of another study.MethodsPhysical examination and echocardiography were performed in all cats. The POC ELISA was assessed visually as either positive or negative by a reader blinded to the echocardiographic findings, and results were analyzed relative to quantitative assay results.ResultsTwenty‐six cats were diagnosed with underlying cardiac disease, and 27 cats were considered free of cardiac disease. Cats with cardiac disease included: 21 with hypertrophic cardiomyopathy, 2 with unclassified cardiomyopathy, 2 with restrictive cardiomyopathy, and 1 with 3rd degree atrioventricular (AV) block. The POC ELISA differentiated cats with cardiac disease with a sensitivity of 65.4% and specificity of 100%.Conclusions and Clinical ImportanceThe POC NT‐proBNP ELISA performed moderately well in a selected population of cats. A negative test result cannot exclude the presence of underlying cardiac disease, and a positive test result indicates that cardiac disease likely is present, but further diagnostic investigation would be indicated for a definitive diagnosis.

A study on the zinc sulfide dissolution kinetics with biological and chemical ferric reagents

The leaching kinetics of a sphalerite concentrate containing 38.25% zinc was studied in the presence of biological and chemical ferric reagents. To produce the biological ferric reagent (BFR), a pyrite concentrate sample was oxidized to ferric ions by iron and sulfur oxidizing bacteria, and this pregnant leach solution was then applied as oxidizing reagent in ZnS leaching. This process is commonly referred to as two-step bioleaching. This biological reagent contained 12.75g/l ferric and its pH was 0.86. The chemical ferric reagents (CFR) were made by dissolution of Fe2(SO4)3 and FeCl3 salts in deionized water. Leaching experiments were carried out at different temperatures to study the mechanism of ZnS dissolution and its kinetics. The kinetic modeling of ZnS dissolution with BFR followed the interfacial transfer and diffusion across the product layer mechanism within the first minutes (about 60min) while it changed to the diffusion-control mechanism after passing this initial period. On the other hand, the ZnS dissolution in presence of ferric sulfate was described by a diffusion mechanism. The surface analysis by SEM and FTIR confirmed that sulfur layer formation on the mineral surfaces could prevent the solvent diffusion to the minerals surface, and consequently it controls the dissolution reaction. The highest zinc recoveries were 70%, 99% and 83% in presence of biological ferric reagent, ferric sulfate and ferric chloride at 90°C after 200min, respectively. The zinc recovery for one-step bioleaching was 90% and was achieved after 20days at 35°C, by iron and sulfur oxidizing bacteria.

Analytical Tools to Improve Optimization Procedures for Lateral Flow Assays.

Immunochromatographic or lateral flow assays (LFAs) are inexpensive, easy to use, point-of-care medical diagnostic tests that are found in arenas ranging from a doctor’s office in Manhattan to a rural medical clinic in low resource settings. The simplicity in the LFA itself belies the complex task of optimization required to make the test sensitive, rapid and easy to use. Currently, the manufacturers develop LFAs by empirical optimization of material components (e.g., analytical membranes, conjugate pads and sample pads), biological reagents (e.g., antibodies, blocking reagents and buffers) and the design of delivery geometry. In this paper, we will review conventional optimization and then focus on the latter and outline analytical tools, such as dynamic light scattering and optical biosensors, as well as methods, such as microfluidic flow design and mechanistic models. We are applying these tools to find non-obvious optima of lateral flow assays for improved sensitivity, specificity and manufacturing robustness.

Molecularly Imprinted Plasmonic Substrates for Specific and Ultrasensitive Immunoassay of Trace Glycoproteins in Biological Samples.

Assays of glycoproteins hold significant biological importance and clinical values, for which immunoassay has been the workhorse tool. As immunoassays are associated with disadvantages such as poor availability of high-specificity antibodies, limited stability of biological reagents, and tedious procedure, innovative alternatives that can overcome these drawbacks are highly desirable. Plasmonic immunosandwich assay (PISA) has emerged as an appealing alternative to immunoassay for fast and sensitive determination of trace glycoproteins in biosamples. Plasmonic substrates play key roles in PISA, not only in determining the specificity but also in greatly influencing the detection sensitivity. Herein, we report a new type of molecularly imprinted plasmonic substrates for rapid and ultrasensitive PISA assay of trace glycoproteins in complex real samples. The substrates were fabricated from glass slides, first coated with self-assembled monolayer (SAM) of gold nanoparticles (AuNPs) and then molecularly imprinted with organo-siloxane polymer in the presence of template glycoproteins. The prepared molecularly imprinted substrates exhibited not only a significant plasmonic effect but also excellent binding properties, ensuring the sensitivity as well as the specificity of the assay. Alkaline phosphatase (ALP) and α-fetoprotein (AFP), glycoproteins that are routinely used as disease markers in clinical diagnosis, were used as representative targets. The limit of detection (LOD) was 3.1 × 10-12 M for ALP and 1.5 × 10-14 M for AFP, which is the best among the PISA approaches reported. The sample volume required was only 5 μL, and the total time required was within 30 min for each assay. Specific and ultrasensitive determination of ALP and AFP in human serum was demonstrated. Because many disease biomarkers are glycoproteins, the developed PISA approach holds great promise in disease diagnostics.

RATA: A method for high-throughput identification of RNA bound transcription factors.

Long non-coding RNAs (lncRNAs) regulate critical cellular processes and their dysregulation contributes to multiple diseases. Although only a few lncRNAs have defined mechanisms, many of these characterized lncRNAs interact with transcription factors to regulate gene expression, suggesting a common mechanism of action. Identifying RNA-bound transcription factors is especially challenging due to inefficient RNA immunoprecipitation and low abundance of many transcription factors. Here we describe a highly sensitive, user-friendly, and inexpensive technique called RATA (RNA-associated transcription factor array), which utilizes a MS2-aptamer pulldown strategy coupled with transcription factor activation arrays for identification of transcription factors associated with a nuclear RNA of interest. RATA requires only ~5 million cells and standard molecular biology reagents for multiplexed identification of up to 96 transcription factors in 2–3 d. Thus, RATA offers significant advantages over other technologies for analysis of RNA-transcription factor interactions.

Monoclonal antibodies in immunodiagnostic assays: a review of recent applications

Monoclonal antibodies (mAbs) have proven to be effective biological reagents in the immunodiagnostic assays. This is due to their binding accuracy to many pathogens, thus, making them valuable research tools. Since the discovery of hybridoma technology by Kohler and Milstein, the use of monoclonal antibodies produced by the hybrid cells have been employed in diagnosis of several diseases. Monoclonal antibody production has several procedures with considerable variations, but the principles remain the same. Improvements in the field of cell culture technology have led to the production of improved qualities of monoclonal antibodies. In general, these antibodies are important biomedical reagents used in research, especially in the field of laboratory diagnostics for a number of different types of diseases in humans and animals. Some of the areas where application of monoclonal antibodies triumph are herein discussed. This review is aimed to assess various diagnostic assay techniques where monoclonal antibodies are applied in order to provide a first-hand information, especially for beginners in monoclonal antibody production, characterisation, evaluation and /or its applications in research and diagnosis.Keywords: Monoclonal antibodies, immunodiagnostic, assays, recent applications

Fat, oil and grease reduction in commercial kitchen ductwork: A novel biological approach

Recent research has characterised emissions upon cooking a variety of foods in a commercial catering environment in terms of volume, particle size and composition. However, there has been limited focus on the deposition of solid grease in commercial kitchen ductwork, the sustainability of these systems and their implications on the heat recovery potential of kitchen ventilation extract air.This paper reviews the literature concerning grease, commonly referred to as Fat, Oils and Grease (FOG) abatement strategies and finds that many of these systems fall short of claimed performances. Furthermore these technologies often add to the energy cost of the operation and reduce the potential application of heat recovery in the ventilation ductwork. The aim of this study was to develop and evaluate a novel FOG removal system, with a focus on low environmental impact.The novel FOG removal system, utilises the biological activity of Bacillus subtilis and associated enzymes. The biological reagent is delivered via a misting system. The temperature, relative humidity and FOG deposit thickness were measured in the ductwork throughout a 3month trial period. FOG deposit thickness was reduced by 47% within 7weeks. The system was found to be effective at reducing the FOG deposit thickness with minimal energy cost and impact upon the kitchen and external environment. Internal ductwork operating temperature was measured with respect to future heat recovery potential and a reduction of 7°C was observed.

Application of rapid biological monitoring for bacillus stearothermophilus spore in the management of surgical instruments

Objective To compare the applications of four types of bacillus stearothermophilus spore reagents, in order to provide basis for the safe management of surgical instruments. Methods From September to December 2015, four types of bacillus stearothermophilus spore reagents that were often used in the disinfection supply center of Jiaozuo People's Hospital of He'nan province were investigated. They were SSI.K31 type bacillus stearothermophilus spore tablet of Beijing Sihuan (common bacterial tablet) , self-contained Bacillus stearothermophilus spore reagent of Beijing Sihuan (48 h self-contained agent) , 1292 biological reagent of American 3M company (3M1292) , and 1492 biological reagent of American 3M company (3M1492) . They were cultured by steam sterilization of 132℃ for 480 seconds. The culture results were obtained at different time according to the different types of reagents. The number of vacuum and sterilization time were adjusted. The spore killing situation was recorded and analyzed. Results In the experimental tube culture of four types bacillus stearothermophilus spore reagents, the difference occurred in the common bacterial tablets because they were susceptible to other factors. The culture results of three self-contained reagents were in agreement. The time of common spore tablets was the longest. The time of 3M1492 was the shortest. Conclusions For common bacterial tablets, 7 days is needed for the bacterial culture. In the transplantation process there is potential contamination chance, which might result in false positive results. The application of self-contained spore biological reagents can avoid false positive results and it is convenient and fast. The super rapid biological monitoring can interpret monitoring results in the shortest time so that the seamless joint of the surgical instruments can be achieved, which speeds up the turnover and produces higher practical value. Key words: Disinfection; Environmental monitoring; Bacillus stearothermophilus; Surgical instruments

AN ATTEMPT TO PRODUCE RECOMBINANT HORSE INSULIN

The present study aimed to produce horse insulin by use of recombinant DNA technology. Because the hormones such as somatotropin, prolactin and leptin, which are practically applicable in veterinary medicine, are also peptides, they can be obtained on the basis of this model technology. To isolate total RNA, horse pancreas samples were collected in RNA stabilizing solution from local abattoirs, sent to the laboratory, and stored the samples in the freezer at -80°C. Molecular biological reagents and kits for total RNA extraction,one step RT (reverse transcription)-PCR reactions, cloning, expression, and purification were used to produce equine proinsulin. Primers were designed from NCBI gene bank data and synthesized in Macrogen Inc, Korea. Obtained equine proinsulin was processed by enzymes trypsin and carboxypeptidase B to form mature insulin. The purity of insulin was assessed by SDS-PAGE. Molecular weight of insulin is approximately 6.0 kDa. The hormone was also measured by ELISA test and it was equal to 55 IU per ml of solution. Finally it can be concluded equine insulin with 55 IU/ml activity can be produced by recombinant DNA technology.

A Do-it-Yourself Protocol for Making Desired Mutant Proteins

The generation of a mutant protein is often crucial to the elucidation of the pathogenesis of a genetic disease. This requires site-directed mutagenesis of an expression vector containing the gene of interest. Current site-directed mutagenesis procedures are derivatives of either a restriction site elimination method or a PCR-based mutagenesis method, which involve a commercial kit manufactured by any one of a number of biotechnology companies. A new efficient economical site-directed mutagenesis procedure is described here to provide researchers an additional choice that does not to rely on a commercial kit. This novel method has been validated, and requires only basic molecular biological techniques and reagents available in most laboratories even in developing countries. The mutation efficiency of this new method is usually greater than 80%. In case several different mutants of a protein are required for elucidation of the protein structure-function relationships, this method has distinct advantages since multiple mutations can be derived from the initial single strand DNA template.

A microfluidic microinjector for toxicological and developmental studies in Drosophila embryos.

Microinjection is an established and reliable method to deliver biological reagents such as transgenic constructs and drugs, to specific locations inside organisms such as the Drosophila embryo and C. elegans worm. In this paper, a simple compliant mechanism based PDMS-microinjection system has been demonstrated. Unlike conventional microinjectors, this unique system could allow one to precisely insert a long taper microneedle laterally and at various positions inside the length of the Drosophila embryo (up to 250 μm) with the resolution of 5 μm. Volumes as low as 30 pL with accuracy of ±10 pL were delivered inside the embryo via pressure pulses. The device has been used to study the effect of toxins on cardiogenesis in Drosophila embryos. Using this device, we demonstrate that the cardioblast (CB) migration velocity is modified in a dose sensitive manner to varying doses of injected sodium azide (NaN3) and, for the first time, quantify the effect of the toxin on heart assembly. Injection with 40 pL of NaN3 was shown to decrease CB migration velocity and filopodia number at concentrations above 10 mM, while embryos injected with the tracer Rhodamine B (0 mM NaN3) displayed no significant difference when compared to uninjected embryos. This device can be potentially used for other embryonic assays, which require accurate delivery of the reagents to a specific location within the embryo.

Dilute Russell Viper Venom Time Interpretation and Clinical Correlation: A Two-Year Retrospective Institutional Review

Background:In 2009, the International Society for Thrombosis and Hemostasis (ISTH) issued interpretative guidelines for the performance and interpretation of lupus anticoagulant testing, including the dilute Russell Viper venom test (RVVT). At UCSF, we noted that the manufacturer guidelines approved by the FDA were not equivalent to these ISTH guidelines. As a result, we report RVVT results as positive if both sets of criteria are met, equivocal if only one set of criteria is met, and negative if neither the manufacturer nor the ISTH criteria are met.Study design:We performed a retrospective review of all RVVT testing at UCSF performed between 6/4/2011 and 7/3/2013. Patients who had a RVVT test performed within that time period were included in the study. For patients with a prolonged RVVT screening time, we gathered records of their previous and subsequent antiphospholipid syndrome (APS) testing at UCSF as well as any history of prolonged aPTT. These laboratory data were correlated with any clinical history of clotting, embolism, bleeding, pregnancy morbidity, systemic lupus erythematosus (SLE) and/or death. Clinical history was obtained through review of the UCSF electronic medical record. This study was approved by the UCSF institutional review board (Human Research Protection Program, Committee on Human Research). UCSF uses Precision Biologic (Halifax, Nova Scotia, Canada) LA Check and LA Sure reagents for the RVVT.Findings:21.6% of RVVT tests showed a prolonged clotting time. Of these, 12% met only the ISTH criteria for the diagnosis of LA, 11% met only the manufacturer criteria, 37% were positive by both sets of criteria, and 40% were not positive by either set of criteria. When comparing the manufacturer and ISTH criteria for RVVT positivity, the latter were more predictive of a concomitant positive Staclot LA (Diagnotica Stago, Parsippany, NJ) result (Risk ratio 1.806 (95th CI 1.133-2.877)). When comparing the manufacturer and ISTH criteria for RVVT positivity, the latter was non-significantly associated with a positive aPL immunoassay result (Risk ratio 6.682 (95th CI 0.8965-49.8)). Patients with a positive RVVT result by only ISTH criteria, only manufacturer criteria, or both did not differ in their relative risk for thrombosis, pregnancy morbidity, systemic lupus erythematosus, or death.If prolonged activated partial thromboplastin time was the only reason for testing, none of these patients subsequently were diagnosed with APS.Patients with an initial positive RVVT who were retested according to ISTH recommendations had a significantly increased risk of being diagnosed with APS during our study period (RR 7.827, 95th CI 1.89-32.41) as compared to those patients without an initial positive RVVT. This indicates an RVVT sensitivity of 0.846 and specificity of 0.7 for a diagnosis of APS.Of 26 patients who had a positive RVVT (regardless of Staclot LA result) and had repeat testing, 11 (42%) went on to meet APS criteria. Of 35 patients who had a positive Staclot LA (regardless of RVVT result) and had repeat testing, 13 (37%) went on to meet APS criteria. In contrast, very few patients with only a positive RVVT or only a positive Staclot-LA were diagnosed with APS (2 patients out of 47). Those patients who tested positive on both RVVT and Staclot LA were significantly more likely to have an APS diagnosis than those positive with Staclot LA alone (RR 6.531, 95th CI 1.69-25.25).Conclusion: This observational study should help laboratorians and physicians better interpret the clinical significance of RVVT results. The ISTH guidelines for RVVT interpretation appear to better correlate with Staclot LA positivity. ISTH and manufacturer criteria do not differ in prediction of subsequent thrombosis, pregnancy morbidity, SLE, or death. Whereas the frequently used term "triple-positivity" in antiphospholipid testing implies significant clinical APS risk and is defined by a single positive lupus anticoagulant in the presence of both anticardiolipin and anti-β2-glycoprotein antibodies, we show that those patients with combined positivity for two lupus anticoagulant assays (both RVVT and Staclot LA) have a 6.5-fold higher risk of subsequent APS diagnosis than those with single lupus anticoagulant positivity. DisclosuresWool:Inova Diagnostics: Honoraria.

The archaeal "7 kDa DNA-binding" proteins: extended characterization of an old gifted family.

The “7 kDa DNA-binding” family, also known as the Sul7d family, is composed of chromatin proteins from the Sulfolobales archaeal order. Among them, Sac7d and Sso7d have been the focus of several studies with some characterization of their properties. Here, we studied eleven other proteins alongside Sac7d and Sso7d under the same conditions. The dissociation constants of the purified proteins for binding to double-stranded DNA (dsDNA) were determined in phosphate-buffered saline at 25 °C and were in the range from 11 μM to 22 μM with a preference for G/C rich sequences. In accordance with the extremophilic origin of their hosts, the proteins were found highly stable from pH 0 to pH 12 and at temperatures from 85.5 °C to 100 °C. Thus, these results validate eight putative “7 kDa DNA-binding” family proteins and show that they behave similarly regarding both their function and their stability among various genera and species. As Sac7d and Sso7d have found numerous uses as molecular biology reagents and artificial affinity proteins, this study also sheds light on even more attractive proteins that will facilitate engineering of novel highly robust reagents.

Novel Wire Planning Schemes for Pin Minimization in Digital Microfluidic Biochips

Digital microfluidic biochips (DMFBs), a second-generation lab-on-chip device has developed in recent years as a feasible alternative to conventional laboratory procedure for biochemical analysis and diagnostic applications. These devices enable the precise manipulation of nanoliter volumes of biological fluids and chemical reagents within a 2-D rectangular array of electrodes. Increasing number of control pin requirements together with high wire planning complexity becomes a major issue for integrated execution of multiple bioassays within a single DMFB layout. In this paper, we propose new techniques for interconnection wire routing for actuating the electrodes operating at identical time sequence. Here, we propose three different algorithms to develop feasible wire plans for a given layout with an aim to minimize the overall number of control pin count. Multiple actuation on the same pin termed multiphasing is proposed to resolve the issue of wire planning for handling the issue of cross contamination at any particular site. The proposed techniques are implemented in layouts using testbenches for benchmark suite III and selective testbenches for benchmark suite I. The results obtained through simulation show encouraging improvement over contemporary contributions.

Specific Recognition of a Single-Stranded RNA Sequence by a Synthetic Antibody Fragment

Antibodies that bind RNA represent an unrealized source of reagents for synthetic biology and for characterizing cellular transcriptomes. However, facile access to RNA-binding antibodies requires the engineering of effective Fab libraries guided by the knowledge of the principles that govern RNA recognition. Here, we describe a Fab identified from a minimalist synthetic library during phage display against a branched RNA target. The Fab (BRG) binds with 20nM dissociation constant to a single-stranded RNA (ssRNA) sequence adjacent to the branch site and can block the action of debranchase enzyme. We report the crystal structure in complex with RNA target at 2.38Å. The Fab traps the RNA in a hairpin conformation that contains a 2-bp duplex capped by a tetraloop. The paratope surface consists of residues located in four complementarity-determining regions including a major contribution from H3, which adopts a helical structure that projects into a deep, wide groove formed by the RNA. The amino acid composition of the paratope reflects the library diversity, consisting mostly of tyrosine and serine residues and a small but significant contribution from a single arginine residue. This structure, involving the recognition of ssRNA via a stem–loop conformation, together with our two previous structures involving the recognition of an RNA hairpin loop and an RNA tertiary structure, reveals the capacity of minimalist libraries biased with tyrosine, serine, glycine, and arginine to form binding surfaces for specific RNA conformations and distinct levels of RNA structural hierarchy.

Sensitive detection of proteins in polyacrylamide gel via isatoic anhydride derivatization: Introduction of a low-cost fluorescent prelabeling procedure.

Here, we introduce isatoic anhydride as a sensitive and commodious fluorescent prelabel for detection of proteins in one-dimensional polyacrylamide gels. High reactivity of isatoic anhydride with nucleophiles in mild alkaline environments makes it an appropriate tag for labeling of biomolecules. In this study, we show that preelectrophoresis labeling of proteins with isatoic anhydride for few minutes at room temperature allows detection of 2-4 ng of standard proteins, BSA and lysozyme, per band. Proteins were successfully labeled in the presence of a wide range of common biological reagents and in crude cell extract. The labeled proteins have the same electrophoretic migration in comparison to unlabeled proteins; however the application of saturation labeling method results in slight band broadening. Compatibility of the method with downstream processes was assessed by tryptic digestion of labeled proteins and study of peptide mixture using gel electrophoresis which revealed partial digestion of labeled proteins due to lysine modification. The present procedure is sensitive, rapid, and inexpensive and is a promising alternative for current protein staining procedures, where downstream processes are not desired.

Optimizing a Method for the Quantification by Quantitative Real-Time Polymerase Chain Reaction of Host Cell DNA in Plasmid Vector Batches Used in Human Gene Therapy.

Gene therapy products are very complex advanced therapy medicinal products produced using different processes that require many chemical and biological reagents and production intermediates, such as producing cells. The quantification of residual impurities in gene therapy vectors is a major quality control step when these vectors are used for therapeutic purposes, whether or not they are derived from viruses. Indeed, in nonviral gene therapy products, particularly plasmid vectors used to transfer genetic material, the presence of host-cell DNA (HCDNA) from the bacterial cells used for the vector production is an important concern because of the risk of immunogenicity and insertional mutagenesis. Several methods have been developed to quantify residual HCDNA, but real-time quantitative polymerase chain reaction (qPCR) seems to be most suitable because it allows detecting traces of "contaminating" DNA. The French National Agency for Medicines and Health Products Safety (ANSM) ensures the quality and safety of gene transfer medicinal products and must be able to quantify, in its own laboratories, the amount of HCDNA present in plasmid vector batches. Therefore, we developed and validated a qPCR method to quantify at the femtogram level the presence of Escherichia coli residual DNA in plasmid vectors. This approach uses the capillary-based LightCycler 1.5 System (Roche) with SYBR Green I, a primer pair against the E. coli 23S ribosomal RNA gene and different concentrations of a linearized plasmid that contains the 23S target sequence, as standard. This qPCR method is linear on an 8-decade logarithmic scale, accurate, reproducible, and sensitive (quantification of up to 10 copies of 23S target sequence per reaction, or 1.4 E. coli genome, or 7 fg of bacterial DNA). This technique allows ensuring that batches of plasmid vectors to be used in clinical trials comply with the specifications on HCDNA content.

An Extrapolation of a Radical Equation More Accurately Predicts Shelf Life of Frozen Biological Matrices.

Arrhenius modeling of analyte recovery at increased temperatures to predict long-term colder storage stability of biological raw materials, reagents, calibrators, and controls is standard practice in the diagnostics industry. Predicting subzero temperature stability using the same practice is frequently criticized but nevertheless heavily relied upon. We compared the ability to predict analyte recovery during frozen storage using 3 separate strategies: traditional accelerated studies with Arrhenius modeling, and extrapolation of recovery at 20% of shelf life using either ordinary least squares or a radical equation y = B1x(0.5) + B0. Computer simulations were performed to establish equivalence of statistical power to discern the expected changes during frozen storage or accelerated stress. This was followed by actual predictive and follow-up confirmatory testing of 12 chemistry and immunoassay analytes. Linear extrapolations tended to be the most conservative in the predicted percent recovery, reducing customer and patient risk. However, the majority of analytes followed a rate of change that slowed over time, which was fit best to a radical equation of the form y = B1x(0.5) + B0. Other evidence strongly suggested that the slowing of the rate was not due to higher-order kinetics, but to changes in the matrix during storage. Predicting shelf life of frozen products through extrapolation of early initial real-time storage analyte recovery should be considered the most accurate method. Although in this study the time required for a prediction was longer than a typical accelerated testing protocol, there are less potential sources of error, reduced costs, and a lower expenditure of resources.

Diagnosis of Dermatophytosis Using Molecular Biology.

Identification of fungi in dermatological samples using PCR is reliable and provides significantly improved results in comparison with cultures. It is possible to identify the infectious agent when negative results are obtained from cultures. In addition, identification of the infectious agent can be obtained in 1day. Conventional and real-time PCR methods used for direct fungus identification in collected samples vary by DNA extraction methods, targeted DNA and primers, and the way of analysing the PCR products. The choice of a unique method in a laboratory is complicated because the results expected from skin and hair sample analysis are different from those expected in cases of onychomycosis. In skin and hair samples, one dermatophyte among about a dozen possible species has to be identified. In onychomycosis, the infectious agents are mainly Trichophyton rubrum and, to a lesser extent, Trichophyton interdigitale, but also moulds insensitive to oral treatments used for dermatophytes, which renders fungal identification mandatory. The benefits obtained with the use of PCR methods for routine analysis of dermatological samples have to be put in balance with the relative importance of getting a result in a short time, the price of molecular biology reagents and equipment, and especially the time spent conducting laboratory manipulations.