A simple,rapid, and sensitive DNA assay procedure

A simple and rapid assay for quantitative determinations of DNA in crude homogenates is described. The method is based on the enhancement of fluorescence seen when bisbenzimidazole (Hoechst 33258) binds to DNA. Crude homogenates in which chromatin has been dissociated with high salt buffer can be assayed directly and reliably in a few minutes. The dissociation of chromatin is critical to accurate determinations of DNA in biological materials using this method. The assay can detect as little as 10 ng of DNA with rather unsophisticated instrumentation.     

A simple, sensitive, and rapid method for detecting seed contaminated with highly virulent Leptosphaeria maculans.

A primer-directed DNA amplification polymerase chain reaction assay for detection of seed contaminated with highly virulent Leptosphaeria maculans was developed. The primers were derived from a 5,238-bp repetitive sequence present only in the highly virulent isolates of the fungus. A procedure for isolating DNA from organisms infesting germinating seed was also developed. Seeds were added to liquid fungal minimal medium, and the culture was incubated for 3 days at room temperature with shaking. The organisms were collected from the cultures by centrifugation and lysed with a combination of sodium dodecyl sulfate and proteinase K. The DNA was extracted with organic solvents and with a high-salt-cetyltrimethylammonium bromide solution. It was also precipitated with a low-salt-cetyltrimethylammonium bromide solution. The extensive treatments used for minimizing polysaccharide contamination greatly improved the reliability of the assay. The minimum contamination level (2 of 1,000 seeds) that was tested was successfully detected with this DNA isolation procedure. The reliability of the assay was 96% at the 1 to 2% level of seed contamination. The described method is less laborious and requires only 4 to 5 days for completion in comparison to the 11 to 22 days required for the currently employed methods. In addition, large sample sizes can be easily handled, thus reducing the probability of contaminated seed escaping detection.     

A simple and rapid method for determining the linearity of a flow cytometer amplification system

We describe a simple and rapid method for determining the linearity of a flow cytometer amplification system. The method is based on a fundamental characteristic of linear amplifiers: The difference between two amplified signals increases linearly with increasing amplifier gain. Two populations of beads or cells, differing slightly in fluorescence intensity, are analyzed by the flow cytometer at increasing photomultiplier tube high-voltage settings. The distribution of the populations' mean difference versus mean position is a straight line intersecting the origin for linear amplifiers. Although some types of nonlinearities cannot be detected with this technique, deviations from linearity indicate nonlinear components in the flow cytometer amplification system. The correlation coefficient is used to quantify degree of nonlinearity. We also describe a method for amplifier nonlinearity compensation.     

A simple, rapid, and sensitive procedure for the assay of endoproteases using Coomassie brilliant blue G-250

A rapid colorimetric method for the assay of proteolytic enzymes based on the binding of Coomassie brilliant blue G-250 to unhydrolyzed protein substrate is described. Considerable assay time is saved since the method does not require the separation of the hydrolyzed products from the undergraded protein substrate. The procedure is applicable to crude as well as purified preparations of various proteolytic enzymes and compares well with the procedure of M. L. Anson.     

A rapid, simple, and humane method for submandibular bleeding of mice using a lancet

Methods for obtaining blood samples from mice tend to be difficult, inhumane, or both. The authors describe an inexpensive, disposable, single-use lancet for submandibular bleeding of mice that allows investigators to quickly draw 0.2-0.5 ml of blood without the use of anesthesia.     

A simple, rapid, and sensitive fluorescence assay for microsomal triglyceride transfer protein

Microsomal triglyceride transfer protein (MTP) is critical for the assembly and secretion of apolipoprotein B (apoB) lipoproteins. Its activity is classically measured by incubating purified MTP or cellular homogenates with donor vesicles containing radiolabeled lipids, precipitating the donor vesicles, and measuring the radioactivity transferred to acceptor vesicles. Here, we describe a simple, rapid, and sensitive fluorescence assay for MTP. In this assay, purified MTP or cellular homogenates are incubated with small unilamellar donor vesicles containing quenched fluorescent lipids (triacylglycerols, cholesteryl esters, and phospholipids) and different types of acceptor vesicles made up of phosphatidylcholine or phosphatidylcholine and triacylglycerols. Increases in fluorescence attributable to MTP-mediated lipid transfer are measured after 30 min. MTP's lipid transfer activity could be assayed using apoB lipoproteins but not with high density lipoproteins as acceptors. The assay was used to measure MTP activity in cell and tissue homogenates. Furthermore, the assay was useful in studying the inhibition of the cellular as well as purified MTP by its antagonists. This new method is amenable to automation and can be easily adopted for large-scale, high-throughput screening.     

A rapid,simple and sensitive loop-mediated isothermal amplification method to detect Anaplasma bovis in sheep and goats samples

A loop-mediated isothermal amplification (LAMP) technique has been widely used in detecting the nucleic acid of various pathogenic bacteria. In this study, a set of four LAMP primers was designed to specifically test Anaplasma bovis. The LAMP assay was performed at 62 °C for 60 min in a water bath. The specificity was confirmed by amplifying A. bovis isolate, while no cross reaction was observed with other five pathogens (Anaplasma bovis, Anaplasma phagocytophilum, Theileria luwenshuni, Babesia motasi and Schistosoma japonicum). The sensitivity of LAMP was 5 × 10⁰ copies/μL, 100 times more than that of conventional PCR (5 × 10² copies/μL). Of 120 blood DNA extracted from sheep and goats field samples, 81 (67.5%), 22 (18.3%) and 43 (35.8%) were positively detected by LAMP, conventional PCR and nested PCR, respectively. The findings indicated that the developed LAMP assay is a new convenient tool for rapid and cost-effective detection of A. bovis.     

A simple and rapid spectrofluorimetric method for determining the pharmacokinetics and metabolism of rhaponticin in rat plasma,feces and urine using a cerium probe

Rhaponticin (RH) demonstrates a variety of pharmacological activities, including antitumor, antithrombotic and antioxidant effects. It is essential to establish a simple, rapid and reliable analytical method for determining the pharmacokinetics of RH. A simple cerium ion (Ce³⁺) probe method was developed and validated to determine RH in rat plasma, feces and urine. The fluorescence intensities of the cerium ion (Ce³⁺) were quenched by addition of RH, along with a remarkable red shift. Spectral data revealed that fluorescence quenching of Ce³⁺ by RH was due to the formation of a Ce³⁺–RH complex. Using to the Stern–Volmer equation, the binding parameters for interactions between Ce³⁺ and RH were obtained. Based on these, a rapid and simple spectrofluorimetric method was developed to determine the metabolism and pharmacokinetics of RH using a Ce³⁺ probe. The assay was linear over the concentration range of 0.11–9.52, 0.25–8.87 and 0.18–9.10 μM for plasma, feces and urine, respectivelyand RH recoveries were found to be 98.24 ± 0.8, 97.78 ± 1.2 and 97.54 ± 0.8% for plasma, feces and urine, respectively. The relative standard deviations were < 9.5%. The spectrofluorimetric method was simple and rapid for quantitative determination of RH and its metabolism, and was affordable for most laboratories because of the fluorescence spectroscopy and low equipment cost. These pharmacokinetic, bioavailability and metabolism studies of RH will provide helpful information for the development of suitable dosage forms and clinical references on rational administration. Copyright © 2013 John Wiley & Sons, Ltd.     

A simple and rapid method for calculating identity-by-descent matrices using multiple markers

A fast, partly recursive deterministic method for calculating Identity-by-Descent (IBD) probabilities was developed with the objective of using IBD in Quantitative Trait Locus (QTL) mapping. The method combined a recursive method for a single marker locus with a method to estimate IBD between sibs using multiple markers. Simulated data was used to compare the deterministic method developed in the present paper with a stochastic method (LOKI) for precision in estimating IBD probabilities and performance in the task of QTL detection with the variance component approach. This comparison was made in a variety of situations by varying family size and degree of polymorphism among marker loci. The following were observed for the deterministic method relative to MCMC: (i) it was an order of magnitude faster; (ii) its estimates of IBD probabilities were found to agree closely, even though it does not extract information when haplotypes are not known with certainty; (iii) the shape of the profile for the QTL test statistic as a function of location was similar, although the magnitude of the test statistic was slightly smaller; and (iv) the estimates of QTL variance was similar. It was concluded that the method proposed provided a rapid means of calculating the IBD matrix with only a small loss in precision, making it an attractive alternative to the use of stochastic MCMC methods. Furthermore, developments in marker technology providing denser maps would enhance the relative advantage of this method.     

A simple, rapid method for demonstrating bacterial flagella

We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.     

A simple,rapid, and sensitive method for measuring protein concentration in subcellular membrane fractions prepared by sucrose density ultracentrifugation

The Multiskan spectrophotometric system and Coomassie brilliant blue G-250 protein-dye binding method have been used together to measure NaOH-solubilized protein in subcellular membrane fractions prepared from isolated rat adipose cells. Forty-eight samples can be read in duplicate within 1 min. Sucrose in concentrations up to 0.7 m interfere only moderately with the assay. A rapid and convenient method is, therefore, now available for multiple protein determinations following sucellular fractionation on sucrose gradients.