PathogenMip assay: a multiplex pathogen detection assay

The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The assay was validated by performing pyrosequencing and Microarray chip detection in parallel experiments. HPV plasmids were used to validate sensitivity and selectivity of the assay. In addition, 20 genomic DNA extracts from primary tumors were genotyped with the PathogenMip Assay results and were in 100% agreement with conventional sequencing using an L1-based HPV genotyping protocol. The PathogenMip Assay is a widely accessible protocol for producing and using highly discriminating probes, with experimentally validated results in pathogen genotyping, which could potentially be applied to the detection and characterization of any microbe.     

Evaluating assay precision

* When evaluating the precision of a method it is necessary to assess the repeatability (within-run) and the total or within-laboratory precision. * It is insufficient to assess repeatability in a single run. * Clinical and Laboratory Standards Institute (CLSI) document EP05-A2 describes the protocols for determining the precision of a method. The precision of a method should be tested at at-least two levels; each run in duplicate, with two runs per day over 20 days. CLSI document EP15-A2 describes the protocols that should be undertaken by the user to verify precision claims by a manufacturer. Precision claims by a manufacturer should be tested at at-least two levels, by running three replicates over five days. * A spreadsheet for assisting with the calculations described in this article is available from the AACB web-site.     

Quantitative agar-invasion assay

A new method for quantification of yeast invasion into the agar medium was developed. Classical agar-invasion assays have been the methods of choice for determination of yeast invasion, but their main disadvantage is the lack of quantification. Our new Quantitative yeast agar-invasion test allows for quantitative measurements and enables sorting strains by their degree of invasiveness. The invasion abilities were measured for 10 clinical and non-clinical Saccharomyces cerevisiae strains and a strain of Candida albicans. Finally, the correlation between the degrees of strains invasiveness and their reported virulence was observed, proposing our assay as a method for quick determination of yeast virulence potential.     

Plant viability assay

There is no single method in the long list given in Table 1 which can be used as an unequivocal criterion of viability. Many of the present methods of assay do correlate to various degrees with the final performance of the cell, tissue, organ, or the plant as a whole. Use of parallel viability tests indicating different cell functions is highly recommended. Great care should be taken to interpret the results of these assays. With several parallel tests, the validity of the interpretation can be enhanced, and in many cases, the interpretation may change considerably, depending upon the results from other tests, Since active transport systems have been implicated as one of the primary sites of freezing injury, more effort needs to be devoted to standardize viability assays based on this cell property. In general, the most popular viability assays for plants are based on biophysical rather than biochemical or metabolic functions. A specific test may be suited to a certain material more than another, yet our goal should be to devise a unique assay which will reflect the threshold of vitality versus death.     

Cholesterol efflux assay

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare. Excessive cholesterol is more common problem. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required. The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.     

A high pressure liquid chromatography-based assay for glutathione-S-transferase class distinction assay

In order to expedite the process of classification of the members of the family of glutathione-S-transferases (GSTs) high performance liquid chromatography with photodiode array detection (HPLC-PDA) was used as a means for measuring enzymatic activity. The GST chosen for the development of the HPLC-PDA technique was from equine liver (E-GST). The characterizing substrates, ethacrynic acid (EA) and bromosulfophthalein (BSP), along with previously gathered characterization data allowed for the distinction of α, μ or π-class enzymes. In an initial characterization of the previously unclassified E-GST it was determined that the enzyme was of the π-class with specific activities of 0.062, ± 0.0015 μmol min^− 1 mg^− 1 and 0.0019, ± 0.00064 μmol min^− 1 mg^− 1 for EA and BSP, respectively. Finally, the activity of the E-GST with the EA and BSP substrates, was measured by HPLC-PDA, and was found to be 0.027, ± 0.003 μmol min^− 1 mg^− 1 and 0.002, ± 0.0005 μmol min^− 1 mg^− 1, respectively. While the HPLC-PDA data do not mirror the spectrophotometric results quantitatively the overall response by the E-GST was the same. In general, the E-GSTs were shown to belong to the π-class when characterized by HPLC-PDA due to an EA specific activity greater than 0.01 μmol min^− 1 mg^− 1 and a negligible BSP activity (≤ 0.002 μmol min^− 1 mg^− 1).     

Enzyme-linked coagulation assay: a clot-based, solid-phase assay for thrombin

A new, solid-phase microtiter plate assay for thrombin has been developed, using fibrinogen bound to wells of a microtiter plate and peroxidase-fibrinogen in solution as an indicator system. When small amounts of thrombin are added to the mixture, peroxidase-fibrin and plate-bound fibrin are formed, and the peroxidase-fibrin binds to the plate-bound fibrin. The amount of peroxidase-fibrin binding is proportional to the thrombin concentration and time of incubation. Using this assay, thrombin was measured at concentrations as low as 0.25 ng/ml (0.006 nM) in 150 microliter of sample. In the presence of the specific inhibitors benzamidine and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, the thrombin activity is reduced, at relative concentrations of inhibitors consistent with their affinities and mechanisms of action. The enzyme-linked coagulation assay is generally useful as a highly sensitive and convenient alternative to conventional "clot-based" tests of coagulation.     

A high pressure liquid chromatography-based assay for glutathione-S-transferase class distinction assay

In order to expedite the process of classification of the members of the family of glutathione-S-transferases (GSTs) high performance liquid chromatography with photodiode array detection (HPLC-PDA) was used as a means for measuring enzymatic activity. The GST chosen for the development of the HPLC-PDA technique was from equine liver (E-GST). The characterizing substrates, ethacrynic acid (EA) and bromosulfophthalein (BSP), along with previously gathered characterization data allowed for the distinction of alpha, mu or pi-class enzymes. In an initial characterization of the previously unclassified E-GST it was determined that the enzyme was of the pi-class with specific activities of 0.062, +/-0.0015 micromol min(-1) mg(-1) and 0.0019, +/-0.00064 micromol min(-1) mg(-1) for EA and BSP, respectively. Finally, the activity of the E-GST with the EA and BSP substrates, was measured by HPLC-PDA, and was found to be 0.027, +/-0.003 micromol min(-1) mg(-1) and 0.002, +/-0.0005 micromol min(-1) mg(-1), respectively. While the HPLC-PDA data do not mirror the spectrophotometric results quantitatively the overall response by the E-GST was the same. In general, the E-GSTs were shown to belong to the pi-class when characterized by HPLC-PDA due to an EA specific activity greater than 0.01 micromol min(-1) mg(-1) and a negligible BSP activity (相似文献   

Fluorimetric assay of renin

A simple fluorimetric assay was set up to test renin within 2 h. N-acetyltetradecapeptide was synthesized and used as substrate. It was demonstrated that N-acetyl-angiotensin I and Leu-Val-Tyr-Ser were the two peptides obtained after hydrolysis by renin. Fluorescamine reaction reacted with the free NH2 of the tetrapeptide generated to induce a fluorimetric reaction detected at 395--405 nm. The Michaelis constant of the reaction was 1.87 . 10(-5) M. With this method as little as one milliGoldblatt Unit (mG.U.) of hog renin could be detected and the generation of tetrapeptide was linear with respect to the renin concentration up to 20 mG.U. The fluorimetric assay was applied to the detection of renin during its purification and to the characterization of renin inhibitors.     

Fast chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) is a widely used method to explore in vivo interactions between proteins and DNA. The ChIP assay takes several days to complete, involves several tube transfers and uses either phenol–chlorophorm or spin columns to purify DNA. The traditional ChIP method becomes a challenge when handling multiple samples. We have developed an efficient and rapid Chelex resin-based ChIP procedure that dramatically reduces time of the assay and uses only a single tube to isolate PCR-ready DNA. This method greatly facilitates the probing of chromatin changes over many time points with several antibodies in one experiment.