One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus,Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae

Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV), Peste de petits ruminants virus (PPRV), Pasteurella multocida (PM) and Mycoplasma capricolum ssp. capripneumonia (Mccp) in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%–4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI) were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s) by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in 17 samples, PPRV in 45, and PM in six samples. In addition, three samples showed a co-infection of PPRV and PM. Overall, the one-step multiplex RT-qPCR assay developed will be a valuable tool for rapid detection of individual and co-infections of the targeted pathogens with high specificity and sensitivity.     

Quantitative assays for uracil-DNA glycosylase of high sensitivity

We have developed a sensitive fluorometric assay using bisulfite deaminated (C----U), covalently-closed circular PM2 DNA as the substrate. We describe a reliable way to prepare this substrate without nicking the PM2 DNA. Methods, which depend on toluenization of the cells, are described for reproducibly and quantitatively assaying uracil-DNA glycosylase. The sensitivity is such that only 200 EL4 mouse thymoma cells or 30,000 Escherichia coli cells are needed for each point in a kinetic assay.     

Detection and quantification of methyl tert-butyl ether-degrading strain PM1 by real-time TaqMan PCR.

The fuel oxygenate methyl tert-butyl ether (MTBE), a widely distributed groundwater contaminant, shows potential for treatment by in situ bioremediation. The bacterial strain PM1 rapidly mineralizes and grows on MTBE in laboratory cultures and can degrade the contaminant when inoculated into groundwater or soil microcosms. We applied the TaqMan quantitative PCR method to detect and quantify strain PM1 in laboratory and field samples. Specific primers and probes were designed for the 16S ribosomal DNA region, and specificity of the primers was confirmed with DNA from 15 related bacterial strains. A linear relationship was measured between the threshold fluorescence (C(T)) value and the quantity of PM1 DNA or PM1 cell density. The detection limit for PM1 TaqMan assay was 2 PM1 cells/ml in pure culture or 180 PM1 cells/ml in a mixture of PM1 with Escherichia coli cells. We could measure PM1 densities in solution culture, groundwater, and sediment samples spiked with PM1 as well as in groundwater collected from an MTBE bioaugmentation field study. In a microcosm biodegradation study, increases in the population density of PM1 corresponded to the rate of removal of MTBE.     

A colloidal gold probe-based silver enhancement immunochromatographic assay for the rapid detection of abrin-a

High-affinity anti-abrin-a monoclonal and polyclonal antibodies were used to develop a sandwich immunochromatographic assay and silver enhancement technology was used to further increase the sensitivity. Using a matrix of double distilled water or soybean milk with added abrin-a, the visual detection limit was found to be 10 ng mL(-1). The detection limit was 0.1 ng mL(-1) for abrin-a, an increase in sensitivity of 100-fold when the silver enhancement technology was employed. The assay was portable and very simple to perform and the detection was completed within 20 min without complicated handling procedures. There was no significant cross-reactivity with several homologous toxins and associated agglutinin. The assay reagents could be stored for 12 weeks at 4°C without significant loss of activity. These characteristics make the strip assay to be an ideal candidate for the development of a rapid toxin detection kit.     

Detection and Quantification of Methyl tert-Butyl Ether-Degrading Strain PM1 by Real-Time TaqMan PCR

The fuel oxygenate methyl tert-butyl ether (MTBE), a widely distributed groundwater contaminant, shows potential for treatment by in situ bioremediation. The bacterial strain PM1 rapidly mineralizes and grows on MTBE in laboratory cultures and can degrade the contaminant when inoculated into groundwater or soil microcosms. We applied the TaqMan quantitative PCR method to detect and quantify strain PM1 in laboratory and field samples. Specific primers and probes were designed for the 16S ribosomal DNA region, and specificity of the primers was confirmed with DNA from 15 related bacterial strains. A linear relationship was measured between the threshold fluorescence (C_T) value and the quantity of PM1 DNA or PM1 cell density. The detection limit for PM1 TaqMan assay was 2 PM1 cells/ml in pure culture or 180 PM1 cells/ml in a mixture of PM1 with Escherichia coli cells. We could measure PM1 densities in solution culture, groundwater, and sediment samples spiked with PM1 as well as in groundwater collected from an MTBE bioaugmentation field study. In a microcosm biodegradation study, increases in the population density of PM1 corresponded to the rate of removal of MTBE.     

Use of a cell transformation assay with established cell lines, and a metabolic cooperation assay with V79 cells for the detection of tumour promoters: a review

Extensive studies on the safety evaluation of chemicals have indicated that a considerable number of non-genotoxic chemicals are carcinogenic. Tumour promoters are likely to be among these non-genotoxic carcinogens, and their detection is considered to be an important approach to the prevention of cancer. In this review, the results are summarised for in vitro transformation assays involving established cell lines, and for an assay for inhibition of gap junctional intercellular communication for the detection of tumour promoters, which involves V79 cells. Although the number of chemicals examined is still too small to permit a full evaluation of the correlation between in vitro cell transformation and in vivo carcinogenicity, it is clear that the sensitivity of the focus formation assay is very high. In the case of the metabolic cooperation assay, the sensitivity appears to be rather poor, but the assay can be considered to be useful because of its simple procedure and its considerable database. These in vitro assays for tumour promoters are recommended as useful tools for the detection of non-genotoxic carcinogens.     

Effect of the freeze-drying process on the phenotypic diversity of Pseudomonas putida strains isolated from the interior of healthy roots of Sida hermaphrodita: Phenotype microarrays (PMs)

The objective of this study was to research the effect of the freeze-drying process on the metabolic changes of Pseudomonas putida strains (E41, E42, R85) isolated from the interior of Sida hermaphrodita roots with the use of the phenotypic microarrays (PM) technology.The proposed method of the freeze-drying process with inulin as component lycoprotectant demonstrated a high bacterial survival ratio (BSR) immediately after freeze-drying and storage after 12 months. While, after 360 days of freeze-drying BSR decreased to value of 74.38.Pseudomonas putida strains were assayed on microplates PM1-PM5, and PM9-PM13 testing 664 different substrates. However, no significant differences in the use of C substrates were observed either before or after the freeze drying process. An insignificant negative effect of the freeze-drying on the use of these substrates was observed. The utilization of N, P and S sources was low or showed no metabolic activity for most of the compounds after freeze-drying. The freeze-drying process increased the sensitivity of the bacteria to antibiotics and selected chemicals.In this study, the freeze-drying process decreased the metabolic activities of the tested strains and their resistance to antibiotics and chemicals.     

Usefulness of a Nested-polymerase chain reaction for molecular diagnosis of human T-cell lymphotropic virus type I/II

This study aimed at implementing a Nested-polymerase chain reaction (Nested-PCR) for the molecular diagnosis of human T-cell lymphotropic virus type I/II (HTLV-I and HTLV-II) infections in peripheral blood mononuclear cells of infected subjects in Argentina. The sensitivity and specificity of the assay for the detection of regional strains were assessed by comparing them with the molecular assay of reference PCR-hybridization. The Nested-PCR detected 1 MT-2 cell (> or = 8 proviral copies)/1x10(6) non-infected cells showing high sensitivity for provirus detection. While both molecular assays showed high specificity (100%) for HTLV-I and HTLV-II detection, the sensitivity values differed: 100% for Nested-PCR and 67% for PCR-hybridization assay. Moreover, this technique showed less sensitivity for the detection of DNA sequences of HTLV-II (33%) than for the detection of DNA sequences of HTLV-I (75%). The high sensitivity and specificity of the Nested-PCR for regional strains and its low costs indicate that this assay could replace the PCR-hybridization assay for the molecular diagnosis of HTLV-I/II infections. It will be interesting to assess the usefulness of this assay as a tool for the molecular diagnosis of HTLV-I/II infections in other developing countries. Other studies that include a greater number of samples should be conducted.     

Development of an oligonucleotide microarray for the detection and monitoring of marine dinoflagellates

Harmful Algal Blooms (HABs), mainly caused by dinoflagellates and diatoms, have great economic and sanitary implications. An important contribution for the comprehension of HAB phenomena and for the identification of risks related to toxic algal species is given by the monitoring programs. In the microscopy-based monitoring methods, harmful species are distinguished through their morphological characteristics. This can be time consuming and requires great taxonomic expertise due to the existence of morphologically close-related species. The high throughput, automation possibility and specificity of microarray-based detection assay, makes this technology very promising for qualitative detection of HAB species. In this study, an oligonucleotide microarray targeted to the ITS1-5.8S-ITS2 rDNA region of nine toxic dinoflagellate species/clades was designed and evaluated. Two probes (45-47 nucleotides in length) were designed for each species/clade to reduce the potential for false positives. The specificity and sensitivity of the probes were evaluated with ITS1-5.8S-ITS2 PCR amplicons obtained from 20 dinoflagellates cultured strains. Cross hybridization experiments confirmed the probe specificity; moreover, the assay showed a good sensitivity, allowing the detection of up to 2 ng of labeled PCR product. The applicability of the assay with field samples was demonstrated using net concentrated seawater samples, un-spiked or spiked with known amounts of cultured cells. Despite the general application of microarray technology for harmful algae detection is not new, a peculiar group of target species/clades has been included in this new-format assay. Moreover, novelties regarding mainly the probes and the target rDNA region have allowed sensitivity improvements in comparison to previously published studies.     

Application and evaluation of the alamarblue assay for cell growth and survival of fibroblasts

Summary Cell proliferation assays are essential to developing an understanding of the molecular mechanisms that modulate cell growth and differentiation. In this paper, we describe the application of alamarBlue, a new and versatile metabolic dye, for the detection of Swiss 3T3 fibroblast proliferation and/or survival. As a redox indicator, alamarBlue is reduced by reactions innate to cellular metabolism and, therefore, provides an indirect measure of viable cell number. Various assay parameters were optimized for a 96-well format to achieve a detectable range of fibroblast cell number from 100 to 20 000 cells/well, which is similar to that obtained with traditional (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and [³H]thymidine assay techniques. Standard (reference) curves generated with a known fibroblast stimulator were used to facilitate quantitation and comparison of unknown test substances. The alamarBlue assay offers the advantages of technical simplicity, freedom from radioisotopes, versatility in detection, no extraction, and excellent reproducibility and sensitivity. We anticipate that this simple and versatile alamarBlue assay, when used alone or in conjunction with other bioassays, will be a useful tool for investigating the complex mechanisms of cellular proliferation.     

Evaluation of rapid alternative methods for drug susceptibility testing in clinical isolates of Mycobacterium tuberculosis

A study was carried out to compare the performance of a commercial method (MGIT) and four inexpensive drug susceptibility methods: nitrate reductase assay (NRA), microscopic observation drug susceptibility (MODS) assay, MTT test, and broth microdilution method (BMM). A total of 64 clinical isolates of Mycobacterium tuberculosis were studied. The Lowenstein-Jensen proportion method (PM) was used as gold standard. MGIT NRA, MODS, and MTT results were available on an average of less than 10 days, whereas BMM results could be reported in about 20 days. Most of the evaluated tests showed excellent performance for isoniazid and rifampicin, with sensitivity and specificity values > 90%. With most of the assays, sensitivity for ethambutol was low (62-87%) whereas for streptomycin, sensitivity values ranged from 84 to 100%; NRA-discrepancies were associated with cultures with a low proportion of EMB-resistant organisms while most discrepancies with quantitative tests (MMT and BMM) were seen with isolates whose minimal inhibitory concentrations fell close the cutoff MGIT is reliable but still expensive. NRA is the most inexpensive and easiest method to perform without changing the organization of the routine PM laboratory performance. While MODS, MTT, and BMM, have the disadvantage from the point of view of biosafety, they offer the possibility of detecting partial resistant strains. This study shows a very good level of agreement of the four low-cost methods compared to the PM for rapid detection of isoniazid, rifampicin and streptomycin resistance (Kappa values > 0.8); more standardization is needed for ethambutol.     

A real‐time PCR assay for improved rapid,specific detection of Cryphonectria parasitica

Cryphonectria parasitica, an ascomycete fungus, is the causal agent of chestnut blight. This highly destructive disease of chestnut trees causes significant losses, and is therefore a regulated pathogen in Europe. Existing methods for the detection of C. parasitica include morphological identification following culturing, or PCR; however, these are time‐consuming resulting in delays to diagnosis. To allow improved detection, a new specific real‐time PCR assay was designed to detect C. parasitica directly from plant material and fungal cultures, and was validated according to the European Plant Protection Organisation (EPPO) standard PM 7/98. The analytical specificity of the assay was tested extensively using a panel of species taxonomically closely related to Cryphonectria, fungal species associated with the hosts and healthy plant material. The assay was found to be specific to C. parasitica, whilst the analytical sensitivity of the assay was established as 2 pg µL^?1 of DNA. Comparative testing of 63 samples of naturally infected plant material by the newly developed assay and traditional morphological diagnosis demonstrated an increased diagnostic sensitivity when using the real‐time PCR assay. Furthermore the assay is able to detect both virulent and hypovirulent strains of C. parasitica. Therefore the new real‐time PCR assay can be used to provide reliable, rapid, specific detection of C. parasitica to prevent the accidental movement of the disease and to monitor its spread.     

The application of CRISPR‐Cas for single species identification from environmental DNA

We report the first application of CRISPR‐Cas technology to single species detection from environmental DNA (eDNA). Organisms shed and excrete DNA into their environment such as in skin cells and faeces, referred to as environmental DNA (eDNA). Utilising eDNA allows noninvasive monitoring with increased specificity and sensitivity. Current methods primarily employ PCR‐based techniques to detect a given species from eDNA samples, posing a logistical challenge for on‐site monitoring and potential adaptation to biosensor devices. We have developed an alternative method; coupling isothermal amplification to a CRISPR‐Cas12a detection system. This utilises the collateral cleavage activity of Cas12a, a ribonuclease guided by a highly specific single CRISPR RNA. We used the target species Salmo salar as a proof‐of‐concept test of the specificity of the assay among closely related species and to show the assay is successful at a single temperature of 37°C with signal detection at 535 nM. The specific assay, detects at attomolar sensitivity with rapid detection rates (<2.5 hr). This approach simplifies the challenge of building a biosensor device for rapid target species detection in the field and can be easily adapted to detect any species from eDNA samples from a variety of sources enhancing the capabilities of eDNA as a tool for monitoring biodiversity.     

Validation of a reverse transcription nested polymerase chain reaction (RT-nPCR) to detect bovine viral diarrhea virus (BVDV) associated with in vitro-derived bovine embryos and co-cultured cells

Sensitive RT-nPCR assays can be used for the rapid detection of viruses. The objective of this research was to validate an RT-nPCR assay for detection of BVDV associated with various samples collected from an IVF system. In 12 research replicates, we maintained matured COCs as negative controls or exposed them to 1 of 4 noncytopathic strains (SD-1, NY-1, CD-87, or PA-131) of BVDV for 1 h immediately before IVF. After 4 d of IVC, we harvested groups of 5 nonfertile ova or degenerated embryos (NFD) and some associated cumulus cells and transferred developing embryos and the remaining cumulus cells into secondary IVC drops. On the seventh d of IVC, cumulus cells, groups of 5 washed NFD and groups of 5 developed, washed embryos were harvested. We also collected single developed embryos after washing, washing with trypsin, washing and cryopreservation in ethylene glycol, or washing with trypsin and cryopreservation in ethylene glycol. All washes were performed according to International Embryo Transfer Society standards. Developed embryos and NFD were sonicated prior to assay. All samples were assayed for BVDV using virus isolation and RT-nPCR. The virus isolation and RT-nPCR assays determined that all negative control samples were BVDV-free. Virus was detected in association with all exposed cumulus cells and groups of developed embryos using both virus isolation and RT-nPCR. Results from viral assays of other exposed samples indicate enhanced sensitivity of the RT-nPCR assay. The RT-nPCR assay used in this research exhibited acceptable sensitivity, specificity, predictive value and repeatability for rapid detection of BVDV associated with the various samples obtained from an IVF system.     

Transcriptomic Analyses of the Biological Effects of Airborne PM2.5 Exposure on Human Bronchial Epithelial Cells

Epidemiological studies have associated high levels of airborne particulate matter (PM) with increased respiratory diseases. In order to investigate the mechanisms of air pollution-induced lung toxicity in humans, human bronchial epithelial cells (16HBE) were exposed to various concentrations of particles smaller than 2.5 μm (PM2.5) collected from Beijing, China. After observing that PM2.5 decreased cell viability in a dose-dependent manner, we first used Illumina RNA-seq to identify genes and pathways that may contribute to PM2.5-induced toxicity to 16HBE cells. A total of 539 genes, 283 up-regulated and 256 down-regulated, were identified to be significantly differentially expressed after exposure to 25 μg/cm² PM2.5. PM2.5 induced a large number of genes involved in responses to xenobtiotic stimuli, metabolic response, and inflammatory and immune response pathways such as MAPK signaling and cytokine-cytokine receptor interaction, which might contribute to PM2.5-related pulmonary diseases. We then confirmed our RNA-seq results by qPCR and by analysis of IL-6, CYP1A1, and IL-8 protein expression. Finally, ELISA assay demonstrated a significant association between exposure to PM2.5 and secretion of IL-6. This research provides a new insight into the mechanisms underlying PM2.5-induced respiratory diseases in Beijing.     

Highly sensitive and specific detection of viable Escherichia coli in drinking water

A highly sensitive and specific assay method was developed for the detection of viable Escherichia coli as an indicator organism in water, using nucleic acid sequence-based amplification (NASBA) and electrochemiluminescence (ECL) analysis. Viable E. coli were identified via a 200-nt-long target sequence from mRNA (clpB) coding for a heat shock protein. In the detection assay, a heat shock was applied to the cells prior to disruption to induce the synthesis of clpB mRNA and the mRNA was extracted, purified, and finally amplified using NASBA. The amplified mRNA was quantified with an ECL detection system after hybridization with specific DNA probes. Several disruption methods were investigated to maximize total RNA extracted from viable cells. Optimization was also carried out regarding the design of NASBA primer pairs and detection probes, as well as reaction and detection conditions. Finally, the assay was tested regarding sensitivity and specificity. Analysis of samples revealed that as few as 40 E. coli cells/mL can be detected, with no false positive signals resulting from other microorganisms or nonviable E. coli cells. Also, it was shown that a quantification of E. coli cells was possible with our assay method.     

A novel approach combining the Calgary Biofilm Device and Phenotype MicroArray for the characterization of the chemical sensitivity of bacterial biofilms

A rapid method for screening the metabolic susceptibility of biofilms to toxic compounds was developed by combining the Calgary Biofilm Device (MBEC device) and Phenotype MicroArray (PM) technology. The method was developed using Pseudomonas alcaliphila 34, a Cr(VI)-hyper-resistant bacterium, as the test organism. P. alcaliphila produced a robust biofilm after incubation for 16 h, reaching the maximum value after incubation for 24 h (9.4 × 10⁶ ± 3.3 × 10⁶ CFU peg^?1). In order to detect the metabolic activity of cells in the biofilm, dye E (5×) and menadione sodium bisulphate (100 μM) were selected for redox detection chemistry, because they produced a high colorimetric yield in response to bacterial metabolism (340.4 ± 6.9 Omnilog Arbitrary Units). This combined approach, which avoids the limitations of traditional plate counts, was validated by testing the susceptibility of P. alcaliphila biofilm to 22 toxic compounds. For each compound the concentration level that significantly lowered the metabolic activity of the biofilm was identified. Chemical sensitivity analysis of the planktonic culture was also performed, allowing comparison of the metabolic susceptibility patterns of biofilm and planktonic cultures.     

Establishment of cell-free electrophysiology for ion transporters: application for pharmacological profiling

Ion transporters are emerging targets of increasing importance to the pharmaceutical industry because of their relevance to a wide range of numerous indications of cardiovascular, metabolic, and inflammatory diseases. However, traditional ion transporter assay technologies using radioactive or fluorescent ligands and substrates or manual patch clamping suffer from several problems: limited sensitivity and robustness, significant numbers of false positives and false negatives, and cost. The authors describe a novel method for the measurement of ion transporters using cell-free electrophysiology based on the SURFE (2) R (surface electrogenic event reader) technology platform. The main advantages of the method described here are high sensitivity and simple handling. Material for assays is mainly a simple membrane preparation, which can be stored over weeks and months. Thus, the application of the method does not depend on a permanently running cell-culture lab. The application of the technology itself uses a bench-top system and chips loaded with membrane fragments. The SURFE (2) R technology was used to establish an Na+/Ca2+-exchanger assay. The assay performance, as judged by the Z' value of 0.73 and the signal-to-background ratio of 7.6, suggests that this is a reliable and robust assay. The authors compared the technology with patch-clamp experiments: The measurement of activity of 17 different inhibitors and the determination of an IC (50)value indicated a good correlation between SURFE (2) R technology and patch clamp results. Using the SURFE (2) R technology, results were obtained with 20 times higher throughput and required less-qualified personnel compared with manual patch clamping.     

Ultrasensitive detection of cortisol with enzyme fragment complementation technology using functionalized nanowire

Cortisol is a member of the glucocorticoid hormone family and a key metabolic regulator. Increased intracellular cortisol levels have been implicated in type 2 diabetes, obesity, and metabolic syndrome. Cortisol is an important bio-marker of stress and its detection is also important in sports medicine. However, rapid methods for sensitive detection of cortisol are limited. Functionalized gold nanowires were used to enhance the sensitivity and selectivity of cortisol detection. Gold nanowires are used to improve the electron transfer between the electrodes. Moreover, the large surface to volume ratio, small diffusion time and high electrical conductivity and their aligned nature will enhance the sensitivity and detection limit of the biosensor several fold. The biosensor was fabricated using, aligned gold (Au) nanowires to behave as the working electrode, platinum deposited on a silicon chip to function as the counter electrode, and silver/silver chloride as reference electrode. The gold nanowires were coupled with cortisol antibodies using covalent linkage chemistry and a fixed amount of 3alpha-hydroxysteroid dehydrogenase was introduced into the reaction cell during each measurement to convert (reduce) ketosteroid into hydroxyl steroid. Furthermore, the micro-fluidic, micro-fluid part of the sensor was fabricated using micro-electro-mechanical system (MEMS) technology to have better control on liquid flow over Au nanowires to minimize the signal to noise ratio. The biosensor was characterized using SEM, AFM and FTIR technique. The response curve of the biosensor was found to be linear in the range of 10-80 microM of cortisol. Moreover, the presence of hydrocortisone is sensitively detected in the range of 5-30 microM. It is concluded that the functionalized gold nanowires with micro-fluidic device using enzyme fragment complementation technology can provide an easy and sensitive assay for cortisol detection in serum and other biological fluids.