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opencc-by-4.0May 2022View details →

Development of thrips barcode database and multiplex real-time PCR assay for quarantine and agriculture pest species

Thrips (Order Thysanoptera) species are agriculturally important as plant sap sucking pests and vectors of several plant diseases. They are very small insects and commonly associated with imported commodities at New Zealand border in all life stages. Morphological identification of thrips is mainly performed on adults, but the available identification keys for immature stages do not include many species and are inadequate, thus DNA barcode was regularly used for thrips identification, here, we have generated DNA barcode data for over 29 thrips species from over 100 individuals.  At New Zealand border, Frankliniella occidentalis is the dominant species intercepted, followed by F. panamensis, Thrips palmi and T. tabaci and several other thrips species. Hence, we have also developed a multiplex real time PCR assay, targeting the four thrips species to facilitate the identification of quarantine interceptions with more accurate and faster diagnostic method for any developmental stages. The DNA barcode database further assists in thrip identification. The assay showed high specificity for all the four target species and could detect 10 copies/ µL of the target DNA. Linear responses and high correlation coefficients between the amount of DNA and Cq values for each species were also achieved. The method was tested on single egg, larva and adult and proved to be applicable for all life stages of the four species. This study has demonstrated the assay is a useful biosecurity tool for rapid and reliable identification of the target thrips species.

Fig 4 in Development of specific PCR assays for the detection of Cryptocaryon irritans

Fig 4 Agarose gel electrophoresis of PCR products amplified from the simulated water body using Cryptocaryon irritans-specific primer set P1/S15. Lanes 1 and 10 represent a DNA size marker, lanes 2 to 7 represent C. irritans from the infected water, lane 8 represents the uninfected water, and lane 9 represents no-DNA control

Fig 1 in Development of specific PCR assays for the detection of Cryptocaryon irritans

Fig 1 Agarose gel electrophoresis of PCR products amplified using conserved primer set P1/NC2 (a), Cryptocaryon irritans-specific primer set P1/S15 (b), and Ichthyophthirius multifiliis-specific primer set S01/S02 (c). Lanes 1 and 11 represent a DNA size marker. Lanes 2 to 9 represent C. irritans, Pseudokeroronpsis rubra, Pseudokeroronpsis carnae, Euplotes sp. 1, I. multifiliis, Pseudourostyla cristata, Paramecium caudaium (cf. Table 1), and host (fish) DNA, respectively. Lane 10 represents no-DNA control

Fig 2 in Development of specific PCR assays for the detection of Cryptocaryon irritans

Fig 2 Agarose gel electrophoresis of PCR products amplified from mixed genomic DNA of C. irritans and I. multifiliis. Lanes 1 and 7 represent a DNA size marker. Lane 2 presents the mixture of C. irritans and I. multifiliis DNA amplified using the primer set P1/S15 and lane 3 represents the genomic DNA of I. multifiliis. Lane 4 represents the mixed genomic DNA of C. irritans and I. multifiliis amplified using the primer set S01–S02. Lane 5 represents the genomic DNA of C. irritans using the primer set S01–S02. Lane 6 represents no-DNA control

Fig 3 in Development of specific PCR assays for the detection of Cryptocaryon irritans

Fig 3 Evaluation of the sensitivity of the specific PCR assay for Cryptocaryon irritans (primer set P1/S15) by agarose gel electrophoresis. Lanes 1 and 13 represent a DNA size marker. Lane 2 represents DNA undiluted (450 ng/µl), and lanes 3 to 11 represent C. irritans DNA diluted for 1:50 (9 ng/µl), 1:100 (4.5 ng/µl), 1:200 (2.25 ng/µl), 1:400 (1.13 ng/µl), 1:800 (0.56 ng/µl), 1:103 (0.45 ng/µl), 1:104 (45 pg/µl), 1:105 (4.5 pg/µl), and 1:106 (0.45 pg/µl), respectively. Lane 12 represents no-DNA control

opencc-by-4.0Jul 2019View details →

HDAC11 target engagement assay development in cells - CETSA

Methods Permeabilized CETSA HEK293T cells were seeded in 6-well plates (8 x 105 cells/well) in DMEM supplemented with 10% FBS , penicillin (100 U/mL) and streptomycin (100 µg/mL). 4 h after seeding cells were transfected with 0.2 µg of N-terminally NanoLuc-tagged HDAC11 and 1.8 µg of empty pcDNA3.1 vectors using X-tremeGENE™ HP transfection reagent, following manufacturer instructions. Next day cells were harvested using Trypsin-EDTA,  and resuspended in pre-warmed Opti-MEM (no phenol-red) at a concentration of 1.2 x 105 cells/ml. A 10x stock solution of digitonin (500 µg/ml, Sigma) and 100x stock solution of protease inhibitor cocktail (cOmplete™, Mini, EDTA-free, Sigma) was prepared in Opti-MEM and DMSO, respectively. The protease inhibitor / DMSO stock solution was used as vehicle to prepare 100x stock solutions of all test compounds. The 10x digitonin stock solution and 100x test compound / vehicle stock solution were pre-dispensed into 96-well PCR reaction plates  at 10 µl/well and 1 µl/well, respectively. Next, 89 µl/well of the cell suspension was added to the pre-loaded PCR plates. Plates were then covered with adhesive plate sealing tape and were incubated at 37°C for 1 h. Immediately prior to heating the PCR plates, a 5x substrate stock solution was prepared by diluting NanoBRET NanoGlo Substrate (Promega) 1:100 into Opti-MEM. The thermal cycler  was allowed to reach the designated temperature prior to placement of the PCR plate into the heating block. For Tm determination experiments, duplicate plates containing a fixed concentration of test compounds (30 µM) as well as vehicle control were prepared to perform a 11 point temperature gradient spanning 37-68.5°C for 3 minutes. The plates were allowed to cool at room temperature for 3 minutes after the heat treatment followed by addition of 25 µl 5x substrate stock solution per well. To measure NanoLuc activity 100 µl of sample were transferred from the PCR plate to a white 96-well plate. Total luminescence was measured immediately after substrate addition with CLARIOstar microplate reader (BMG Labtech). Non-permeabilized CETSA HEK293T cells were seeded in 6 well plates (8 x 105 cells/well) in DMEM supplemented with 10% FBS , penicillin (100 U/mL) and streptomycin (100 µg/mL). 4 h after seeding cells were transfected with 0.2 µg of N-terminally NanoLuc tagged HDAC11 and 1.8 µg of empty pcDNA3.1 vectors using X-tremeGENE™ HP transfection reagent, following manufacturer instructions. Next day cells were tretaed with compounds/DMSO for 6 h. Cells were harvested using Trypsin-EDTA and resuspended in pre-warmed Opti-MEM (no phenol-red) at a concentration of 1 x 105 cells/ml.  Next, 100 µl/well of the cell suspension was added to the pre-loaded PCR plates.  Immediately prior to heating the PCR plates, a 5x substrate stock solution was prepared by diluting NanoBRET NanoGlo Substrate (Promega) 1:100 into Opti-MEM. The thermal cycler  was allowed to reach the designated temperature prior to placement of the PCR plate into the heating block. For Tm determination experiments, duplicate plates containing a fixed concentration of test compounds (30 µM) as well as vehicle control were prepared to perform a 11 point temperature gradient spanning 37-72°C for 3 minutes. The plates were allowed to cool at room temperature for 3 minutes after the heat treatment followed by addition of 25  µl 5x substrate stock solution per well. To measure NanoLuc activity 100 µl of sample were transferred from the PCR plate to a white 96-well plate. Total luminescence was measured immediately after substrate addition with CLARIOstar microplate reader (BMG Labtech).

opencc-by-4.0Dec 2019View details →

Development of a high-throughput small molecule screening assay for phenotypical characterization of lysosomal storage disorder-affected cells, with infantile cystinosis as a proof of principle

Together with the Pivot Park Screening Centre we performed a drug screen on CTNS-/- proximal tubule cells. For this we developed an assay to evaluate LC3-II positive puncta, and which may be applied for any disease in which autophagy plays an important role. The screen was optimized by the hotel for a 384 well format, making it useful for high throughput screening. The screen was performed with 1280 compounds from the Prestwick library.

dryad36/100

Data from: Development and validation of a RAD-Seq target-capture based genotyping assay for routine application in advanced black tiger shrimp (Penaeus monodon) breeding programs

Background The development of genome-wide genotyping resources has provided terrestrial livestock and crop industries with the unique ability to accurately assess genomic relationships between individuals, uncover the genetic architecture of commercial traits, as well as identify superior individuals for selection based on their specific genetic profile. Utilising recent advancements in de-novo genome-wide genotyping technologies, it is now possible to provide aquaculture industries with these same important genotyping resources, even in the absence of existing genome assemblies. Here, we present the development of a genome-wide SNP assay for the Black Tiger shrimp (Penaeus monodon) through utilisation of a reduced-representation whole-genome genotyping approach (DArTseq). Results Based on a single reduced-representation library, 31,262 polymorphic SNPs were identified across 650 individuals obtained from Australian wild stocks and commercial aquaculture populations. After filtering to remove SNPs with low read depth, low MAF, low call rate, deviation from HWE, and non-Mendelian inheritance, 7,542 high-quality SNPs were retained. From these, 4,236 high-quality genome-wide loci were selected for bates-probe development and 4,194 SNPs were included within a finalized target-capture genotype-by-sequence assay (DArTcap). This assay was designed for routine and cost effective commercial application in large scale breeding programs, and demonstrates higher confidence in genotype calls through increased call rate (from 80.2 ± 14.7 to 93.0% ± 3.5%), increased read depth (from 20.4 ± 15.6 to 80.0 ± 88.7), as well as a 3-fold reduction in cost over traditional genotype-by-sequencing approaches. Conclusion Importantly, this assay equips the P. monodon industry with the ability to simultaneously assign parentage of communally reared animals, undertake genomic relationship analysis, manage mate pairings between cryptic family lines, as well as undertake advance studies of genome and trait architecture. Critically this assay can be cost effectively applied as P. monodon breeding programs transition to undertaking genomic selection.

opencc-zeroAug 2020View details →

opencc-by-4.0Nov 2020View details →

Development and Optimization of an IQF USP5 Activity Assay

Development and optimization of a fluorescence based deubiquitinase (DUB) activity assay for USP5 with internally quenched fluorophore pair (IQF) substrates: di-ubiquitin lysine linked K48 and K63 (Ub2K48, Ub2K63). The inhibitory capability of USP5 zinc-finger ubiquitin binding domain (ZnF-UBD) ligands was investigated.

opencc-by-4.0Dec 2017View details →

USP5 Zf-UBD Purification & Fluorescence Polarization Displacement Assay Development

USP5 zinc-finger ubiquitin binding domain (Zf-UBD) expression and purification from E. coli, and screening conditions for a fluorescence polarization (FP) displacement assay with a FITC-labelled ubiquitin peptide.

opencc-by-4.0Feb 2018View details →

The process of HDAC11 Assay Development: using kit

Preliminary tests to check if the Fluor-de-Lys-green HDAC activity kit could be used for developing a high-throughput HDAC11 assay have been performed here using controls and recombinantly purified HDAC11.

opencc-by-4.0Feb 2018View details →

USP5 Zf-UBD Differential Scanning Fluorimetry Assay Development

Development of DSF assay for USP5 Zf-UBD to determine optimal protein screening conditions and significance of thermal shift of USP5 Zf-UBD in the presence of a ubiquitin RLRGG peptide

opencc-by-4.0Apr 2018View details →

The process of HDAC11 Assay Development: enzyme stability

Before performing the kinetic study for calculating the Km for HDAC11, it is important to know the duration of the stability of the protein under the assay conditions. This is being analyzed here.   Note: 1. In the assay buffer, BSA conc. is 0.5 mg/ml (instead of 0.5%).           2. In the 7.5 ul developer solution, 40 uM of TSA (Trichostatin A) is also included.

opencc-by-4.0Apr 2018View details →

The process of HDAC11 Assay Development: buffer screening

A buffer screen at various pH was performed to pick the buffer that gives the best activity for HDAC11.  Note: 1. In the assay buffer, BSA conc. is 0.5 mg/ml (instead of 0.5%).           2. In the 7.5 ul developer solution, 40 uM of TSA (Trichostatin A) is also included.

opencc-by-4.0Jun 2018View details →

The process of HDAC11 Assay Development: linearity and Km calculation

With previously optimized conditions, the linearity range for HDAC11 was obtained and the Km for the substrate was calculated.   Note: 1. In the assay buffer, BSA conc. is 0.5 mg/ml (instead of 0.5%).           2. In the 7.5 ul developer solution, 40 uM of TSA (Trichostatin A) is also included.

opencc-by-4.0Jun 2018View details →

The process of HDAC11 Assay Development: Buffer optimization again

The pH range used earlier was expanded for further optimization to check if the activity could improve.   Note: 1. In the assay buffer, BSA conc. is 0.5 mg/ml (instead of 0.5%).           2. In the 7.5 ul developer solution, 40 uM of TSA (Trichostatin A) is also included.

opencc-by-4.0Jul 2018View details →

The process of HDAC11 Assay Development: Effect of DMSO

For the purpose of compound library screenings, the compounds would be made available in DMSO and thus, when they would be incubated with the protein, the protein solution will now contain DMSO as well. It is important to determine the concentration of DMSO that could be tolerated by the protein such that the protein stays active. Hence, the effect of varying concentration of DMSO was analyzed on HDAC11 activity.

opencc-by-4.0Jul 2018View details →

The process of HDAC11 Assay Development: Effect of Detergent

The presence of detergent can prevent the sticking of the fluorophore to the sides of the wells of the plate and thus improve the signals. Hence, the effect of detergent was observed on the overall signals and the activity of HDAC11.