Recombinase polymerase amplification-nucleic acid lateral flow immunoassays for Newcastle disease virus and infectious bronchitis virus detection.

Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) are two poultry pathogens affecting the respiratory tract of chickens, and cause major economic losses in the industry. Rapid detection of these viruses is crucial to inform implementation of appropriate control measures. The objective of our study is developing a simple, rapid and field applicable recombinase polymerase amplification (RPA)-nucleic acid lateral flow (NALF) immunoassay for detection of NDV and IBV. Isothermal amplification of the matrix protein (M) gene of NDV and the nucleoprotein (N) gene of IBV was implemented via recombinase polymerase amplification at 38 °C for 40 min and 20 min, respectively using modified labeled primers. NALF device used in this study utilizes antibodies for detection of labeled RPA amplicons. The results revealed that RPA-NALF immunoassays can detect both viruses after 40 min at 38 °C and only NDV after 20 min. The limit of detection (LOD) was 10 genomic copies/RPA reaction. The assays results on clinical samples collected from diseased chicken farms demonstrated a good performance in comparison with quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). The assays established in this study can facilitate rapid, on-site molecular diagnosis of suspected cases of ND and IB viral infections as the results can be detected by the naked eye without the need for measuring fluorescence. Furthermore, the NALF device could be adapted to detect other infectious agents.

RPA using a multiplexed cartridge for low cost point of care diagnostics in the field.

A point of care device utilising Lab-on-a-Chip technologies that is applicable for biological pathogens was designed, fabricated and tested showing sample in to answer out capabilities. The purpose of the design was to develop a cartridge with the capability to perform nucleic acid extraction and purification from a sample using a chitosan membrane at an acidic pH. Waste was stored within the cartridge with the use of sodium polyacrylate to solidify or gelate the sample in a single chamber. Nucleic acid elution was conducted using the RPA amplification reagents (alkaline pH). Passive valves were used to regulate the fluid flow and a multiplexer was designed to distribute the fluid into six microchambers for amplification reactions. Cartridges were produced using soft lithography of silicone from 3D printed moulds, bonded to glass substrates. The isothermal technique, RPA is employed for amplification. This paper shows the results from two separate experiments: the first using the RPA control nucleic acid, the second showing successful amplification from Chlamydia Trachomatis. Endpoint analysis conducted for the RPA analysis was gel electrophoresis that showed 143 base pair DNA was amplified successfully for positive samples whilst negative samples did not show amplification. End point analysis for Chlamydia Trachomatis samples was fluorescence detection that showed successful detection of 1 copy/µL and 10 copies/µL spiked in a MES buffer.

A Simple, Low-Cost Platform for Real-Time Isothermal Nucleic Acid Amplification.

Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings.

The zinc cluster protein Sut1 contributes to filamentation in Saccharomyces cerevisiae.

Sut1 is a transcriptional regulator of the Zn(II)(2)Cys(6) family in the budding yeast Saccharomyces cerevisiae. The only function that has been attributed to Sut1 is sterol uptake under anaerobic conditions. Here, we show that Sut1 is also expressed in the presence of oxygen, and we identify a novel function for Sut1. SUT1 overexpression blocks filamentous growth, a response to nutrient limitation, in both haploid and diploid cells. This inhibition by Sut1 is independent of its function in sterol uptake. Sut1 downregulates the expression of GAT2, HAP4, MGA1, MSN4, NCE102, PRR2, RHO3, and RHO5. Several of these Sut1 targets (GAT2, HAP4, MGA1, RHO3, and RHO5) are essential for filamentation in haploids and/or diploids. Furthermore, the expression of the Sut1 target genes, with the exception of MGA1, is induced during filamentous growth. We also show that SUT1 expression is autoregulated and inhibited by Ste12, a key transcriptional regulator of filamentation. We propose that Sut1 partially represses the expression of GAT2, HAP4, MGA1, MSN4, NCE102, PRR2, RHO3, and RHO5 when nutrients are plentiful. Filamentation-inducing conditions relieve this repression by Sut1, and the increased expression of Sut1 targets triggers filamentous growth.

Modular development of a prototype point of care molecular diagnostic platform for sexually transmitted infections.

This paper presents the design of a modular point of care test platform that integrates a proprietary sample collection device directly with a microfluidic cartridge. Cell lysis, within the cartridge, is conducted using a chemical method and nucleic acid purification is done on an activated cellulose membrane. The microfluidic device incorporates passive mixing of the lysis-binding buffers and sample using a serpentine channel. Results have shown extraction efficiencies for this new membrane of 69% and 57% compared to the commercial Qiagen extraction method of 85% and 59.4% for 0.1ng/µL and 100ng/µL salmon sperm DNA respectively spiked in phosphate buffered solution. Extraction experiments using the serpentine passive mixer cartridges incorporating lysis and nucleic acid purification showed extraction efficiency around 80% of the commercial Qiagen kit. Isothermal amplification was conducted using thermophillic helicase dependant amplification and recombinase polymerase amplification. A low cost benchtop real-time isothermal amplification platform has been developed capable of running six amplifications simultaneously. Results show that the platform is capable of detecting 1.32×10(6) of sample DNA through thermophillic helicase dependant amplification and 1×10(5) copy numbers Chlamydia trachomatis genomic DNA within 10min through recombinase polymerase nucleic acid amplification tests.