Rapid Identification of Pseudomonas fluorescens Harboring Thermostable Alkaline Protease by Real-time Loop-mediated Isothermal Amplification

Thermostable alkaline protease (TAP) harbored by Pseudomonas fluorescens decomposes protein in milk and dairy products, leading to milk and dairy product spoilage during storage. Thus, a specific, sensitive, rapid, and simple method is required to detect TAP-harboring P. fluorescens. Two sets of primers targeting the aprX and gyrB genes of P. fluorescens were designed. The detection system and conditions were optimized, and a real-time loop mediated isothermal amplification (real-time LAMP) method was developed for the simultaneous detection of TAP-harboring P. fluorescens in two separate reaction tubes. The phylogenetic tree targeting aprX showed that P. fluorescens and P. lurida clustered on the same branch. The phylogenetic tree targeting gyrB showed that P. fluorescens clustered on the same branch with 95% confidence value, whereas P. lurida clustered on different branches. DNA of 16 strains of P. fluorescens and 34 strains of non-P. fluorescens was detected by real-time LAMP. TAP-harboring P. fluorescens can only be identified when the real-time LAMP detection results of both aprX and gyrB are positive. The dissociation temperatures of aprX and gyrB in the real-time LAMP amplified products were approximately 90.0 °C and 88.0 °C, respectively. The detection limits of the real-time LAMP targeting aprX and gyrB were 4.9 CFU/reaction in pure culture and 2.2 CFU/reaction in skimmed milk. The coefficient of variation of the repeatability test was less than 2%, indicating that the established real-time LAMP of P. fluorescens targeting gyrB and aprX has good stability and repeatability. Two-hundred raw milk samples were tested for the presence of TAP-harboring P. fluorescens by real-time LAMP in 3 h, and the coincidence rate of the results with those obtained using the traditional method, which takes at least 5-7 d, was 100%. Real-time LAMP will be a practical and effective method for accurate and rapid identification of TAP-harboring P. fluorescens in raw milk.

Space-Time Loop Tiling for Dynamic Programming Codes

We present a new space-time loop tiling approach and demonstrate its application for the generation of parallel tiled code of enhanced locality for three dynamic programming algorithms. The technique envisages that, for each loop nest statement, sub-spaces are first generated so that the intersection of them results in space tiles. Space tiles can be enumerated in lexicographical order or in parallel by using the wave-front technique. Then, within each space tile, time slices are formed, which are enumerated in lexicographical order. Target tiles are represented with multiple time slices within each space tile. We explain the basic idea of space-time loop tiling and then illustrate it by means of an example. Then, we present a formal algorithm and prove its correctness. The algorithm is implemented in the publicly available TRACO compiler. Experimental results demonstrate that parallel codes generated by means of the presented approach outperform closely related manually generated ones or those generated by using affine transformations. The main advantage of code generated by means of the presented approach is its enhanced locality due to splitting each larger space tile into multiple smaller tiles represented with time slices.

Development of a real-time loop mediated isothermal amplification assay for Stromatinia cepivora in response to an outbreak in Northern Idaho

Stromatinia cepivora is the causal agent of white rot disease of Allium species. In 2018, white rot was observed in Boundary county in Northern Idaho in garlic and onion plants in a variety of home and market garden operations. As the university diagnostic lab for Idaho is situated in Parma within a regulated area for Stromatinia cepivora, a point of care (POC) assay using real-time loop mediated isothermal amplification (LAMP) was developed to minimize the amount of material potentially sent to the diagnostic lab. The LAMP assay was used with a BioRanger platform and although the limit of detection was one hundred times less than TaqMan, it was capable of detecting a single sclerotia. This study demonstrates the rapid development and deployment of a POC suitable LAMP assay. Despite limitations in sensitivity and dynamic range compared to real-time PCR, POC LAMP assays are advantageous where biosecurity concerns prohibit the movement of material suitable for diagnosis as well as facilitating engagement with growers.