Paper based point-of-care testing disc for multiplex whole cell bacteria analysis

Point-of-care testing (POCT) of infectious bacterial agents offers substantial benefits for disease diagnosis, mainly by shortening the time required to obtain results and by making the test available bedside or at remote care centers. Immunochromatographic lateral flow biosensors offer a low cost, highly sensitive platform for POCT. In this article, we describe the fabrication and testing of a multiplex immuno-disc sensor for the specific detection of Pseudomonas aeruginosa and Staphylococcus aureus. Antibody conjugated gold nanoparticles were used as the signaling agents. The detection range of the bacteria lies within 500-5000 CFU/ml. The advantage of the immuno-disc sensor is that it does not require any preprocessing of biological sample and is capable of whole cell bacterial detection. We also describe the design and fabrication of a compact portable device which converts the color intensity of the gold nanoparticles that accumulate at the test region into a quantitative voltage reading proportional to the bacterial concentration in the sample. The combination of the immuno-disc and the portable color reader provides a rapid, sensitive, low cost, and quantitative tool for the detection of a panel of infectious agents present in the patient sample.     

Effect of infection by Metarhizium anisopliae (Hypocreales: Clavicipitaceae) on the feeding and oviposition of the pea leafminer Liriomyza huidobrensis (Diptera: Agromyzidae) on different host plants

The effect of fungal infection by Metarhizium anisopliae on feeding and oviposition of adult Liriomyza huidobrensis was examined on three host plants, faba bean (Vicia faba), French bean (Phaseolus vuklgaris) and snow pea (Pisum sativum) in the laboratory. Flies were contaminated with dry conidia and allowed to feed and oviposit on the different host plants. Mortality in L. huidobrensis varied between 14% and 20% in the controls and between 77% and 100% in fungal treatments 120 h post-infection for the three host plants. L. huidobrensis made more punctures (47.3–52.6 cm^?2) in the control than in the fungal treatments (23.1–26.9 cm^?2) for the three host plants. The cumulative average number of punctures cm^?2/female by L. huidobrensis was higher in the controls than in fungal treatments from 72 h post-treatment in faba bean (12.2 vs. 8.2) and French bean (14.8 vs. 8.9), and from 48 h post-inoculation in snow pea (8.5 vs. 5.7). Female L. huidobrensis laid more eggs in the control (0.6–6.1) than in fungal treatments (0.2–1.5) across the host plants tested. The cumulative mean number of eggs cm^?2/female was significantly higher in the controls than in fungal treatments from 48 h post-treatment in faba bean (0.4 vs. 0.2) and French bean (0.1 vs. 0), and 96 h post-inoculation in snow pea (0.2 vs. 0.1). The host plant did not affect the average total number of punctures but had a significant effect on egg laying, with faba bean harboring greater number of eggs in both control and fungal treatments. A proper timeline application of the fungus before onset of feeding and oviposition peaks will be crucial in field suppression of the pest using M. anisopliae. In addition, a great consideration must be given to the target host plants prior to application of the fungus.     

Tradescantia micronucleus bioassay

Four coded chemicalsm azidoglycerol (AG), N-methyl-N-nitrosourea (MNU), sodium azide (NaN₃), and maleic hydrazide (MH), were tested with the Tradescantia micronucleus (Trad-MCN) bioassay by five independent laboratories from five different countries. The purpose of this international collaborative study was to evaluate four plant bioassays, of which the Trad-MCN assay was one, for their sensitivity, efficiency and reliability. The study was carried out under the sponsorship of the International Programme on Chemical Safety. All laboratories adhered to a standard Trad-MCN protocol which suggested that three replicate tests be conducted with each chemical. The results reported by all laboratories, although not equal, showed good agreement among the laboratories. In fact, all five laboratories obtained positive results with MH and MNU, while four of the five laboratories achieved positive results with NaN₃. AG was tested in only three laboratories. Two reported negative results, while one reported positive results but only at a single high dose. The data from this study suggest that under normal conditions, the Trad-MCN bioassay is an efficient and reliable short-term bioassay for clastogens. It is suitable for the rapid screening of chemicals, and also is specially qualified for in situ monitoring of ambient pollutants.     

Tradescantia stamen hair mutation bioassay

The Tradescantia stamen hair mutation (Trad-SH) assay (clone 4430) was evaluated for its efficiency and reliability as a screen for mutagens in an IPCS collaborative study on plant systems. Four coded chemicals, i.e. azidoglycerol (AG, 3-azido-1,2-propanediol), N-methyl-N-nitrosourea (MNU), sodium azide (NaN₃) and maleic hydrazide (MH) were distributed by the Radian Corporation to the five laboratories in five different countries for testing mutagenicity. Pink mutations were scored between the 7th and 14th day according to a standard protocol. Test results from the five individual laboratories were analyzed and compared after decoding. One out of the two laboratories that conducted tests on AG demonstrated that AG is a mutagen with genetically effective doses ranging from 50 to 100 μg/ml. MH yielded positive responses in all laboratories but no linear dose-response pattern was observed. The effective dose range for MH was between 1 and 45 μg/ml. The mutagenicity of MNU was reported by five laboratories in the dose range between 10 and 80 μg/ml. NaN₃, which exhibited a relatively high degree of toxicity, elicited a positive mutagenic response in three of the five laboratories in which it was tested. As with MNU the effective dose for NaN₃ ranged between 3 and 80 μg/ml. The results from the current study substantiate the Trad-SH assay as a reliable system for screening chemicals for their potential mutagenic effects. Although the study was carried out exclusively under laboratory conditions, a survey of the current literature would indicate that the Trad-SH assay could be an effective in situ monitor of gaseous, liquid, and radioactive pollutants as well.     

Genetic diversity among varieties and wild species accessions of pea (Pisum sativum L.) based on molecular markers, and morphological and physiological characters.

Random amplified polymorphic DNA, simple sequence repeat, and inter-simple sequence repeat markers were used to estimate the genetic relations among 65 pea varieties (Pisum sativum L.) and 21 accessions from wild Pisum subspecies (subsp.) abyssinicum, asiaticum, elatius, transcaucasicum, and var. arvense. Fifty-one of these varieties are currently available for growers in western Canada. Nei and Li's genetic similarity (GS) estimates calculated using the marker data showed that pair-wise comparison values among the 65 varieties ranged from 0.34 to 1.00. GS analysis on varieties grouped according to their originating breeding programs demonstrated that different levels of diversity were maintained at different breeding programs. Unweighted pair-group method arithmetic average cluster analysis and principal coordinate analysis on the marker-based GS grouped the cultivated varieties separately from the wild accessions. The majority of the food and feed varieties were grouped separately from the silage and specialty varieties, regardless of the originating breeding programs. The analysis also revealed some genetically distinct varieties such as Croma, CDC Handel, 1096M-8, and CDC Acer. The relations among the cultivated varieties, as revealed by molecular-marker-based GS, were not significantly correlated with those based on the agronomic characters, suggesting that the 2 systems give different estimates of genetic relations among the varieties. However, on a smaller scale, a consistent subcluster of genotypes was identified on the basis of agronomic characters and their marker-based GS. Furthermore, a number of variety-specific markers were identified in the current study, which could be useful for variety identification. Breeding strategies to maintain or enhance the genetic diversity of future varieties are proposed.     

Classification and Characterization of Species within the Genus Lens Using Genotyping-by-Sequencing (GBS)

Lentil (Lens culinaris ssp. culinaris) is a nutritious and affordable pulse with an ancient crop domestication history. The genus Lens consists of seven taxa, however, there are many discrepancies in the taxon and gene pool classification of lentil and its wild relatives. Due to the narrow genetic basis of cultivated lentil, there is a need towards better understanding of the relationships amongst wild germplasm to assist introgression of favourable genes into lentil breeding programs. Genotyping-by-sequencing (GBS) is an easy and affordable method that allows multiplexing of up to 384 samples or more per library to generate genome-wide single nucleotide Polymorphism (SNP) markers. In this study, we aimed to characterize our lentil germplasm collection using a two-enzyme GBS approach. We constructed two 96-plex GBS libraries with a total of 60 accessions where some accessions had several samples and each sample was sequenced in two technical replicates. We developed an automated GBS pipeline and detected a total of 266,356 genome-wide SNPs. After filtering low quality and redundant SNPs based on haplotype information, we constructed a maximum-likelihood tree using 5,389 SNPs. The phylogenetic tree grouped the germplasm collection into their respective taxa with strong support. Based on phylogenetic tree and STRUCTURE analysis, we identified four gene pools, namely L. culinaris/L. orientalis/L. tomentosus, L. lamottei/L. odemensis, L. ervoides and L. nigricans which form primary, secondary, tertiary and quaternary gene pools, respectively. We discovered sequencing bias problems likely due to DNA quality and observed severe run-to-run variation in the wild lentils. We examined the authenticity of the germplasm collection and identified 17% misclassified samples. Our study demonstrated that GBS is a promising and affordable tool for screening by plant breeders interested in crop wild relatives.