Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay

Sensitive, specific, rapid, inexpensive and easy-to-use nucleic acid tests for use at the point-of-need are critical for the emerging field of personalised medicine for which companion diagnostics are essential, as well as for application in low resource settings. Here we report on the development of a point-of-care nucleic acid lateral flow test for the direct detection of isothermally amplified DNA. The recombinase polymerase amplification method is modified slightly to use tailed primers, resulting in an amplicon with a duplex flanked by two single stranded DNA tails. This tailed amplicon facilitates detection via hybridisation to a surface immobilised oligonucleotide capture probe and a gold nanoparticle labelled reporter probe. A detection limit of 1 7 10−11 M (190 amol), equivalent to 8.67 7 105 copies of DNA was achieved, with the entire assay, both amplification and detection, being completed in less than 15 minutes at a constant temperature of 37 \ub0C. The use of the tailed primers obviates the need for hapten labelling and consequent use of capture and reporter antibodies, whilst also avoiding the need for any post-amplification processing for the generation of single stranded DNA, thus presenting an assay that can facilely find application at the point of need

Patterns in recent and Holocene pollen accumulation rates across Europe - the Pollen Monitoring Programme Database as a tool for vegetation reconstruction

The collection of modern, spatially extensive pollen data is important for the interpretation of fossil pollen assemblages and the reconstruction of past vegetation communities in space and time. Modern datasets are readily available for percentage data but lacking for pollen accumulation rates (PARs). Filling this gap has been the motivation of the pollen monitoring network, whose contributors monitored pollen deposition in modified Tauber traps for several years or decades across Europe. Here we present this monitoring dataset consisting of 351 trap locations with a total of 2742 annual samples covering the period from 1981 to 2017. This dataset shows that total PAR is influenced by forest cover and climate parameters, which determine pollen productivity and correlate with latitude. Treeless vegetation produced PAR values of at least 140 grains cm(-2) yr(-1). Tree PAR increased by at least 400 grains cm(-2) yr(-1) with each 10% increase in forest cover. Pollen traps situated beyond 200 km of the distribution of a given tree species still collect occasional pollen grains of that species. The threshold of this long-distance transport differs for individual species and is generally below 60 grains cm(-2) yr(-1). Comparisons between modern and fossil PAR from the same regions show similar values. For temperate taxa, modern analogues for fossil PARs are generally found downslope or southward of the fossil sites. While we do not find modern situations comparable to fossil PAR values of some taxa (e.g. Corylus), CO2 fertilization and land use may cause high modern PARs that are not documented in the fossil record. The modern data are now publicly available in the Neotoma Paleoecology Database and aid interpretations of fossil PAR data.Peer reviewe

Finding Larger Transnational Media Markets : Media Practices of the Vietnamese Diasporic Community

Addressing a concern about the absence of Vietnamese migrants in the Czech media landscape, this chapter first reviews various life contexts of the different Vietnamese populations in the Czech Republic (CR) and then discusses how they have generally lacked participation in the Czech media landscape because of their adoption of transnational media practices. This study also demonstrates how the diasporic community has failed to establish a conventional form of diasporic media but instead has found new translocal information outlets on social media. While the old and new first generations have relied more on media outlets from their country of origin, young migrant children have explored media markets beyond the binational border. However, Vietnamese migrants have recently begun to use social media platforms as networked information outlets, reaching a variety of communities and media outlets located in the CR, Vietnam and their own diasporic community

Detection and Identification of Old World Leishmania by High Resolution Melt Analysis

Protozoal parasites of the genus Leishmania are transmitted by sand fly bites to humans and animals. Three major forms of disease are caused by these parasites: cutaneous leishmaniasis, responsible for disfiguring skin wounds; mucocutaneous leishmaniasis, causing non-healing ulceration around the mouth and nose; and the potentially fatal visceral leishmaniasis, involving internal organs such as the spleen and liver. More than 2 million new human infections are caused annually by leishmaniasis globally, it is endemic in more than 88 countries and prevalent also as an imported disease in non-endemic regions due to travel and tourism. Most species of Leishmania that infect humans are zoonotic and transmitted from animal reservoir hosts. As various leishmanial parasites cause disease with similar symptoms, but require different therapeutic regimens and have dissimilar prognoses, reliable, sensitive and rapid diagnostic assays are needed. This study focuses on the five main species that cause leishmaniasis in the Old World. It presents a new assay for rapid detection, species identification and quantification of leishmanial parasites in clinical samples, reservoir hosts and sand flies. This technique could be especially valuable in regions where several leishmanial species exist, in non-endemic regions where infected patients require a rapid diagnosis, and for epidemiological host and vector studies leading to prevention programs

Distinct genetic control of parasite elimination, dissemination, and disease after Leishmania major infection

Elimination of pathogens is the basis of host resistance to infections; however, relationship between persisting pathogens and disease has not been clarified. Leishmania major infection in mice is an important model of host–pathogen relationship. Infected BALB/c mice exhibit high parasite numbers in lymph nodes and spleens, and a chronic disease with skin lesions, splenomegaly, and hepatomegaly, increased serum IgE levels and cytokine imbalance. Although numerous gene loci affecting these disease symptoms have been reported, genes controlling parasites’ elimination or dissemination have never been mapped. We therefore compared genetics of the clinical and immunologic symptomatology with parasite load in (BALB/c × CcS-11) F2 hybrids and mapped five loci, two of which control parasite elimination or dissemination. Lmr5 influences parasite loads in spleens (and skin lesions, splenomegaly, and serum IgE, IL-4, and IFNγ levels), and Lmr20 determines parasite numbers in draining lymph nodes (and serum levels of IgE and IFNγ), but no skin or visceral pathology. Three additional loci do not affect parasite numbers but influence significantly the disease phenotype—Lmr21: skin lesions and IFNγ levels, Lmr22: IL-4 levels, Lmr23: IFNγ levels, indicating that development of L. major-caused disease includes critical regulations additional to control of parasite spread

Genetic Control of Resistance to Trypanosoma brucei brucei Infection in Mice

Trypanosoma brucei are extracellular protozoa transmitted to mammalian host by the tsetse fly. They developed several mechanisms that subvert host's immune defenses. Therefore analysis of genes affecting host's resistance to infection can reveal critical aspects of host-parasite interactions. Trypanosoma brucei brucei infects many animal species including livestock, with particularly severe effects in horses and dogs. Mouse strains differ greatly in susceptibility to T. b. brucei. However, genes controlling susceptibility to this parasite have not been mapped. We analyzed the genetic control of survival after T. b. brucei infection using CcS/Dem recombinant congenic (RC) strains, each of which contains a different random set of 12.5% genes of their donor parental strain STS/A on the BALB/c genetic background. The RC strain CcS-11 is even more susceptible to parasites than BALB/c or STS/A. In F2 hybrids between BALB/c and CcS-11 we detected and mapped four loci, Tbbr1-4 (Trypanosoma brucei brucei response 1–4), that control survival after T. b. brucei infection. Tbbr1 (chromosome 3) and Tbbr2 (chromosome 12) have independent effects, Tbbr3 (chromosome 7) and Tbbr4 (chromosome 19) were detected by their mutual inter-genic interaction. Tbbr2 was precision mapped to a segment of 2.15 Mb that contains 26 genes