New biomolecular tools for aerobiological monitoring: Identification of major allergenic Poaceae species through fast real‐time PCR

Abstract

Grasses (Poaceae) are very common plants, which are widespread in all environments and urban areas. Despite their economical importance, they can represent a problem to humans due to their abundant production of allergenic pollen. Detailed information about the pollen season for these species is needed in order to plan adequate therapies and to warn allergic people about the risks they take in certain areas at certain moments. Moreover, precise identification of the causative species and their allergens is necessary when the patient is treated with allergen‐specific immunotherapy. The intrafamily morphological similarity of grass pollen grains makes it impossible to distinguish which particular species is present in the atmosphere at a given moment. This study aimed at developing new biomolecular tools to analyze aerobiological samples and identifying major allergenic Poaceae taxa at subfamily or species level, exploiting fast real‐time PCR. Protocols were tested for DNA extraction from pollen sampled with volumetric and gravimetric methods. A fragment of the matK plastidial gene was amplified and sequenced in Poaceae species known to have high allergological impact. Species‐ and subfamily‐specific primer–probe systems were designed and tested in fast real‐time PCRs to evaluate the presence of these taxa in aerobiological pollen samples. Species‐specific systems were obtained for four of five studied species. A primer–probe set was also proposed for the detection of Pooideae (a grass subfamily that includes also major cereal grains) in aerobiological samples, as this subfamily includes species carrying both grass allergens from groups 1 and 5. These, among the 11 groups in which grass pollen allergens are classified, are considered responsible for the most frequent and severe symptoms.

Assessment of ITS1, ITS2, 5′-ETS, and trnL-F DNA Barcodes for Metabarcoding of Poaceae Pollen

“…The effectiveness of the protocols for sample preparation for HTS highly depends, among other things, on the quality and quantity of the DNA. Various methods for pollen DNA extraction involve using commercial solutions such as column-based and DNA binding with magnetic beads purification methods after preliminary homogenization of the pollen sample with metal beads [47,48]. We propose a protocol based on a classical CTABlysis extraction method [49] with modifications that achieve results similar to commercial kits for pollen DNA extraction.…”

Pollen Production of Selected Grass Species in Russia and India at the Levels of Anther, Flower and Inflorescence

“…The determination of the precise flowering times of different species and their pollen concentrations may be very important for allergy sufferers as, despite the lack of detailed studies, it is assumed that different grass species may have pollen grains with different degrees of allergenicity [ 44 ]. Refining data on grass pollination period down to the species level is possible either on the basis of phenological observations [ 29 ] or by metabarcoding [ 45 , 46 , 47 , 48 ]. Both approaches need data on the pollen production of different species to determine the impact of individual species and validate the results.…”

AeRobiology: The computational tool for biological data in the air

“…New aerobiological methods with automatic sampling devices have been developed over the last years and new devices are under improvement (Crouzy, Stella, Konzelmann, Calpini, & Clot, ; Kawashima et al, ; Oteros et al, ). Such novel techniques will require new methods for analysing the huge amount of real‐time aerobiological data provided (Ghitarrini et al, ). In any case, conventional and real‐time methods need faster techniques for analysing the aerobiological data.…”