Quantitative DNA Analyses for Airborne Birch Pollen

Abstract

Birch trees produce large amounts of highly allergenic pollen grains that are distributed by wind and impact human health by causing seasonal hay fever, pollen-related asthma, and other allergic diseases. Traditionally, pollen forecasts are based on conventional microscopic counting techniques that are labor-intensive and limited in the reliable identification of species. Molecular biological techniques provide an alternative approach that is less labor-intensive and enables identification of any species by its genetic fingerprint. A particularly promising method is quantitative Real-Time polymerase chain reaction (qPCR), which can be used to determine the number of DNA copies and thus pollen grains in air filter samples. During the birch pollination season in 2010 in Mainz, Germany, we collected air filter samples of fine (<3 μm) and coarse air particulate matter. These were analyzed by qPCR using two different primer pairs: one for a single-copy gene (BP8) and the other for a multi-copy gene (ITS). The BP8 gene was better suitable for reliable qPCR results, and the qPCR results obtained for coarse particulate matter were well correlated with the birch pollen forecasting results of the regional air quality model COSMO-ART. As expected due to the size of birch pollen grains (~23 μm), the concentration of DNA in fine particulate matter was lower than in the coarse particle fraction. For the ITS region the factor was 64, while for the single-copy gene BP8 only 51. The possible presence of so-called sub-pollen particles in the fine particle fraction is, however, interesting even in low concentrations. These particles are known to be highly allergenic, reach deep into airways and cause often severe health problems. In conclusion, the results of this exploratory study open up the possibility of predicting and quantifying the pollen concentration in the atmosphere more precisely in the future.

Fig 1. DNA quantification via qPCR measurements.

DNA concentrations in copies m^-3 (cp m^-3) are given for the multi-copy ITS region and single-copy BP8 gene for coarse (a) and fine particle filter samples (b) DNA copies m^-3 are given for ITS quantification on the primary vertical axis, and on the secondary vertical axis for the BP8 gene. The x-axis shows the time, 2010-03-16 / 2010–0608, during the birch-specific pollination period.

Fig 2. Comparison between qPCR measurements and COSMO-ART simulation.

(a) DNA concentrations in copies m^-3 (cp m^-3) of coarse particle filter samples are given for multi-copy ITS region (cp m^-3 air: primary vertical axis) and simulations of pollen concentrations from COSMO-ART (sum of pollen grains per m³ air: on secondary vertical axis) during the birch-specific main-pollination period in 2010 (2010-03-30 / 2010-04-27, x-axis) for Mainz (b) Quantification results for single-copy BP8 gene (cp m^-3 air: primary y axis) and pollen concentrations from COSMO-ART for Mainz and Löwenstein, as well as pollen concentrations averaged hourly in pollen m^-3 sampled via Burkard traps (secondary y axis) are summed corresponding to the sampling interval of the appropriate filter sample(y-axis) during the birch-specific main-pollination period in 2010 (2010-03-30 / 2010-04-27, x-axis). Pollen counts were provided from nearest available pollen measurement station in Löwenstein, Baden-Wurttemberg about 130 kilometers south-east of Mainz.