Biomolecular identification of allergenic pollen: a new perspective for aerobiological monitoring?
BACKGROUND
Accurate and updated information on airborne pollen in specific areas can help allergic patients. Current monitoring systems are based on a morphologic identification approach, a time-consuming method that may represent a limiting factor for sampling network enhancement.


OBJECTIVE
To verify the feasibility of developing a real-time polymerase chain reaction (PCR) approach, an alternative to optical analysis, as a rapid, accurate, and automated tool for the detection and quantification of airborne allergenic pollen taxa.


METHODS
The traditional cetyl trimethyl ammonium bromide-based method was modified for DNA isolation from pollen. Taxon-specific DNA sequences were identified via bioinformatics or literature searches and were PCR amplified from the matching allergenic taxa; based on the sequences of PCR products, complementary or degenerate TaqMan probes were developed. The accuracy of the quantitative real-time PCR assay was tested on 3 plant species.


RESULTS
The setup of a modified DNA extraction protocol allowed us to achieve good-quality pollen DNA. Taxon-specific nuclear gene fragments were identified and sequenced. Designed primer pairs and probes identified selected pollen taxa, mostly at the required classification level. Pollen was properly identified even when collected on routine aerobiological tape. Preliminary quantification assays on pollen grains were successfully performed on test species and in mixes.


CONCLUSIONS
The real-time PCR approach revealed promising results in pollen identification and quantification, even when analyzing pollen mixes. Future perspectives could concern the development of multiplex real-time PCR for the simultaneous detection of different taxa in the same reaction tube and the application of high-throughput molecular methods.
Longhi
Sara
S
IASMA Research and Innovation Centre, Fondazione Edmund Mach, Trento, Italy.
Cristofori
Antonella
A
Gatto
Pamela
P
Cristofolini
Fabiana
F
Grando
Maria Stella
MS
Gottardini
Elena
E
D052179:Antigens, Plant; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D018744:DNA, Plant / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D004784:Environmental Monitoring / Q000379:methods*; D007558:Italy; D015336:Molecular Probe Techniques; D018515:Plant Leaves / Q000737:chemistry / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D011058:Pollen / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000276:immunology; D016133:Polymerase Chain Reaction
D052179:Antigens, Plant; D017931:DNA Primers; D015342:DNA Probes; D018744:DNA, Plant; D010940:Plant Proteins