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

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Protocol publication

[…] Three pairs of primers, directed to different parts of the plant genome, were found in bibliography (Cuénoud et al., ; Drumwright, Allen, Huff, Ritchey, & Cahoon, ; Mason‐Gamer, Weil, & Kellogg, ) and used to perform a first screening of the five species of interest. They were directed to the nuclear waxy gene (F‐for/K‐bac), and the plastidial rbcL (rbcl‐F/rbcl‐R) and matK (matK 390‐F/matK 1326‐R) genes, which have been often targeted in barcoding approaches (Table ). Primers (Sigma‐Aldrich) were used to amplify leaf DNA under the published conditions. All the PCR products were purified using the illustra GFX PCR DNA and GEL Band Purification kit (GE Healthcare), and Sanger sequenced. Sequences were aligned using MEGA6 Software (Tamura, Stecher, Peterson, Filipski, & Kumar, ). matK sequences, of higher quality, were analyzed to identify polymorphic regions. New primer pairs with different levels of discriminating power (subfamily/species) were designed using Primer3 (Untergrasser et al., ). The matk‐PGP TaqMan probe, for real‐time PCR assay, was designed using Primer Express software 3.01 (Thermo Fisher Scientific), to hybridize a conserved trait of matK sequences from the five studied species. A 6‐carboxyfluorescein (FAM) dye was linked at the 5′‐end and a non–fluorescent minor groove binder quencher (MGB) at the 3′‐end of the probe. Probe was purchased from Thermo Fisher Scientific. The oligonucleotide sequences are listed in Table . The alignment of Poaceae matK fragment on which the sequences were studied is shown in Figure . […]

Pipeline specifications

Software tools MEGA, Primer3, Primer Express
Application qPCR
Organisms Homo sapiens