IASMA Research and Innovation Centre, Fondazione Edmund Mach, Trento, Italy.
Ann Allergy Asthma Immunol. 2009 Dec;103(6):508-14. doi: 10.1016/S1081-1206(10)60268-2.
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.
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.
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.
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.
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.
准确且最新的特定地区 airborne pollen 信息可以帮助过敏患者。当前的监测系统基于形态识别方法，这是一种耗时的方法，可能成为采样网络增强的限制因素。
验证开发实时聚合酶链反应（PCR）方法的可行性，该方法替代光学分析，作为一种快速、准确、自动化的工具，用于检测和定量 airborne allergenic pollen 分类群。
对花粉进行 DNA 分离的传统十六烷基三甲基溴化铵法进行了改良。通过生物信息学或文献搜索鉴定分类群特异性 DNA 序列，并从匹配的过敏原分类群中进行 PCR 扩增；基于 PCR 产物序列，开发了互补或简并 TaqMan 探针。对 3 种植物物种进行了定量实时 PCR 检测的准确性测试。
改良的 DNA 提取方案的设置使我们能够获得高质量的花粉 DNA。鉴定并测序了分类群特异性核基因片段。设计的引物对和探针识别了选定的花粉分类群，大多数情况下都达到了所需的分类水平。即使在常规空气生物学带上收集花粉，也能正确识别花粉。对测试物种和混合物进行了初步的花粉粒定量检测。
实时 PCR 方法在花粉鉴定和定量方面显示出有希望的结果，即使在分析花粉混合物时也是如此。未来的研究方向可能涉及开发用于在同一反应管中同时检测不同分类群的多重实时 PCR，以及应用高通量分子方法。
