title: qPCR standard operating procedure for measuring microorganisms in dust from dwellings in large cohort studies.
abstract: The aim of the present study was to assess performance, feasibility and relevance of a Standard Operational Procedure (SOP) for large-scale use in the microbial analysis of children's indoor environments. We analyzed dust settled on Electrostatic Dust Fall Collectors (EDCs) by using qPCR which targeted 6 molds, 3 bacteria and 1 mite, chosen for their involvement in allergic or inflammatory processes. Six types of commercialized electrostatic wipes were tested for their releasing capacity of fungal DNA from fungal spores captured by the wipes. Specificity, repeatability and detection limits of the qPCR procedure were tested using calibrated microbial suspensions. The feasibility and relevance of this sampling and analysis method were assessed in a 75-home pilot study. Our result showed that one specific make of wipe was more effective than the others in releasing fungal DNA. qPCR procedure showed good repeatability. The quantification limit was about 5 fg DNA/μL for all species except Penicillium chrysogenum (0.5 fg DNA/μL) and Dermatophagoïdes pteronyssinus (10 fg DNA/μL). No cross-reactivity was observed. DNA concentrations in the 53/75 homes participating in the pilot study were between 0 and 24 625, 0 and 69 738 equivalent cells per cm(2) for the fungi and bacteria, and between 0 and 1 equivalent mites per cm(2) for D. pteronyssinus. Using the SOP described, we were able to classify the 53 dwellings from the least to the most contaminated according to the quantity of DNA measured for each species. Our SOP measured fungi, bacteria and mites using a cost-efficient, discreet and well-accepted sampling method with just one qPCR tool. The whole procedure can be used for microbial analysis in large cohort studies such as the ELFE study ("Etude Longitudinale Française depuis l'Enfance") and could help improve our understanding of the interactions between the environment, allergic diseases and child development.
authors: Chrono-Environnement Research Team UMR/CNRS-6249-Franche-Comté University, Besançon, France; Department of Parasitology and Mycology, University Hospital, Besançon, France.
authors: Emeline
authors: E
authors: Scherer
authors: Steffi
authors: S
authors: Rocchi
authors: Gabriel
authors: G
authors: Reboux
authors: Stéphanie
authors: S
authors: Vandentorren
authors: Sandrine
authors: S
authors: Roussel
authors: Mallory
authors: M
authors: Vacheyrou
authors: Chantal
authors: C
authors: Raherison
authors: Laurence
authors: L
authors: Millon
mesh_terms: D016902:Air Pollution, Indoor / Q000032:analysis*; D000485:Allergens / Q000032:analysis; D000818:Animals; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002648:Child; D004269:DNA, Bacterial / Q000032:analysis; D004271:DNA, Fungal / Q000032:analysis; D004391:Dust / Q000032:analysis*; D004784:Environmental Monitoring / Q000379:methods*; D005658:Fungi / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006798:Housing; D006801:Humans; D008969:Molecular Sequence Data; D010865:Pilot Projects; D016133:Polymerase Chain Reaction / Q000379:methods*; D039981:Pyroglyphidae / Q000235:genetics / Q000378:metabolism
chemical_list: D000485:Allergens; D004269:DNA, Bacterial; D004271:DNA, Fungal; D004391:Dust
keywords: Chronic exposure; Dust mites; Environmental bacteria; Environmental fungi; Indoor microorganisms; Wipes