Citation:

HAUGLAND, R. A. AND S. J. VESPER. GENETIC-BASED ANALYTICAL METHODS FOR BACTERIA AND FUNGI. Chapter 7, C.S. Yang and P. Heinsohn (ed.), Sampling and Analysis of Indoor Micoorganisms. John Wiley & Sons Incorporated, New York, NY, , 133-152, (2007).

Impact/Purpose:

1. Develop and publish a standard method or guidance document for QPCR analysis of microorganisms in environmental samples (air and water filtrates and dust).a standard method or guidance document for QPCR analysis of microorganisms in environmental samples (air and water filtrates and dust). Publication will involve a consensus standards organization. 2. Use QPCR methods to monitor childhood exposures to mold as a part of field studies, in order to establish whether a relationship exists between molds encountered in indoor environments and asthma-related health problems.

Description:

In the past two decades, advances in high-throughput sequencing technologies have lead to a veritable explosion in the generation of nucleic acid sequence information (1). While these advances are illustrated most prominently by the successful sequencing of the human genome, they have also factored heavily in our current knowledge of nucleic acid sequences from a variety of microorganisms. Concurrent with this growth in sequencing activity has been the development of a variety of techniques for the amplification and manipulation of nucleic acids. The combination of these technologies is currently supporting a significant shift away from the use of traditional culture-based analyses for the detection and characterization of microorganisms and towards the use of new analytical methods based on genetic composition, i.e. nucleic acids, (45, 113). This shift is evident in a number of fields including the microbiological analyses of indoor environments.

Genetic analysis methods for bacterial and fungal microorganisms are presently becoming increasingly more widespread in their applications, not only in research settings, but also in clinical and environmental testing laboratories. Advantages of these methods over culture and phenotypic analyses can include higher speed, sensitivity and accuracy in the detection and identification of microorganisms as well as better resolution between similar organisms and an ability to detect non-cultivatable or fastidious organisms. Through their potential for automation, these methods also offer the possibility for less reliance on analyst training and decreased labor investments. As will be discussed in more detail below, current limitations of many of these genetic analysis methods can include on-going uncertainty of the extent to which available sequence information circumscribes the genetic variability of different target microbial groups, technical and quality control issues and higher costs related to expenditures for instruments and reagents. It is reasonable to expect, however, that each of these limitations will decrease in the future making genetic microbial testing methods an increasingly attractive option for many clinical and environmental applications.

The first section of this chapter provides an overview (with references for additional reading) of currently available genetic based analytical techniques that may be useful for investigations of bacterial and fungal microorganisms in the indoor environment. These techniques are grouped into four general categories: 1) in vitro nucleic acid amplification; 2) hybridization probes; 3) nucleic acid sequencing; and 4) microbial strain typing. The second section provides example applications of techniques within each of these categories for the study of indoor microbiology. The third section provides a synopsis of quality assurance issues and presently accepted quality control measures for laboratories performing genetic analysis methods, focusing primarily on methods involving nucleic acid amplification techniques. Other current technical limitations of these methods and their outlook for the future are also discussed.

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