Chapter 2. Quality in the Analytical Molecular Biology Laboratory

White-Nose Syndrome Diagnostic Laboratory Network handbook

“…With techniques such as qPCR that are highly sensitive, extra care must be taken to prevent false positives related to laboratory cross-contamination. Good laboratory practices related to space, equipment, and workflow can help minimize the possibility of contamination during sample processing (table 4; Conraths and Schares, 2006;Hopkins, 2008). Physical separation between workspaces in the laboratory, unidirectional workflows that control movement of personnel and equipment, and air flow controls that minimize aerosols are some of the fundamental strategies to minimize contamination in a molecular diagnostic laboratory (table 4; figs.…”

“…Physical separation between workspaces in the laboratory, unidirectional workflows that control movement of personnel and equipment, and air flow controls that minimize aerosols are some of the fundamental strategies to minimize contamination in a molecular diagnostic laboratory (table 4; figs. 9 and 12; Hopkins, 2008). Additionally, most laboratories have systems in place for detecting and investigating potential contamination issues when suspect results arise.…”

“…after minor aerosolization events. For this reason, the most critical spaces to keep physically isolated from those used to manipulate clean reagents are those where amplicons are generated, analyzed (for example, by electrophoresis), or purified (Persing, 1991;Hopkins, 2008). Careful use of equipment, reagents, and consumables can also help reduce the possibility of laboratory contamination.…”

“…By categorizing the various steps of the qPCR process as either "clean" or "dirty," it is possible to eliminate unnecessary movement of instruments and materials between so-designated spaces. Pipettes, tips, plastics, and bench-top instruments such as centrifuges or vortexers should be reserved within the spaces provided for "clean" and "dirty" activities without the need for being transported from one part of the laboratory to another; for example, equipment used to make qPCR master mix should always remain in that "clean" space, whereas other instruments for adding template nucleic acid to the qPCR reaction plate are kept in a different space (Conraths and Schares, 2006;Hopkins, 2008). Positive controls, primers, probes, and other qPCR reagents should be aliquoted for single use whenever possible to avoid potentially introducing contaminants through repeated pipetting (Persing, 1991;Hopkins, 2008).…”

“…Pipettes, tips, plastics, and bench-top instruments such as centrifuges or vortexers should be reserved within the spaces provided for "clean" and "dirty" activities without the need for being transported from one part of the laboratory to another; for example, equipment used to make qPCR master mix should always remain in that "clean" space, whereas other instruments for adding template nucleic acid to the qPCR reaction plate are kept in a different space (Conraths and Schares, 2006;Hopkins, 2008). Positive controls, primers, probes, and other qPCR reagents should be aliquoted for single use whenever possible to avoid potentially introducing contaminants through repeated pipetting (Persing, 1991;Hopkins, 2008). Using a BSC can help prevent dispersing aerosolized microorganisms or contaminants, and changing laboratory coats and shoe coverings when entering a "clean" space from a "dirty" space can help reduce the chance of personnel inadvertently contaminating reactions via their clothing (Hopkins, 2008).…”