Biosafety Requirements in LPA Facility Design

There are two types of risk while setting up a Polymerase Chain Reaction (PCR) facility for Line Probe Assay (LPA):

1. Biohazard risk: The potential that a laboratory worker will become infected when working with live M. tuberculosis.
2. Bio-risk: The potential that specimens or reagents will become contaminated with DNA, amplified products (amplicons) or exogenous contaminants that lead to false-positive PCR results.

Precautionary measures to reduce the biohazard and bio-risk in DNA molecular procedures are critical and drive the general facility design.

LPA Facility Design and Layout

There should be four separate rooms for:

• DNA extraction
• Reagent preparation for PCR - the pre-amplification step
• PCR amplification
• Hybridization and detection

In case of space constraints, one room can be used for both hybridization and amplification.

Movement in the LPA Facility (Figure 1 and 2)

• There should be a unidirectional flow of lab staff, as well as the tools used.
• Never reverse the flow.

For example:

1. In the morning, commence work in the reagent preparation room as it is “the ultra-clean room”. From this room, one may proceed to either the specimen preparation room, the PCR amplification room, or the PCR amplification and detection room.
2. If one starts work in the specimen preparation room they should not enter the reagent preparation room, but they may work in the PCR amplification room, or the PCR amplification and detection room.
3. If one starts working in the amplification or detection room they should not enter the reagent preparation room.

Figure 1: LPA Facility Layout showing Maintenance of Unidirectional Movement and Flow of Tools

Figure 2: Requirements to Maintain the Unidirectional Flow of Tools and Staff in the LPA Laboratory

Resources

• Molecular Detection of Drug-resistant Tuberculosis by Line Probe Assay.

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