A0

During PCR Reagent preparation, if DNA fragments from the lab environment, such as a DNA template amplified in a previous qPCR experiment, enter the qPCR reaction or reagents (even in small quantities), they can be amplified during the reaction. This contamination and non-specific amplification can cause misleading results, such as false positives. Hence, contamination needs to be rigorously controlled.

Do’s and Don’ts for Contamination Control during PCR Reagent Preparation 

Do’s​

In a Line Probe Assay (LPA) lab, subsequent to DNA extraction, reagents are prepared for PCR amplification using the checklist and worksheets.

Preparation Steps

• Prepare a written checklist of materials and tools needed.
• Prepare a worksheet with a list of specimens (identifiers) to be tested.
• Prepare worksheet to calculate the volume master mix ingredients (put on the wall near work area). ​

Setting up the Workplace

Contamination of the Polymerase Chain Reaction (PCR) can occur from previously processed specimens. Hence, it is important to take precautions to avoid contamination.

To ensure contamination control during DNA extraction in a Line Probe Assay (LPA) setting, follow the do's and don’ts elaborated below.

DO's​

Key Features of Vortex Mixer

It is used in the TB laboratory to mix specimens with decontamination solutions and to homogenize solutions of TB bacilli for DNA extraction and during the preparation of the PCR Master Mix.

• During the DNA extraction step:
  • Growth from liquid culture is vortexed to break up clumped Mycobacterium tuberculosis
  • Homogenize bacilli
  • Mix specimens with decontamination solutions​
• Gentle vortex is used to mix reagents in PCR master mix tube.

The design of the LPA facility includes the availability of different rooms to perform different steps. Each room has a set of specific equipment.

There should be a minimum of 4 separate rooms to carry out procedures. The details of these rooms and equipment are elaborated below:

1. DNA Extraction Room: For DNA extraction from decontaminated samples. Table 1 shows the equipment in the DNA extraction room.

Importance of Electrical Supply and Backup Power in the Line Probe Assay (LPA) Facility

• Reagents used in LPA are to be stored at 4°C or -20°C. Hence, require uninterrupted electricity supply/ backup power.
• Amplification and hybridization procedures must be conducted under closely monitored temperature conditions.
• Uninterrupted Power Supply (UPS) connection is required during PCR amplification and use of the automated hybridization systems to avoid interruption of the procedure and subsequent loss of results.

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.

DNA extraction for Line Probe Assay (LPA) from clinical specimens can be performed in either a BSL-2 or BSL-3 laboratory, while DNA extraction from mycobacterial cultures must be performed in a BSL-3 laboratory. Only after heat-killing of the organism and DNA isolation can the sample be considered non-infectious and moved to the LPA laboratory.

The subsequent steps, i.e., amplification and post-amplification, only require a BSL-1 laboratory.

Aspects of biosafety in LPA laboratory include:

After inoculating the tubes, they have to be incubated in the BACTEC MGIT 960 instrument to check if there is mycobacterial growth after the stipulated time.

The steps for incubating MGIT tubes for TB cultures are as follows:

1. Enter the inoculated MGIT tubes into BACTEC MGIT 960 instrument.

2. Scan the tube bar code and specimen bar code (if available​) (Figure 1).

Figure 1: Scanning the tube bar code

Mycobacteria Growth Indicator Tube (MGIT) contains a modified Middlebrook 7H9 broth base. When supplemented with MGIT Growth Supplement and PANTA, it provides an optimum medium for the growth of a majority of mycobacterial species. All types of specimens, pulmonary as well as extra-pulmonary (except blood), can be inoculated into MGIT for primary isolation of mycobacteria.

The steps for inoculating MGIT tubes are elaborated below: