A0

Each strip for Second Line - Line Probe Assay (SL - LPA) has a total of 27 reaction zones (Figure below).

Figure: Key Zones of SL - LPA Evaluation Strip; Source: GenoType MTBDRsl VER 2.0 kit, Instructions for Use.

Evaluation of Assay Strip for SL- LPA Zones:

Each strip for First Line – Line Probe Assay (FL - LPA) has a total of 27 reaction zones (Figure below).

The Key Zones Include:

Conjugate Control (CC)

• Indicates the efficiency of conjugate binding and substrate reaction

Amplification Control (AC)

• Develops when control amplicon binds indication correct procedure was followed
• The absence of AC zone indicates an invalid test

M. tuberculosis Complex (TUB)

Do’s and Don’ts for Contamination Control 

DO’s

• Label the tools and instruments “For use in Hybridization Area”.​
• Change gloves if they get contaminated amplicon.​
• Decontaminate trays before reuse​.
• Keep cleaned strip trays and tweezers in a clean and sealed plastic bag.​
• Always clean work bench and tools with 1% freshly prepared sodium hypochlorite solution followed by 70% alcohol​.
• Establish weekly laboratory cleaning protocols for floors, doors, walls.

General Instructions

Test Reagents Kits Used for First Line (FL) and Second Line (SL) Line Probe Assay (LPA) 

• For FL LPA- kit used is MTBDR plus version 2.0  
• For SL LPA- kit used is MTBDRsl version 2.0  

Components of kits are shown in the following table. 

Purpose of DNA extraction 

• Extract DNA from cellular components 

For Line Probe Assay (LPA), DNA is extracted from:  

• Clinical specimen​ 
• Culture isolates on Loewenstein-Jensen (LJ) solid medium  
• Bacteria growth in liquid media​ (Mycobacteria Growth Indicator Tube (MGIT), BD Diagnostics) 

Extraction of DNA from a clinical specimen and culture isolates (LJ, liquid) is shown in the figure below: 

Uses of Twincubator in Line Probe Assay (LPA):

• Different steps of the hybridization (shown in figure below) and detection procedure with DNA strips in reverse blot hybridization assays.
• In the amplification/ post-amplification area.

Figure: Twincubator used in Hybridization

Features of Twincubator: ​

In the Line Probe Assay (LPA) lab, the water bath is used for warming reagents used in the Hybridization Room.

It is electrically heated and electronically controlled.

Components:

• Recessing push/ turn control for simple operation of the bath
• Visual temperature indication​
• Push button to set desired temperature​
• Electrically heated and electronically controlled​
• Water trough​

Installation of Water Bath (Figure):

Thermocycler is also known as Polymerase Chain Reaction (PCR) Machine.

Use of Thermocycler

Thermal cycler is used for amplification of nucleic acid by PCR​.

The thermocycler should be placed in the amplification/ post-amplification area. 

The features of a thermocycler are listed below.

Polymerase Chain Reaction (PCR) hood is a workstation enclosed by three sides.

Use of Polymerase Chain Reaction (PCR) Hood:

• Used in the Master Mix Reagent Preparation and DNA Amplification Room
• Ultraviolet (UV) light (Figure) in PCR hood denatures nucleic acids​
• Used to decontaminate reagents and equipment prior to PCR reaction​
• Provides protection against exogenous DNA, contaminants or inhibitors from entering the reaction tubes