First Line - Line Probe Assay (FL - LPA) can detect the mutations that are most frequently identified in resistant strains. 

However, there are certain limitations of the test as follows:

xamples of banding patterns for First Line - Line Probe Assay (FL - LPA) are shown in figure below.

Description

• All wild type bands display signal, mark as “+” against Wild Type (WT) column of the respective gene
• If at least one of the wild type bands is absent, mark as “-“ against WT column
• Similarly, “+” or “-” for Mutation (MUT) probes

Predicting Drug Resistance in Special Cases

Zone developed/ not developed (failed) in Wild Type (WT) and Mutation (MUT) probes are used to predict drugs resistance in Second Line - Line Probe Assay (SL - LPA).

For Fluoroquinolones (FLQ)

Tables 1 and 2 show mutations in gyrA, gyrB genes and corresponding WT, MUT and responsible codons and mutations:

• gyrA WT1-3: gyrA wild type probes; gyrA MUT1-2, 3A-3D: gyrA mutation probes
• gyrB WT: gyrB wild type probes; gyrB MUT1-2: gyrB mutation probes

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: