Steps Involved in Proficiency Testing (PT):

A batch of samples is defined as a group of samples that are processed, amplified and hybridized:

• At the same time
• Under the same conditions
• Using the same PCR master mix, thermal cycler and hybridization solutions
• On the same hybridization platform.

QC during the DNA extraction procedure

The quality concepts of the Line Probe Assay (LPA) laboratory include:

Issue #1:

Absence of Conjugate Control (CC) and Amplification Control (AC) in a negative control (Figure 1).

Figure 1: Absence of CC and AC in a Negative Control

Table 1 below lists the reasons and troubleshooting for the absence of CC and AC in a negative control​.

There could be overall weak or no signals on the strip, including or except, the Conjugate Control (CC) zone.

Figure: LPA showing No Signal Except CC 

Reasons and Troubleshooting for No Signal Except CC are given in the table below:

The limitations of Second Line Probe Assay (SL-LPA) are:

he examples of banding patterns for Second Line - Line Probe Assay (SL - LPA) are shown in the 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 probes (MUT).

Notes

• Bands with intensities similar or better than Amplification Control (AC) zone are considered.
• Not all bands of strip show same signal strength

Special Scenarios

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

For Rifampicin

Table 1 shows mutations in rpoB gene and corresponding WT, MUT and responsible codons and mutations.

• rpoB WT1-8: rpoB wild type probes
• rpoB MUT1-3: rpoB mutation probes

Table 1: Mutations in the rpoB gene and corresponding wild type and mutation band; Source: GenoType MTBDR plus ver 2.0 kit, Instructions for Use.

Reporting Drug-resistance in Special Cases