Hybridization Setup in LPA and Genotypic DST

Hybridization Setup in LPA and Genotypic DST

Learning Objectives

Hybridization Setup in LPA and Genotypic DST

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Content

The steps listed below need to be followed when setting up for the hybridisation procedure.

Workstation Set-up

Reagent Preparation

Kit Component 1 is used for this step.
Hybridization reagent preparation and steps involved in hybridization procedure remains the same in First Line (FL) and Second Line (SL) Line Probe Assay (LPA).
Bring out the reagent kit from the refrigerator.
Pre-warm the hybridisation and stringent solution in a water bath, as these 2 solutions form crystals due to the kit’s storage at 2 - 8ºC.

Reagents Required for Hybridization

The steps for preparing these reagents are shown in the tables below.

Table 1: Preparation of Hybridization and Stringent Solutions
PREPARATION OF HYBRIDIZATION SOLUTION	PREPARATION OF STRINGENT SOLUTION
Pre-warm hybridization solution in a water bath. Check the hybridization solution for precipitate. If the solution is consistent, without any precipitate, aliquot the calculated amount to a falcon tube and label it as “HYB". Remember: Hybridization solution should be warm when adding it to the Hybridization tray.	Pre-warm stringent solution in a water bath. Check the stringent bottle for precipitate. If the solution is consistent, without any precipitate, aliquot the calculated amount to a falcon tube and label it as “STR”.

Table 2: Preparation of Conjugate and Substrate Solutions
PREPARATION OF CONJUGATE SOLUTION	PREPARATION OF SUBSTRATE SOLUTION
Aliquot calculated conjugate diluent to a 50 ml falcon using a micropipette. Add calculated conjugate concentrate to it, using a micropipette. Mix well. Label it as “CONJUGATE”.	Aliquot calculated substrate diluent to a 50 ml falcon using a micropipette. Add calculated substrate concentrate to it, using a micropipette. Mix well. Cover the falcon tube with aluminium foil as the substrate is photosensitive.

Table 3: Preparation of RIN Solution and Distilled Water
PREPARATION OF RIN SOLUTION	FOR DISTILLED WATER
Aliquot calculated RIN solution to a 50 ml falcon tube. Label it as "RIN".	Aliquot the calculated amount of distilled water required in a falcon tube. Label it as "DW".