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Sequence of Donning of Personal Protective Equipment (PPE): (How to put on PPE)

1. Footwear
2. Gown
3. N-95 respirator
4. Goggles and face shield
5. Gloves

Donning must be done before entering the patient/ laboratory area.

Sequence of Doffing of PPE: (How to safely remove PPE)

During hybridization, single-stranded amplicons bind to probes and highly specific binding is ensured to produce even staining in the presence of stringent buffer and temperature conditions. However, high/ dark backgrounds in strips may be due to the following reasons:

• CON-C and/or SUB-C used too concentrated​
• Washing steps not performed with necessary care
• Wash solutions were too cold​

Solution: Repeat reverse hybridization step.​

During hybridization, single-stranded amplicons bind to probes and highly specific binding is ensured to produce even staining in the presence of stringent buffer and temperature conditions. However, uneven staining can occur due to the following reasons:

There could be overall weak or no signals on the strip including or except for the conjugate control zone.

Weak or no signal​ including conjugate control (CC) zone could be due to:

• Too low room temperature
• Reagents not equilibrated to room temperature (20-22°C)
• Hybridization solutions not pre-warmed
• Too high or too low incubation temperature
• No or too little amount of CON-C and/or SUB-C used

Solution: Repeat reverse hybridization.

General instructions when conducting hybridization using a twincubator are:

• Thoroughly clean the workbench with disinfectant​ (1% sodium hypochlorite).
• Prepare reagents as per the calculation table.
• Using clean/ new tweezers place strips onto a clean sheet of paper and label them as in the worksheet.​
• Use a clean/ new tray.

Steps of Hybridization Using Twincubator​ 

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

Workstation Set-up

Deoxyribonucleic Acid (DNA) hybridization is based on complementary strands of single-stranded DNA (ssDNA) hybridizing or binding to each other to form double-stranded DNA (dsDNA).

When an HIV-positive person dies from TB, the underlying cause is classified as HIV with TB as a contributory cause. However, the milestones and targets for reductions in TB deaths set at the End TB Strategy are for the combined total of deaths in HIV-positive and HIV-negative people.

Contamination can happen in the amplification room due to exogenous DNA and spillover from last amplification or contaminants or inhibitors entering the reaction tubes.

Do’s and Don’ts for Contamination Control in the Amplification Room 

Do’s

In the Line Probe Assay (LPA) labs, subsequent to the preparation of the Polymerase Chain Reaction (PCR) Master mix, DNA amplification is carried out.

The DNA amplification steps remain the same in both First Line - Line Probe Assay (FL-LPA) and Second Line - Line Probe Assay (SL-LPA). In DNA amplification, there is the addition of DNA to the master mix, and the amplification of DNA using PCR machine.

The procedure for amplification is shown below. Play the video below to know more.