LPA Troubleshooting for No Signal

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.

Figure: No Signal including CC Zone

Points to Note

• A higher incubation temperature may prevent the binding of the amplicons to the probes.
• A higher temperature during the stringent wash step may result in weak intensities of the banding patterns.
• The temperature of the hybridizer should be monitored if such problems are seen.
• If the problem persists and the thermostat on the hybridizer gives an appropriate reading of 45ºC, and a digital thermometer reads a different temperature that is above or below 45ºC; the problem is likely with the machine’s internal temperature probe and should be reported to the manufacturer and corrected.

Do’s and Don'ts to Avoid Weak or No Signals

• Perform LPA at the appropriate room temperature.
• Bring reagents to room temperature before use (20-22°C).
• The green and red hybridization solutions must be pre-warmed to 45ºC.
• Follow instructions to add the exact volume of reagents.
• The central heating/cooling thermostat must be appropriately adjusted.
• Do not use pipettes that are not calibrated.
• Do not miss any step in the hybridization process.

Resources

• GenoType MTBDR plus ver 2.0 Kit, Instructions for Use.
• GenoType MTBDRsl VER 2.0 Kit, Instructions for Use.
• GLI Training Package on LPA.

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