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LPA Troubleshooting for No Signal
Learning ObjectivesLPA Troubleshooting for No Signal
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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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