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Troubleshooting

Troubleshooting

System inside GeneProof

Troubleshooting guide is divided according to the main themes. This division will allow for electronization and implementation into the GeneProof Web site, including a continuous update. Information sources for the updates are OTRS, discussion with customers and others. Troubleshooting data are updated repeatedly.

The troubleshooting logic copy the diagnostics process in standard molecular diagnostic laboratories. The general topic is always written in capital letters, which is then further expand according to the specific problem.

GENERAL – issue with low or no fluorescence

Low fluorescence

Possible Cause Solution
Degraded MasterMix Store at -20 °C, thaw at 2 - 8 °C no longer than 30 min. Vortex and short spin before use.
Unverified cycler Use verified cycler. List of verified cyclers here.
Incorrect consumables Use recommended consumables. List of consumables in device manual.
Incorrect transport medium Use TE buffer or Nuclease-free water.
Air bubbles in PCR mix solution Spin PCR strip before PCR.

No fluorescence

Possible cause Solution
Incorrect PCR setup Contact support. Templates can be provided.
Incorrect consumables Use recommended consumables. List of consumables in device manual.
Incorrect transport medium Use TE buffer or Nuclease-free water.

 

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SAMPLE – issue with low fluorescence

Possible Cause Solution
Insufficient extraction Use validated extraction.
Unvalidated specimen Check list of validated specimen in IFU.
Mishandling with sample Check sampling and sample storage in IFU.
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SAMPLE – issue with non-sigmoidal curves

Possible Cause Solution
Invalid curves caused by software Contact support.
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SAMPLE – false positive sample

Possible Cause Solution
Contamination Clean workspace.
Cross-talk Contact support.
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INTERNAL CONTROL – fail of internal control

Possible Cause Solution
Incorrect volume Add 10 % of elution volume to sample before extraction.
Degraded IC Store at -20 °C, thaw at 2 - 8 °C no longer than 15 min.
Insufficient extraction Use validated extraction.
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INTERNAL CONTROL – fail of internal control + positive sample

Possible cause Solution
Possible competition Normal - still valid result.
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INTERNAL CONTROL – fail of internal control + negative sample

Possible cause Solution
Failed extraction Use validated extraction. Repeat whole analysis.
PCR inhibition Repeat analysis of failed samples.
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NEGATIVE CONTROL – positive signal

Possible Cause Solution
Contamination Clean workspace.
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POSITIVE CONTROL – negative signal

Possible Cause Solution
Degraded Positive Control Store at -20 °C, thaw at 2 - 8 °C no longer than 30 min. Vortex and short spin before use.
Degraded MasterMix Store at -20 °C, thaw at 2 - 8 °C no longer than 30 min. Vortex and short spin before use.
All

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POSITIVE CONTROL – signal in HEX channel

Possible Cause Solution
Cross-talk Set threshold (TH) above curve in HEX.
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CALIBRATORS – issue with abnormal Ct

Possible Cause Solution
Incorrect pipetting Repeat PCR. Valid calibration curve values - R^2 ≥ 0,99, SLOPE between -3,8 to -3,1.
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