5. How to determine false negative and false positive results?
2026-03-24 11:18
5.1 False Negative Judgment
There are many causes for false negative results, mainly characterized by no FAM amplification curve, including sample inhibition, nucleic acid extraction failure, gene mutation and instrument malfunctions. Most mainstream domestic PCR reagents do not have internal control monitoring function, and the FAM amplification curve for target gene detection is used for both quantification and qualitative analysis (negative/positive judgment), so the quality of FAM amplification curve is critical.
For reagents with competitive internal control monitoring function, the following four scenarios may occur:
① No FAM amplification but normal internal control amplification: Normal result, judge the sample as negative;
② No FAM amplification and no internal control amplification: Abnormal result, possibly caused by nucleic acid extraction failure or inhibition, retest is mandatory;
③ Normal FAM amplification and normal internal control amplification: Normal result, as the target nucleic acid and internal control are amplified in the same reaction system;
④ Normal FAM amplification but no internal control amplification: Normal result, highly positive samples will inhibit internal control amplification, leading to weak or negative internal control results.
5.2 False Positive Judgment
False positive results are relatively rare in PCR detection, caused by reagent contamination, aerosol contamination, operational contamination, amplification product contamination, non-specific amplification, consumable contamination, etc. Therefore, it is recommended to set negative controls to monitor the presence of contamination.
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