What should be done if the amplification curve of a molecular diagnostic kit rises late, and the negative control also shows a slight rise?

First, suspect extraction inhibitors. Even if the nucleic acid concentration of sputum samples is normal, inhibitors such as mucin and polysaccharides may remain. The positive nucleic acid template can be diluted 1:2 and 1:5 and rerun. If the Ct value does not shift back proportionally or the signal rises significantly, it indicates the presence of inhibitors. A peak appearing 2-3 cycles late already requires investigation, and it is not recommended to issue a report directly. For handling, the number of wash steps can be increased, magnetic bead extraction can be used instead, or an amplification system with better tolerance to inhibitors can be selected. If necessary, re-extract the same batch of samples and then run them on the instrument again.

The negative control showed slight elevation after 38 cycles. First, check the melting curve to determine whether it is primer dimers or nonspecific amplification; meanwhile, set up 3-5 replicate wells for the NTC and negative control. If the melting peak is inconsistent with that of the positive product, it is mostly background or dimers; if it has the same peak as the positive, be alert to aerosol contamination. For handling, implement zoning, change the water or primers/probes, inspect the pipettes and clean bench, and a UNG system is better. If CT exceeds the cutoff but the trend is abnormal, it should also be recorded; do not judge as negative based only on the result.

Don't focus only on the kit. Although instrument calibration has been performed, the heated lid temperature, reaction tube lot, and sealing film integrity can also cause an overall Ct shift to the right. Check whether the final reaction volume is too low, whether the sealing film is leaking, and whether the ROX reference is abnormal. If the same lot of reagent is subjected to temperature fluctuations during storage or repeated freeze-thaw cycles, its performance can also decline. Take a new lot or an unopened kit from the same lot and run it in parallel with the same quality control sample; if the new kit performs normally, it indicates that the current kit may have storage or transport issues. If the new kit also shows a right shift, then investigate the extraction and template.