Which batch has a more stable lytic effect of proteinase K in virus nucleic acid extraction?

200 samples per day, changing suppliers cannot be based solely on price. I suggest identifying 2-3 companies first, using small package samples, and conducting parallel extraction using the same virus quality control sample or positive throat swab according to your extraction kit, with a focus on Ct values, repeatability, and RNA integrity after extraction. Protease K has both recombinant and natural sources, and the recombinant batch is more stable. The risk of animal contamination is also low, making it suitable for clinical samples. When testing, prepare 20 mg/mL according to the quality or the recommended concentration of the reagent kit. Do not replace based on volume, as different brands have different specific activities and formulas. We also need to set up a negative control without proteinase K to confirm the necessity of lysis. In terms of delivery time, domestic small packaging is generally available in stock at relatively low prices; Imported small package cold chain may take 1-2 weeks and the price is several times higher. It is recommended to include logistics records and arrival temperatures in the evaluation, as a cold chain break may affect activity. The final choice should be based on the stability of Ct values and supply continuity of your own sample, rather than solely relying on promotional data.

Adjust the incubation time and temperature according to your lysis buffer system. Common ranges are 55–65°C for 10–30 minutes; for throat swab samples, I usually start with 56°C for 15 minutes. You can set a temperature gradient of 50, 55, 60, and 65°C, and time points of 10, 20, and 30 minutes, keep the subsequent steps fixed, and compare Ct values and RNA yield. Longer time or higher temperature is not necessarily better, especially for RNA testing, where you need to prevent prolonged high temperature from affecting RNA integrity. After lysis, proteinase K must be thoroughly inactivated; many workflows use 95°C for 5–10 minutes or column removal, otherwise residual activity will inhibit qPCR. Throat swab pretreatment is also critical: the swab must be eluted thoroughly, and for samples with a lot of mucus, you can briefly centrifuge and take the supernatant before lysis; excessively high viscosity will slow proteinase K digestion. During optimization, pay attention to whether the lysis buffer changes from turbid to clear, and to the CV of Ct values across replicate wells. I generally consider a Ct value CV of less than 0.5 acceptable; if it exceeds that, check the lysis step and the enzyme lot.

I mainly look at the specific activity, purity, and nuclease residue of proteinase K, especially the residual DNase/RNase, which has a direct impact on the Ct value of virus nucleic acid extraction. Different brands may have different definitions of active units, and we cannot just compare numbers. We need to clarify the detection substrate and unit. In terms of batch stability, I suggest that the supplier provide historical CoA to observe the fluctuation range of specific activity, and request a nucleic acid enzyme residue detection report or statement. After arrival, perform parallel extraction with the same positive throat swab sample, repeat at least 3 times, and compare Ct values and amplification curves. Extract 200 throat swabs per day, buy small packages such as 100 mg or 1 g test batches first, do not switch to large quantities as soon as they arrive. There is a significant difference in the price of small packaging between imported and domestically produced products. The cold chain delivery time for imported products may be 1-4 weeks, while domestically produced products have more stock and lower prices, but it is important to test their viability. Cheap but not completely cracked, and a higher Ct value later becomes more troublesome.