Nucleic acid isolation from respiratory samples

General recommendations for sample preparation

Swabs (e.g., nasopharyngeal or oropharyngeal)

• Dry swabs (without viral transport media)
  Rinse swabs with moderate shaking in 400–500 μl of sterile PBS for 30 min to release sample material from the swab (swab heads should be completely submerged in PBS). Transfer the appropriate sample volume of the rinse solution (e.g., 150–400 μl) to a suitable reaction container/tube according to the corresponding kit's user manual and proceed with the standard protocol, starting with the sample lysis step.
• Wet swabs (with viral transport media)
  Rinse swabs for 30 min with moderate shaking in viral transport media to release sample material from the swab. Transfer the appropriate sample volume (e.g., 150–400 μl) to a suitable reaction container/tube according to the corresponding kit's user manual and proceed with the standard protocol, starting with the sample lysis step.

Sputum/bronchoalveolar lavage (nonviscous)

Nonviscous, clear, and homogenous sputum and bronchoalveolar lavage samples can be used directly for nucleic acid extraction. Transfer the appropriate sample volume (e.g., 150–400 μl) to a suitable reaction container/tube according to the corresponding kit's user manual and proceed with the standard protocol, starting with the sample lysis step.

Sputum/bronchoalveolar lavage (viscous)

Viscous sputum and bronchoalveolar lavage samples should be liquefied before subjecting them to the nucleic extraction procedure. Transfer the appropriate sample volume (e.g., 150–400 μl) to a suitable reaction container/tube according to the corresponding kit's user manual. Add the respective amount of lysis reagents (e.g., lysis buffer, proteinase K, carrier RNA) to the sample and incubate at 70°C for 10 min with moderate shaking. Check if the sample is liquefied, allow the sample to cool down, and proceed with the binding step. If the sample is not liquefied after the heat incubation, follow the recommended guidelines of the CDC for processing sputum samples. Transfer the appropriate volume of the liquefied sample (e.g., 150–400 μl) to a suitable reaction container/tube according to the corresponding kit's user manual and proceed with the standard protocol, starting with the sample lysis step.

Recommended sample volumes for viral nucleic acid extraction