Processing of Sputum Sample before Truenat Testing

The sputum sample needs to be processed to liquefy and lyse the specimen before Truenat testing. 

• Sputum samples contain Polymerase Chain Reaction (PCR) inhibitors but PCR amplification requires pure DNA from sputum samples.
• The sputum sample should be homogenized and pipettable before DNA extraction can begin.
• Pre-treatment also decontaminates specimen for storage/ transportation/ extraction.

The Trueprep AUTO MTB Sample pre-treatment pack is used for processing the specimen. It digests sputum, releases bacteria, and removes PCR inhibitors.

Steps for specimen pre-treatment include:

1. Open a Trueprep AUTO MTB sample pre-treatment kit which contains a graduated 1 ml transfer pipette, lysis buffer bottle and liquefaction buffer bottle.
2. The sample and all reagents including the liquefaction buffer,  lysis buffer should be at room temperature before starting the steps of pre-treatment.
3. Switch on the Trueprep AUTO v2 sample prep device.
4. Label lysis buffer bottle with corresponding sample number and date of extraction.
5. Add 2 drops of liquefaction buffer to the sputum sample, swirl mix, incubate for 10 minutes at room temperature. If the sample is not pipettable after 10 minutes, incubate for another 5 minutes and swirl at 2 minutes intervals.
6. Transfer 0.5 ml of the liquefied sputum sample from the sample container to the corresponding lysis buffer bottle using the 1 ml graduated transfer pipette provided.
7. Dispose of the used transfer pipette into the container filled with freshly prepared 1% sodium hypochlorite.
8. Add 2 drops of liquefaction buffer into the lysis buffer bottle; swirl gently to mix and incubate the lysis buffer bottle at room temperature for 3-5 minutes.
9. Ensure that the sample has completely liquefied.
10. Sputum may be stored in lysis buffer for up to 1 week at 30°C with no degradation of DNA.

Resources

• Trueprep AUTO MTB Sample Pre-treatment Pack.
• Practical Guide to Implementation of Truenat™ Tests for the Detection of TB and Rifampicin Resistance.

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