Ch 02: Specimen processing for Truenat

The sputum sample needs to be processed to liquefy and lyse the specimen before Truenat testing. This is because of the following reasons:

• 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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The extra-pulmonary samples (Non-sputum) can also be processed and used for MTB and Rif diagnosis using Truenat. The processing of various extra-pulmonary samples is described below.

 

Reagents required:

Trueprep AUTO MTB Sample Pre-treatment Pack for sample processing

 

• Reagents stable for two years (2-40°C) or one month (temperatures up to 45°C)
• Avoid exposure to light or elevated temperatures
• Do not freeze

 

Steps in processing of non-sputum samples

Non-sputum samples like tissue biopsy should not be processed at the Designated Microscopy Centre (DMC), but should be transferred to a higher centre.

 

General Instructions

1. Disinfect work surfaces with freshly prepared 1% bleach, followed by 70% alcohol.
2. Process fresh specimens immediately or store frozen at -20^oC. Avoid more than three freeze thaws.
3. Bring frozen samples and refrigerated reagents to room temperature (20-30°C).
4. Open the Trueprep AUTO MTB sample pre-treatment pack that contains:
   • Liquefaction buffer bottle
   • Lysis buffer bottle
   • Graduated 1 ml transfer pipette.
5. Processing is to be done as instructed for each sample type.
6. Non-sputum sample is stable for 3 days at up to 40°C and 1 week at 30°C in the lysis buffer.

 

A. Processing for Bronchoalveolar Lavage (BAL), Pleural fluid, Peritoneal fluid 

 

Equipment: Centrifuge and centrifuge tubes (10-15 ml volume)

1. Add 5-10 ml specimen in a centrifuge tube.
2. Centrifuge at 4000x for 5 minutes to concentrate the sample.
3. Discard the supernatant until 500 μl remains.
4. Add two drops of the liquefaction buffer to the concentrated sample (500 μl).
5. Transfer the contents to a labelled lysis buffer tube.
6. Incubate for five minutes and proceed for DNA extraction.

 

B. Processing for Pus, Abscess, Lymph node aspirate, Cerebrospinal Fluid (CSF)

Equipment: Centrifuge tubes (1.5 ml volume)

1. Add 0.5 ml specimen in a 1.5 ml tube.
2. Add two drops of liquefaction buffer.
3. Transfer the contents to a labelled lysis buffer tube.
4. Incubate for 5 minutes and proceed for DNA extraction.

​

C. Processing for Tissue/ Biopsy Samples 

Equipment: Mortar and pestle (for tissue homogenization)

1. Homogenize sample by using 100 μl lysis buffer in mortar and pestle.
2. Add two drops of liquefaction buffer.
3. Transfer the contents to a labelled lysis buffer tube.
4. Incubate for five minutes.
5. Use only clear fluid for DNA extraction.

The processed samples will be used for DNA extraction and PCR amplification for diagnosis of MTB and Rif resistance.

Resources

 

• Trueprep AUTO MTB Sample Pre-treatment Pack.

 

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Truenat Mycobacterium tuberculosis (MTB) and Truenat MTB Rifampicin (Rif) Dx Assays amplify target genes through Polymerase Chain Reaction (PCR).

 

The presence of mutant gene is detected using probe melt assay.

 

The target genes include:

• Truenat MTB - ribonucleoside-diphosphate reductase gene nrdZ (the precursor for DNA synthesis) 
• Truenat MTB Rif Dx assay – Rifampicin Resistance Determining Region (RRDR) region of the RNA polymerase Beta (rpoB) gene for resistance to Rif (between codon positions 509 and 533)

Figure: Image showing RRDR of the rpoB gene of M. tuberculosis; the target gene of Truenat MTB Rif Dx assay includes codons 509-533

 

 

 

Resources

 

• Truenat MTB Pack Insert.
• Truenat MTB Rif Pack Insert.
• Practical Guide to Implementation of TrueNAT Tests for the Detection of TB and Rifampicin Resistance.

 

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Equipment and Consumables

• Trueprep AUTO v2 Universal Cartridge Based Sample Prep Device
• Trueprep AUTO v2 Universal Cartridge Based Sample Prep Kit

 

Steps in DNA extraction (Molbio Trueprep Auto Universal Cartridge Based Sample Prep Device)

1. Wear gloves, mask.

2. Insert the plug-in connector to attach the reagent pack to the Trueprep device (Figure 1).

Figure 1: Trueprep Cartridge Based Sample Prep Kit attached to the Trueprep Auto Device using the Plug-in Connector; Source: Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance

 

 

3. Disinfect the work surfaces with the freshly prepared 1% bleach, followed by 70% alcohol.

4. Open the cartridge pouch, take out the cartridge.

5. Label the cartridge (Patient ID, Date), place it on the cartridge stand.

6. Open the black cap of the cartridge sample chamber.

7. Using a pipette transfer 3 ml contents of the lysis buffer bottle (processing step) in the sample chamber.

8. Discard the pipette and lysis buffer bottle (1% sodium hypochlorite).

9. Re-cap the sample chamber.

10. Press the “Power” button to switch on the Trueprep device.

11. Press “Eject” to eject the cartridge holder (Figure 2), pull out gently.

Figure 2: Top view of the Trueprep AUTO Universal Cartridge-based Sample Prep Device; Source: Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin Resistance

 

 

12. Insert the cartridge in the cartridge holder (sample chamber to the right when seen from the front).

13. Push to close the cartridge holder (the click sound confirms the correct cartridge loading).

14. Press the “Start” button (Figure 2).

15. DNA extraction begins, reagents from bottles are automatically added to the cartridge (pre-programmed) (Figure 2).

16. On completion (18-20 min), the cartridge holder automatically ejects.

17. Remove the cartridge, place it on the cartridge stand.

18. Label the ECT tube (Elute Collection Tube) with Patient ID & Date.

19. Pierce the covering of elute compartment in the cartridge with a filter barrier pipette tip, aspirate elute (DNA) into the ECT tube.

20. Discard the used pipette tips, used cartridge (1% sodium hypochlorite).

21. DNA can be used immediately for amplification/ stored in fridge (4°C for up to 24 hours) or -20°C (for up to 1 year).

Resources

 

• Practical Guide to Implementation of TrueNAT Tests for the Detection of TB and Rifampicin Resistance.
• Operation of Molbio Trueprep AUTO Universal Sample Prep Device.
• Molbio Trueprep Auto Universal Cartridge-based Sample Prep Device.

 

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