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  • Truenat

    Content

    Truenat is an indigenous rapid molecular test platform that is currently under use in NTEP for diagnosis of TB and Rif Resistance. It is a platform utilising real-time Polymerase Chain Reaction (PCR) technology built into micro-PCR chips.

    Testing on Truenat involves three components:

    1. Workstation (consisting of 2 devices)
      • Trueprep AUTO Universal Cartridge-based Sample Prep Device for the automated extraction and purification of DNA
      • Truelab Real-time micro PCR Analyzer for performing real-time PCR. It is available as 1 (Uno), 2 (Duo) or 4 (Quattro) chip ports.
    2. Cartridge and Chip
    3. Reagent kits (Sample Pre-treatment and Prep kits)

      Figure: Truenat  Source: MolBio Products.

      Test results for MTB detection and Rif Resistance has a turn around time of 1-2 hours. Depending on the micro-PCR chips used various tests can be performed using Truenat. Truenat MTB micro-PCR chips detect Mycobacterium tuberculosis bacteria for TB diagnosis. Truenat MTB RIF micro-PCR chip is used as a reflex test to detect resistance to Rifampicin (RIF), the first-line drug for TB treatment

      Truenat has many advantages. Truenat is designed to be mobile and is battery operated (~8 hours on full charge). It can be deployed in peripheral laboratories and microscopy centres with minimal or no added facilities and hence it is more point-of-care. Biosafety requirements are similar to smear microscopy. However, it is multi staged and partially automated, requiring the presence of a Lab Technician through out the test.

      Resources

      1. Truenat MTB Kit Insert.
      2. Trueprep AUTO Universal Cartridge-based Sample Prep Device.
      3. Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin-resistance, 2021.

       

      Assessment

      Question​

      Answer 1​

      Answer 2​

      Answer 3​

      Answer 4​

      Correct answer​

      Correct explanation​

      Page id​

      Part of Pre-test​

      Part of Post-test​
      Truenat is used in NTEP for: MTB detection Rif Resistance Detection INH resistance Detection MTB and Rif Resistance Detection 4 Truenat is used for MTB and Rif Resistance detection in NTEP   Yes Yes

      The Truelab Analyzer is available in how many chip ports?

      2 (Duo)

      1 (Uno), 2 (Duo) and 4 (Quattro)

      1 (Uno)

      4 (Quattro)

      2

      The Truelab Analyzer is available as 1 (Uno), 2 (Duo) and 4 (Quattro) chip ports.

      ​

      		 Yes Yes

    • Truenat Cartridge and Inside Demonstration

      Content

      Truenat cartridges are components of Trueprep AUTO v2 Universal Cartridge Based Sample Prep Kit. As shown in the figure below, each cartridge is:

      • Used with Trueprep Device for DNA extraction and purification
      • Disposable
      • Single-use
      • Preloaded with Internal Positive Control.

      Truenat cartridges are used with Trueprep devices for DNA extraction and purification. The components of the cartridge are illustrated in the following figure and include:

      • Sample Chamber - Processed samples (pretreated with lysis buffer) are added to the sample chamber of the cartridge for processing on the Trueprep device.
      • Matrix Chamber - DNA released by chemical and thermal lysis cells binds to the proprietary matrix here.
      • Elute Chamber - Trapped DNA is washed with buffers to remove Polymerase Chain Reaction (PCR) inhibitors and is eluted from the matrix using the elution buffer. The purified DNA is collected from here.
      • Waste generated is contained within the dump region inside the cartridge.

      Figure: Inside of a TrueNAT Cartridge; Source: Practical Guide to Implementation of TrueNAT Tests for the Detection of TB and Rifampicin Resistance

      Resources

    • Consumables Required at a Truenat Lab

      Content

      Consumables required for Truenat test provided by the manufacturer include:

       

      Trueprep AUTO MTB Sample Pre-treatment Pack for sample processing

      1. Liquefaction buffer
      2. Lysis buffer
      3. Disposable transfer pipette (graduated) - 1ml

      Trueprep AUTO v2 Universal Cartridge Based Sample Prep Kit for DNA extraction

      1. The Reagent Pack contains the following reagents
        1. Wash Buffer A
        2. Wash Buffer B
        3. Elution Buffer
        4. Priming Waste
      2. The Cartridge Pack contains the following
        • Cartridge
        1. Elute collection tube
        2. Elute collection tube label
        3. Disposable transfer pipette
      3. Disposable Transfer Pipettes (graduated) - 3 ml
      4. Reagent Reset Card

      Truenat MTB Chip-based Real-Time PCR test for Mycobacterium tuberculosis

      1. Truenat MTB micro-PCR chip
      2. Microtube with freeze-dried PCR reagents
      3. DNase & RNase free pipette tip

      Truenat MTB-RIF Dx Chip-based Real-Time PCR Test for Rifampicin Resistant Mycobacterium tuberculosis

      1. Truenat MTB-RIF Dx micro-PCR chip
      2. Microtube with freeze-dried PCR reagents
      3. DNase & RNase free pipette tip

      Other Consumables

      1. Gloves
      2. Masks
      3. Sodium hypochlorite
      		  

       

         

          Resources

           

           

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        1. Overview of the Truenat Testing Process

          Content

          The Truenat Assay Technology works on Real-time Polymerase Chain Reaction (PCR).

           

          The key steps include:

          1. Collection of specimens from a presumptive TB/known TB Patient (for UDST).
          2. Liquefaction and lysis of specimen using the Trueprep AUTO MTB sample pre-treatment pack (20 minutes)
          3. Extraction and purification of DNA using Trueprep AUTO v2 Universal Cartridge-based sample prep kit and Trueprep AUTO v2 Universal Cartridge-based sample prep device (20 minutes)
          4. Amplification of extracted DNA by the Truelab Real-time micro-PCR analyzer using freeze dry PCR reagent in microtubes on a Truenat MTB chip (35 minutes)
          5. DNA from positive test results is tested using Truenat MTB RIF Dx chip as a reflex test (55 minutes)

           

          The process flow from sample to result for Truenat Assay Technology is described in the figure below.

           

          Figure: Process flow from sample to result for Truenat Assay Technology; Source: Practical Guide to Implementation of Truenat™ Tests for the Detection of TB and Rifampicin Resistance.

           

          Resources

           

           

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        2. Truenat MTB and RIF Assay as a TB Diagnostic Test

          Content

           

          The tests are performed using Trueprep AUTO Sample Pre-treatment and Prep kits and Truenat micro-PCR chips.

           

          To detect M. tuberculosis, the Truenat MTB chip amplifies a portion of the ribonucleoside-diphosphate reductase gene, nrdB with a Limit of Detection (LOD) of about 100 Colony Forming Units (CFU)/ml sputum sample.

           

          DNA extraction and detection of M. tuberculosis takes approximately one hour.

           

          When M. tuberculosis is detected, as a follow-on test, a small volume of the already extracted DNA is used to test for resistance to Rif using Truenat MTB Rif Dx chip.

           

          Mutations associated with Rif-resistance within the RRDR region of rpoB gene are detected using a probe melt assay and read using the Truelab micro-PCR Analyzer.

           

          The detection of Rif-resistance takes an additional one hour.

           

          Truenat Testing Capacity

          • The Truelab Analyzer is available as 1 (Uno), 2 (Duo) or 4 (Quattro) chip ports (figure below).
          • The different ports can be used independently to test multiple samples.
          • In an eight-hour daily work shift, the estimated throughput of Truelab Analyzer Uno, Duo or Quattro can be 7-9, 15-18, 30-36 specimens, respectively.

           

          Results Reporting 

          A Truelab micro PCR printer (figure below) is used to print test results. It can also be connected via SIM card/ Wi-Fi/ Bluetooth to transmit results.

           

          Figure: Different Equipments used in the Truenat Workstation

           

           

          Resources

           

           

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        3. Inbuilt Controls used in TrueNAT 

          Content

          Truenat tests use Internal Quality Control to assess test validity using Internal Positive Control (IPC).

           

          • The cartridge contains pre-loaded IPC.
          • IPC is part of the full process control.
          • IPC undergoes all processing steps along with specimen from extraction to amplification.
          • IPC assesses the validity of the test run from sample processing to result and is illustrated in following figures (Figure 1(A) and Figure 1(B)).

           

          Figure 1(A): Amplification of IPC in Valid Test; Source: MolBio Diagnostics Pvt. Ltd.

           

           

          Figure 1(B): Invalid Test with Failure of IPC Amplification; Source: MolBio Diagnostics Pvt. Ltd.

           

           

          Resources

           

           

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