Repurposing Residual Eluate from TrueNat™ / TruePrep™ MTB/RIF/MTB Plus Testing for Zoonotic Tuberculosis Surveillance Using Species-Specific Real-Time PCR

Abstract

This protocol describes a novel, resource-efficient strategy to extend diagnostic output from the WHO-endorsed TrueNat™ platform by utilizing residual DNA eluate—typically discarded post-MTBC detection—for species-specific identification of zoonotic TB pathogens. By applying a two-step real-time PCR based on Masanga et al. (2023), we enable downstream screening for M. bovis, M. caprae, and M. orygis from the same clinical specimen, aligning with One Health surveillance objectives.

Background

TrueNat™ is extensively used in India’s National TB Elimination Program (NTEP) for detecting Mycobacterium tuberculosis complex and Rifampicin resistance. It generates ~90 µL of DNA eluate, of which only 12 µL is used for diagnosis. The remaining eluate remains underutilized despite retaining high-quality DNA suitable for further molecular assays. This protocol outlines a practical workflow to harness this eluate for zoonotic MTBC subspecies identification, bridging a critical diagnostic gap.

Materials and Reagents

Equipment

• Real-time PCR system (e.g., Bio-Rad CFX96, Applied Biosystems QuantStudio)
• Class II Biosafety Cabinet (for eluate handling)
• Microcentrifuge and PCR-grade pipettes
• Cold storage (4 °C or –20 °C freezer for eluates and primers)
• Vortex and mini-spin centrifuge
• PCR tubes or optical plates compatible with your qPCR platform

Procedure Overview

Step 1: Eluate Collection and Storage

1. Following TrueNat assay completion, collect the residual eluate (~78 µL) using a PCR-grade pipette.
2. Transfer to a sterile, nuclease-free microcentrifuge tube.
3. Store at 4 °C for up to 48 hours, or –20 °C for longer durations. Avoid freeze-thaw cycles.

Step 2: Primer Design and Reaction Setup

1. Initially, use specific primers targeting IS1081, MTBC Animal and MTBC Human strains; if the MTBC Animal target is positive, proceed with primers specific to Mycobacterium bovis, Mycobacterium caprae, and Mycobacterium orygis as described by Masanga et al. (2023).

    

2. Prepare PCR reactions (total volume: 20 µL) for the first set in multiplex format, containing:

• 10 µL qPCR master mix
• 0.5 µL primer-probe mix - IS1081 (10 µM) - FAM
• 0.5 µL primer-probe mix - MTBC Animal (10 µM) - TxRed
• 0.5 µL primer-probe mix - MTBC Human (10 µM) - Cy5
• 5 µL eluate DNA
• 3.5 µL nuclease-free water

3. Prepare PCR reactions (total volume: 20 µL) for the second set in multiplex format - if MTBC Animal is detected, containing:

• 10 µL qPCR master mix
• 0.5 µL primer-probe mix - M orygis (10 µM) - FAM
• 0.5 µL primer-probe mix - M caprae (10 µM) - TxRed
• 0.5 µL primer-probe mix - M bovis (10 µM) - Cy5
• 5 µL eluate DNA
• 3.5 µL nuclease-free water

Step 3: Amplification Protocol

1. Run each target in multiplex.
2. Suggested thermocycling conditions:

• Initial denaturation: 95 °C for 3 min
• 40 cycles of:
  • Denaturation: 95 °C for 15 sec
  • Annealing/Extension: 60 °C for 30 sec - Camera detection ON

Step 4: Controls and Verification

• Include NTC (no template control), positive control DNA, and extraction negative controls.
• Confirm probe-specific signal.

Data Analysis

• Positive amplification in IS1081 region indicates MTBC presence (see Masanga et al, 2023 for full decision matrix)
• Ct values <30 are considered positive; optimize based on lab threshold.
• Compare with known control profiles for confirmation.
• Optional: calculate diagnostic yield using retrospective eluates from known TB cases.

Anticipated Results

When applied to residual DNA eluates from the TrueNat platform, this protocol is expected to:

• Detect MTBC subspecies (e.g., M. bovis, M. caprae, M. orygis) with species-specific real-time PCR signals
• Generate amplification curves with Ct values <30 for positive samples, matching the expected fluorophore signals
• Demonstrate compatibility of eluate quality with secondary PCR, even after 24–48 hours of storage at 4 °C
• Identify zoonotic strains in previously classified TB-positive eluates, enabling retrospective surveillance opportunities

We anticipate allowing accurate species identification based on the decision matrix in Masanga et al. (2023). Positive results can prompt source-tracing efforts and policy-level interventions.

Troubleshooting

Acknowledgments

This protocol was conceptualized in alignment with India’s One Health vision and the National TB Elimination Program. We thank colleagues at Mahatma Gandhi Institute of Medical Sciences - MGIMS, Datta Meghe Institute of Higher Education and Research - DMIHER (both Sawangi and Wanadongri campus) and District TB Officer - DTO, Wardha, Maharashtra for access to TrueNat workflows and initial feedback on eluate stability. We acknowledge Masanga et al. (2023) for providing a robust species-specific PCR platform, which forms the basis of our screening strategy. This protocol is intended to serve as an open-access framework for scaling low-cost zoonotic TB surveillance.