(H-295) RT-PCR Detection of Fragmented Tuberculosis DNA in Urine

Introduction:: Tuberculosis (TB) was the leading cause of death from an infectious disease prior to the COVID-19 pandemic with an estimated 10.6 million cases in 2021 that resulted in 1.6 million deaths. Low- and middle-income countries are especially at risk, accounting for 98% of reported TB cases [1]. TB detection remains a significant diagnostic challenge. A standard of care in many settings is sputum smear microscopy. However, sputum is difficult to produce for those infected with HIV and children. The WHO has endorsed the PCR-based Cepheid GeneXpert MTB/RIF assay as a frontline diagnostic test for TB in endemic settings. Despite having a limit of detection of ~1,000 M.tb bacilli/mL in real world settings, GeneXpert is not ideal because it has a sensitivity of ~70% in smear negative TB patients and is relatively expensive (~$15/test after subsidies) [2]. Urine is an ideal patient sample because it is easily collected and available in large volumes [3]. Several TB urine-based diagnostic assays are currently approved by the WHO but suffer from poor sensitivity due to low biomarker concentrations and DNases which degrade cell free DNA into sequence fragments too short to be detected by PCR [4]. The steps in Figure 1 illustrate a novel method to capture and detect TB DNA fragments from high volume urine samples using magnetic-beads functionalized with synthetic RNA complementary to the TB biomarker IS6110. The captured DNA fragments are extended along the RNA by reverse transcription and then this cDNA is detected by PCR.

Materials and Methods:: A biotin modified RNA Ultramer^TM was synthesized (Integrated DNA Technologies, Inc.) with complementarity to a 105 base pair portion of the TB IS6110 region. A similar RNA strand using the sequence for the mouse gene GAPDH was synthesized for use as an internal spike control. PCR primers and probes were optimized for a high analytical sensitivity. In Figure 1 A, using 10⁸ copies of the RNA capture template and 10⁴ copies of a short IS6110 DNA fragment, reverse transcription was completed at 55ºC for 1 hour using Luna Universal Probe One-Step Reaction kit (New England Bio Labs, Inc., Cat. E3006). In Figure 1 C, the RT enzyme was then inactivated for 5 minutes at 95ºC. In Figure 1 D, a primer and probe mixture was added to the sample and qPCR was completed by cycling the samples between 95ºC for 15 seconds to melt and 60ºC for 30 seconds to anneal/extend while acquiring on the probe-specific fluorescence channels.

Results, Conclusions, and Discussions:: In preliminary studies, we were able to easily detect 10⁴ copies/reaction of TB DNA fragments, a copy number likely to be found in an infected individual’s urine sample [4]. A critical feature of this design is the inactivation of the reverse transcriptase enzymes after the RT step (Figure 1 C). If not inactivated, the addition of primers and a probe will produce a false positive during PCR (Figure 1 D). This inactivation was conformed since the no fragment samples tested negative (Figure 2).

TB RNA is not found in urine and the use of a synthetic RNA capture template is a novel approach to detect TB DNA fragments. This method has the potential for detecting small fragments of TB DNA concentrated from large volumes of urine. An advantage of this technology is the use of existing RT-qPCR kits. A disadvantage is that the current method requires opening the sample tub between steps C & D (Figure 1). Methods to implement this overall design without tube opening would be more desirable and are currently under development.


Acknowledgements (Optional): :

References (Optional): : [1] Tuberculosis. 27 Oct 2022. https://www.who.int/news-room/fact-sheets/detail/tuberculosis.

[2] Stevens, W. S., Scott, L., Noble, L., Gous, N., & Dheda, K. (2017). Impact of the GeneXpert MTB/RIF Technology on Tuberculosis Control. Microbiology Spectrum, 5, 1.

[3] Green, C., Huggett, J. F., Talbot, E., Mwaba, P., Reither, K., and Zumla, A. I. (2009) Rapid diagnosis of tuberculosis through the detection of mycobacterial DNA in urine by nucleic acid amplification methods. The Lancet. Infectious diseases 9, 505-511

[4] Amy Oreskovic, Norman D. Brault, Nuttada Panpradist, James J. Lai, Barry R. Lutz, (2019) Analytical Comparison of Methods for Extraction of Short Cell-Free DNA from Urine. The Journal of Molecular Diagnostics, 21, 1067-1078