Simplified Co-extraction of total Nucleic Acids from Respiratory Samples for detection of Mycobacterium tuberculosis and SARS-CoV-2 optimized for compatibility across Diagnostic Platforms

Tuberculosis (TB) and COVID-19 are leading infectious diseases with high mortality, caused by Mycobacterium tuberculosis ( Mtb ) and SARS-CoV-2 (SC2) , respectively. Co-infection is common but is often undiagnosed as it is challenging to process both pathogens from a single sample. In this study, we present a simple and efficient method for co-extracting nucleic acids (NA) from these two distinct respiratory pathogens for downstream diagnostic testing. We evaluated three different nucleic acid amplification (NAA)-based platforms, LightCycler480 (LC480) qPCR, Qiacuity digital PCR (dPCR), and Cytation3 for CRISPR-Cas13a-based SHINE-TB/SC2 detection assays. Chelex-100 chelating resin-based boiling preparation method was optimized for Mtb NA extraction from saliva and sputum. Saliva showed compatibility with all three platforms, with sensitivity as low as 100 CFU/ml (or 2 genomic copies/µl). This method worked well for sputum using dPCR at 100% (21/21) positivity, though the CRISPR-based SHINE-TB assay showed more variability and sensitivity to sputum inhibitor carry-over, resulting in an 81% positive rate (17/21). Diluting sputum with TE buffer (1:1) improved the detection (2/4). Extraction efficiency of our method was 48%, 62.2%, 86.4% and 99.3% for concentrations 10 ⁵ , 10 ⁴ , 10 ³ and 10 CFU/ml, respectively. The dynamic range for Mtb spiked in pooled sputum showed 100% detection (N=8) at ≥10 ³ CFU/ml with all three methods. Dual-pathogen co-extraction and detection of SC2 (10 ⁵ PFU/ml) and Mtb (10 ⁵ CFU/ml) in salivary sputum was successful using CRISPR-Cas13a assays. We have developed a rapid and efficient co-extraction method for multi-pathogen testing across diagnostic platforms and believe this is the first protocol optimized to co-extract Mtb and SARS-CoV-2 from a single sample.