Single-tube, nested, reverse transcriptase PCR for detection of viable Mycobacterium tuberculosis

Abstract

Several problems remain before molecular biology-based techniques, such as PCR, are widely accepted for the detection of infectious agents. Among the most formidable of these problems are the inability of the tests to distinguish between viable and nonviable organisms. We approached this problem by using the fact that bacterial mRNA has an extremely short half-life, averaging only a few minutes. We reasoned that by targeting bacterial mRNA by a reverse transcriptase PCR (RT-PCR), a positive signal would indicate the presence of a recently viable organism. To test our hypothesis, we chose to target the mRNA coding for the ubiquitous 85B antigen of mycobacteria. After partially sequencing the gene coding for 85B, we developed primers that were specific for Mycobacterium tuberculosis. In a single-tube, nested, RT-PCR (STN RT-PCR), these primers detected fewer than 40 CFU in spiked sputum samples and as few as 12 CFU in clinical sputum specimens. The sensitivity of STN RT-PCR with smear-negative samples was as good as that of culture. The specificity was 100%. More importantly, when M. tuberculosis was cultured with and without 1 g of isoniazid per ml, this assay could distinguish between those cultures which contained the antibiotic and those which did not. Subcultures on Lowenstein-Jensen agar confirmed the viability assessments of the STN RT-PCR. Control experiments demonstrated that isoniazid did not inhibit the RT-PCR. In addition, when an IS6110-targeted, DNA PCR was used to examine the same samples, all samples through 13 days (the last sample) continued to be positive, irrespective of whether isoniazid was present, thereby demonstrating the superiority of an mRNA target in the detection of mycobacterial viability.

PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings

“…128,129 In some clinical situations, detection of RNA species by RT-PCR has been shown to correlate well with the presence of viable organisms and has been effectively used to monitor antibiotic therapy. [130][131][132][133][134] Clinical application of RNA-based approaches will need further improvement, however, because they have been hampered in development by difficulties in extracting detectable concentrations of intact RNA from small numbers of bacteria.…”

Elimination of guttural pouch infection and inflammation in asymptomatic carriers ofStreptococcus equi

“…bi. PCR gave positive results for 71 weeks with samples from patients regarded as treated successfully for tuberculosis (Jou et al 1997). A pragmatic approach was taken in the control of the outbreak as it was highly unlikely that these horses posed an infectious risk, given the absence of pouch pathology, the intensity of previous treatment and the subsequent inability to culture S. equi on repeated swabs or lavages over a prolonged period.…”

Day zero quantitative mRNA analysis as a prognostic marker in pulmonary tuberculosis category II patients on treatment

“…Following reverse transcription, amplification was performed in a nested manner using primers described by NT Jou et al. [8]. The additional details of PCR amplification are provided in the online data supplement.…”

“…RNA was extracted as per the protocol published by Jou et al. [8] with certain modifications. To rule out any contaminating DNA, the RNA was treated with RNase – free DNase I (New England Biolabs Inc., Ipswich, MA, USA).…”