Evaluation of Peptide Nucleic Acid Probe-based Real-time PCR for Detection of Mycobacterium tuberculosis Complex and Nontuberculous Mycobacteria in Respiratory Specimens

Abstract

BackgroundA peptide nucleic acid (PNA) probe-based real-time PCR (PNAqPCR™ TB/NTM detection kit; PANAGENE, Korea) assay has been recently developed for the simultaneous detection of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) in clinical specimens. The study was aimed at evaluation of the performance of PNA probe-based real-time PCR in respiratory specimens.MethodsTo evaluate potential cross-reactivity, the extracted DNA specimens from Mycobacterium species and non-mycobacterial species were tested using PNA probe-based real-time PCR assay. A total of 531 respiratory specimens (482 sputum specimens and 49 bronchoalveolar washing fluid specimens) were collected from 230 patients in July and August, 2011. All specimens were analyzed for the detection of mycobacteria by direct smear examination, mycobacterial culture, and PNA probe-based real-time PCR assay.ResultsIn cross-reactivity tests, no false-positive or false-negative results were evident. When the culture method was used as the gold standard test for comparison, PNA probe-based real-time PCR assay for detection of MTBC had a sensitivity and specificity of 96.7% (58/60) and 99.6% (469/471), respectively. Assuming the combination of culture and clinical diagnosis as the standard, the sensitivity and specificity of the new real-time PCR assay for detection of MTBC were 90.6% (58/64) and 99.6% (465/467), respectively. The new real-time PCR for the detection of NTM had a sensitivity and specificity of 69.0% (29/42) and 100% (489/489), respectively.ConclusionsThe new real-time PCR assay may be useful for the detection of MTBC in respiratory specimens and for discrimination of NTM from MTBC.

Artificial Nucleic Acid Probes and Their Applications in Clinical Microbiology

“…Their second product, the PANA qPCR TB/NTM Detection kit, is a simple, one-stop kit which extracts the microbial DNA from several different clinical sample types and using standard qPCR methods, where the molecular beacon has been replaced with a PNA probe, enables the discrimination of TB from NTM in roughly 3 hours while achieving >99% specificity for both targets. However, in one study, sensitivity reached only 69% for NTM but overall, this kit demonstrated similar performance characteristics to other nucleic acid amplification-based assays (Choi et al, 2012). A more recent study comparing the Cobas TaqMan MTN assay (Roche Diagnostics) with Panagene's system found that the performance of the PNA-based assay is superior to the TaqMan assay (Kim et al, 2013).…”

“…Choi and co-workers developed a highly specific assay that utilised fluorescent PNAs to detect and discriminate among M. tuberculosis and non-tuberculous mycobacteria in clinical specimens. Application of the assay to 531 patient specimens, demonstrated no Type-I or Type-II errors and a specificity of 100% and 99.6%, for non-tuberculous Mycobacteria and M. tuberculosis, respectively (Choi et al, 2012). Though conceptually similar to standard real-time PCR-based approaches using molecular beacons, there are differences.…”

“…In addition, PNAs have found diagnostic uses as fluorescent probes used in PCR in place of ssDNAs (Choi et al, 2012) and as probes for in situ assays (Bonvicini et al, 2006;Wilson, Hall, & Procop, 2010), and microarrays (Choi, Kim, & Park, 2009;Raymond et al, 2005) proving that PNAs have application in range of assays and offer unique advantages in the next generation of diagnostic platforms.…”

Evaluation of Peptide Nucleic Acid Probe-Based Fluorescence In Situ Hybridization for the Detection ofMycobacterium tuberculosisComplex and Nontuberculous Mycobacteria in Clinical Respiratory Specimens

“…However, the AFB stain yields poor positive predictive values for TB in clinical settings in which NTM is frequently isolated, because it does not allow differentiation MTB from NTM [5]. Recently, several investigators have used peptide nucleic acid (PNA) probes for the identification of MTB and NTM in various specimens, in- cluding cultures, sputum, and tissue [6][7][8][9][10][11][12]. PNA probes are artificially synthesized DNA analogues with an uncharged peptide backbone, which have more favorable hybridization properties and chemical, thermal, and biological stability than previous probes [13,14].…”

Development of a Peptide Nucleic Acid-Based Multiprobe Real-Time PCR Method Targeting the hsp65 Gene for Differentiation among Mycobacterium abscessus Strains

“…A PNA probe-based real-time PCR assay has been developed for the diagnosis of mycobacterial infections, particularly for distinguishing between Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM) in clinical specimens (13,14 T , and M. xenopi ATCC 19250 T ), were used in this study. All clinical isolates were collected from the Asan Medical Center (Seoul, Republic of Korea) during the period from January 2004 to June 2011.…”