Comparison of diagnostic performance of three real-time PCR kits for detecting Mycobacterium species

Abstract

Purpose: PCR is widely used for rapidly and accurately detecting Mycobacterium species. The purpose of this study was to assess the diagnostic performance of three real-time PCR kits and evaluate the concordance with two older PCR methods.

Materials and methods: Using 128 samples, the five PCR methods were assessed, including an in-house PCR protocol, the COBAS Amplicor MTB, the COBAS TaqMan MTB, the AdvanSure TB/NTM real-time PCR, and the Real-Q M. tuberculosis kit. The discrepant results were further examined by DNA sequencing and using the AdvanSure Mycobacteria Genotyping Chip for complete analysis.

Results: For Mycobacterium tuberculosis (MTB) detection, all five kits showed 100% matching results (positive; N = 11 and negative; N = 80). In non-tuberculous mycobacterium (NTM) discrimination, the AdvanSure yielded two true-positive outcomes from M. intracellulare and one false positive outcome, while the Real-Q resulted in one true-positive outcome and one false negative outcome for each case and another false negative result using the provided DNA samples.

Conclusion: Real-time PCR, yielded results that were comparable to those of the older PCR methods for detecting MTB. However, there were disagreements among the applied kits in regard to the sample test results for detecting NTM. Therefore, we recommend that additional confirmatory measures such as DNA sequencing should be implemented in such cases, and further research with using a larger numbers of samples is warranted to improve the detection of NTM.

Fig. 1

In-house MTB PCR. Lane 1, size marker; Lane 2, internal control; Lane 7, positive; Lane 3-6, 8, negative. MTB, Mycobacterium tuberculosis.

Fig. 2

Two discrepant cases reaffirmed by sequencing and using the AdvanSure Mycobacteria Genotyping Chip (LG Life science, Seoul, Korea). (A) For 37 DNA samples provided, the AdvanSure kit detected all mycobacterial species and Real-Q kit showed a negative result for M. lentiflavum (target sequence; IS6110 region). (B) In contrast with the one false positive result from the AdvanSure kit, the Real-Q missed one true positive M. intracellulare in the clinical samples (target sequence; 16S-23S internal transcribed spacer region).