Diagnostic evaluation of an in-house developed single-tube, duplex, nested IS6110 real-time PCR assay for rapid pulmonary tuberculosis diagnosis

Kenneth Siu Sing Leung, Gilman Kit Hang Siu, Kingsley King Gee Tam, Pak Leung Ho, Samson Sai Yin Wong, Eunice Ka Chun Leung, Shi Hui Yu, Oliver Chiu Kit Ma, Wing Cheong Yam

Research output: Journal article publicationJournal articleAcademic researchpeer-review

4 Citations (Scopus)


Objective: To perform a prospective evaluation on the diagnostic performance of an in-house developed, duplex nested IS6110 real-time Polymerase-Chain-Reaction (PCR) assay (IS6110-qPCR assay) for rapid pulmonary TB diagnosis. Methods: A total of 503 sputum specimens were prospectively collected from July 2016 to November 2016. Diagnostic accuracy and optimal cut-off Cycle-threshold (Ct) value for IS6110-qPCR assay was determined by Receiver Operating Characteristic (ROC) curve. Using the optimal cut-off Ct, diagnostic performance of IS6110-qPCR assay was assessed with reference to both bacteriological and clinical information. Meanwhile, limit of detection (LOD) was calculated using Mycobacterium tuberculosis H37Rv as reference strain. Result: ROC curve analysis of IS6110-qPCR assay showed a high Area Under the Curve (AUC) value (0.948) with optimal Ct value at 24.140. Prospective analysis of IS6110-qPCR assay with cut-off Ct = 24.140 showed a high overall sensitivity and specificity of 97.2% and 99.7%, respectively. No cross reactivity was observed among all non-tuberculous mycobacteria specimens in this study. LOD analysis on MTB-spiked sputum showed an average detection limit of 5.0 CFU/mL at Ct = 23.18 (±SD, 0.57). Conclusion: IS6110-qPCR assay is a highly accurate and cost-effective assay developed for primary screening of suspected TB cases, which is particularly suitable for regions with limited resources but high TB burden.

Original languageEnglish
Pages (from-to)120-125
Number of pages6
Publication statusPublished - Sep 2018


  • Diagnostic
  • IS6110
  • Mycobacterium tuberculosis
  • Real-time PCR

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Microbiology (medical)
  • Infectious Diseases

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