A novel method for diagnosis of smear-negative tuberculosis patients by combining a random unbiased Phi29 amplification with a specific real-time PCR

Yu Pang; Jie Lu; Jian Yang; Yufeng Wang; Chad Cohen; Xin Ni; Yanlin Zhao

doi:10.1016/j.tube.2015.03.011

A novel method for diagnosis of smear-negative tuberculosis patients by combining a random unbiased Phi29 amplification with a specific real-time PCR

Tuberculosis (Edinb). 2015 Jul;95(4):411-4. doi: 10.1016/j.tube.2015.03.011. Epub 2015 Apr 17.

Authors

Yu Pang¹, Jie Lu², Jian Yang³, Yufeng Wang¹, Chad Cohen⁴, Xin Ni⁵, Yanlin Zhao⁶

Affiliations

¹ National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.
² Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, Beijing, China.
³ Shaanxi Tuberculosis Dispensary, Shaanxi, China.
⁴ McGill International TB Centre, Montreal, Quebec, Canada.
⁵ Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, Beijing, China. Electronic address: [email protected].
⁶ National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China. Electronic address: [email protected].

PMID: 25957821
DOI: 10.1016/j.tube.2015.03.011

Abstract

In this study, we develop a novel method for diagnosis of smear-negative tuberculosis patients by performing a random unbiased Phi29 amplification prior to the use of a specific real-time PCR. The limit of detection (LOD) of the conventional real-time PCR was 100 colony-forming units (CFU) of MTB genome/reaction, while the REPLI real-time PCR assay could detect 0.4 CFU/reaction. In comparison with the conventional real-time PCR, REPLI real-time PCR shows better sensitivity for the detection of smear-negative tuberculosis (P = 0.015).

Keywords: Diagnostics; REPLI real-time PCR; Smear negative; Tuberculosis.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bacteriological Techniques*
Colony Count, Microbial
DNA, Bacterial / genetics*
DNA-Directed DNA Polymerase / metabolism*
Humans
Limit of Detection
Mycobacterium tuberculosis / genetics*
Mycobacterium tuberculosis / growth & development
Mycobacterium tuberculosis / isolation & purification
Predictive Value of Tests
Prospective Studies
Real-Time Polymerase Chain Reaction*
Sputum / microbiology
Tuberculosis, Pulmonary / diagnosis*
Tuberculosis, Pulmonary / genetics
Tuberculosis, Pulmonary / microbiology

Substances

DNA, Bacterial
DNA-Directed DNA Polymerase