소변검체에서 두 유전자 단일 튜브 이중 실시간 중합효소연쇄반응 검사를 이용한 활동성 결핵의 진단

Abstract

Abstracts Background: Tuberculosis (TB) is a global health problem and the leading cause of morbidity and mortality worldwide. A delay in TB diagnosis will affect the control of the disease. Therefore, rapid, easy to perform, highly sensitive, and more accurate diagnostic tools during the early stages of infection are crucial. However, conventional smear/culture methods using sputum samples have well-known limitations of low sensitivity and prolonged result time. Furthermore, some pulmonary TB (PTB) patients cannot expectorate sputum. In addition, the diagnosis of extrapulmonary TB (EPTB) needs invasive procedures. Thus, non-sputum diagnostic methods are urgently needed. Advancements in real-time polymerase chain reaction (PCR) in detecting Mycobacterium tuberculosis (MTB)-specific trans-renal DNA (Tr-DNA) from urine, which are small DNA fragments from cells dying throughout the body, have recently been made. However, Tr-DNA-based studies have found that the sensitivity and specificity were 0.5 (95% CI: 0.36 – 0.72) and 0.94 (95% CI: 0.78 – 0.99), respectively, for a combination of PTB and EPTB specimens. Therefore, the development of more sensitive methods remains a critical goal. Purpose: The aim of this study was to develop a new highly sensitive nucleic acid amplification test (NAAT) using duplex, one-tube nested, real-time PCR for the rapid and accurate detection of MTB in urine specimens. Methods: Duplex, one-tube nested, real-time PCR was designed with two sequential reactions with two sets of primers and dual probes for the insertion sequences of IS6110 and rpoB in MTB. The analytical sensitivity of the assay was determined through a standard curve of 10-fold dilutions [10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, and 10 fg] of DNA isolated from the MTB H37Rv strain. Clinical specimens were used in a preliminary study to evaluate the sensitivity and specificity of the newly developed test. Sputum specimens were provided from 26 patients with culture-positive PTB and 108 healthy subjects, and urine specimens from 45 patients with culture-positive PTB and 30 healthy subjects. Results: The Ct (cycle threshold) values for MTB H37Rv by rpoB single real-time PCR, IS6110 single real-time PCR, rpoB one-tube nested real-time PCR, IS6110 one-tube nested real-time PCR, and rpoB and IS6110 duplex, one-tube nested, real-time PCR ranged from 17.36 to 37.18, 14.95 to 34.88, 8.44 to 27.92, 5.83 to 26.36, and 4.99 to 24.99, respectively. To increase the sensitivity, the reaction protocol for the duplex, one-tube nested, real-time PCR was modulated from 10 and 40 cycle to 15 and 25 cycle and, as a result, the minimum limit of detection was changed from 10fg to 1fg, respectively. Overall, duplex, one-tube nested, real-time PCR with 15 and 25 cycle was the most sensitive assay for detecting MTB DNA. The sensitivity of the Real MTB-ID (M&D, Korea) assay, and duplex, one-tube nested, real-time PCR test was 46.2% (12/26) and 100% (26/26), respectively, for sputum specimens. In comparison, the sensitivity of the AdvanSureTM TB/ Nontuberculous mycobacterium (NTM) real-time PCR (LG Lifescience, Korea) assay, and the duplex, one-tube nested, real-time PCR test was 6.7% (3/45) and 51.1% (23/45), respectively, for urine specimens. Conclusions: IS6110 and rpoB gene one-tube nested real-time PCR was useful for detecting MTB in urine specimens due to its relatively high sensitivity and simple manipulation compared to conventional methods.