Development of a highly sensitive one-tube nested real-time PCR for detecting Mycobacterium tuberculosis

Choi, Yeji; Jeon, Bo Young; Shim, Tae Sun; Jin, Huang; Cho, Sang Nae; Lee, Hyeyoung

doi:10.1016/j.diagmicrobio.2014.08.009

Cited by 16 publications

(12 citation statements)

References 21 publications

(26 reference statements)

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“…tuberculosis genomic DNA, which is less than the amount of DNA in a single bacterial cell (approximately 5 fg) [ 24 ]. In the present study, this OTNPCR-LFST was 100 times more sensitive than CPCR-LFST, which is consistent with previous reports showing that nested PCR greatly increases sensitivity compared to traditional PCR [ 13 – 15 ]…”

Section: Discussionsupporting

confidence: 93%

“…Nested PCR usually takes up to 5–6 hours to complete. Investigators have attempted to develop one-tube or single-tube nested PCR technologies, in which both outer and inner primers are contained in a closed tube, the initial PCR cycles are initialed at high annealing temperatures, and the later cycles are performed at low annealing temperatures, thus reducing carry-over contamination and maintaining high sensitivity[ 13 – 20 ]. However, to date, this technique has been susceptible to inhibition by complex, labor-intensive operation for gel electrophoresis detection or increased equipment cost for real-time fluorescence detection, which greatly diminishes its routine use.…”

Section: Introductionmentioning

confidence: 99%

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Novel approach based on one-tube nested PCR and a lateral flow strip for highly sensitive diagnosis of tuberculous meningitis

Sun

Chen

et al. 2017

PLoS ONE

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Rapid and sensitive detection of Mycobacterium tuberculosis (M. Tb) in cerebrospinal fluid is crucial in the diagnosis of tuberculous meningitis (TBM), but conventional diagnostic technologies have limited sensitivity and specificity or are time-consuming. In this work, a novel, highly sensitive molecular diagnostic method, one-tube nested PCR-lateral flow strip test (OTNPCR-LFST), was developed for detecting M. tuberculosis. This one-tube nested PCR maintains the sensitivity of conventional two-step nested PCR and reduces both the chance of cross-contamination and the time required for analysis. The PCR product was detected by a lateral flow strip assay, which provided a basis for migration of the test to a point-of-care (POC) microfluidic format. The developed assay had an improved sensitivity compared with traditional PCR, and the limit of detection was up to 1 fg DNA isolated from M. tuberculosis. The assay was also specific for M. tuberculosis, and no cross-reactions were found in other non-target bacteria. The application of this technique to clinical samples was successfully evaluated, and OTNPCR-LFST showed 89% overall sensitivity and 100% specificity for TBM patients. This one-tube nested PCR-lateral flow strip assay is useful for detecting M. tuberculosis in TBM due to its rapidity, high sensitivity and simple manipulation.

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Section: Discussionsupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Novel approach based on one-tube nested PCR and a lateral flow strip for highly sensitive diagnosis of tuberculous meningitis

Sun

Chen

et al. 2017

PLoS ONE

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“…In recent years, polymerase chain reaction (PCR) and other methods for amplifying DNA and RNA have been developed, which enable TB diagnosis in several hours 5 – 7 . For example, Xpert MTB/RIF (Cepheid, Sunnyvale, CA, USA), a rapid automatic system based on real-time PCR and molecular beacon technology, used to detect M. tb genes from untreated sputum samples in less than 2 h 8 , 9 .The real-time fluorescence quantitative PCR (qPCR) was used to quantify BCG DNA in skin biopsy specimens of the BCG-vaccinated adults 10 .…”

Section: Introductionmentioning

confidence: 99%

Detection of circulating Mycobacterium tuberculosis -specific DNA by droplet digital PCR for vaccine evaluation in challenged monkeys and TB diagnosis

Song

Tan

Zhang

et al. 2018

Emerging Microbes & Infections

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Mycobacterium tuberculosis (M. tb) is emerging as a more serious pathogen due to the increased multidrug-resistant TB and co-infection of human immunodeficiency virus (HIV). The development of an effective and sensitive detection method is urgently needed for bacterial load evaluation in vaccine development, early TB diagnosis, and TB treatment. Droplet digital polymerase chain reaction (ddPCR) is a newly developed sensitive PCR method for the absolute quantification of nucleic acid concentrations. Here, we used ddPCR to quantify the circulating virulent M. tb-specific CFP10 (10-kDa culture filtrate protein, Rv3874) and Rv1768 DNA copy numbers in the blood samples from Bacille Calmette-Guerin (BCG)-vaccinated and/or virulent M. tb H37Rv-challenged rhesus monkeys. We found that ddPCR was more sensitive compared to real-time fluorescence quantitative PCR (qPCR), as the detection limits of CFP10 were 1.2 copies/μl for ddPCR, but 15.8 copies/μl for qPCR. We demonstrated that ddPCR could detect CFP10 and Rv1768 DNA after 3 weeks of infection and at least two weeks earlier than qPCR in M.tb H37Rv-challenged rhesus monkey models. DdPCR could also successfully quantify CFP10 and Rv1768 DNA copy numbers in clinical TB patients’ blood samples (active pulmonary TB, extrapulmonary TB (EPTB), and infant TB). To our knowledge, this study is the first to demonstrate that ddPCR is an effective and sensitive method of measuring the circulating CFP10 and Rv1768 DNA for vaccine development, bacterial load evaluation in vivo, and early TB (including EPTB and infant TB) diagnosis as well.

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“…The nucleic acid amplification test IS6110 has shown high levels of sensitivity to detect the presence of Mtb . One-tube nested RT-PCR is 100 times more sensitive in comparison to conventional RT-PCR ( Choi et al, 2014 ). In another study, the culture and mpt64RT-PCR demonstrated the same sensitivity (90.3%) in sputum samples.…”

Section: Current Status Of Known Biomarkers For Diagnostic Assaysmentioning

confidence: 99%

Recent Trends in System-Scale Integrative Approaches for Discovering Protective Antigens Against Mycobacterial Pathogens

et al. 2018

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Mycobacterial infections are one of the deadliest infectious diseases still posing a major health burden worldwide. The battle against these pathogens needs to focus on novel approaches and key interventions. In recent times, availability of genome scale data has revolutionized the fields of computational biology and immunoproteomics. Here, we summarize the cutting-edge ‘omics’ technologies and innovative system scale strategies exploited to mine the available data. These may be targeted using high-throughput technologies to expedite the identification of novel antigenic candidates for the rational next generation vaccines and serodiagnostic development against mycobacterial pathogens for which traditional methods have been failing.

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Development of a highly sensitive one-tube nested real-time PCR for detecting Mycobacterium tuberculosis

Cited by 16 publications

References 21 publications

Novel approach based on one-tube nested PCR and a lateral flow strip for highly sensitive diagnosis of tuberculous meningitis

Novel approach based on one-tube nested PCR and a lateral flow strip for highly sensitive diagnosis of tuberculous meningitis

Detection of circulating Mycobacterium tuberculosis -specific DNA by droplet digital PCR for vaccine evaluation in challenged monkeys and TB diagnosis

Recent Trends in System-Scale Integrative Approaches for Discovering Protective Antigens Against Mycobacterial Pathogens

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