Novel Wide-Range Quantitative Nested Real-Time PCR Assay for Mycobacterium tuberculosis DNA: Development and Methodology

Abstract

Previously, we designed an internally controlled quantitative nested real-time (QNRT) PCR assay forTuberculous meningitis (TBM) is the severest form of infection of Mycobacterium tuberculosis, causing death or severe neurological defects in more than half of those affected in spite of antituberculosis treatment (25). The diagnosis of TBM remains a complex issue because the most widely used conventional bacteriological detection methods, such as direct smear for acid-fast bacilli and culture for M. tuberculosis, are unable to rapidly detect M. tuberculosis with sufficient sensitivity in the acute phase of TBM (7,8,11,12,18,19,21,22,23,25). At present, the detection of M. tuberculosis DNA in cerebrospinal fluid (CSF) by use of PCR is widely used as a more rapid, sensitive, and specific diagnostic method (1,7,8,10,11,12,15,17,18,19,21,22,23,26). Recently, we designed a novel internally controlled quantitative nested real-time PCR (QNRT-PCR) assay based on TaqMan PCR (Applied Biosystems) (22). This novel assay technique combines the high sensitivity of nested PCR with the accurate quantification of real-time PCR (22, 23). However, this original QNRT-PCR (OR-QNRT-PCR) assay is still unstable and has many points that should be improved (22,23).In this study, to reliably detect M. tuberculosis DNA in CSF samples with a wider detection range, we attempted to improve on the OR-QNRT-PCR technique; therefore, a new internal control for use as a "calibrator" was prepared. We named this improved assay technique wide-range QNRT-PCR (WR-QNRT-PCR) and examined its ability to quantitatively detect M. tuberculosis DNA in samples. In this paper, the development and methodology of the WR-QNRT-PCR assay are stated. MATERIALS AND METHODSThis study was approved by the Nihon University Institutional Review Board. Preparation of the new internal control (plasmid) for use as a calibrator. For the WR-QNRT-PCR assay, two types of the original plasmid, wild plasmids (W-plasmids) and new-mutation plasmids (NM-plasmids), were prepared for a quantitative detection of M. tuberculosis DNA, and this was done as well for the OR-QNRT-PCR assay (22, 23).W-plasmid, which was inserted into a 239-bp DNA fragment of the gene sequence encoding the MPB64 protein of M. tuberculosis (MPT64; GenBank accession no. NC_000962) (22, 23) into pCR 2.1 vector (Invitrogen Corp., San Diego, CA) was constructed for use as the standard template by the previously reported procedure (22,23).NM-plasmid was developed based on the previously reported M-plasmid (22, 23) for use as a new internal-control "calibrator" in the WR-QNRT-PCR assay. In NM-plasmid, a total of four regions, where two pairs of (outer and inner) forward and reverse primers annealed, were replaced with the artificial random nucleotides added to the TaqMan probe annealing region in the M-plasmid (Fig. 1). The sequences of the artificial random nucleotides were set to have the same nucleotide composition as MPT64 of wild M. tuberculosis. Replacing procedures

Gastrointestinal tuberculosis: a diagnosis not to miss

“…Progress has been made in this field with the identification of their role alongside other tests (cultures, Mantoux and IGRAs), but as yet PCR assays remain a research tool. 6,7 The measurement of adenosine deaminases in both pleural and ascitic fluid has gained wide acceptance, but like PCR techniques, its use still requires further evaluation prior to recommending routine usage. 8 A therapeutic trial of standard quadruple antituberculous drugs for a six-month regimen was used in many of these cases, with successful outcomes for all.…”

Novel Wide-Range Quantitative Nested Real-Time PCR Assay for Mycobacterium tuberculosis DNA: Clinical Application for Diagnosis of Tuberculous Meningitis

“…In the preliminary experiments, the WR-QNRT-PCR assay demonstrated significantly improved quantitative accuracy and had a wide detection range (1 to 10 5 copies) compared to what was seen for the OR-QNRT-PCR assay (17).…”

“…In 2006, we designed a novel internally controlled quantitative nested real-time PCR (QNRT-PCR) assay based on TaqMan PCR (Applied Biosystems) (15). Moreover, based on this original QNRT-PCR (OR-QNRT-PCR) assay, an improved wide-range QNRT-PCR (WR-QNRT-PCR) assay was developed (17). For use as a "calibrator" in WR-QNRT-PCR assay, a new internal control was constructed (17).…”

“…Moreover, based on this original QNRT-PCR (OR-QNRT-PCR) assay, an improved wide-range QNRT-PCR (WR-QNRT-PCR) assay was developed (17). For use as a "calibrator" in WR-QNRT-PCR assay, a new internal control was constructed (17). In the preliminary experiments, the WR-QNRT-PCR assay demonstrated significantly improved quantitative accuracy and had a wide detection range (1 to 10 5 copies) compared to what was seen for the OR-QNRT-PCR assay (17).…”

Meningitis tuberculosa: claves para su diagnóstico y propuestas terapéuticas

“…Las nuevas técnicas de RPC en tiempo real pueden llegar a ser la principal prueba diagnóstica para la MTBC en el futuro próximo 35 . En Chile se dispone de Amplicor MTBC ® que amplifi ca la secuencia de inserción IS6110 y ha sido evaluado especialmente en muestras respiratorias como muy efi ciente; sin embargo, no existe la sufi ciente experiencia local con muestras extra-pulmonares 36 .…”