Automated System for Capture and Detection of Nucleic Acids

Brown, Alexander; Akinsanya, A.A.; Barker, Susan J.; Brophy, Michael H.; Dobb, A.K.; Doyle, Shawn M.; Hudson, I.R.; Minter, Stephen J.; Wraith, M.J.; Oultram, John D.

doi:10.2144/99271pf01

Cited by 3 publications

(3 citation statements)

References 4 publications

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“…As little as 10 4 copies or 5 fg each of CymMV and ORSV can be detected simultaneously with either the RdRp or CP gene as the target. This compares favourably with other novel detection techniques such as immuno-capillary zone electrophoresis (Eun and Wong, 1999), molecular beacons (Eun and Wong, 2000) and the DARAS ® system (Brown et al, 1999).…”

Section: Discussionmentioning

confidence: 73%

Simultaneous quantitation of two orchid viruses by the TaqMan® real-time RT-PCR

Eun

Seoh

Wong

2000

Journal of Virological Methods

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Simultaneous quantitation of two orchid viruses, cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV), were carried out using the TaqMan((R)) real-time RT-PCR, a novel detection technique that combines RT-PCR with the power of fluorescent detection. Four TaqMan((R)) probes were synthesized, targeting at the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes of both viruses. The reporter dye FAM (6-carboxyfluorescein) was used to label the 5' terminus of probes specific to CymMV, while TET (tetrachloro-6-carboxyfluorescein) was used for the ORSV probes. TAMRA (6-carboxy-tetramethyl-rhodamine), which was attached at the 3' terminus of each probe, was used as the universal quencher. With increasing amounts of standard RNA templates, the respective threshold cycle (C(T)) values were determined and a linear relationship was established between these C(T) values and the logarithm of initial template amounts. The amounts of starting templates in mixed-infected Oncidium flowers and leaves were estimated from the standard curves. As little as 10(4) copies or 5 fg each of CymMV and ORSV could be detected simultaneously with either the RdRp or CP gene as the target. This system offers a sensitive, high throughput and rapid method for plant virus detection.

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Section: Discussionmentioning

confidence: 73%

Simultaneous quantitation of two orchid viruses by the TaqMan® real-time RT-PCR

Eun

Seoh

Wong

2000

Journal of Virological Methods

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“…Specific PCR products can be detected following agarose gel electrophoresis or in enzyme‐linked immunosorbent assays[5–6], but the need for these relatively time‐consuming and labour‐intensive procedures has limited the introduction of this successful technique into routine diagnostic laboratories. The aim of the present study was to assess whether PCR for rapid diagnosis of meningococcal meningitis could be used in conjunction with a novel automated detection system (DARAS ® Tepnel Life Sciences, Manchester, UK)[9] that combines the amplification potential of PCR with sequence‐specific capture and colorimetric detection of PCR products.…”

Section: Introductionmentioning

confidence: 99%

Use of an automated DNA analysis system (DARAS®) for sequence-specific recognition of Neisseria meningitidis DNA

Seward

Towner

2000

Clinical Microbiology and Infection

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“…8 The key to the system is the use of target-specific microbeads, incorporating covalently attached oligonucleotide probes. PCR samples are automatically analyzed using the precise fluid handling, temperature control and signal detection capabilities of the system.…”

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confidence: 99%

Digoxigenin-end labeled oligonucleotides facilitate the detection of minimal residual disease in acute lymphoblastic leukemia

et al. 2000

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TO THE EDITORRecently, three large prospective trials unequivocally demonstrated that the evaluation of minimal residual disease (MRD) plays a pivotal role as a prognostic parameter in acute lymphoblastic leukemia (ALL). [1][2][3] Individualized therapeutic adaptations based on the MRD status might be of considerable benefit for ALL patients. Several methodologies are available for MRD analyses in ALL patients. [1][2][3][4] Immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements have been utilized as PCR targets. An important PCR strategy for MRD analysis is the amplification of the rearranged Ig or TCR genes and hybridization with 32 P-labeled clone-specific probes in dot blot hybridization. Now that the relevance of MRD monitoring in ALL has been recognized, modification of current techniques or development of new techniques enabling rapid and reliable MRD analysis is relevant. We report on a non-radioactive dot blot analysis taking advantage of digoxigenin (DIG)-labeled oligonucleotides for the rapid detection of MRD. DIG is a steroid hapten which is linked via a spacer arm to a nucleotide. DIG-ddUTP is incorporated at the 3′-end of an oligonucleotide by terminal transferase. After hybridization, the target DNA is detectable by an enzyme-linked immunoassay through an anti-DIG-AP Fab fragment.We applied this technique on 10 adult ALL patients with IGK and/or TCRD gene rearrangements. The patients were treated according to the German multicenter trial of ALL in adults 05/99. 5 High molecular weight DNA was prepared from bone marrow samples obtained at initial diagnosis using standard techniques. IGK and TCRD gene rearrangements were identified and characterized by PCR and sequencing as described. 6,7 Oligonucleotides corresponding to the clonespecific junctional regions of the respective IGK/TCRD genes are shown in Table 1.Ten-fold serial dilutions of initial leukemic DNA into normal buffy coat (BC) DNA were prepared in a range from 10 −1 to 10 −6 . One g of each DNA preparation as well as BC DNA were amplified using a set of outer primers. 6 A negative control (H 2 O) was included in each PCR reaction. PCR was performed using the touch down program with 40 cycles. 7 Five l of the products were spotted in duplicate on to nylon membrane (NYTRAN 0.45, Schleicher and Schuell, Dassel, Germany). The membranes were soaked with denaturing buffer (0.5 M NaOH, 1.5 M NaCl) and neutralizing buffer (0.5 M Tris-HCl, 1.5 M NaCl, pH 7.0) for 2 min each and cross-linked by UV. 100 pmol of each oligonucleotide was 3′-end labeled with DIGddUTP using a DIG oligonucleotide 3′-end labeling kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's instructions. Non-incorporated DIG-ddUTPs were removed by ethanol precipitation and the labeled probe was dissolved into 20 l of distilled water (final concentration 5 pmol/ l). One pmol of the DIG-labeled oligonucleotide was spotted on to a nylon membrane, which was subsequently processed in the immunological detection step to verify the labeling eff...

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Automated System for Capture and Detection of Nucleic Acids

Cited by 3 publications

References 4 publications

Simultaneous quantitation of two orchid viruses by the TaqMan® real-time RT-PCR

Simultaneous quantitation of two orchid viruses by the TaqMan® real-time RT-PCR

Use of an automated DNA analysis system (DARAS®) for sequence-specific recognition of Neisseria meningitidis DNA

Digoxigenin-end labeled oligonucleotides facilitate the detection of minimal residual disease in acute lymphoblastic leukemia

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