Evaluation of the performance of LNA and MGB probes in 5′-nuclease PCR assays

Letertre, Capucine; Perelle, Sylvie; Dilasser, Françoise; Arar, Khalil; Fach, Patrick

doi:10.1016/j.mcp.2003.08.004

Cited by 64 publications

(51 citation statements)

References 9 publications

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“…To increase specificity and to investigate the sensitivity at higher melting temperatures, we compared modified TaqMan probes with higher melting temperatures with conventional TaqMan probes (24 ). Adjusting the PCR protocols to the different probe technologies revealed that an annealing temperature of 60°C increased assay specificity.…”

Section: Assay Developmentmentioning

confidence: 99%

High-Volume Extraction of Nucleic Acids by Magnetic Bead Technology for Ultrasensitive Detection of Bacteria in Blood Components

Störmer

Kleesiek

Dreier³

2007

Clinical Chemistry

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Background: Nucleic acid isolation, the most technically demanding and laborious procedure performed in molecular diagnostics, harbors the potential for improvements in automation. A recent development is the use of magnetic beads covered with nucleic acid-binding matrices. We adapted this technology with a broadrange 23S rRNA real-time reverse transcription (RT)-PCR assay for fast and sensitive detection of bacterial contamination of blood products. Methods: We investigated different protocols for an automated high-volume extraction method based on magnetic-separation technology for the extraction of bacterial nucleic acids from platelet concentrates (PCs). We added 2 model bacteria, Staphylococcus epidermidis and Escherichia coli, to a single pool of apheresisderived, single-donor platelets and assayed the PCs by real-time RT-PCR analysis with an improved primerprobe system and locked nucleic acid technology. Coamplification of human ␤ 2 -microglobulin mRNA served as an internal control (IC). We used probit analysis to calculate the minimum concentration of bacteria that would be detected with 95% confidence. Results: For automated magnetic bead-based extraction technology with the real-time RT-PCR, the 95% detection limit was 29 ؋ 10 3 colony-forming units (CFU)/L for S. epidermidis and 22 ؋ 10 3 CFU/L for E. coli. No false-positive results occurred, either due to nucleic acid

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Section: Assay Developmentmentioning

confidence: 99%

High-Volume Extraction of Nucleic Acids by Magnetic Bead Technology for Ultrasensitive Detection of Bacteria in Blood Components

Störmer

Kleesiek

Dreier³

2007

Clinical Chemistry

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“…The modified oligonucleotides having methyl bridge (or locked) between the 2' oxy and 4' carbon of deoxyribose sugar have interesting properties that not only guarantee their complementary base-pairing but also increase their T m up to about 3 o C to 8 o C per base, compared to non-modified oligonucleotide [14]. In this study, we designed our probes to have "locked" nucleotides at the mutant position and its neighbouring positions.…”

Section: Discussionmentioning

confidence: 99%

Sensitive quantification of mitochondrial mutation using new Taqman probes

et al. 2014

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AbstractThe A3243G mitochondrial mutation is the major cause of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). The severity of the disease is correlated with the heteroplasmy level of the mutation. Here we describe for the first time the validation of a real-time polymerase chain reaction (PCR) assay with Taqman locked nucleic acid (LNA) fluorescent (FAM for mutant, HEX for wild type) probes for quantification of heteroplasmy levels in a total of 18 family members from 5 Vietnamese MELAS patients carrying A3243G. Almost no background of FAM signals was detected in normal samples, indicating that the probes were allele-specific. Standard curves indicate sensitive detection at 0.1% mutants and high reliability with R2 > 0.985. The correlation line between measured % mutant and expected % mutant was highly reliable, with a slope of 0.993 and R2 of 0.998. All positive A3243G mutant samples pre-screened by PCR-restriction fragment length polymorphism (RFLP) were confirmed, and their heteroplasmy levels quantified to be from 3.68 to 80.85%. The heteroplasmy levels in patients were higher than in their family members and generally correlated well with the severity of their clinical symptoms. Overall, this work is the first demonstration of the application of LNA probes for sensitive and highly reliable quantification of heteroplasmy levels in human mitochondria.

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“…Two important chemistries that are used in qPCR systems are TaqMan and LNA probes that specifically bind to the target sequence (Gachon et al, 2004). Locked nucleic acid (LNA) is the analogue of a nucleic acid described by the Wengel and Imanishi laboratories (Letertre et al, 2003). The C3′-endo type sugars are conformationally locked by a methylene bridge between 2′ oxygen and 4′ carbon of the ribose ring (Jepsen et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

“…The flexibility of the ribofuranose ring is reduced by this methylene bridge causing the formation of a rigid bicyclic monomer. This also increases the local organization of the phosphate backbone and result in strong hybridization between two DNA strands (Letertre et al, 2003;Reynisson et al, 2006). To the best of our knowledge, the studies on direct quantitative detection of S. aureus in Turkish cheese samples are very limited.…”

Section: Introductionmentioning

confidence: 99%