Quantification of HIV-1 Using Multiple Quantitative Polymerase Chain Reaction Standards and Bioluminometric Detection

Nygren, Malin; Ronaghi, Mostafa; Nyrén, Pål; Albert, Jan; Lundeberg, Joakim

doi:10.1006/abio.2000.4871

Cited by 17 publications

(7 citation statements)

References 40 publications

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“…Among those, methods to detect pyrophosphate ion derived from nucleic acid amplification (or phosphate ion hydrolyzed from pyrophosphate ion) are included (10,12). However, when amplification reaction is conducted with PCR, pyrophosphate ion or phosphate ion is produced only to orders of within a few tenths of M. For this reason, special coloration reagents or enzymes are required to detect produced pyrophosphate ion.…”

Section: Discussionmentioning

confidence: 98%

Detection of Loop-Mediated Isothermal Amplification Reaction by Turbidity Derived from Magnesium Pyrophosphate Formation

Mori

Nagamine

Tomita

et al. 2001

Biochemical and Biophysical Research Communications

1,524

1,070

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Section: Discussionmentioning

confidence: 98%

Detection of Loop-Mediated Isothermal Amplification Reaction by Turbidity Derived from Magnesium Pyrophosphate Formation

Mori

Nagamine

Tomita

et al. 2001

Biochemical and Biophysical Research Communications

1,524

1,070

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“…Furthermore, although a quantitative assay for P. piscicida (6) has been developed, the costly probes, reagents, and equipment required may not render this method accessible to most laboratories. The development of quantitative assays, such as the quantitative competitive PCR (QC-PCR), that only require the construction of an internal standard for the competitive amplification of the experimental DNA mixture provides inexpensive yet useful and reliable alternatives (43,59).…”

mentioning

confidence: 99%

Characterization of the rRNA Locus of Pfiesteria piscicida and Development of Standard and Quantitative PCR-Based Detection Assays Targeted to the Nontranscribed Spacer

Saito

Drgon

Robledo

et al. 2002

Appl Environ Microbiol

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Pfiesteria piscicida is a heterotrophic dinoflagellate widely distributed along the middle Atlantic shore of the United States and associated with fish kills in the Neuse River (North Carolina) and the Chesapeake Bay (Maryland and Virginia). We constructed a genomic DNA library from clonally cultured P. piscicida and characterized the nontranscribed spacer (NTS), small subunit, internal transcribed spacer 1 (ITS1), 5.8S region, ITS2, and large subunit of the rRNA gene cluster. Based on the P. piscicida ribosomal DNA sequence, we developed a PCR-based detection assay that targets the NTS. The assay specificity was assessed by testing clonal P. piscicida and Pfiesteria shumwayae, 35 additional dinoflagellate species, and algal prey (Rhodomonas sp.). Only P. piscicida and nine presumptive P. piscicida isolates tested positive. All PCR-positive products yielded identical sequences for P. piscicida, suggesting that the PCR-based assay is species specific. The assay can detect a single P. piscicida zoospore in 1 ml of water, 10 resting cysts in 1 g of sediment, or 10 fg of P. piscicida DNA in 1 g of heterologous DNA. An internal standard for the PCR assay was constructed to identify potential false-negative results in testing of environmental sediment and water samples and as a competitor for the development of a quantitative competitive PCR assay format. The specificities of both qualitative and quantitative PCR assay formats were validated with >200 environmental samples, and the assays provide simple, rapid, and accurate methods for the assessment of P. piscicida in water and sediments.

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“…So far, the method has been used for applications where sequence primers can be designed to produce a read length sufficient for a correct result. Examples include single nucleotide polymorphism (SNP) analysis [25][26][27][28][29], identification and subtyping of bacteria [25], mutation detection in disease genes [30][31], and quantification of and mutation detection in HIV-1 [32][33]. The pyrosequencing technology has been further developed and is today also available as a tool for large-scale sequencing (genome Sequencer FLX, 454 Life Science/Roche).…”

Section: Pyrosequencingmentioning

confidence: 99%

Real-time polymerase chain reaction followed by fast sequencing allows rapid genotyping of microbial pathogens

Wahab

Ankarklev

Lebbad

et al. 2010

Scandinavian Journal of Infectious Diseases

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In this study we describe a novel protocol for rapid molecular analysis of patient samples using a combination of real-time polymerase chain reaction (PCR) and Sanger sequencing. This would normally take 2 working days in the diagnostic laboratory, but using this protocol the process can be completed within 3 h using equipment normally found in the laboratory. The innovative steps in this protocol are the sequencing of the product generated in the diagnostic real-time PCR, addition of a sequencing tail to the PCR primer, which increases the quality of the sequence without loss of sensitivity or specificity, and optimization of the hands-on and instrument steps using modern reagents. The read length of the sequencing step is routinely 250 nucleotides, which is substantially longer than existing rapid sequencing methods, increasing the chances of covering several genetic markers within 1 analysis. As proof of the concept, we used the detection and genotyping of the intestinal parasite Giardia lamblia, but the protocol can be applied to any PCR and sequence-based analysis.

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Quantification of HIV-1 Using Multiple Quantitative Polymerase Chain Reaction Standards and Bioluminometric Detection

Cited by 17 publications

References 40 publications

Detection of Loop-Mediated Isothermal Amplification Reaction by Turbidity Derived from Magnesium Pyrophosphate Formation

Detection of Loop-Mediated Isothermal Amplification Reaction by Turbidity Derived from Magnesium Pyrophosphate Formation

Characterization of the rRNA Locus of Pfiesteria piscicida and Development of Standard and Quantitative PCR-Based Detection Assays Targeted to the Nontranscribed Spacer

Real-time polymerase chain reaction followed by fast sequencing allows rapid genotyping of microbial pathogens

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