scholarly journals Homogeneous Real-Time Detection of Single-Nucleotide Polymorphisms by Strand Displacement Amplification on the BD ProbeTec ET System

2003 ◽  
Vol 49 (10) ◽  
pp. 1599-1607 ◽  
Author(s):  
Sha-Sha Wang ◽  
Keith Thornton ◽  
Andrew M Kuhn ◽  
James G Nadeau ◽  
Tobin J Hellyer
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Dna Sequencing ◽  
Real Time ◽  
Whole Blood ◽  
Nucleotide Polymorphisms ◽  
Strand Displacement ◽  
Strand Displacement Amplification ◽  
Single Nucleotide ◽  
Buccal Swabs ◽  
Real Time Detection

Abstract Background: The BD ProbeTec™ ET System is based on isothermal strand displacement amplification (SDA) of target nucleic acid coupled with homogeneous real-time detection using fluorescent probes. We have developed a novel, rapid method using this platform that incorporates a universal detection format for identification of single-nucleotide polymorphisms (SNPs) and other genotypic variations. Method: The system uses a common pair of fluorescent Detector Probes in conjunction with unlabeled allele-specific Adapter Primers and a universal buffer chemistry to permit analysis of multiple SNP loci under generic assay conditions. We used Detector Probes labeled with different dyes to facilitate differentiation of two alternative alleles in a single reaction with no postamplification manipulation. We analyzed six SNPs within the human β2-adrenergic receptor (β2AR) gene, using whole blood, buccal swabs, and urine samples, and compared results with those obtained by DNA sequencing. Results: Unprocessed whole blood was successfully genotyped with as little as 0.1–1 μL of sample per reaction. All six β2AR assays were able to accommodate ≥20 μL of unprocessed whole blood. For the 14 individuals tested, genotypes determined with the six β2AR assays agreed with DNA sequencing results. Conclusion: SDA-based allelic differentiation on the BD ProbeTec ET System can detect SNPs rapidly, using whole blood, buccal swabs, or urine.

scholarly journals Clinical Applications of Whole-Blood PCR with Real-Time Instrumentation

2005 ◽  
Vol 51 (11) ◽  
pp. 2025-2030 ◽  
Author(s):  
Alison Castley ◽  
Melinda Higgins ◽  
John Ivey ◽  
Cyril Mamotte ◽  
David C Sayer ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Real Time ◽  
Whole Blood ◽  
Detection System ◽  
Melting Curve ◽  
Factor V Leiden ◽  
Melting Curve Analysis ◽  
Factor V ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide

Abstract Background: As the genetic basis of many human diseases is being discovered, there is increasing need for the detection of single-nucleotide polymorphisms/mutations in medical laboratories. We describe an innovative approach that combines PCR amplification directly on whole blood and real-time detection PCR technology (WB-RTD PCR). Methods: We compared WB-RTD PCR with the method for extracted DNA-RTD PCR for the detection of mutations in the prothrombin (n = 94), factor V Leiden (n = 49), and hemochromatosis (n = 22) genes. Mutation detection on the Roche LightCycler was based on use of fluorescence resonance energy transfer (FRET) probes and melting curve analysis. We also compared the WB-RTD PCR on the LightCycler and the ABI Prism™ 7700 sequence detection system with minor groove– binding nonfluorescent quencher probes. Results: We obtained complete concordance between both methods in assigning genotypes. We also demonstrated that the WB-RTD PCR method can be performed on real-time PCR instruments from Applied Biosystems and the LightCycler. Omission of the need for DNA extraction and gel electrophoresis allowed substantial labor and cost savings with this method. Conclusion: This approach has applications for testing other medically relevant single-nucleotide polymorphisms.

scholarly journals Description and Validation of a Novel Real-Time RT-PCR Enterovirus Assay

2008 ◽  
Vol 54 (2) ◽  
pp. 406-413 ◽  
Author(s):  
Weston C Hymas ◽  
Wade K Aldous ◽  
Edward W Taggart ◽  
Jeffery B Stevenson ◽  
David R Hillyard
Keyword(s):  
Sequence Analysis ◽  
Single Nucleotide Polymorphisms ◽  
Real Time ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Single Nucleotide ◽  
The Real ◽  
Nontranslated Region

Abstract Background: Enteroviruses are a leading cause of aseptic meningitis in adult and pediatric populations. We describe the development of a real-time RT-PCR assay that amplifies a small target in the 5′ nontranslated region upstream of the classical Rotbart enterovirus amplicon. The assay includes an RNA internal control and incorporates modified nucleotide chemistry. Methods: We evaluated the performance characteristics of this design and performed blinded parallel testing on clinical samples, comparing the results with a commercially available RT-PCR assay (Pan-Enterovirus OligoDetect kit) that uses an enzyme immunoassay–like plate end detection. Results: We tested 778 samples and found 14 discrepant samples between the 2 assays. Of these, the real-time assay detected 6 samples that were negative by the OligoDetect kit, 5 of which were confirmed as positive by sequence analysis using an alternative primer set. Eight discrepant samples were positive by the OligoDetect kit and real-time negative, with 6 confirmed by sequencing. Overall, detection rates of 97% and 96% were obtained for the OligoDetect kit and real-time assays, respectively. Sequence analysis revealed the presence of a number of single nucleotide polymorphisms in the targeted region. The comparative sensitivities of the 2 assays were equivalent, with the limit of detection for the real-time assay determined to be approximately 430 copies per milliliter in cerebrospinal fluid. Conclusions: This novel real-time enterovirus assay is a sensitive and suitable assay for routine clinical testing. The presence of single nucleotide polymorphisms can affect real-time PCR assays.

Loss of heterozygosity testing using real-time PCR analysis of single nucleotide polymorphisms

2005 ◽  
Vol 132 (3) ◽  
pp. 200-204 ◽  
Author(s):  
Tamara Čačev ◽  
Mladen Jokić ◽  
Radan Spaventi ◽  
Krešimir Pavelić ◽  
Sanja Kapitanović
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Real Time ◽  
Loss Of Heterozygosity ◽  
Real Time Pcr ◽  
Pcr Analysis ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide

Short PNA molecular beacons for real-time PCR allelic discrimination of single nucleotide polymorphisms

2004 ◽  
Vol 18 (2) ◽  
pp. 117-122 ◽  
Author(s):  
Kenneth Petersen ◽  
Ulla Vogel ◽  
Eszter Rockenbauer ◽  
Kirsten Vang Nielsen ◽  
Steen Kølvraa ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Real Time ◽  
Real Time Pcr ◽  
Molecular Beacons ◽  
Nucleotide Polymorphisms ◽  
Allelic Discrimination ◽  
Single Nucleotide

scholarly journals Real-Time PCR Assays of Single-Nucleotide Polymorphisms Defining the Major Brucella Clades

2008 ◽  
Vol 46 (7) ◽  
pp. 2474-2474
Author(s):  
J. T. Foster ◽  
R. T. Okinaka ◽  
R. Svensson ◽  
K. Shaw ◽  
B. K. De ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Real Time ◽  
Real Time Pcr ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Pcr Assays
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