A flexible bioluminescent‐quantitative polymerase chain reaction assay for analysis of competitive PCR amplicons

Abstract

Aequorin-based flash-type bioluminescent methods can detect nucleic acid molecules in the attomolar range (10(-18)) enabling improved monitoring of the polymerase chain reaction (PCR) at cycles previously considered too low for product detection. The high sensitivity of bioluminescence (BL) was used to examine the efficiency of the PCR and to assess the effect of substrate variation during the linear phase of amplification. Primer efficiency was dependent on initial template concentration, in a manner indicative of a two-component reaction. However, the rate of amplicon formation was significantly impaired at low template levels and could not be overcome by excess primer. The PCR was directly dependent upon nucleotide concentration, which was independent of template concentration. Conditions were identified for optimal linear amplification and detection using BL. Accurate quantitative analysis was performed using competitive coamplification of a specific target standard sequence containing identical target primer recognition sites and novel internal sequences. Quantitation was most accurate when target molecule was similar in concentration to the internal standard. The Bioluminescent Quantitative-PCR (BLQ-PCR) assay has the potential to eliminate processing variability. We demonstrated high quantitative potential with a broad dynamic range. Overall, the BLQ-PCR assay is flexible and a viable alternative to contemporary Q-PCR techniques.

A Role for Complement C5 in Organism Containment and Granulomatous Response during Murine Tuberculosis

“…Bioluminescent hybridization immunoassay Bioluminescent detection has been optimized for low cycle optimal for measurement of PCR products derived from early phase log amplification [20, 21]. This property of detecting product during the early amplification phase translates into quantitative advantages; high sensitivity for low copy number and increased range in product detection maximize accurate quantitation of product [22]. Five µl of biotinylated PCR product was denatured by the addition of 1/4 volume (1.25 µl) denaturation buffer (1 m NaOH, 200 m m EDTA) for 5 min at room temperature.…”

Bioluminescent Quantitation and Detection of Gene Expression During Infectious Disease

“…This conclusion was also noted in a competitive analysis of amplicon production where the inherent sensitivity of aequorin was used to monitor the efficiency of PCR during low cycle amplification. These studies have helped to define reaction conditions for maximizing the concentration of the primer set relative to template material for use in competitive quantitative analysis [40]. The parameters defined are easily applied to similar systems for target DNA amplification.…”