Pyrophosphate detection method using 5-Br-PAPS to detect nucleic acid amplification - Application to LAMP method.

Genetic testing has been increasingly used in several fields. In many applications, nucleic acid amplification technology is required. However, current methods to detect nucleic acid amplification require expensive reagents and special equipment or exhibit limited sensitivity, which hinders their use. To address this issue, this study reports an assay method for detecting occurrence of acid amplification in post-amplification samples using pyrophosphate, a highly sensitive byproduct of nucleic acid amplification. The method proposed requires two reagents and an automated analyzer. First, hydrogen peroxide is derived from pyrophosphate, an indicator of nucleic acid amplification, and the oxidizing power of hydrogen peroxide is used to produce Fe (III) from Fe (II). The specific metal chelator 5-Br-PAPS forms a complex with the trivalent iron produced, resulting in a highly sensitive coloration. The within-run reproducibility of our method (n = 20) was less than 3.67% at each concentration tested, and the detection limit was 0.075 µmol/L, sufficient for quantitative analysis. The technique described could detect pyrophosphate in a sample that was amplified using the loop-mediated isothermal amplification method after only 10 min. Therefore, the proposed method has the potential to be a new, rapid, and simple detection technique for amplified nucleic acids.

Development of a protein assay with copper chelator chromeazurol B, based on the biuret reaction.

This study aimed to provide a novel and highly sensitive protein assay based on the biuret reaction and using chromeazurol B, a metal chelate compound. The method consists of two reagents and an automated analyzer. First, a complex of copper and protein (biuret reaction) is formed. Second, a chelating reagent containing chromeazurol B forms a three-dimensional complex of protein, copper, and chromeazurol B at neutral pH, resulting in highly sensitive coloration. The intra-assay (n = 20) variation for the three levels was 3.54 % or lower at each concentration. Each response with α, ß-, and γ-globulin was 103.8 % and 104.3 %, respectively, against albumin. The molar absorption coefficient (ε) of the present method was 2.5 × 105 m2/mol against human albumin, higher than that of the commercially available Lowry method (ε = 8.7 × 104 m2/mol), which is based on the same principle. The correlation test for the pyrogallol method with 30 urine samples showed good performance (r = 0.961). The method described here (the Biuret-based CAB method) is a more sensitive and rapid assay than the Lowry method, and it may also be applied to biological samples because of its similar reactivity towards various proteins.