Loopamp fluorescent detection reagent

The amplification products were detected using two methods; a real-time turbidimeter (La-320C; Eiken Chemical Co., Ltd.) at 650 nm and visual observation. Pyrophosphate ions are released during the LAMP reaction process and they form a white magnesium phosphate precipitate. This can be monitored using a real-time turbidimeter, drawing reaction curves every 6 sec with Mg²⁺ ions in the reaction buffer (13 (link)). For visual detection, 1 µl of Loopamp Fluorescent Detection reagent (Eiken Chemical Co., Ltd.), containing a metal indicator, was added into the reaction system prior to amplification. The reaction buffer initially turned orange because the calcein was quenched by Mn²⁺ ions. Then, during amplification the calcein was displaced by pyrophosphate ions from the calcein/Mn²⁺ complex, and the color changed from orange to green. By contrast, if no amplification occurred, no color change was observed.

A set of five primers was manually designed to target the nucleotide sequence of P. shigelloides ATCC 51903, based on the mechanism of CPA (27 (link)). The sequences and locations of the primers within hugA are presented in Table II and Fig. 1. CPA reactions were performed using the Loopamp kit (Eiken Chemical Co., Ltd., Tokyo, Japan) in a final volume of 20 µl containing 2.4 mM cross primer As, 1.44 mM each of primers 2a and 3a, 0.3 mM each of displacement primers 4s and 5a, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 4 mM MgSO4, 10 mM (NH4)2SO4, 0.1% Tween 20, 0.8 M betaine, 1.4 mM deoxynucleoside triphosphates (dNTPs), 1 µl of Bst DNA polymerase (8 U µl⁻¹), 1 µl Loopamp Fluorescent Detection Reagent (Eiken Chemical Co., Ltd.) and 1 µl DNA template. The reaction mixture was incubated in an LA320 Real-Time Turbidimeter (Teramecs Co., Ltd., Kyoto, Japan) at 63°C for 60 min, and then heated at 95°C for 5 min to terminate the reaction. Amplified products were directly detected by observing a colour change from orange to green by the naked eye, or by electrophoresis on 2% agarose gels using staining with GoldenView reagent. Furthermore, real-time monitoring of the CPA reaction was performed by recording the optical density at 650 nm every 6 sec using the LA-320C Real-Time Turbidimeter. A positive reaction was defined as a turbidity cut-off value of >0.1 within 60 min.