Improved Visual Detection of speB Gene in Streptococcus pyogenes Isolates by Real-time Loop-Mediated Isothermal Amplification Turbidimetry Method

Abstract

Background: Group A Streptococcus (GAS) causes a wide array of clinical manifestations ranging from mild pharyngitis to suppurative and non-suppurative severe debilitating diseases. Hence, a simple, rapid detection method with high sensitivity and specificity is needed. Objectives: This study embarked on the visual detection of the streptococcal pyrogenic exotoxin B (speB) gene by real-time turbidimetry and loop-mediated isothermal amplification (RT-LAMP) methods. The real-time monitoring of the sigmoidal graph generated from a turbidimetry method was incorporated in the assay. Methods: The amplification of the speB gene was virtually observed in real-time monitoring of the graph (sigmoidal curve) generated via a turbidimeter, thus providing a “guide” to accurately estimate the time to positivity for the gene detection. Results: The targeted gene was detected at 15 min but was optimally amplified within 45 min at an isothermal temperature of 63°C with 100% specificity using an established set of primers. The formation of sigmoidal curves was correlated with other visual observations by the naked eye (from orange to green), ultra-violet light (green fluorescence), and agarose gel electrophoresis. The improved detection limit of the real-time RT-LAMP assay was also observed compared to conventional PCR assay (0.001 pg/µL versus 1 ng/µL). Conclusions: The improved visual detection of RT-LAMP assay could provide additional insight for rapid, cost-effective, and reliable identification of GAS via speB gene detection in low or middle-income countries. It could also be a very important tool to improve the healthcare management of patients infected with GAS in the future.