improved visual detection of speb gene in streptococcus pyogenes isolates by real-time loop-mediated isothermal amplification turbidimetry method

Background: groupa 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.