Abstract
Streptococcus agalactiae is a major pathogenic bacterium causing perinatal infections in humans. In the present study, a novel real-time fluorescence loop-mediated isothermal amplification technology was successfully developed and evaluated for the detection of S. agalactiae in a single reaction. Six specific primers were designed to amplify the corresponding six regions of fbs B gene of S. agalactiae, using Bst DNA polymerase with DNA strand displacement activity at a constant temperature for 60 min. The presence of S. agalactiae was indicated by the fluorescence in real-time. Amplification of the targeted gene fragment was optimized with the primer 1 in the current setup. Positive result was only obtained for Sa by Real-LAMP among 10 tested relevant bacterial strains, with the detection sensitivity of 300 pg/µl. Real-LAMP was demonstrated to be a simple and rapid detection tool for S. agalactiae with high specificity and stability, which ensures its wide application and broad prospective utilization in clinical practice for the rapid detection of S. agalactiae.
Highlights
Streptococcus agalactiae (Sa), a facultative anaerobic Gram-positive Streptococcus, was reported to colonize the human gastrointestinal and genitourinary tract of up to 30% of healthy human adults
No reaction peak in the Real-loop-mediated isothermal amplification (LAMP) without ring primer could be visually detected until 63 min after initiation
A rapid, specific, sensitive and cost-effective diagnostic methodology was developed in the present study based on Real-time LAMP technology
Summary
Streptococcus agalactiae (Sa), a facultative anaerobic Gram-positive Streptococcus, was reported to colonize the human gastrointestinal and genitourinary tract of up to 30% of healthy human adults. Sa was regarded as a major pathogenic bacterial strain for perinatal infections in pregnant women and their newborns as early as in 1970s. It was the leading cause for sepsis and meningitis in infants and young children, and has been a serious concern in perinatal medicine. The reaction rate was further increased two to three times by Nagamine et al [3], and two loop primers were specially designed and adopted. This significantly increased the detection sensitivity as compared with conventional PCR, and the reaction time was greatly reduced. A novel real-time LAMP (Real Time-LAMP or Real-LAMP) was developed, License 4.0 (CC BY)
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