Loop-Mediated Isothermal Amplification Method Targeting the lytA Gene for Detection of Streptococcus pneumoniae

Abstract

It is difficult to separate Streptococcus pneumoniae from the genotypically similar species Streptococcus mitis and Streptococcus oralis, which are commensals of the human oral cavity. A novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63°C) with high specificity, efficiency, and rapidity, was examined regarding its applicability for detecting S. pneumoniae. An S. pneumoniae-specific LAMP primer targeting the lytA gene was designed. The primer specificity was validated using 10 Streptococcus and 7 non-Streptococcus species. Within 60 min, the assay could detect 10 or more copies of purified S. pneumoniae DNA with a sensitivity 1,000 times that of conventional PCR. Clinical isolates of 21 other strains (3 S. oralis, 17 S. mitis, and 1 Streptococcus species) that harbor virulencefactor-encoding genes (lytA or ply) were tried to differentiate S. pneumoniae. The detection of S. pneumoniae in clinical isolates was more selective using the LAMP method than using conventional PCR. Therefore, LAMP appears to be a sensitive and reliable means of diagnosing S. pneumoniae infection.Streptococcus pneumoniae is a common major human pathogen associated with community-acquired pneumonia, septicemia, meningitis, and otitis media (2). While traditional antimicrobial therapy has proven to be an effective treatment, the emergence of antimicrobial-resistant strains has resulted in an increasing number of cases (10). The isolation and identification of pneumococcus is complicated by contamination with alpha-hemolytic streptococci belonging to the normal flora. Pneumococcal detection using classical techniques, including growth-based assays, colonial morphology, optochin susceptibility, bile solubility, and serology, can be time-consuming and can produce equivocal results. Sensitive and specific assays that can be completed promptly in the clinical laboratory are essential for early diagnosis and effective antibiotic therapy. Molecular assays are inherently valuable because detection can be achieved with enhanced sensitivity and specificity and detection is not diminished with nonviable organisms.The molecular methods that have been used to date (3, 6, 17) are expensive and complicated to perform. Varying degrees of success have been realized using PCR-based assays for detecting S. pneumoniae with primers specific to repetitive regions or to genes encoding rRNA (7, 9, 12), pneumolysin (18,19,23), or autolysin (5, 14, 18). Autolysin and pneumolysin, which are encoded by the lytA and ply genes, respectively, represent potential targets for the specific detection of S. pneumoniae. Both genes are essential for pathogenesis and are well-characterized virulence markers (1). The restricted allelic variation of lytA (4, 21) makes this gene an ideal target for specific identification in clinical and epidemiological studies. For example, the gene may allow the differentiation of S. pneumoniae from the genotypically similar species Streptococcus mitis and...

A Loop-Mediated Isothermal Amplification (LAMP) Assay for Strongyloides stercoralis in Stool That Uses a Visual Detection Method with SYTO-82 Fluorescent Dye

“…The F2 and B2 primers were used for PCR amplification of a portion of the LAMP product. 29 However, in this case, the PCR amplified LAMP product sequence was identical to the original Strongyloides sequence targeted by the LAMP primers, and not particular to the LAMP reaction. Analytical specificity and sensitivity.…”

“…This was based on PCR of the LAMP product using primers F2 and B2. 29 HotStarTaq DNA polymerase and PCR buffer (Qiagen) were used according to instructions from the manufacturer.…”

Rapid identification of transgenic cotton (Gossypium hirsutum L.) plants by loop-mediated isothermal amplification

“…High specificity is assured since target recognition is mediated by four distinct primers targeting six different sequences (Notami et al 2000;Nagamine et al 2002;Fukuta et al 2004). In some studies, LAMP was even shown to be more selective (Seki et al 2005) and less sensitive to background DNA (Notami et al 2000) than PCR. LAMP has high amplification efficiency and with a lower amount of sample DNA (1-10 ng) (Guan et al 2010, it yields DNA in quantities of more than 500 g/ml (Morriset et al 2008).…”

Novel primers for increased specificity and sensitivity for the detection ofStaphylococcus aureusby real-time LAMP

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