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 degrees 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 virulence-factor-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.

FIG. 1.

(A) Nucleotide sequences of S. pneumoniae R6 autolysin gene used for LAMP primer. The sequences used for LAMP primers are shown in boldface. *, consensus sequence among S. pneumoniae strains. (B) Schematic representation of primers used in this study.

FIG. 2.

Sensitivities of electrophoretic analysis of LAMP-amplified products. Lanes M, 100-bp ladder used as a size marker; lane 1, 10⁶ copies of genomic DNA from S. pneumoniae ATCC 6305; lane 2, 10⁵ copies; lane 3, 10⁴ copies; lane 4, 10³ copies; lane 5, 10² copies; lane 6, 10 copies; lane 7, 1 copy; lane 8, no template; lane 9, LAMP product from lane 1 after TasI digestion. The digested fragments were 102 and 111 bp.

FIG. 3.

(A) Real-time sensitivity of S. pneumoniae LAMP, as monitored by the measurement of turbidity. Shown from left to right in the figure are the curves of decreasing concentrations (1,000,000 to 1) of bacteria. The detection limit was 10 copies. (B) The relation between the threshold times (Tt) of each sample and the log of the amount of initial template DNA.