Simultaneous detection of Streptococcuspneumoniae and prevention of carryover contamination using multiple cross displacement amplification with Antarctic thermal sensitive uracil-DNA-glycosylase and a lateral flow biosensor.

Abstract

Streptococcus pneumoniae is an important clinical pathogenic bacterium that is the primary cause of meningitis, septicemia and community-acquired pneumonia. The mortality rate of pneumococcal disease is high, especially in children younger than 5-years-old. Rapid and accurate detection of S.pneumoniae is critical for clinical diagnosis. A ply gene-based multiple cross displacement amplification (MCDA) assay, amplifying DNA under 65°Cfor 40min, was established to detect S.pneumoniae. Antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) was applied to prevent carryover contamination. A lateral flow biosensor (LFB) was used to indicate the MCDA results. The ply-MCDA assay could detect as low as 10 fg of S. pneumoniae DNA and 447 colony forming units (CFU)/mL of spiked sputum samples. The analytical sensitivity of the ply-MCDA assay to detect clinical specimens was 100 times higher than that of PCR. The specificity of the ply-MCDA assay was evaluated using 15 S.pneumoniae strains and 25 non-S. pneumoniae strains, which confirmed the high selectivity of the ply-MCDA assay for S.pneumoniae. The AUDG enzyme could effectively eliminate carryover contamination and thus prevented false-positive results. In conclusion, ply-AUDG-MCDA-LFB is a simple, rapid and accurate method to detect S.pneumoniae.