Highly Sensitive and Rapid Identification of Streptococcus agalactiae Based on Multiple Cross Displacement Amplification Coupled With Lateral Flow Biosensor Assay

Streptococccus agalactiae (S. agalactiae) is an important neonatal pathogen that is associated with mortality and morbidity. Therefore, we developed a rapid, accurate, and sensitive method based on multiple cross displacement amplification (MCDA) for the detection of the target pathogen. Four sets of MCDA primers were designed for targeting the S. agalactiae-specific groEL gene, and one set of MCDA primers with the optimum amplification efficiency was screened for establishing the S. agalactiae-MCDA assay. As a result, the newly-developed assay could be conducted at a fixed temperature (61°C) for only 30 min, eliminating the use of complex instruments. A portable and user-friendly nanoparticle-based lateral flow biosensor (LFB) assay was employed for reporting MCDA results within 2 min. Our results suggested that the detection limit of the S. agalactiae-MCDA-LFB assay is 300 fg per reaction, and no cross-reaction occurred with non-S. agalactiae strains. For 260 vaginal and rectal swabs, the detection rate of the MCDA-LFB assay was 7.7%, which was in accordance with the reference method of enrichment/qPCR, and higher by 4.6% than the CHROMagar culture. Moreover, the total procedure time of the MCDA-LFB assay was around 50 min, including sample collection, template preparation, MCDA reaction, and result reporting. Therefore, the MCDA-LFB assay is superior to enrichment/qPCR and CHROMagar culture and has great promise for point-of-care testing of S. agalactiae from vaginal and rectal swabs of pregnant women in resource-limited settings.