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

Xueqin Cheng,Ling Xie,Zhifang Yu,Meifang Wang,Jing Yang,Dexi Liu,Yulong Gu,Tao Ren,Zhiqian Dou,Yijun Tang,Yan Liu

doi:10.3389/fmicb.2020.01926

Abstract

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.

Highlights

Streptococccus agalactiae is a common neonatal pathogen that can cause severe early-onset diseases, such as pneumonia, sepsis, and, less frequently, meningitis (Boyer and Gotoff, 1985; McGee et al, 2010)
The only instrument required for the multiple cross displacement amplification (MCDA) assay is a thermostatic apparatus, which is achieved in remote districts, unlike qPCR that depends on expensive thermal cycling equipment or a CO2 incubator that is required for the culture method
The prevalence of S. agalactiae by CHROMagar culture was 3.1%, which was similar to the results previously reported (Ji et al, 2019). This may be due to the fact that Staphylococcus species and Enterococcus species displayed purple colonies on the CHROMagar plates, which may lead to the missed detection of S. agalactiae. All these results demonstrated that the MCDA-lateral flow biosensor (LFB) assay is a rapid, sensitive, and specific technique for detecting S. agalactiae directly from vaginal and rectal swabs

Summary

Introduction

Streptococccus agalactiae is a common neonatal pathogen that can cause severe early-onset diseases, such as pneumonia, sepsis, and, less frequently, meningitis (Boyer and Gotoff, 1985; McGee et al, 2010). Due to the vertical transmission of S. agalactiae from carrier mothers to newborn infants, the maternal rectovaginal colonization becomes the most important risk element of earlyonset disease. Timely detection of the maternal S. agalactiae carrier state can help clinicians to prevent the occurrence of vertical transmission during labor (Verani et al, 2010). According to some reported studies in mainland China, the maternal S. agalactiae colonization rate is about 3.7–14.52%, and the incidence rate of invasive S. agalactiae diseases in infants is estimated to be between 0.55 and 1.79 per 1,000 live births, with a fatality risk around 6.775%, which is still a substantial burden on developing countries such as China (Huang et al, 2019). Prenatal screening and early diagnosis of S. agalactiae in actively laboring women in China are needed to help prevent the occurrence of severe disease in infants

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