Visual multiple cross displacement amplification for the rapid identification of S. agalactiae immediately from vaginal and rectal swabs

Xueqin Cheng,Yijun Tang,Xiaobing Li,Meifang Wang,Jing Yang,Yulong Gu,Fenglin Cai,Dexi Liu,Zhiqian Dou

doi:10.1186/s13568-020-01168-3

Abstract

Streptococcus agalactiae (S. agalactiae) is an important pathogen that can lead to neonatus and mother infection. The current existing techniques for the identification of S. agalactiae are limited by accuracy, speed and high-cost. Therefore, a new multiple cross displacement amplification (MCDA) assay was developed for test of the target pathogen immediately from vaginal and rectal swabs. MCDA primers screening were conducted targeting S. agalactiae pcsB gene, and one set of MCDA primers with better rapidity and efficiency was selected for establishing the S. agalactiae-MCDA assay. As a result, the MCDA method could be completed at a constant temperature of 61 °C, without the requirement of special equipment. The detection limit is 250 fg (31.5 copies) per reaction, all S. agalactiae strains displayed positive results, but not for non-S. agalactiae strains. The visual MCDA assay detected 16 positive samples from 200 clinical specimen, which were also detected positive by enrichment/qPCR. While the CHROMagar culture detected 6 positive samples. Thus, the MCDA assay is prefer to enrichment/qPCR and culture for detecting S. agalactiae from clinical specimen. Particularly, the whole test of MCDA takes about 63.1 min, including sample collection (3 min), DNA preparation (15 min), MCDA reaction (45 min) and result reporting (6 s). In addition, the cost was very economic, with only US$ 4.9. These results indicated that our S. agalaciae-MCDA assay is a rapid, sensitive and cost-efficient technique for target pathogen detection, and is more suitable than conventional assays for an urgent detection, especially for 'on-site' laboratories and resource-constrained settings.

Highlights

Streptococcus agalactiae is a gram-positive group B Streptococcus (GBS) that is associated with the asymptomatic colonization of human urogenital and gastrointestinal tracts (Kwatra et al 2016; Vieira et al 2019)
Primers screening In order to achieve the optimum amplification situation of S. agalactiae, three sets of multiple cross displacement amplification (MCDA) primers targeting pcsB gene were designed
Confirmation of MCDA products Three groups of genomic DNA were prepared to confirm the feasibility of the secreeded MCDA primers, including the first group of S. agalactiae ATCC12386, the second group of S. pyogenes, and the third group of S. pneumoniae

Summary

Introduction

Streptococcus agalactiae is a gram-positive group B Streptococcus (GBS) that is associated with the asymptomatic colonization of human urogenital and gastrointestinal tracts (Kwatra et al 2016; Vieira et al 2019). This opportunistic pathogen can cause serious infections in susceptible hosts, especially in neonates, Cheng et al AMB Expr (2021) 11:9 agalactiae significantly reduced in western countries (Verani et al 2010). Pregnant women who screened positive and received antibiotic treatment whose babies subsequently still infected with S. agalactiae. Rapid screening techniques at the time of delivery were needed to do to prevent the occurrence of serious early-onset disease

Methods

Results

Conclusion