Investigation ofStreptococcus agalactiaeusingpcsB‐based LAMP in milk, tilapia and vaginal swabs in Haikou, China

Abstract

Aims To develop a pcsB‐based Loop‐mediated isothermal amplification (LAMP) method for the detection of Streptococcus agalactiae (GBS) in milk, tilapia and vaginal swabs. Methods and Results The sensitivity of the LAMP method using real‐time turbidity monitoring was 1 pg of template within 1 h at 64°C, 100‐fold higher than conventional PCR. The sensitivity of visual detection dropped an order of magnitude using SYBR Green I or hydroxynaphthol blue. The validity of the visual LAMP assay was assessed by the detection of GBS in 180 vaginal swabs from one hospital, 14 brain tissues samples of diseased tilapias from two fishponds and fresh milk of 67 dairy cattle from one farm. In total, 17 samples (4 vaginal swabs, 13 tilapia brain tissues but no milk sample) tested positive for GBS. Subsequent bacterial identification confirmed the specificity and reliability of the LAMP method. Molecular serotyping and multilocus sequence typing demonstrated that all 13 tilapia GBS isolates were identical (serotype Ia, ST7), whereas the four human GBS isolates were more diverse and could be classified into two serotypes (Ia, III) and four sequence types (ST19, ST23, ST24, ST862). Virulence gene testing showed that only the bac, rib and lmb genes were not present in all isolates. Antimicrobial susceptibility profiles of the isolates were basically consistent with their genotypes, except for sulphonamide and fluoroquinolone. Conclusions We developed a reliable pcsB‐based LAMP assay for GBS detection. Our results demonstrated that the prevalence of GBS was 92·9% among diseased tilapia, 2·2% among female patients and 0% on a dairy farm in Hainan. Significance and Impact of the Study The pcsB‐based LAMP method is suitable for GBS detection and contains great potential of application in dairy industry, aquiculture and clinical.

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

“…The minimum detection limit of the S. agalactiae -MCDA-LFB assay is 300 fg, which is 333-fold more sensitive than the S. agalactiae -PCR assay, the detection threshold of which was 100 pg ( Pu et al, 2019 ). Apart from its excellent sensitivity, the MCDA-LFB assay also has high specificity that can clearly differentiate S. agalactiae from other Streptococcus and non- Streptococcus species.…”

“…Moreover, the sensitivity of qPCR is controversial, as it varied from 62.5 to 98.5% compared to that of the bacterial culture method ( Verani et al, 2010 ). The popular loop-mediated isothermal amplification (LAMP) assay has also been developed for detecting S. agalactiae ( Kimura et al, 2013 ; Pu et al, 2019 ), and the LAMP products were usually analyzed by a turbidimeter or colorimetric indicator. However, the results obtained by a turbidimeter inevitably suffer from background interference, and the results are somewhat subjective when using colorimetric indicator with the naked eye, especially when using a limited quantity of DNA, usually leading to false-negative results.…”

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

“…The whole experiment only depends on a simple heater, with the weight of 1 kg, which is very easy to achieve in resource-limited restricts, not like an expensive thermal cycler that required for qPCR. The detection limit of the MCDA assay was 250 fg, which is more sensitive than 100 pg of PCR and 1 pg of LAMP (Pu et al 2019). The MCDA assay depends on ten specific primers recognizing 10 different regions of pcsB sequence to accomplish amplification.…”

LAMP-Based Point-of-Care Biosensors for Rapid Pathogen Detection

“…Garg et al [ 40 ] introduced successful LAMP-based turbidimetric detection of Mycobacterium leprae targeting the 16s rRNA gene. Likewise, similar studies reported the detection of multiple pathogens—for instance, Clostridium botulinum [ 41 ], Streptococcus pneumoniae [ 42 ], and Streptococcus agalactiae [ 43 ]. However, this method provides low sensitivity, as the faint product can easily be misjudged.…”