Development of a loop-mediated isothermal amplification assay for the detection of Streptococcus agalactiae in bovine milk

Abstract

Streptococcus agalactiae is a well-characterized bovine mastitis pathogen that is known to be highly contagious and capable of spreading rapidly in affected dairy herds. Loop-mediated isothermal amplification (LAMP) is a novel molecular diagnostic method that has the capability to provide rapid, cost-effective screening for pathogens to support on-farm disease control and eradication programs. In the current study, a LAMP test was developed to detect S. agalactiae in milk. The assay was validated on a bank of existing clinical mastitis milk samples that had previously been identified as S. agalactiae positive via traditional microbiological culture techniques and PCR. The LAMP assay was conducted on bacterial colonies and DNA extracted from milk in tube- and plate-based formats using multiple detection platforms. The 1-h assay conducted at 64 °C exhibited repeatability (coefficient of variation) of 2.07% (tube) and 8.3% (plate), sensitivity to ~20 pg of extracted DNA/reaction, and specificity against a panel of known bacterial mastitis pathogens. Of the 109 known S. agalactiae isolates assessed by LAMP directly from bacterial cells in culture, 108 were identified as positive, in accordance with PCR analysis. The LAMP analysis from the corresponding milk samples indicated that 104 of these milks exhibited a positive amplification curve. Although exhibiting some limitations, this assay provides an opportunity for rapid screening of milk samples to facilitate on-farm management of this pathogen.

An update on environmental mastitis: Challenging perceptions

“…A relatively novel mastitis-diagnostic with potential for short time-to-result is loop-mediated isothermal amplification (LAMP). LAMP primers are available for S. aureus (Sheet et al, 2016) and major streptococci (Bosward et al, 2016;Wang & Liu, 2015), and implementation as pregnancy-test-like lateral flow device is possible (Cornelissen et al, 2016). A major challenge for on-farm molecular detection of pathogens or AMR genes is the large number of bacterial species and resistance genes that may be present in milk or mastitis pathogens.…”

Indirect enzyme-linked immunosorbent assay method based on Streptococcus agalactiae rSip-Pgk-FbsA fusion protein for detection of bovine mastitis

“…Although numerous pathogenic microorganisms have been implicated in BM, Streptococcus agalactiae (SA) is the main causal pathogen of this disease (Bramley and Schultze 1991, Baseggio et al 1997, Pinto et al 2013, Wang et al 2015a, Bosward et al 2016. The detection rate of SA in mastitis milk samples from cows in Inner Mongolia of China was reported to be 70%, posing great hazards to human and animal health (Andersen et al 2003, Duarte et al 2004, Verhoeven et al 2014.…”

Quantification of microRNAs directly from body fluids using a base-stacking isothermal amplification method in a point-of-care device

“…This allows greater distinction between levels of microRNA. The coefficient of variation among all tested samples was also low (mean CV = 2.6%), which is near or below the CV observed in other studies using isothermal amplification following DNA extraction (Bosward et al 2016; Brotons et al 2016) or directly from groundwater samples (Stedtfeld et al 2016). …”