Rapid detection of Streptococcus uberis in raw milk by loop-mediated isothermal amplification

Abstract

A loop-mediated isothermal amplification (LAMP) method to detect Streptococcus uberis in raw milk was developed and evaluated. Three genes (sodA, pauA, cpn60) were assessed for their suitability as targets in LAMP. The analytical sensitivity was 120, 120, and 12 fg per assay for the sodA, pauA, and cpn60 assays, respectively, with a detectable signal within 8 min for the highest concentration (12ng/assay) and ~60 min for the lowest concentrations. The LAMP assays correctly identified 7 Strep. uberis strains among a set of 83 mastitis pathogens. To enable DNA isolation from raw milk, a new method was used in which a pretreatment with a cocktail of lysing enzymes was performed before an established procedure. This method resulted in an analytical sensitivity of 48 cfu/assay for the sodA LAMP assay using raw milk spiked with Strep. uberis, corresponding to 2.4×10(4) cfu/mL milk. For raw milk samples from cows experimentally infected with Strep. uberis, results of enumeration were largely reflected by results of LAMP. Evaluation of the sodA LAMP assay with 100 raw milk field samples, of which 50 were Strep. uberis culture-negative and 50 Strep. uberis culture-positive, showed that the assay had a diagnostic sensitivity of 96.0% and a diagnostic specificity of 96.0%. In conclusion, the described LAMP assay may offer a simple alternative for convenient and sensitive detection of S. uberis in raw milk, provided a compatible rapid DNA isolation procedure is available.

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.…”

Development of a novel loop-mediated isothermal amplification assay for the detection of lipolytic Pseudomonas fluorescens in raw cow milk from north China

“…Comparing this to the existing literature, 2 studies have previously reported that the results produced from the LAMP assay in detecting P. fluorescens in pure culture and in artificially contaminated milk samples were of a similar order of magnitude (Cho et al, 2014;Ye et al, 2015). Three out of 5 published studies have suggested that the detection limit of the method is lower in pure culture than in milk (Yang et al, 2011;Wang et al, 2015;Cornelissen et al, 2016). The DNA extraction method used is an important factor that may affect the results obtained (Sowmya et al, 2012).…”

“…The culture-dependent method for the detection of microorganisms in biological samples is both laborious and time consuming, and can thus result in a missed opportunity to control and improve the quality of raw cow milk. Culture-independent methods have been shown to be useful for the rapid and specific detection of microorganisms (Cho et al, 2014;Bosward et al, 2016;Cornelissen et al, 2016). As a nucleic acid-based amplification method, loop-mediated isothermal amplification (LAMP) has been developed for the rapid detection of a small amount of microorganisms.…”

“…Four to 6 specific primers have been designed for use in LAMP, together with a strand-displacing Bst DNA polymerase that can amplify the target DNA by up to 10 9 copies per hour (Notomi et al, 2000). In addition, LAMP proceeds under isothermal conditions and does not require expensive and sophisticated instruments (Cornelissen et al, 2016). Positive LAMP reactions can be judged by the naked eye following DNA amplification with a thermal cycler and gel electrophoresis (Kumar and Mondal, 2015).…”

Development of an isothermal amplification-based assay for the rapid visual detection of Salmonella bacteria

“…However, current detection methods require expensive instruments or inconvenient, time-consuming procedures that severely limit their suitability for point-of-use detection (Rohde et al, 2016). Recombinase polymerase amplification is a recently described isothermal amplification technique that can amplify target DNA to detectable levels in less time and at a lower constant temperature than other isothermal amplification procedures, such as helicase-dependent amplification (Ramalingam et al, 2009), loop-mediated isothermal amplification (Cornelissen et al, 2016), and rolling circle amplification (Zhu et al, 2014). In addition, RPA is tolerant to impure samples (Krõlov et al., 2014).…”