Design and application of a loop-mediated isothermal amplification assay for the rapid detection of Staphylococcus pseudintermedius

Abstract

Abstract. Staphylococcus pseudintermedius is a commensal and opportunistic pathogen of dogs. It is mainly implicated in canine pyoderma, as well as other suppurative conditions of dogs. Although bacterial culture is routinely used for clinical diagnosis, molecular methods are required to accurately identify and differentiate S. pseudintermedius from other members of the Staphylococcus intermedius group. These methods, owing largely to their cost, are not easy to implement in nonspecialized laboratories or veterinary practices. In the current study, loop-mediated isothermal amplification (LAMP), a novel isothermal nucleic acid amplification procedure, was employed to develop a rapid, specific, and sensitive S. pseudintermedius assay. Different detection strategies, including the use of a lateral flow device, were evaluated. The assay was evaluated for crossreactivity against 30 different bacterial species and validated on a panel of 108 S. pseudintermedius isolates, originating from different dog breeds and locations within the United Kingdom. The assay was specific, showing no cross-reactivity during in silico and in vitro testing. When tested using DNA extracts prepared directly from 35 clinical surgical site swabs, the assay could detect S. pseudintermedius in less than 15 min, with a diagnostic sensitivity of 94.6%, superior to that of a polymerase chain reaction method. The LAMP assay also had an analytical sensitivity in the order of 10 1 gene copies, and the amplified products were readily detected using a lateral flow device. The LAMP assay described in the present study is simple and rapid, opening up the possibility of its use as a diagnostic tool within veterinary practices.

DNA Microsystems for Biodiagnosis

“…Since this analytical method is still mainly used in research laboratories, new approaches are being investigated to make it practical, such as developing a portable detection prototype based on Arduino, an open electronics micro controller board [81]. Currently, LAMP is more accurate than quantitative-PCR for the detection of meningococcal infections in children [82], gene doping therapies [83], peanut allergens in processed food [84], acute viral necrobiotic infections in scallops [85], and Staphylococcus pseudintermedius bacterial infections in canines [86].…”

Enterohaemorrhagic and other Shiga toxin-producingEscherichia coli(STEC): Where are we now regarding diagnostics and control strategies?

“…The efficiency and speed of LAMP technology means that tests can detect STEC infections and provide information on, for example, AMR profiles, in <15 min, directly from a swab, with excellent specificity and sensitivity (Diribe, Thomas, AbuOun, Fitzpatrick, & La Ragione, ; Diribe et al., ).…”

“…Recent advances in genome sequencing technology enable primers to be designed on pan‐genomes generated from multiple strains, thus increasing specificity. LAMP can detect between 5 and 10 organisms in a swab sample and is therefore considered to have superior sensitivity to culture or PCR‐based diagnostics (Diribe et al., ; La Ragione, R.M., personal communication). LAMP assays have been designed on a number of targets, including rfbe for Enterohemorrhage E. coli, Z3276 gene for the detection of E. coli O157:H7 and stx1 with stx2 for Shiga toxins.…”

Development of Loop-Mediated Isothermal Amplification Rapid Diagnostic Assays for the Detection of Klebsiella pneumoniae and Carbapenemase Genes in Clinical Samples

“…LAMP is a single-step nucleic acid amplification technique that is rapid, sensitive, and cost-effective. This technique can amplify a few copies of DNA into billions of copies within 30 min ( Notomi et al, 2000 ; Diribe et al, 2014 ) and has been used to detect a wide range of viral, bacterial, and parasitic pathogens ( Wong Y.-P. et al, 2018 ; Li et al, 2019 ; Tian et al, 2019 ; Vergara et al, 2020 ). LAMP is less sensitive to PCR inhibitors in poorly or nonprocessed samples, such as blood, sputum, and urine, making it suitable for use as a point-of-care diagnostic ( Notomi et al, 2000 ; Nagamine et al, 2002 ; Kaneko et al, 2005 ; Parida et al, 2008 ; Francois et al, 2011 ; Dhama et al, 2014 ; Gadkar et al, 2018 ; Moehling et al, 2021 ).…”