Use of a recombinase polymerase amplification commercial kit for rapid visual detection of Pasteurella multocida

Abstract

Background Pasteurella multocida (P. multocida) is a bacterium that causes bovine respiratory disease (BRD) and haemorrhagic septicaemia (HS) in cattle, buffaloes and bison. Rapid point-of-care diagnosis or regular testing of Pasteurellosis, therefore, could contribute greatly to early detection, and screening infected animal is important. Up to now, there are no published reports on the use of recombinase polymerase amplification (RPA) combined with a lateral flow dipstick (LFD) for P. multocida detection. Results This study proposes a promising isothermal detection method for P. multocida with the potential to be developed as an on-site test for Pasteurellosis. The method includes an RPA combined with LFD . First, the analytical sensitivity and specificity of P. multocida RPA-LFD were tested. The RPA-LFD, performed at 39 °C, successfully detected P. multocida DNA in 30 min, with a detection limit of up to 120 copies per reaction. Then, the practicability of RPA-LFD was analysed using 62 nasal swabs and 33 fresh lungs samples from 17 different dairy farms. Compared to real-time quantitative PCR (qPCR), the RPA-LFD assay yielded a clinical specificity of 95.15%, positive predictive value (PPV) of 95.15% and 0.958 kappa coefficient. Compared with the culture method, it achieved 100% sensitivity, 67.20% specificity and a 0.572 kappa coefficient. Conclusions These results combined with the simple conditions required for the performance of the RPA-LFD assay, have demonstrated the effectiveness and practicability of the method for development into a regular on-site protocol for the diagnosis of Pasteurellosis.

A Sensitive and Accurate Recombinase Polymerase Amplification Assay for Detection of the Primary Bacterial Pathogens Causing Bovine Respiratory Disease

“…bovis assays, respectively, and 134 genome copies for ICE. Limits of detection were similar to other published RPA and multiplex RPA assays (27,32,35,36). Sensitivity of RPA depends greatly on primer and probe design, but design software and recommendations are currently lacking (19).…”

“…Beker et al (13) developed a multiplex PCR assay targeting four conserved core genes required for integration and maintenance of ICE structures within the Pasteurellaceae family and demonstrated relevance of this assay to detecting these elements in P. multocida and M. haemolytica (13). Furthermore, RPA has recently been utilized for detection of P. multocida in cattle (32). However, to our knowledge, this is the first study to develop and apply RPA for detecting four major bacterial BRD pathogen species in multiplex and real-time formats, and BRD pathogen-associated with ICEs in bovine DNPS.…”

Fast, simple and highly specific molecular detection of Vibrio alginolyticus pathogenic strains using a visualized isothermal amplification method

“…The RPA-LFD method has been applied for the detection of many pathogens, including goose parvovirus [33], Burkholderia mallei [34], Trichinella spp. [35], Mycoplasma bovis [36], Candidatus Liberibacter asiaticus [37], Pasteurella multocida [38] and Phytophthora soja [39].…”

A rapid field-based assay using recombinase polymerase amplification for identification of Thrips palmi, a vector of tospoviruses

“…This technique has been successfully used for diagnosis of animal and human diseases including porcine coronavirus in piglets, Pasteurella multocida in cattle, Schistosoma haematobium in humans, and zoonotic diseases caused by Mycobacterium avium subsp. paratuberculosis (Rosser et al 2015 ; Hansen et al 2016 ; Zhao et al 2019 ; Ma et al 2019a , b ) . RPA has also been used in the diagnosis of plant pathogens including banana bunchy top virus, little cherry virus 2, and citrus greening bacterium (Mekuria et al 2014 ; Londono et al 2016 ; Kapoor et al 2017 ; Ghosh et al 2018 ).…”