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

Abstract

Rapid and accurate diagnosis of bovine respiratory disease (BRD) presents a substantial challenge to the North American cattle industry. Here we utilize recombinase polymerase amplification (RPA), a fast and sensitive isothermal DNA-based technology for the detection of four BRD pathogens (Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycoplasma bovis), genes coding antimicrobial resistance (AMR) and integrative conjugative elements (ICE) which can harbor AMR genes. Eleven RPA assays were designed and validated including: a) one conventional species-specific multiplex assay targeting the 4 BRD pathogens, b) two species-specific real-time multiplex RPA assays targeting M. haemolytica/M. bovis and P. multocida/H. somni, respectively with a novel competitive internal amplification control, c) seven conventional assays targeting AMR genes (tetH, tetR, msrE, mphE, sul2, floR, erm42), and d) one real-time assay targeting ICE. Each real-time RPA assay was tested on 100 deep nasopharyngeal swabs (DNPS) collected from feedlot cattle previously assessed for targets using either culture methods and/or polymerase chain reaction (PCR) verification (TC-PCR). The developed RPA assays enabled sensitive and accurate identification of BRD agents and AMR/ICE genes directly from DNPS, in a shorter period than TC-PCR, showing considerable promise as a tool for point-of-care identification of BRD pathogens and antimicrobial resistance genes.

Mycoplasma bovis Infections—Occurrence, Diagnosis and Control

“…Traditionally, culture is used for the confirmation of BRD infections, but the incubation period for each examined bacterial pathogens is different and samples inoculated onto agar plates are often overgrown with other, fast growing bacteria. For that reason, the multiplex real-time PCRs used by the laboratories [ 49 , 50 , 76 ] are the most suitable for simultaneous direct detection of M. bovis and other pathogens involved in BRD, such as P. multocida , M. haemolytica and H. somni , in contrast to methods not dedicated for different pathogen identification in mixed infections such as one-target PCR, traditional culture or MALDI-TOF MS [ 77 ]. When using one target PCR, there is no information about the involvement of other pathogens in the disease, different bacteria have various growth requirements and slow growing bacteria can be easily overgrown by others, and MALDI-TOF MS is not able properly detect all organisms from polymicrobial samples.…”

Detection of blueberry stunt phytoplasma in Eastern Canada using cpn60-based molecular diagnostic assays

“…Therefore, other tests must be undertaken to confirm sequence identity or quantify pathogen load. Recently described, alternative isothermal amplification methods such as Recombinase Polymerase Amplification have been shown to generate results with comparable speed and sensitivity, but simpler primer design than LAMP 42 .…”

Molecular Detection of Blueberry Stunt Phytoplasma in Eastern Canada: a Multi-year Study

“…Therefore, other tests must be undertaken to con rm sequence identity or quantify pathogen load. Recently described, alternative isothermal ampli cation methods such as Recombinase Polymerase Ampli cation have been shown to generate results with comparable speed and sensitivity, but simpler primer design than LAMP 37 .…”

“…Samples that were discordant (LAMP positive/nested PCR negative) were re-analyzed using BBSP-speci c real-time qPCR as described here (Table S1) as well as using nested PCR targeting the ribosomal protein (rp) operon 15 . For rp operon ampli cation, primers rpF1/rpR1 37 were used in the rst round, then the PCR product was diluted 1:30 and 2 µl used in a second round with primers rp(I)FIA/rp(I)RIA 38 . PCR and thermal cycling conditions for rp (both rounds) were as reported by Lee et al 38 , except 1U Taq polymerase (Invitrogen) was used per reaction.…”