Loop-Mediated Isothermal Amplification for the Detection of Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in Bovine Nasal Samples

Abstract

This work develops a loop-mediated isothermal amplification (LAMP) assay that detects the presence of bacterial pathogens (Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni) for bovine respiratory disease complex (BRD) in crude nasal samples. Diagnosing BRD involves a physical examination of cattle expressing physical symptoms linked to the disease. Unfortunately, these symptoms are not unique to BRD alone and do not identify specific pathogens to treat. Nucleic acid-based diagnostics, like polymerase chain reaction (PCR), identify BRD pathogens by amplifying species-specific genes present in samples. However, PCR-based approaches require (i) expensive equipment for successful operation and (ii) DNA extractions to remove PCR inhibitors in samples. LAMP offers an accurate, inhibitor-resistant approach to detecting BRD pathogens in a point-of-care format. This developed LAMP assay is 97% accurate in pure DNA samples, 99% sensitive, and 89% specific in DNA-spiked bovine nasal samples (with 10 4 DNA copies/reaction).

A paper-based colorimetric molecular test for SARS-CoV-2 in saliva

“…We evaluated several metal-ion-chelating, DNA intercalating, and pH-sensitive colorimetric reporters for their ability to produce a robust and discernible colorimetric response during the RT-LAMP reaction using primer sets our lab had previously developed to target Histophilus somni ( Mohan et al, 2021 ). Some of the investigated metal-ion-chelating and DNA intercalating indicators were able to produce a colorimetric response in solution but were unable to produce a colorimetric response on paper ( Fig.…”

On-farm colorimetric detection of Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in crude bovine nasal samples

“…Glycerol stocks of P. multocida , M. haemolytica , and H. somni isolates were obtained from Purdue University’s Indiana Animal Disease Diagnostic Laboratory (ADDL), as previously described [ 16 ]. These isolates were originally cultured by ADDL as a part of routine diagnostic testing from lung/nasopharyngeal sample submissions and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis.…”

“…P. multocida , M. haemolytica , and H. somni were isolated and final DNA concentrations were quantified according to Mohan et al [ 16 ]. Specifically, all pathogens were streaked on tryptic soy agar plates supplemented with defibrinated sheep blood (blood agar).…”

“…The colorimetric assay was conducted by modifying the previously published procedure [ 16 ]. Specifically, in the colorimetric assay, the New England Biolabs’ Warmstart Colorimetric LAMP 2 × Master Mix was used.…”

“…We have recently designed a LAMP assay to detect the presence of Pasteurella multocida , Mannheimia haemolytica , and Histophilus somni from bovine nasal samples using a fluorescence reporter with 89% analytical specificity and 99% analytical sensitivity [ 16 ]. In the current work, we report a colorimetric assay that can be conducted on the farm to detect these bacterial pathogens.…”