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Development of loop-mediated isothermal amplification–based diagnostic assays for detection of Pasteurella multocida and hemorrhagic septicemia–associated P multocida serotype B:2

Moustafa, A.M. and Bennett, M.D. (2017) Development of loop-mediated isothermal amplification–based diagnostic assays for detection of Pasteurella multocida and hemorrhagic septicemia–associated P multocida serotype B:2. American Journal of Veterinary Research, 78 (2). pp. 134-143.

Link to Published Version: http://dx.doi.org/10.2460/ajvr.78.2.134
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Abstract

OBJECTIVE To develop 2 rapid loop-mediated isothermal amplification (LAMP) assays for detection of Pasteurella multocida DNA (Pm-LAMP assay) and P multocida DNA from strains associated with hemorrhagic septicemia (HS) in cattle and buffalo (HS-LAMP assay).

SAMPLE Solutions containing 16 P multocida strains and 9 other bacterial species at various concentrations.

PROCEDURES Optimal conditions were determined for running the Pm-LAMP and HSLAMP assays. The assays were then used to detect DNA of the test organisms. Results of LAMP assays were validated against conventional PCR assays designed for specific detection of P multocida and the B:2 serotype of HS-associated strains.

RESULTS Following incubation of sample reaction mixtures for 27 minutes, specificity and sensitivity of the HS-LAMP assay at template DNA amounts as low as 5 pg were 93% and 97%, respectively. When duplicates of each sample were incubated for 28 minutes (a positive result defined as positive results for both reactions of a given sample), specificity and sensitivity of the HSLAMP assay in the same conditions increased to 100%. The best specificity and sensitivity of Pm-LAMP single (93% and 91%) and duplicate (97% and 98%) reactions at template DNA amounts as low as 10 pg were achieved at 33 and 34 minutes, respectively.

CONCLUSIONS AND CLINICAL RELEVANCE These preliminary findings suggested the developed HS-LAMP assay had high sensitivity and specificity for detection of HS-associated P multocida. Additional research is needed to determine the accuracy of the assay for use on clinical specimens obtained in HS-endemic countries such as Pakistan and Thailand.

Publication Type: Journal Article
Murdoch Affiliation: School of Veterinary and Life Sciences
Publisher: American Veterinary Medical Association
Copyright: © 2017 American Veterinary Medical Association
URI: http://researchrepository.murdoch.edu.au/id/eprint/35736
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