title: Development of loop-mediated isothermal amplification-based diagnostic assays for detection of Pasteurella multocida and hemorrhagic septicemia-associated P multocida serotype B:2.
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 HS-LAMP 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 HS-LAMP 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.
authors.forename: Ahmed M
authors.initials: AM
authors.lastname: Moustafa
authors.forename: Mark D
authors.initials: MD
authors.lastname: Bennett
mesh_terms: D000818:Animals; D002020:Buffaloes; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000382:microbiology; D006483:Hemorrhagic Septicemia / Q000175:diagnosis / Q000662:veterinary*; D021141:Nucleic Acid Amplification Techniques / Q000662:veterinary*; D016979:Pasteurella multocida / Q000145:classification / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D065288:Serogroup