Development of Loop-Mediated Isothermal Amplification Assay Based on 6b Gene Sequence for Rapid Detection of Pasteurella multocida B:2

Abstract

Accurate, sensitive and rapid diagnostic methods are required for early management of outbreaks and for implementing control programme for haemorrhagic septicaemia (HS). The objectives of the present study were to develop a loop-mediated isothermal amplification assay for the detection of HS-causing type B strains of Pasteurella multocida (HSPmB-LAMP) and to compare its specificity, sensitivity and other features with those of conventional HS causing P. multocida type B-specific polymerase chain reaction (HSPmB-PCR). Six LAMP primers targeting HS causing P. multocida type B-specific 6b gene sequence (GenBank accession no: AF016260.1) were designed and used for optimization of HSPmB-LAMP. The HSPmB-LAMP reaction gave consistent results when the reaction mixture contained 6 mM MgSO4, 0.8 M betaine and loop primers at 63 °C for 75 min in a water bath or a thermo-cycler. The LAMP products were detected as ladder like patterns in agarose gel electrophoresis and also by picogreen fluorescence method. The detection limit of the HSPmB-LAMP assay was 1 pg of genomic DNA template, about ten times lower than that of conventional HSPmB-PCR. The developed assay was specific for P. multocida B:2 and gave no false amplification with P. multocida type A and various other bacteria. Thus a rapid, highly sensitive, specific and economical HSPmB-LAMP assay for diagnosis of HS caused by P. multocida B:2 has been developed for the first time.