Loop-mediated isothermal amplification assay for the detection of Salmonella spp. in pig feces

Abstract : Salmonella are causative agents of gastroenteritis and systemic disease in animals. The invA gene was selectedas a target sequence of loop-mediated isothermal amplification (LAMP) assay for diagnosis of Salmonella infection.The detection limits for broth dilution, spiked feces and enrichment were 10 4 , 10 5 and 10 2 CFUs/mL, respectively.The LAMP assay developed in the present study may be a reliable method for detection of Salmonella spp. in pig feces.Keywords : detection limit, invA, loop-mediated isothermal amplification assay, Salmonella Salmonella (S.) are gram-negative bacilli belong to thefamily of Enterobacteriaceae and comprise a number ofclosely related serotypes, many of which are responsible forvarious illness and death in animals [5, 8]. Although most ofthe Salmonella cause gastrointestinal disorder, some Serovarsuch as Cholerasuis, Abortusequi, Gallinarum produce sys-temic diseases [3]. It is important that the identification ofcausative agent is simple and rapid. A variety of methodssuch as isolation and PCR have been applied for salmonello-sis in animal and human beings. Although isolation of agentsis golden method for diagnosis of the disease, it is laboriousand time consuming. Thus, molecular diagnostics such asPCR have been applied to detect Salmonella spp. in fecesand pork [1]. However, there are shortcomings such as carry-over contaminations and non-specific reactions. Recently,loop-mediated isothermal amplification (LAMP) has beendeveloped and used as an alternative for molecular diagnosis[13]. LAMP is a nucleic acid amplification technique thatrelies on an auto-cycling strand displacement DNA synthesisunder isothermal condition [10, 11, 12, 13]. Although thereare some reports for inspections such as eggs and pork [14,19], there are few reports for developing LAMP for diagnos-tic purpose. Therefore, LAMP assay was developed for ampli-fying invA gene to detect the Salmonella spp. and evaluatedfor the detection limit and specificity. Nineteen strains containing 6 Salmonella spp. (Cholerasuis,Derby, Enteritidis, Montevideo, Schwarzenground, Typhimu-rium) and 13 non-Salmonella spp. (Acinetobacter haemolyticusATCC17906, Acinetobacter johnsonii ATCC17909, Acineto-bacter junii ATCC17098, Alcaligenes xylosoxydans ATCC15173,Burkholderia cepacia ATCC25416, Comamonas testosteroniATCC11996, Enterobacter cloacae ATCC13047, Escheri-chia coli ATCC25922, Kocuria rosea ATCC186, Micrococ-cus luteus ATCC4698, Ochrobactrum anthropi ATCC49188,Serratia marcescens ATCC13880, Stenotrophomonas malto-philia ATCC13637) were used. All strains of Salmonellawere from field isolates and serogrouped by O and H anti-body (Becton, Dickson and Company, USA) except S. Chol-eraesuis (ATCC 10708). The primer sequence is shown inTable 1. Salmonella invasion protein gene (invA) was tar-geted for the LAMP assay. Each primer was designed usingPrimerExplorer V3 software (Eiken Chemical, Japan). Theprimers consisted of forward outer (F3), backward outer(B3), forward inner (FIP), backward inner (BIP), loop for-ward (LF), and loop backward (LB). The reaction was car-ried out in a 25µL volume containing 15µL of IsothermalMaster Mix (OptiGene, UK), 50µM of the primers FIP andBIP, 25µM of the primers LF and LB, 5µM of the primersF3 and B3 and 2µL of DNA template. The amplified signalwas detected by Genie (OptiGene) under the 63