Highly Sensitive and Specific Detection and Serotyping of Five Prevalent Salmonella Serovars by Multiple Cross-Displacement Amplification

Abstract

Salmonella is a common cause of foodborne disease worldwide, including Australia. More than 85% of outbreaks of human salmonellosis in Australia were caused by five Salmonella serovars. Rapid, accurate, and sensitive identification of Salmonella serovars is vital for diagnosis and public health surveillance. Recently, an isothermal amplification technique, termed multiple cross-displacement amplification (MCDA), has been employed to detect Salmonella at the species level. Herein, seven MCDA assays were developed and evaluated for rapid detection and differentiation of the five most common Salmonella serovars in Australia: Typhimurium, Enteritidis, Virchow, Saintpaul, and Infantis. MCDA primer sets were designed by targeting seven serovar/lineage-specific gene markers identified through genomic comparisons. The sensitivity and specificity of the seven MCDA assays were evaluated using 79 target strains and 32 nontarget strains. The assays were all highly sensitive and specific to target serovars, with the sensitivity ranging from 92.9% to 100% and the specificity from 93.3% to 100%. The limit of detection of the seven MCDA assays was 50 fg per reaction (10 copies) from pure DNA, and positive results were detected in as little as 8 minutes. These seven MCDA assays offer a rapid, accurate, and sensitive serotyping method. With further validation in clinically relevant conditions, these assays could be used for culture-independent serotyping of common Salmonella serovars directly from clinical samples.