Asymmetric helicase-dependent amplification combined with lateral flow assay (HDA-LFA) for the detection of S. Paratyphi A

Abstract

IntroductionS. Paratyphi A is a causative agent of paratyphoid fever with milder symptoms compared to typhoid fever. Culture method is the gold standard used for diagnosis and the disadvantages of the method is less sensitive, laborious and requires 3 – 5 days for identification of the bacteria. Therefore, a rapid and user-friendly asymmetric helicase-dependent amplification combined with lateral flow assay (HDA-LFA) was developed for the detection of S. Paratyphi A. ObjectivesTo determine the detection limit, sensitivity and specificity of the HDA-LFA for the detection of S. Paratyphi A using spiked stool samples. MethodsA pair of S. Paratyphi A HDA-LFA primer was designed to amplify a 93 bp intergenic region of S. Paratyphi A and a 123 bp competitive internal amplification control. The lateral flow strips were assembled after lining with antibodies using Biodot dispensing reagent. The asymmetric HDA assay was carried out using heating block for 1 hour at 65oC and the amplified products were then detected via LFA and analysed by naked eyes within 15 minutes.Results and discussion: The limit of detection for S. Paratyphi A HDA-LFA using spiked stool sample was at 102 CFU/ml while at 103 CFU/ml when using agarose gel electrophoresis method. The assay was also validated using stool samples spiked with 25 S. Paratyphi A and 75 non-S. Paratyphi A isolates and revealed 100% sensitivity and specificity without any inhibition. ConclusionThe assay is found to be rapid, sensitive and specific and has the potential to be an alternative DNA-based detection method for laboratories with limited access to specific equipment such as thermal cycler.