The evaluation of a multiplex PCR-DNA dipstick assay with culture method and EZ Typhi Carrier DNA assay to detect Salmonella Typhi in well water samples

Abstract

IntroductionSalmonella Typhi is the causative agent for typhoid fever that infects people especially in underdeveloped and developing countries. Even though culture method is the gold standard for detection of this pathogen, there are some limitations such as time consuming and less sensitive that leads to the development of a more sensitive and efficient method such as polymerase chain reaction (PCR). ObjectiveTo evaluate the sensitivity of a multiplex PCR-DNA dipstick assay incomparison with culture method and EZTyphi Carrier DNA assay for the detection of S.Typhi in well water samples. MethodsA total of 580 well water samples were collected and filtered using filter membrane. Bacteria trapped on the membrane was enriched in a selective Selenite F and evaluated using these three methods; (i) Culture method: Bacteria culture was plated on a selective media agar plate and further tested by biochemical tests and serotyping (test was performed within 3 days); (ii) EZTyphi Carrier DNA assay: Extracted DNA from bacteria culture was amplified using two sets of primers targeted for S. Typhi (1238 bp) and internal amplification control (IAC) (810 bp) (3 hours); (iii) Multiplex PCR-DNA dipstick assay: Extracted DNA was amplified using four sets of primers targeted for S. Typhi (70 bp), S. Paratyphi A (93 bp), pan-Salmonella (146 bp) and IAC (123 bp) (2 hours). Results & DiscussionFifteen out of 580 water samples were positive S.Typhi by multiplex PCR-DNA dipstick assay while one sample was positiveS. Typhi by EZTyphi Carrier DNA and culture method. This finding proved that the multiplex PCR-DNA dipstick was superior to culture method and PCR-agarose gel electrophoresis since it has higher positivity compared to the other two methods. ConclusionA rapid and sensitive multiplex PCR-DNA dipstick assay can be used to detect S. Typhi in well water especilally in typhoid endemic area.