The Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Salmonella enterica serovar Typhi

Abstract

Typhoid fever remains a public health threat in many countries. A positive result in traditional culture is a gold-standard for typhoid diagnosis, but this method is time consuming and not sensitive enough for detection of samples containing a low copy number of the target organism. The availability of the loop-mediated isothermal amplification (LAMP) assay, which offers high speed and simplicity in detection of specific targets, has vastly improved the diagnosis of numerous infectious diseases. However, little research efforts have been made on utilizing this approach for diagnosis of Salmonella enterica serovar Typhi by targeting a single and specific gene. In this study, a LAMP assay for rapid detection of S. Typhi based on a novel marker gene, termed STY2879-LAMP, was established and evaluated with real-time PCR (RT-PCR). The specificity tests showed that STY2879 could be amplified in all S. Typhi strains isolated in different years and regions in China, whereas no amplification was observable in non-typhoidal strains covering 34 Salmonella serotypes and other pathogens causing febrile illness. The detection limit of STY2879-LAMP for S. Typhi was 15 copies/reaction in reference plasmids, 200 CFU/g with simple heat-treatment of DNA extracted from simulated stool samples and 20 CFU/ml with DNA extracted from simulated blood samples, which was 10 fold more sensitive than the parallel RT-PCR control experiment. Furthermore, the sensitivity of STY2879-LAMP and RT-PCR combining the traditional culture enrichment method for simulated stool and blood spiked with lower S. Typhi count during the 10 h enrichment time was also determined. In comparison with LAMP, the positive reaction time for RT-PCR required additional 2-3 h enrichment time for either simulated stool or blood specimens. Therefore, STY2879-LAMP is of practical value in the clinical settings and has a good potential for application in developing regions due to its easy-to-use protocol.

Fig 1. Target sequences of primers for STY2879-LAMP.

Nucleotide sequence of STY2879 gene used for designing the inner and outer primers. The outer primers consist of B3 and the sequence (F3) complementary to F3c, respectively. Two inner primers were labeled as forward inner primer (FIP) and the backward inner primer (BIP), respectively, and each contains two distinct sequences corresponding to the sequence and complementary sequence of the target gene.

Fig 2. The real-time amplification of the STY2879 gene by LAMP.

A. The real-time sensitivity of STY2879-LAMP as monitored by measurement of turbidity (optimal density at 650 nm). A turbidity of ≥ 0.1 was considered to be positive for STY2879-LAMP. Ten-fold serial dilutions of the reference plasmid pEASY-T1-STY2879 ranging from 1.5 × 10⁵ to 10⁰ copies/reaction were tested. B. The relationship between the threshold time (Tt) of each sample and the log copies/reaction. The standard curve was drawn on the basis of three independent repeats and the linear relationship R² = 0.9804. C. A direct visual detection by the naked eye with the addition of the FDR dye, which was added prior to the amplification. The original orange color of the FDR changed to green in the case of a positive amplification under natural light, whereas the original orange color was retained for a negative reaction.

Fig 3. The amplification of STY2879-LAMP in detecting S. Typhi in simulated human stool samples.

The assay was performed using simple heat-treated DNA samples from pre-enrichment simulation stool specimens spiked with different levels of S. Typhi (CT18) ranging from 2×10⁷～2×10¹ CFU/g (sample 1–7); 8 is the negative control without the target DNA. The amplification curves of sample 1 and 2 are overlapping.

Fig 4. The sensitivity of the STY2879-LAMP assay in human simulated blood samples.

The STY2879-LAMP was performed using simulated blood samples spiked with serially diluted S. Typhi strain CT18. The sensitivity of the STY2879-LAMP assay was monitored by a real-time measurement of turbidity. The corresponding curves of decreasing concentrations of CT18 are shown from sample 1 to 7 (1–7: from 2×10⁶–2× 10⁰ CFU/mL); 8 is the negative control.