Rapid and SensitiveSalmonellaTyphi Detection in Blood and Fecal Samples Using Reverse Transcription Loop-Mediated Isothermal Amplification

Abstract

Typhoid fever caused by Salmonella enterica serovar Typhi remains a significant public health problem in developing countries. Although the main method for diagnosing typhoid fever is blood culture, the test is time consuming and not always able to detect infections. Thus, it is very difficult to distinguish typhoid from other infections in patients with nonspecific symptoms. A simple and sensitive laboratory detection method remains necessary. The purpose of this study is to establish and evaluate a rapid and sensitive reverse transcription-based loop-mediated isothermal amplification (RT-LAMP) method to detect Salmonella Typhi infection. In this study, a new specific gene marker, STY1607, was selected to develop a STY1607-RT-LAMP assay; this is the first report of specific RT-LAMP detection assay for typhoid. Human-simulated and clinical blood/stool samples were used to evaluate the performance of STY1607-RT-LAMP for RNA detection; this method was compared with STY1607-LAMP, reverse transcription real-time polymerase chain reaction (rRT-PCR), and bacterial culture methods for Salmonella Typhi detection. Using mRNA as the template, STY1607-RT-LAMP exhibited 50-fold greater sensitivity than STY1607-LAMP for DNA detection. The STY1607-RT-LAMP detection limit is 3 colony-forming units (CFU)/mL for both the pure Salmonella Typhi samples and Salmonella Typhi-simulated blood samples and was 30 CFU/g for the simulated stool samples, all of which were 10-fold more sensitive than the rRT-PCR method. RT-LAMP exhibited improved Salmonella Typhi detection sensitivity compared to culture methods and to rRT-PCR of clinical blood and stool specimens from suspected typhoid fever patients. Because it can be performed without sophisticated equipment or skilled personnel, RT-LAMP is a valuable tool for clinical laboratories in developing countries. This method can be applied in the clinical diagnosis and care of typhoid fever patients as well as for a quick public health response.

Asymmetric Membrane for Digital Detection of Single Bacteria in Milliliters of Complex Water Samples

“…Primers specific to the gene marker STY1607 were used to detect the corresponding mRNA as well as DNA. 61 Due to the variations of mRNA copies from cell to cell, it is hard to quantify target cells by detecting the number of mRNAs. However, mRT-LAMP circumvents these difficulties as each Salmonella bacterium was encapsulated inside a single pore, and thus, the contained nucleic acids, no matter how many, were amplified, resulting in a bright fluorescence.…”

“…The primers for E. coli were designed to be specific to a conserved region on the malB gene, 67 whereas primers for Salmonella were specific to gene marker STY1607. 61 Their sequence is shown in the Supporting Information . Primer specificity has already been demonstrated and published.…”

“…Primer specificity has already been demonstrated and published. 61 , 67 Thus, no selectivity tests (toward other bacteria) were conducted in this study.…”

Enteric Fever in Patients of Lahore: Association of Positive Blood Cultures with the Typhidot Immunoassay Tests and Widal Agglutination

“…In China, India, Nepal, Thailand and the United States, the incidence of relative parathyroid fever has also increased which was previously less common and caused mild diseases than typhoid fever. Incomplete protection due to replacement of the bivalent TA vaccine with monovalent vaccines effective only against S. typhi (9). Men are more likely to be infected than women, which is in line with previous studies, suggesting that this trend remains unchanged, suggesting that men are more likely to be infected by Salmonella (10).The gold standard in the diagnosis of typhoid fever is the isolation of S. typhi and S. Para typhi from patients' body fluids, especially blood and bone marrow (11).…”

A hydrogel beads based platform for single-cell phenotypic analysis and digital molecular detection

“…For gdLAMP assays, 7.5 w/v% PEG hydrogel was added as 10× PEG monomers. The primers, ordered from IDT with the sequences shown in Supplementary Table S1, were targeting a 196 bp region within the S. Typhi specific gene STY1607 (39). For gdLAMP and ddLAMP assays, harvested DNA was serial diluted 5, 20, 50, 100, and 200 times.…”