Reverse‐Transcriptase Loop‐Mediated Isothermal Amplification as a Rapid Screening/Monitoring Tool for Salmonella Enterica Detection in Liquid Whole Eggs

Abstract

Reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) is a novel molecular detection method that is specific, fast, and simple. It is based on reverse transcription followed by DNA amplification using the Bst DNA polymerase large fragment requiring one temperature and a simple waterbath, without the need for any expensive equipment. Detection is by turbidity or agarose gel electrophoresis. Our objective was to apply this LAMP-based technology to rapidly and sensitively detect Salmonella enterica serovar Enteritidis in liquid whole eggs (LWEs) within 1 d. LWE were inoculated with S. Enteritidis and stomached in tetrathionate broth (TTB), and spread-plated on Xylose lysine tergitol 4 agar either immediately or after 6, 12, or 16-h enrichment. RNA was extracted from 5-mL TTB and the RT-LAMP assay was carried out using invA primers. After 16 and 12-h enrichment, improved Salmonella detection up to 10⁰ to 10¹ and 10⁴ CFU/25 mL LWE, respectively was obtained. Without enrichment, Salmonella could be detected at 10⁷ CFU/25 mL; however, after 6-h enrichment a 1-log improvement to 10⁶ CFU/25 mL was obtained. This RT-LAMP assay appears to be suitable as a potential screening/monitoring tool for Salmonella enterica from LWE products in routine settings with results obtainable within 24-h, which is significantly faster than traditional cultural assays.

Detecting Salmonella Serovars in Shell Eggs by Loop-Mediated Isothermal Amplification

“…However, the in situ procedure was rather lengthy, including fixation for 8 h, permeabilization with lysozyme for 10 min, followed by fluorescence microscopic examination (38). Recently, Techathuvanan et al (27,29) coupled reverse transcription with LAMP1 (RT-LAMP) to detect viable Salmonella in pork and eggs. In spiked liquid whole eggs, the detection limit was 108 CFU/25 ml without enrichment and 16 h of enrichment was required for samples spiked at 10° CFU/ 25 ml (29).…”

“…LAMP assays have been applied previously to detect Salmonella serovars in various food samples, including eggs (21,29,38), milk (15,26), pork (27,37), and produce (7,39), usually with overnight enrichment. An earlier survey in Japan showed that LAMP was as sensitive as the culture, but clearly more sensitive than PCR, in detecting Salmo nella serovars from 110 naturally contaminated liquid eggs (21).…”

“…Salient features of LAMP include being isothermal (therefore no need for a thermal cycler), rapid, specific, sensitive, and robust (e.g., tolerance to biological substances) (13). Several LAMP assays have been applied to detect Salmonella in liquid eggs or on egg shells (21,29,38).…”

Application of ethidium bromide monoazide for quantification of viable and dead cells of Salmonella enterica by real-time loop-mediated isothermal amplification

“…Salmonellosis is most often attributed to the consumption of contaminated foods such as poultry, beef, pork, eggs, milk, seafood, nut products, and fresh produce. There is a need for the development of more sensitive, rapid, and inexpensive methods for detection of this pathogen associated with foods (Techathuvanan and D'Souza 2012;Kokkinos et al, 2014 ).…”

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

“…Bst DNA polymerase ensures highly efficient amplification under a wide range of isothermal reaction conditions within 1 h (Mori and Notomi, 2009). RT-LAMP assays using RNA as a template have been widely developed to detect viruses (Teoh et al, 2013;Kasanga et al, 2014) and bacteria, including Salmonella Enteritidis (Techathuvanan et al, 2012) and Salmonella Typhimurium (Techathuvanan et al, 2011). The sensitivity of RT-LAMP is higher or equivalent to routine RT-PCR (Techathuvanan et al, 2011;Liu et al, 2013).…”