Detection of Salmonella enterica in Naturally Contaminated Liquid Eggs by Loop-Mediated Isothermal Amplification, and Characterization of Salmonella Isolates

Abstract

Loop-mediated isothermal amplification (LAMP) assay was effective in detecting Salmonella enterica in naturally contaminated liquid egg samples. Salmonella was detected in 110 samples taken from four eggbreaking plants. The egg samples were pre-enriched in buffered peptone water (BPW) at 37°C for 20 h. The selective enrichment was done in Rappaport-Vassiliadis or tetrathionate broth and plated onto xylose lysine deoxycholate agar and brilliant green agar, modified. In addition, the PCR assay was used to detect Salmonella after pre-enrichment in BPW at 37°C for 20 h. The culture method and PCR assay were compared to the LAMP assay, which was also performed after pre-enrichment in BPW. PCR failed to detect Salmonella in 10% of 110 samples, whereas the culture method and LAMP assay successfully identified Salmonella in all samples. However, the LAMP assay was found to be much more rapid than the culture method and as sensitive in detecting Salmonella from liquid eggs. In all of the egg-breaking plants studied, Salmonella was isolated on most tested days. The positive samples showed that more than 75% of the Salmonella strains had identical genetic patterns when analyzed by pulsed-field gel electrophoresis. This suggests that the same Salmonella strains having survived long periods of time in the plants were contaminating the production line. The LAMP assay is rapid, specific, and sensitive for Salmonella detection in liquid eggs and is able to monitor Salmonella contamination in egg-handling plants more reliably.

Molecular Detection of New Delhi Metallo-Beta-Lactamase-1 (NDM-1) Positive Bacteria from Environmental and Drinking Water Samples by Loop Mediated Isothermal Amplification of bla NDM-1

“…Similar level of sensitivity was reported in detecting Neisseria meningitidis from the cerebrospinal fluid of humans [25], and Arcobacter species from infected chicken tissues [26]. The increased sensitivity of LAMP is attributed to the tolerance of the Bst DNA polymerase to the impurities that co-purify along with the template DNA [27,28], and its ability to accumulate more than 10 9 copies of the amplicons in less than an hour [12]. With specific reference to the LAMP based detection of NDM-1, the current study reports higher sensitivity and lesser reaction time compared with the earlier report [20].…”

Rapid detection of nusG and fadA in Fusobacterium nucleatum by loop-mediated isothermal amplification

“…LAMP uses four to six specific primers that recognize six to eight distinct sequences on the target DNA, thus amplifying the target DNA with high specificity. This method is widely used for clinical diagnosis of epidemic-causing bacteria Ohtsuka et al, 2005), viruses (Okafuji et al, 2005) and parasites (Chen et al, 2011;Kong et al, 2012), as well as for foetal sex identification (Hirayama et al, 2006). LAMP has yet to be used for clinical diagnosis of F. nucleatum.…”

Loop-mediated isothermal amplification assay for the detection of Salmonella spp. in pig feces

“…LAMP is a nucleic acid amplification technique that relies on an auto-cycling strand displacement DNA synthesis under isothermal condition [10,11,12,13]. Although there are some reports for inspections such as eggs and pork [14,19], there are few reports for developing LAMP for diagnostic purpose. Therefore, LAMP assay was developed for amplifying invA gene to detect the Salmonella spp.…”

“…In spiked feces without enrichment, the detection limit was 10 The present study describes the development of Salmonella-specific LAMP assay targeting the InvA gene. The InvA gene has been used as a target in many reports [2,14,19], whereas phoP gene was used for detection of Salmonella in milk and minced pork [7]. However, the phoP gene is present in most Enterobacteriaceae and non-specific amplification might occur when testing samples.…”

“…and more specific than the phoP gene [6]. LAMP assay have been applied to eggs [2,14] and pork [19] to detect the Salmonella spp. The detection limit of Salmonella targeting rfbJ and fim Y was 10 3 CFU/mL in previous reports [15,18] which was lower than that of our LAMP assay.…”