Rapid Detection of Viable Salmonellae in Produce by Coupling Propidium Monoazide with Loop-Mediated Isothermal Amplification

Chen, Siyi; Wang, Fei; Beaulieu, John C.; Stein, R. E.; Ge, Beilei

doi:10.1128/aem.00354-11

Cited by 145 publications

(126 citation statements)

References 47 publications

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“…The three LAMP assays developed here fell within these detection ranges in terms of speed and sensitivity. Numerous other studies also reported the superior sensitivity of LAMP in comparison with PCR (9,16,18); however, few comparisons were made between LAMP and qPCR. Similar to findings of the present study, a recent study on LAMP detection of Salmonella also reported comparable sensitivities between LAMP and qPCR (9).…”

Section: Discussionmentioning

confidence: 99%

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Loop-Mediated Isothermal Amplification Assays for Detecting Shiga Toxin-Producing Escherichia coli in Ground Beef and Human Stools

Wang

Jiang

2012

J Clin Microbiol

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Shiga toxin-producing Escherichia coli (STEC), encompassing E. coli O157 and non-O157 STEC, is a significant cause of foodborne illnesses and deaths in the United States and worldwide. Shiga toxins (encoded by stx) and intimin (encoded by eae) are important virulence factors for STEC strains linked to severe human illnesses such as hemorrhagic colitis and hemolytic-uremic syndrome. In this study, the stx 1 , stx 2 , and eae genes were chosen as targets to design loop-mediated isothermal amplification (LAMP) assays for the rapid, specific, sensitive, and quantitative detection of STEC strains. The assay performances in pure culture and spiked ground beef and human stools were evaluated and compared with those of quantitative PCR (qPCR). No falsepositive or false-negative results were observed among 90 bacterial strains used to evaluate assay specificity. The limits of detection for seven STEC strains of various serogroups (O26, O45, O103, O111, O121, O145, and O157) were approximately 1 to 20 CFU/reaction in pure culture and 10 3 to 10 4 CFU/g in spiked ground beef, which were comparable to the results of qPCR. Standard curves generated suggested good linear relationships between STEC cell numbers and LAMP turbidity signals. When applied in ground beef samples spiked with two low levels (1 to 2 and 10 to 20 CFU/25 g) of STEC cultures, the LAMP assays achieved accurate detection after 6 to 8 h enrichment. The assays also consistently detected STEC in human stool specimens spiked with 10 3 or 10 4 CFU/0.5 g stool after 4 h enrichment, while qPCR required 4 to 6 h. In conclusion, the LAMP assays developed in this study may facilitate rapid and reliable identification of STEC contaminations in high-risk food commodities and also facilitate prompt diagnosis of STEC infections in clinical laboratories.

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Section: Discussionmentioning

confidence: 99%

“…For LAMP specificity, DNA templates of 90 bacterial strains (see the table in the supplemental material) were prepared by heating at 95°C for 10 min as described previously (9). Aliquots (2 l) of each template were subjected to LAMP amplification and repeated twice.…”

Section: Methodsmentioning

confidence: 99%

Loop-Mediated Isothermal Amplification Assays for Detecting Shiga Toxin-Producing Escherichia coli in Ground Beef and Human Stools

Wang

Jiang

2012

J Clin Microbiol

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“…However, the mt COI of C. nozakii in our study have a low GC content (34%). Lower betaine concentration may result in elevated LAMP amplification efficiency when amplifying non-GC-rich target sequences (Chen et al, 2011). The band appeared to be more clear with the addition of 0.4 mM dNTP than with other concentrations ( Figure 1B).…”

Section: Resultsmentioning

confidence: 92%

Development of a rapid assay to detect the jellyfish Cyanea nozakii using a loop-mediated isothermal amplification method

Liu

Dong

Chen

2015

Mitochondrial DNA Part A

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Blooms of the harmful jellyfish Cyanea nozakii, which are a severe nuisance to fisheries and tourisms, frequently occur in the northern East China Sea, Yellow Sea, and Bohai Sea. To provide early warning of this species, a simple and effective molecular method for identifying C. nozakii was developed using the loop-mediated isothermal amplification method (LAMP). The LAMP assay is highly specific and uses a set of four primers that target six different regions on the mitochondrial cytochrome c oxidase subunit I (COI) gene of C. nozakii. The amplification conditions, including the dNTP and betaine concentrations, the inner primer to outer primer concentration ratio, reaction time and temperature, were optimized. The LAMP assay amplified DNA extracted from tissue samples of C. nozakii but did not amplify DNA from other common scyphozoans and hydrozoans collected in the same region. In addition, the LAMP assay was more sensitive than conventional PCR. Therefore, the established LAMP assay is a sensitive, specific, fast, and easily performed method for detection of C. nozakii at different stages in their life cycle.

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“…The concentration of EMA used in the assay is critical in affecting DNA amplification of viable cells. PMA is found to be less effective in differentiating between low number of viable and heat-killed cells of Salmonella [106].…”

Section: Ema/pma-lampmentioning

confidence: 99%

“…EMA and PMA have been reported to combine with LAMP for detection of viable cells of Salmonella [106][107][108][109]. The concentration of EMA used in the assay is critical in affecting DNA amplification of viable cells.…”

Section: Ema/pma-lampmentioning

confidence: 99%

Recent and the Latest Developments in Rapid and Efficient Detection of Salmonella in Food and Water

Hu¹,

Li²

2017

Adv Tech Biol Med

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Salmonella is a leading cause of foodborne diseases. Gastroenteritis caused by nontyphoid Salmonella is still a major infectious disease in the world. About 95% of Salmonella infections are caused by ingestion of contaminated food and water. Rapid, sensitive, and efficient detection and identification of Salmonella from foods and water are critical for minimizing the spread of outbreaks caused by this pathogen. These methods can be applied to track the food source of contamination or by early diagnosis of the infections in a clinical setting. Culture-based methods for detection of Salmonella are laborious and time-consuming, typically taking 5-7 days to obtain a pure culture and serovar identification. In the past few decades, molecular-based technologies have greatly shortened the time for detection and identification of bacterial pathogens from food and water, and drastically increased the specificity and sensitivity of the assays. In this review, we report an update on the development of rapid and efficient methods for the detection and identification of Salmonella in food and water, focusing on the approaches for concentration of Salmonella cells and viable cell detection, as well as the advantages and drawbacks of these methods.

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Rapid Detection of Viable Salmonellae in Produce by Coupling Propidium Monoazide with Loop-Mediated Isothermal Amplification

Cited by 145 publications

References 47 publications

Loop-Mediated Isothermal Amplification Assays for Detecting Shiga Toxin-Producing Escherichia coli in Ground Beef and Human Stools

Loop-Mediated Isothermal Amplification Assays for Detecting Shiga Toxin-Producing Escherichia coli in Ground Beef and Human Stools

Development of a rapid assay to detect the jellyfish Cyanea nozakii using a loop-mediated isothermal amplification method

Recent and the Latest Developments in Rapid and Efficient Detection of Salmonella in Food and Water

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