Dataset Information

Detection of Campylobacter jejuni Based on a Real-Time Fluorescence Loop-Mediated Isothermal Amplification Method.

ABSTRACT: Campylobacter jejuni (C. jejuni), a foodborne pathogenic bacterium, is among the most prevalent causes of human gastroenteritis globally. We developed and evaluated a loop-mediated isothermal amplification (LAMP) method to detect C. jejuni. Outer primers and inner primers were designed based on the hipO gene. The ratio between the concentrations of the inner and outer primers and the reaction temperature were then optimized to achieve optimal assay conditions. The analytical specificity tests showed that, among 12 genera of 74 pure bacterial culture strains, only four C. jejuni isolates could be detected, whereas no amplification was observed in C. coli, C. lari, and the other 11 genera of foodborne pathogens (n = 70). Moreover, the LAMP assay showed a higher analytical sensitivity (34.2 fg μL^-1) than the conventional PCR method (342 fg μL^-1). The limit of detection of C. jejuni based on the LAMP assay was 10³ CFU g^-1 in the artificially spiked samples of chicken meat. In conclusion, the developed LAMP assay will be a powerful and practical tool for the fast, specific, and sensitive detection of C. jejuni.

SUBMITTER: Liu Y

PROVIDER: S-EPMC9453007 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Detection of Campylobacter jejuni Based on a Real-Time Fluorescence Loop-Mediated Isothermal Amplification Method.

Liu Ying Y Xu Meidi M Wang Jiang J Cao Yang Y Wang Tao T Mu Lan L Niu Chao C

BioMed research international 20220831

Campylobacter jejuni (C. jejuni), a foodborne pathogenic bacterium, is among the most prevalent causes of human gastroenteritis globally. We developed and evaluated a loop-mediated isothermal amplification (LAMP) method to detect C. jejuni. Outer primers and inner primers were designed based on the hipO gene. The ratio between the concentrations of the inner and outer primers and the reaction temperature were then optimized to achieve optimal assay conditions. The ana ...[more]

PMID: 36093400

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Project description:Campylobacter jejuni is one of the leading causes of foodborne illness worldwide. C. jejuni is commonly found in poultry. It is the most frequent cause of contamination and thus resulting in not only public health concerns but also economic impacts. To test for this bacterial contamination in food processing plants, this study attempted to employ a simple and rapid detection assay called loop-mediated isothermal amplification (LAMP). The best cutoff value for the positive determination of C. jejuni calculated using real-time LAMP quantification cycle (Cq) was derived from the receiver operating characteristic (ROC) curve modeling. The model showed an area under curve (AUC) of 0.936 (95% Wald CI: 0.903-0.970). Based on Youden's J statistic, the optimal cutoff value which had the highest sensitivity and specificity from the model was calculated as 18.07. The LAMP assay had 96.9% sensitivity, 95.8% specificity, and 93.9 and 97.9% positive and negative predictive values, respectively, compared to a standard culture approach for C. jejuni identification. Among all non-C. jejuni strains, the LAMP assay gave each of 12.5% false-positive results to C. coli and E. coli (1 out of 8 samples). The assay can detect C. jejuni at the lowest concentration of 103 CFU/mL. Our results suggest a preliminary indicator for the application of end-point LAMP assays, such as turbidity and UV fluorescence tests, to detect C. jejuni in field operations. The LAMP assay is an alternative screening test for C. jejuni contamination in food samples. The method provides a rapid detection, which requires only 9 min with a cutoff value of Cq. We performed the extraction of DNA from pure cultures and the detection of C. jejuni using the LAMP assay within 3 h. However, we were not able to reduce the time for the process of enrichment involved in our study. Therefore, we suggest that alternative enrichment media and rapid DNA extraction methods should be considered for further study. Compared to other traditional methods, our proposed assay requires less equipment and time, which is applicable at any processing steps in the food production chain.

Project description:BackgroundDetection of Acinetobacter baumannii has been relying primarily on bacterial culture that often fails to return useful results in time. Although DNA-based assays are more sensitive than bacterial culture in detecting the pathogen, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. In addition, these molecular tools require expensive laboratory instruments. Therefore, establishing molecular tools for field use require simpler molecular platforms. The loop-mediated isothermal amplification method is relatively simple and can be improved for better use in a routine clinical bacteriology laboratory. A simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in the same platform has been developed in recent years. This method is referred to as real-time loop-mediated isothermal amplification. In this study, we attempted to utilize this method for rapid detection of A. baumannii.Methodology and significant findingsSpecies-specific primers were designed to test the utility of this method. Clinical samples of A. baumannii were used to determine the sensitivity and specificity of this system compared to bacterial culture and a polymerase chain reaction method. All positive samples isolated from sputum were confirmed to be the species of Acinetobacter by 16S rRNA gene sequencing. The RealAmp method was found to be simpler and allowed real-time detection of DNA amplification, and could distinguish A. baumannii from Acinetobacter calcoaceticus and Acinetobacter genomic species 3. DNA was extracted by simple boiling method. Compared to bacterial culture, the sensitivity and specificity of RealAmp in detecting A. baumannii was 98.9% and 75.0%, respectively.ConclusionThe RealAmp assay only requires a single unit, and the assay positivity can be verified by visual inspection. Therefore, this assay has great potential of field use as a molecular tool for detection of A. baumannii.

Project description:BackgroundEnterocytozoon hepatopenaei (EHP) is a newly emerged microsporidian parasite that causes retarded shrimp growth in many countries. But there are no effective approaches to control this disease to date. The EHP could be an immune risk factor for increased dissemination of other diseases. Further, EHP infection involves the absence of obvious clinical signs and it is difficult to identify the pathogen through visual examination, increasing the risk of disease dissemination. It is urgent and necessary to develop a specific, rapid and sensitive EHP-infected shrimp diagnostic method to detect this parasite. In the present study, we developed and evaluated a rapid real-time loop-mediated isothermal amplification (real-time LAMP) for detection of EHP.MethodsA rapid and efficient real-time LAMP method for the detection of EHP has been developed. Newly emerged EHP pathogens in China were collected and used as the sample, and three sets of specificity and sensitivity primers were designed. Three other aquatic pathogens were used as templates to test the specificity of the real-time LAMP assay. Also, we compared the real-time LAMP with the conventional LAMP by the serial dilutions of EHP DNA and their amplification curves. Application of real-time LAMP was carried out with clinical samples.ResultsPositive products were amplified only from EHP, but not from other tested species, EHP was detected from the clinical samples, suggesting a high specificity of this method. The final results of this assay were available within less than 45 min, and the initial amplification curve was observed at about 6 min. We found that the amplification with an exponential of sixfold dilutions of EHP DNA demonstrated a specific positive signal by the real-time LAMP, but not for the LAMP amplicons from the visual inspection. The real-time LAMP amplification curves demonstrated a higher slope than the conventional LAMP.DiscussionIn this study, pathogen virulence impacts have been increased in aquaculture and continuous observation was predominantly focused on EHP. The present study confirmed that the real-time LAMP assay is a promising and convenient method for the rapid identification of EHP in less time and cost. Its application greatly aids in the detection, surveillance, and prevention of EHP.

Dataset Information

Detection of Campylobacter jejuni Based on a Real-Time Fluorescence Loop-Mediated Isothermal Amplification Method.

Publications

Detection of <i>Campylobacter jejuni</i> Based on a Real-Time Fluorescence Loop-Mediated Isothermal Amplification Method.

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