A Cutoff Determination of Real-Time Loop-Mediated Isothermal Amplification (LAMP) for End-Point Detection of Campylobacter jejuni in Chicken Meat

Abstract

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.

Progression of LAMP as a Result of the COVID-19 Pandemic: Is PCR Finally Rivaled?

“…Various methods, such as the addition of DMSO (dimethyl sulfoxide) [ 33 , 34 , 35 ] to the reaction mix and the use of OSD (oligonucleotide strand displacement) probes [ 24 , 25 , 26 , 27 , 28 ], have been proven to improve LAMP accuracy. In particular, compartmentalization of the reaction through microfluidics [ 29 , 30 , 31 ] and emulsion [ 32 , 122 , 123 ] techniques have shown substantial success in improving accuracy by limiting the effects of contaminants.…”

“…Both microfluidics [ 29 , 30 , 31 ] and emulsion [ 32 ] reactions have also been applied to LAMP diagnostics to compartmentalize the reactions, once again improving accuracy. Adding dimethyl sulfoxide (DMSO) to the reaction has also been proven to increase LAMP’s analytical specificity and sensitivity [ 33 ] and clinical specificity and sensitivity [ 34 , 35 ]. In addition, in the recent years of the SARS-CoV-2 pandemic, LAMP-centered research has especially exploded.…”

Visual Detection of Chicken Adulteration Based on a Lateral Flow Strip-PCR Strategy

“…Furthermore, the amplification capacity of nucleic acids can improve the detection sensitivity of corresponding DNA analytical methods. Commonly used nucleic acid-based detection methods include polymerase chain reaction (PCR) [ 13 ], real-time fluorescent PCR [ 14 ], digital PCR [ 15 ], multiplex PCR [ 16 ], loop-mediated isothermal amplification (LAMP) [ 17 ], cross-priming amplification (CPA) [ 18 ], and recombinase polymerase amplification (RPA) [ 19 , 20 ]. PCR is considered the “gold standard” for identifying adulterated meat products and has been successfully employed to identify beef, lamb, pork, and chicken [ 21 ].…”