Rapid and Specific Detection of Listeria monocytogenes With an Isothermal Amplification and Lateral Flow Strip Combined Method That Eliminates False-Positive Signals From Primer–Dimers

Abstract

Listeria monocytogenes is an important foodborne pathogenic bacterium that is explicitly threatening public health and food safety. Rapid, simple, and sensitive detection methods for this pathogen are of urgent need for the increasing on-site testing demands. Application of the isothermal recombinase polymerase amplification (RPA) and the lateral flow strip (LFS) in the detection is promising for fast speed, high sensitivity, and little dependency on equipment and trained personnel. However, the simplicity comes with an intrinsic and non-negligible risk, the false-positive signals from primer-dimers. In this study, an improved RPA-LFS system was established for detection of L. monocytogenes that eliminated false-positive signals from primerdimers. Primer candidates were carefully selected from the entire L. monocytogenes genome sequence and rigorously screened for specific amplifications in PCR and RPA reactions. For the optimal primer pairs, probes that matched the targeted fragment sequences, although had the smallest chance to form cross-dimers with the primers, were designed and screened. The intelligent use of the probe successfully linked the positive signal to the actual amplification product. This RPA-LFS system was highly specific to L. monocytogenes and was able to detect as low as 1 colony-forming unit of the bacterium per reaction (50 µl) without DNA purification, or 100 fg of the genomic DNA/50 µl. The amplification could be conducted under the temperature between 37 and 42 • C, and the whole detection finished within 25 min. Test of artificially contaminated milk gave 100% accuracy of detection without purification of the samples. Various food samples spiked with 10 colony-forming unit of L. monocytogenes per 25 g or 25 ml were successfully detected after an enrichment time period of 6 h. The RPA-LFS system established in this study is a rapid, simple, and specific detection method for L. monocytogenes that has eliminated false-positive results from primer-dimers. In

DIRECT DETECTION OF LISTERIA MONOCYTOGENES BY RECOMBINASE POLYMERASE AMPLIFICATION

“…The obtained results agree with the previous studies, showing that RPA assays succeeded to directly detect the target bacteria in simulated clinical samples without the need for genomic DNA extraction (Santiago-Felipe et al, 2015;Geng et al, 2019). The reduced sensitivity of RPA observed with milk samples is mostly due to the matrices of crude samples that potentially affect the amplification process (Miao et al, 2019;L. Wang et al, 2020).…”

A Recombinase Polymerase Amplification and Lateral Flow Strip Combined Method That Detects Salmonella enterica Serotype Typhimurium With No Worry of Primer-Dependent Artifacts

“…In addition to the primer pair, using a probe in the RPA reactions could increase the specificity and reduce the primerdependent artifacts (Dong et al, 2020;Wang et al, 2020). This measure has been proven to be effective in some cases (Kersting et al, 2014).…”

Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe

“…This significantly decreases the formation of labeled by-product amplicons [ 14 ]. Some research proposed a solution for decreasing the false-positive signal of RPA-LFT by the optimization of the probe design [ 15 ], using blocking antiprimers [ 16 ] and the proper combination of probe–primer sets [ 17 , 18 ] to avoid their cross-dimerization. However, there is no research about the direct comparison of RPA-LFT based on the generation of labeled amplicons through forward and reverse primers and through the oligonucleotide probe and reverse primer.…”