Application and Evaluation of the Loop-Mediated Isothermal Amplification assay for the Detection of Aflatoxigenic Fungi Contaminants of Rice Grains in Kenya

Abstract

Aflatoxigenic fungi are most common filamentous fungi that synthesis aflatoxins and represent the major fungal pathogens to agricultural products. Aflatoxins remain a major threat to global food security, these molecules could be resisted into food during processing and in additional may remain within the food chain. Aflatoxins are carcinogenic, hepatotoxic, mutagenic, teratogenic, can inhibit numerous metabolic systems and immunosuppressive properties.Studies of aflatoxigenic strains can help to enhance strategies control and prevent aflatoxigenic fungi contamination and aflatoxins production in foodstuffs. In this study, isolation of Aspergillus species was based on morphological characteristics including the mycelium growth pattern, color, and properties of fruiting bodies of the fungi. The innovated technique loop-mediated isothermal amplification assay was applied to amplify Norsolorinic Acid gene.The loop-mediated isothermal amplification have been optimized by combination of the rapidity, simplicity and specificity to detect the target genomic DNA in the reactions. The amplification curves monitored by Loopamp realtime Turbidi meter were analyzed in order to distinguish aflatoxigenic and non-aflatoxigenic strains.Overall, the results showed that the loop-mediated isothermal amplification method was effective in detecting aflatoxigenic strains with high specificity of 71.5 % and sensitivity under lower concentrations of DNA. In additional, it was faster than the conventional polymerase chain reaction. The loop-mediated isothermal amplification assay described in this study might be a promising tool for prediction potential threats by aflatoxigenic fungi and aflatoxins risk in food and commodities.

Development of PCR, LAMP and qPCR Assays for the Detection of Aflatoxigenic Strains of Aspergillus flavus and A. parasiticus in Hazelnut

“…Both techniques, TaqMan ® qPCR and LAMP, showed high specificity using DNA from pure culture samples, with no amplification of any other fungi commonly found in hazelnuts. In both cases, the diagnostic specificity and sensitivity were higher than in other studies (96% for the LAMP and 84% for the TaqMan ® qPCR), overcoming other assays such as the LAMP designed by Douksouna et al [48], to detect aflatoxigenic molds in rice, with a specificity of 71.5% using pure DNA. In addition, both tests were validated in terms of repeatability and reproducibility following the EPPO PM7/98 validation standards.…”

“…LAMP assays have been designed in other genes such as alpha amylase 1 (amy1) to detect A. flavus or ATP citrate lyase subunit 1 (acl1) to detect A. nomius and A. parasiticus [44] in Brazil nuts, peanuts and green coffee beans, similarly to the qPCR assays. The aflatoxigenic biosynthetic gene cluster has been already exploited to design LAMP assay methods on genes such as aflP, aflD or nor1 to distinguish between aflatoxigenic and non-aflatoxigenic fungi in different food matrices [46,48,50].…”

“…In addition, LAMP assays are less sensitive to inhibitors present in the matrix than other molecular techniques allowing the use of crude extraction based on the alkaline disruption of the membrane of the cells to extract the DNA [68]. Previous LAMP assays did not test the sensitivity of the technique with real samples, artificially or naturally infected samples [48] However, it is essential to evaluate the potential application of the assays in real samples. No inhibitions were shown using hazelnuts samples with any of the two assays.…”

“…Positive results of the LAMP assays were obtained only for the strains with the presence of a 308 bp PCR product checked by means of agarose gel. BLASTn analysis of F3, loopF, loopB, and FIP primers showed similarity at 100% with A. flavus NRRL 3357 (GenBank accession number CP0044620 and XM 002379911) that causes aflatoxin contamination on food and feed [48] and with the strains A. flavus AF36, A. flavus AF13, A. flavus BN008, A. flavus AF70, and other A. parasiticus and A. flavus strains (GenBank accession numbers: AY510455, AY510451, AY510452, AY510453, AY371490, AF515601, respectively), as well, they showed 100% similarity with Aspergillus pseudotamarii (GenBank accession number XM_032052844.1) and A. sojae (CP035528.1). BIP and B3 primers showed 100% similarity with A. flavus NRRL 3357, A. flavus AF36, A. flavus AF13, A. flavus AF70, and A. parasiticus and A. flavus (GenBank accession numbers: CP0044620.1, AY510455, AY510451, AY510452, AY510453, CP051029.1, AF515601, respectively) and approximately at 90% with A. flavus BN008 (AY510452.1).…”

“…More recently, LAMP assays have been designed to differentiate between ochratoxigenic Aspergillus spp. or aflatoxigenic and non-aflatoxigenic, or fumonisin producer strains of A. flavus [44][45][46][47][48][49]. In addition, a novel LAMP assay designed by Niessen et al [50] was developed on the nor1 gene for the detection of aflatoxin producers within Aspergillus section Flavi, showing high sensitivity and specificity.…”