Development of multiplex loop-mediated isothermal amplification assays to detect medically important yeasts in dairy products

Abstract

Rapid detection of yeast contamination is important in the food industry. We have developed loop-mediated isothermal amplification (LAMP) assays to detect the emerging opportunistic pathogenic yeasts: Candida albicans, Candida glabrata, Candida tropicalis, the Candida parapsilosis group, Trichosporon asahii, and Trichosporon mucoides. These yeasts may cause deep-seated candidiasis or trichosporonosis. Four LAMP primer sets specific for Candida were designed to target the internal transcribed spacer 2 (ITS2) region between the 5.8S and 26S rRNA genes, and two LAMP primer sets specific for Trichosporon were designed to target the intergenic spacer 1 (IGS1) region between the 26S and 5S rRNA genes. The LAMP assays could detect these yeasts in a range between 10(0) and 10(3) cells mL(-1) in a contaminated dairy product within 1 h. We also developed multiplex LAMP assays to detect these Candida or Trichosporon species in a single reaction. Multiplex LAMP assays can detect contamination if at least one of the target species is present; they are more time- and cost-efficient than conventional methods and could detect target yeasts with sensitivity close to that of the LAMP assays. Multiplex LAMP assays established in this study can be used as a primary screening method for yeast contamination in food products.

Development of a multiplex loop-mediated isothermal amplification method for the simultaneous detection of Salmonella spp. and Vibrio parahaemolyticus

“…Although there is no theoretical limit to the number of primers to be used in a multiplex LAMP reaction, spurious-prone amplification among primers limits the establishment of specific reactions. A multiplex LAMP assay combining 21 primers targeting four Candida species has been reported21. The high specificity of primers used in this study has been reported and validated previously1012, and there was no spurious amplification products in our multiplex LAMP reaction, which showed 100% inclusivity and 100% exclusivity for the detection of 19 bacterial strains including Salmonella spp.…”

Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens

“…This approach however is difficult for non-culturable organisms and dependent on unique pathogen surface antigens. Turbidity monitoring [ 39 ] to achieve multiplex LAMP has also been reported; however, this approach has poor reproducibility and requires extensive design optimization to achieve differentiable time-to-positivity values.…”

<p>Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections</p>

“…36 LAMP with a detection limit of 1ng has also been successfully used for rapid detection of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, both of which cause periodontitis in humans. 37 LAMP assays for detecting yeasts, 38 influenza viruses 39 and Plasmodium have been developed. 40 Different companies have also designed commercial LAMP kits (Table 2) for the detection of Salmonella, [41][42][43] verotoxin-producing Escherichia coli [44][45][46] and Campylobacter.…”