Colorimetric Point-of-Care Detection of Clostridium tyrobutyricum Spores in Milk Samples

Abstract

Clostridium tyrobutyricum represents the main spoiling agent responsible for late blowing defects (LBD) in hard and semi-hard cheeses. Its spores are resistant to manufacturing procedures and can germinate during the long ripening process, causing the burst of the cheese paste with a consequent undesirable taste. The lower quality of blown cheeses leads to considerable financial losses for the producers. The early identification of spore contaminations in raw milk samples thus assumes a pivotal role in industrial quality control. Herein, we developed a point of care (POC) testing method for the sensitive detection of C. tyrobutyricum in milk samples, combining fast DNA extraction (with no purification steps) with a robust colorimetric loop-mediated isothermal amplification (LAMP) technique. Our approach allows for the sensitive and specific detection of C. tyrobutyricum spores (limit of detection, LoD: ~2 spores/mL), with the advantage of a clear naked-eye visualization of the results and a potential semi-quantitative discrimination of the contamination level. In addition, we demonstrated the feasibility of this strategy using a portable battery-operated device that allowed both DNA extraction and amplification steps, proving its potential for on-site quality control applications without the requirement of sophisticated instrumentation and trained personnel.

LAMP-Based Point-of-Care Biosensors for Rapid Pathogen Detection

“…DNA extraction was achieved by chemical lysis, which eliminates the time-consuming multistep purification process. A LOD of ~2 spores/mL was achieved by direct visualization of colour change from red to yellow in contaminated milk samples with high sensitivity and specificity [ 51 ]. Ye et al [ 52 ] developed a glass fibre paper-based POC diagnostic tool capable of RNA extraction in 5 min, the isothermal amplification of the target gene in just 25 min, and a visual readout of the final product.…”

“…( E ) Workflow of Cap-iLAMP. Reproduced with permission from references [ 49 , 51 , 53 , 60 , 61 ]. Copyright 2021 and 2022 Springer Nature, 2021 American Chemical Society, 2021 MDPI.…”

Detection of butyric spores by different approaches in raw milks from cow, ewe and goat

“…Recently, a promising approach has been developed based on Loop-Mediated Isothermal Amplification (LAMP) (Cecere et al, 2021). In this study, 20 mL of raw milk sample was digested to recover spores, and DNA was extracted by heating.…”

Semiquantitative Determination of Thiocyanate in Saliva Through Colorimetric Assays: Design of CNN Architecture via Input-Aware NAS

“…POC devices have recently attracted a surge in attention in various branches of research and clinical diagnostics [24][25][26]. Several types of POCs have been developed for different applications [27][28][29][30] including lab-on-a-chip [31], lab-on-a-disc [32], microfluidic paper-based analytical [33,34], lateral flow devices [35,36], and isothermal nucleic acid amplification [37].…”