Development of RT-LAMP assay for detection of lead and cadmium toxicity using HepG2 cells

Heavy metals such as lead and cadmium are prevalent in the environment. These are harmful to living beings even at lower concentrations as they persist in the body for years and lead to the development of severe diseases. Therefore, the present work was designed to develop a rapid and field-applicable cell-based assay for quick detection of lead and cadmium in biofluids using an RNA biomarker. The RNA biomarker was identified by analyzing the impact of these heavy metals on the gene expression of candidate genes using HepG2 cells. The results showed that the gene expression of AhR pathway-related genes, apoptosis-related genes, MAPK1, and HMOX1 were significantly increased after lead and cadmium treatments (P < 0.05). Interestingly, the gene expression of HMOX1 was increased linearly up to fivefold in a dose and time-dependent manner in the case of both heavy metals which also correlated with an increased secretion of bilirubin from the cells after 6 h treatment. Therefore, the RT-LAMP assay was developed for lead and cadmium toxicity using HMOX1. The positive amplification was visualized in the form of color change of HNB dye from violet to blue in 30 min. Additionally, standard curves were also prepared for the RT-LAMP color change after treatment with different concentrations of lead and cadmium for their quantification in unknown samples. The developed RT-LAMP assay was also validated using lead and cadmium-spiked milk samples. The ROC curve analysis showed 100% sensitivity and specificity for both heavy metals above their MRL value in infant milk substitutes and infant foods. This assay can be utilized for early detection of heavy metals in common food items such as milk.