MRG Chip: A High-Throughput qPCR-Based Tool for Assessment of the Heavy Metal(loid) Resistome

Bacterial metal detoxification mechanisms have been well
studied
for centuries in pure culture systems. However, profiling metal resistance
determinants at the community level is still a challenge due to the
lack of comprehensive and reliable quantification tools. Here, a novel
high-throughput quantitative polymerase chain reaction (HT-qPCR) chip,
termed the metal resistance gene (MRG) chip, has been developed for
the quantification of genes involved in the homeostasis of 9 metals.
The MRG chip contains 77 newly designed degenerate primer sets and
9 published primer sets covering 56 metal resistance genes. Computational
evaluation of the taxonomic coverage indicated that the MRG chip had
a broad coverage matching 2 kingdoms, 29 phyla, 64 classes, 130 orders,
226 families, and 382 genera. Temperature gradient PCR and HT-qPCR
verified that 57 °C was the optimal annealing temperature, with
amplification efficiencies of over 94% primer sets achieving 80–110%,
with R2 > 0.993. Both computational
evaluation and the melting
curve analysis
of HT-qPCR validated a high specificity. The MRG chip has been successfully
applied to characterize the distribution of diverse metal resistance
determinants in natural and human-related environments, confirming
its wide scope of application. Collectively, the MRG chip is a powerful
and efficient high-throughput quantification tool for exploring the
microbial metal resistome.