| 英文摘要 |
In this study, we adopted the “Tally and Design” strategy to develop q-PCR probes which can serve as an indicator of heavy metal concentrations in the soil. The strategy first involves extracting gene frequencies of candidate microbial heavy metal resistant genes from Third Generation long read metagenome analysis of the soil samples. Subsequently, heavy metal resistant genes that demonstrate strong correlation with concentration of heavy metal are selected as representative genes of respective heavy metal. Homolog q-PCR primers and probes are then designed against these representative genes and validated.
As results shown, correlation analysis between heavy metal concentrations and gene frequencies of soil samples from heavy metal contaminated areas (Tainan City and Changhua) City, and soil samples from non contaminated areas (Tainan City and Nantou City) identified chrF (Chromiun), copG (Copper), nikC (Nickel), pbrR (Lead) and zitB/ybgR (Zinc) as the representative genes. Subsequently, among the 25 sets of qPCR probe were designed and validated, 22 sets demonstrated Ct values that are positively correlated to the concentration of heavy metals.
In conclusion, this study has successfully shown that Third Generation long read metagenome analysis can greatly shorten the development process of qPCR probes. Moving forward, the accuracy of the probes can be further improved by including more samples in the correlation analysis.
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