Citation: SONG Ben-Ru, ZHEN Mei-Nan, LIU Xiao-Mei, TANG Jing-Chun. Real-time Fluorescence Quantitative Polymerase Chain Reaction for Anaerobic Degradation Genes masD and bamA in Petroleum Hydrocarbons[J]. Chinese Journal of Analytical Chemistry, ;2019, 47(2): 207-213. doi: 10.19756/j.issn.0253-3820.181417 shu

Real-time Fluorescence Quantitative Polymerase Chain Reaction for Anaerobic Degradation Genes masD and bamA in Petroleum Hydrocarbons

1.
College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China;
2.
Key Laboratory of Pollution Processes and Environmental Criteria, Ministry of Education, Nankai University, Tianjin 300071, China;
3.
Tianjin Engineering Center of Environmental Diagnosis and Contamination Remediation, Nankai University, Tianjin 300071, China

Received Date: 22 June 2018
Revised Date: 5 December 2018

Fund Project: This work was supported by the National Natural Science Foundation of China (Nos. 41473070, U1806216), the Tianjin S&T Program (Nos. 16YFXTSF00520, 17ZXSTSF00050, 17PTGCCX00240), the Science & Technology Development Project of CNPC (No. 2016D-4610) and the 111 Pprogram, Ministry of Education, China (No. T2017002).

The masD and bamA are key genes for anaerobic degradation of alkanes and aromatic hydrocarbons, respectively. In this study, the SYBR Green I real-time quantitative polymerase chain reaction (Real Time-qPCR) method was established with the two genes as targets. With reference to the GenBank sequence of related degraded oil strains, the anaerobic degradation gene amplification primers masD-f, masD-r and bamA-f, bamA-r for alkanes and aromatic hydrocarbons were designed and synthesized. Two pairs of primers were amplified by conventional PCR to obtain 389 bp and 354 bp fragments, respectively. The amplified products were confirmed as fragments of masD and bamA by being sequenced and queried in the NCBI database. The positive clone plasmid was extracted and serially diluted to construct a real-time qPCR standard curve. The best reaction conditions for the 25-μL amplification system were as follows:0.2 μmol/L pre-primer and post-primer, 12.5 μL of 2×Trans Start Top Green qPCR Super Mix, 52℃ of annealing temperatures for masD and 56℃ for bamA genes. Real Time-qPCR technology showed high sensitivity and repeatability, which was 100 times higher than traditional PCR technology. The quantitative detection of anaerobic genes in the anaerobic degradation system of petroleum hydrocarbons promoted by graphene oxide showed that the addition of different concentrations of graphene oxide increased the copy numbers of bamA gene, but had no significant effect on the copy numbers of masD gene.
- Petroleum hydrocarbon,
- Anaerobic degradation gene,
- masD gene,
- bamA gene,
- Real-time quantitative polymerase chain reaction
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沈阳化工大学材料科学与工程学院沈阳 110142