Establishment of Analysis Method for Detection of Petroleum Degrading Genes AlkB and Nah in Contaminated Soil and Its Application
10.11895/j.issn.0253-3820.140103
- VernacularTitle:污染土壤中主要石油降解基因AlkB和Nah定量检测方法的建立和应用
- Author:
Qinglong LIU
;
Jingchun TANG
;
Xiaotong WAN
- Publication Type:Journal Article
- Keywords:
Hydrocarbon degrading genes;
Fluorescent;
Polymerase chain reaction;
SYBR Green I;
Alkanes monoxygenase degradation;
Naphthalene dioxygenase degradation
- From:
Chinese Journal of Analytical Chemistry
2014;(9):1348-1353
- CountryChina
- Language:Chinese
-
Abstract:
SYBR Green I Real Time-qPCR method was developed to quantify the numbers of copyies of AlkB ( alkanes degradation gene) and Nah ( naphthalene dioxygenase degradation gene) functional degradation gene corresponding to alkanes and aromatic hydrocarbons degradation. Two pairs of primers AlkBf/AlkBr and Nahf/Nahr were designed for AlkB and Nah amplification respectively, according to the nucleotide sequences of related degradation microorganisms published in GenBank. The purified recovery products of traditional PCR were combined with pEASY-T1 vectors and transformed in competent cells to amplify. The recombinant plasmids were extracted and used as positive templates to create standard curve through gradient dilution. The conditions for the real time PCR were as the follows: the final concentration of forward and reverse primers were 0. 2 μmol/L, 2×TransStart Top Green qPCR SuperMix, and the annealing temperatures of AlkB and Nah PCR were 50℃ and 57℃, respectively. The method showed a sensitivity of 100 times higher than that of the traditional PCR method and good repeatability. The numbers of copies of AlkB in three functional regions of an oilfield indicated that oil producing zone with serious oil pollution had the highest AlkB copy numbers, and residential zone with lighter oil pollution had the lowest AlkB copy numbers. Nah degradation gene distribution was more uniform.
