Quantitative real-time PCR (qPCR) method was applied to quantify the functional gene encoding organophosphorus hydrolase for assessing the degradation efficacy by bacterial strains on an organophosphorus compound in in situ soil. The specific primers targeting the organophosphorus hydrolase were designed and tested on reference bacterial strains and in DNA samples extracted from in situ soil samples contaminated by an organophosphorus compound. The established qPCR assay is a practical method for the analysis of in situ soil samples undergoing bioremediation of organophosphorus compounds.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA et al. (1999) In Short Protocols in Molecular Biology, (4th ed). Wiley, USA.
Beller HR, Kane SR, Legler TC, and Alvarez PJJ (2002) A real-time polymerase chain reaction method for monitoring anaerobic, hydrocarbon-degrading bacteria based on a catabolic gene. Environ Sci Technol 36, 3977–3984.
Bird SB, Sutherland TD, Gresham C, Oakeshott J, Scott C, and Eddleston M (2008) OpdA, a bacterial organophosphorus hydrolase, prevents lethality in rats after poisoning with highly toxic organophosphorus pesticides. Toxicology 247, 88–92.
Cébron A, Norini M-P, Beguiristain T, and Leyval C (2008) Real-time PCR quantification of PAH-ring hydroxylating dioxygenase (PAH-RHDá) genes from gram positive and gram negative bacteria in soil and sediment samples. J Microbiol Meth 73, 148–159.
Correa PA, Lin L, Just CL, Hu D, Hornbuckle KC, Schnoor JL et al. (2010) The effects of individual PCB congeners on the soil bacterial community structure and the abundance of biphenyl dioxygenase genes. Environ Int 36, 901–906.
Di Gennaro P, Moreno B, Annoni E, García-Rodríguez S, Bestetti G, and Benitez E (2009) Dynamic changes in bacterial community structure and in naphthalene dioxygenase expression in vermicompost-amended PAH-contaminated soils. J Hazard Mater 172, 1464–1649.
Fajardo C, Saccà M, Gibello A, Martinez-Iñigo M, Nande M, Lobo C et al. (2012) Assessment of s-triazine catabolic potential in soil bacterial isolates applying atz genes as functional biomarkers. Water Air Soil Pollut 223, 3385–3392.
Fierer N, Jackson JA, Vilgalys R, and Jackson RB (2005) Assessment of soil microbial community structure by use of taxon-specific quantitative PCR Assays. Appl Environ Microbiol 71, 4117–4120.
Grimsley JK, Scholtz JM, Pace CN, and Wild JR (1997) Organophosphorus hydrolase is a remarkably stable enzyme that unfolds through a homodimeric intermediate. Biochem 36, 14366–14374.
Harper LL, McDaniel CS, Miller CE, and Wild JR (1988) Dissimilar plasmids isolated from Pseudomonas diminuta MG and a Flavobacterium sp. (ATCC 27551) contain identical opd genes. Appl Environ Microbiol 54, 2586–2589.
Higuchi R, Fockler C, Dollinger G, and Watson R (1993) Kinetic PCR Analysis: real-time monitoring of DNA amplification reactions. Nat Biotech 11, 1026–1030.
Kumar M and Khanna S (2010) Diversity of 16S rRNA and dioxygenase genes detected in coal-tar-contaminated site undergoing active bioremediation. J Appl Microbiol 108, 1252–1262.
Land DJ (1991) 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics, Stackebrandt E and Goodfellow M (eds.), pp. 115–175, Wiley & Sons, UK.
Lee SH, Shin JH, Choi JH, Park JW, Kim JE, and Rhee IK (2004) Isolation and characterization of chlorothalonil-dissipating bacteria from soil. Korean J Microbiol Biotechnol 32, 96–100.
Liu YR, Zheng YM, and He JZ (2012) Toxicity of profenofos to the springtail, Folsomia candida, and ammonia-oxidizers in two agricultural soils. Ecotoxicology 21, 1126–1134.
MAF and NACF (2006) Education book of safety ginseng production and distribution technology. pp. 39–64. Ministry of Agriculture and Forestry and National Agricultural Cooperative Federation, Korea.
Mulbry WW, Karns JS, Kearney PC, Nelson JO, McDaniel CS, and Wild JR (1986) Identification of a plasmid-borne parathion hydrolase gene from Flavobacterium sp. by southern hybridization with opd from Pseudomonas diminuta. Appl Environ Microbiol 51, 926–930.
Mulbry WW, Kearney PC, Nelson JO, and Karns JS (1987) Physical comparison of parathion hydrolase plasmids from Pseudomonas diminuta and Flavobacterium sp. Plasmid 18, 173–177.
Muyzer G, de Waal EC, and Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59, 695–700.
NIAST (2000) Methods of soil and crop plant analysis. National Institute of Agricultural Science and Technology, Korea.
Rodríguez-Cruz MS, Bælum J, Shaw LJ, Sørensen SR, Shi S, Aspray T et al. (2010) Biodegradation of the herbicide mecoprop-p with soil depth and its relationship with class III tfdA genes. Soil Biol Biochem 42, 32–39.
Schollenberger CJ (1927) A rapid approximate method for determining soil organic matter. Soil Sci 24, 65–68.
Serdar CM, Gibson DT, Munnecke DM, and Lancaster JH (1982) Plasmid involvement in parathion hydrolysis by Pseudomonas diminuta. Appl Environ Microbiol 44, 246–249.
Sethunathan N and Yoshida T (1973) A Flavobacterium sp. that degrades diazinon and parathion. Can J Microbiol 19, 873–875.
Shin JH, Kwak YY, Kim WC, So JH, Shin HS, Park JW et al. (2008) Isolation of endosulfan degrading bacteria and their degradation characteristics. Korean J Environ Agric 27, 292–297.
Siddavattam D, Khajamohiddin S, Manavathi B, Pakala SB, and Merrick M (2003) Transposon-like organization of the plasmid-borne organophosphate degradation (opd) gene cluster found in Flavobacterium sp. Appl Environ Microbiol 69, 2533–2539.
Singh BK and Walker A (2006) Microbial degradation of organophosphorus compounds. FEMS Microbiol Rev 30, 428–471.
Singh BK, Walker A, Morgan JAW, and Wright DJ (2003) Effects of soil pH on the biodegradation of chlorpyrifos and isolation of a chlorpyrifosdegrading bacterium. Appl Environ Microbiol 69, 5198–5206.
Sogorb MA, Vilanova E, and Carrera V (2004) Future applications of phosphotriesterases in the prophylaxis and treatment of organophosporus insecticide and nerve agent poisonings. Toxicol Lett 151, 219–233.
Steinberg LM and Regan JM (2009) mcrA-targeted real-time quantitative PCR method to examine methanogen communities. Appl Environ Microbiol 75, 4435–4442.
Stubner S (2004) Quantification of gram-negative sulphate-reducing bacteria in rice field soil by 16S rRNA gene-targeted real-time PCR. J Microbiol Meth 57, 219–230.
Theriot C and Grunden A (2011) Hydrolysis of organophosphorus compounds by microbial enzymes. Appl Microbiol Biotechnol 89, 35–43.
Zhang H, Yang C, Li C, Li L, Zhao Q, and Qiao C (2008) Functional assembly of a microbial consortium with autofluorescent and mineralizing activity for the biodegradation of organophosphates. J Agric Food Chem 56, 7897–7902.
About this article
Cite this article
Kwak, Y., Kim, SJ., Rhee, IK. et al. Application of quantitative real-time polymerase chain reaction on the assessment of organophosphorus compound degradation in in situ soil. J Korean Soc Appl Biol Chem 55, 757–763 (2012). https://doi.org/10.1007/s13765-012-2168-4
- opd gene
- organophosphorus hydrolase
- quantitative real-time polymerase chain reaction