Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction

Abstract

In order to develop a rapid and specific detection test for bacteria in soil, we improved a method based on the polymerase chain reaction (PCR). Each step of the protocol, including direct lysis of cells, DNA purification, and PCR amplification, was optimized. To increase the efficiency of lysis, a step particularly critical for some microorganisms which resist classical techniques, we used small soil samples (100 mg) and various lytic treatments, including sonication, microwave heating, and thermal shocks. Purification of nucleic acids was achieved by passage through up to three Elutip d columns. Finally, PCR amplifications were optimized via biphasic protocols using booster conditions, lower denaturation temperatures, and addition of formamide. Two microorganisms were used as models: Agrobacterium tumefaciens, which is naturally absent from the soil used and was inoculated to calibrate the validity of the protocol, and Frankia spp., an actinomycete indigenous to the soil used. Specific primers were characterized either in the plasmid-borne vir genes for A. tumefaciens or in the variable regions of the 16S ribosomal gene for Frankia spp. Specific detection of the inoculated A. tumefaciens strain was routinely obtained when inocula ranged from 107 to 103 cells. Moreover, the strong correlation we observed between the size of the inocula and the results of the PCR reactions permitted assessment of the validity of the protocol in enumerating the number of microbial cells present in a soil sample. This allowed us to estimate the indigenous population of Frankia spp. at 0.2 x 105 genomes (i.e., amplifiable target sequences) per g of soil.

Community analysis of ammonia-oxidising bacteria, in relation to oxygen availability in soils and root-oxygenated sediments, using PCR, DGGE and oligonucleotide probe hybridisation

“…Previous MPN analyses of the same samples showed less than 10 Q culturable ammonia-oxidising bacteria g 3I dry sediment for some samples [4]. Detection of this number of target cells in a background of at least 10 V nontarget cells compares favourably with previous attempts to detect known numbers of inoculated cells in soils [38,39]. However, as the MPN method detects only culturable cells, this may underestimate the actual number of target cells present.…”

Incorporating polymerase chain reaction-based identification, population characterization, and quantification of microorganisms into aerosol science: A review

“…Indeed, the use of these physical disruption steps has been associated with 99% cell disruption in environmental samples and large increases in DNA yields from soils and aerosol samples (Haugland et al, 2002, Zhou et al, 1996, Miller et al, 1999. Other researchers, however, have cautioned that methods such as bead beating and sonication can shear genomic DNA into fragments (Miller et al, 1999;Picard et al, 1992) and that these fragments may lead to either incomplete PCR amplification or chimera (PCR amplicon composed of DNA from different, multiple microorganisms) production. Miller and coworkers describe speeds and times for bead beating that reduce this fragmentation (Miller et al, 1999), and extraction by sonication is not recommended.…”

Adaptation and population dynamics of Azotobacter vinelandii during aerobic biological treatment of olive-mill wastewater

“…OMW samples (1 ml) were removed each day from the reactor and bacterial cells were collected by centrifugation (4015Ug, 10 min). The pellet was washed three times in distilled water to remove OMW residuals, resuspended in 200 Wl TENP bu¡er (50 mM Tris, 20 mM EDTA disodium salt pH 8.0, 100 mM NaCl, 1% w/v polyvinyl-polypyrolidone) [26], and subjected to six successive thermal shocks (liquid nitrogen 380³C/water bath 80³C). The cell debris was removed by centrifugation at 1925Ug for 5 min.…”

“…A quantitative molecular method, combining the polymerase chain reaction (PCR) with the most probable number (MPN) statistical analysis has been used to estimate speci¢c microbial populations in situ [25,26] and even to show certain dynamic changes [27^29]. To enable the estimation of the population of A. vinelandii strain A during the aerobic treatment of OMW, a quantitative PCR approach based on MPN analysis was optimised, combined with a simple DNA extraction procedure.…”