Rapid method for processing soil samples for polymerase chain reaction amplification of specific gene sequences

Pillai, Suresh D.; Josephson, Karen L.; Bailey, Rachel L.; Gerba, Charles P.; Pepper, Ian L.

doi:10.1128/aem.57.8.2283-2286.1991

Cited by 118 publications

(37 citation statements)

References 11 publications

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“…The PCR has become a powerful tool with which to explore microbial activities and identities in environmental microbiology (Mahbubani et al 1990;Pillai et al 1991;Bej and Mahbubani 1992;Koenraad et al 1995;Juck et al Fig. 2 Analysis of the limit of detection upon seeding environmental water samples with toxigenic Vibrio cholerae following enrichment in CDC broth using the pit-stop semi-nested PCR protocol.…”

Section: Discussionmentioning

confidence: 99%

Detection of toxigenicVibrio choleraefrom environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure

Theron

Cilliers

Preez³

et al. 2000

Journal of Applied Microbiology

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E N T E R . 2000. A pit-stop semi-nested PCR assay for the detection of toxigenic Vibrio cholerae in environmental water samples was developed and its performance evaluated. The PCR technique ampli®es sequences within the cholera toxin operon speci®c for toxigenic V. cholerae. The PCR procedure coupled with an enrichment culture detected as few as four V. cholerae organisms in pure culture. Treated sewage, surface, ground and drinking water samples were seeded with V. cholerae and following enrichment, a detection limit of as few as 1 V. cholerae cfu ml À1 was obtained with ampli®cation reactions from crude bacterial lysates. The proposed method, which includes a combination of enrichment, rapid sample preparation and a pit-stop semi-nested PCR, could be applicable in the rapid detection of toxigenic V. cholerae in environmental water samples.

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Section: Discussionmentioning

confidence: 99%

Detection of toxigenicVibrio choleraefrom environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure

Theron

Cilliers

Preez³

et al. 2000

Journal of Applied Microbiology

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“…330 the use of PCR, very small numbers of N. fowled amebae concentrated directly from the contaminated water might be rapidly identified. As direct detection of N. fowleri DNA can be hampered by environmental components such as humic acids which are negative factors for DNA amplification [19], the highest specificity and sensitivity is required in order to detect low numbers of this pathogenic agent. Work is currently in progress in our, laboratory to assess the capability of the above described PCR system to detect N. fowleri from environmental samples.…”

Section: Discussionmentioning

confidence: 99%

Differentiation of Naegleria fowleri and other naegleriae by polymerase chain reaction and hybridization methods

Sparagano¹

1993

FEMS Microbiology Letters

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In order to detect and identify Naegleria fowleri strains an assay based on the Polymerase Chain Reaction (PCR) was evaluated. The amplified DNA fragments were detected by gel electrophoresis and ethidium bromide staining, followed by Southern blot hybridization with an internal digoxigenin-labeled probe. A set of primers (B1B2) which flank a 678-bp region within a virulence-associated gene, allowed for the highly specific identification of N. fowleri, since Naegleriae (N. lovaniensis, N. australiensis, N. gruberi, N. andersoni and N. jadini) and other Protozoa did not react. These primers did not detect amplification products from various organisms: Gram-positive bacteria, Gram-negative bacteria, algae, yeasts and human DNA. Whereas a second set of primers (A1A2), which flank a different sequence, detected various Naegleriae and Acanthamoebae strains. After 40 amplification cycles, the limit of detection was a single cell (cyst or trophozoite). Thus, the PCR appears to be a rapid and powerful tool for identification and detection of N. fowleri.

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“…This approach enables sensitive detection of any group of bacteria for which probes are available, including those in non-culturable states. The extraction of DNA from soils and the application of DNA probes and PCR amplification has been reported previously (Tsai and Olson 1991;Pillai et al 1991;Selenska and Klingmüller 1992;Smalla et al 1993;Flemming et al 1994). Previous studies on soil DNA extraction and PCR sensitivity have involved 'reconstruction' experiments, adding organisms with marker sequences to soil and sediment samples prior to the extraction of total DNA after a relatively short incubation period (Steffan and Atlas 1988;Picard et al 1992;Tsai and Olson 1992;Erb and Wagner-Döbler 1993;Tebbe and Vahjen 1993;Smalla et al 1993;Flemming et al 1994;Knaebel and Crawford 1995).…”

Section: Introductionmentioning

confidence: 99%

Monitoring genetically modified rhizobia in field soils using the polymerase chain reaction

et al. 1998

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Monitoring genetically modified (GM) bacterial inoculants after field release using conventional culture methods can be difficult. An alternative is the detection of marker genes in DNA extracted directly from soil, using specific oligonucleotide primers with the polymerase chain reaction (PCR). The PCR was used to monitor survival of two GM Rhizobium leguminosarum bv. viciae inoculants after release in the field at Rothamsted. One strain, RSM2004, is marked by insertion of transposon Tn5; the second strain, CT0370, released at the same site, is modified by chromosomal integration of a single copy of the gene from E. coli conferring GUS activity. Both GM strain provide a realistic case study for the development of PCR-based detection techniques. Specific primers were developed to amplify regions of the Tn5 and GUS genetic markers using PCR and conditions optimized for each primer set to routinely detect a signal from 10 fg of purified template DNA, the equivalent of one cell per reaction. Procedures to improve the sensitivity of detection are described, to detect fewer than 50 cells g-1 soil in soil-extracted DNA.

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Rapid method for processing soil samples for polymerase chain reaction amplification of specific gene sequences

Cited by 118 publications

References 11 publications

Detection of toxigenicVibrio choleraefrom environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure

Detection of toxigenicVibrio choleraefrom environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure

Differentiation of Naegleria fowleri and other naegleriae by polymerase chain reaction and hybridization methods

Monitoring genetically modified rhizobia in field soils using the polymerase chain reaction

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