Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-24
2003-03-11
Jones, W. Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S023200, C536S024300, C536S024320, C536S024330
Reexamination Certificate
active
06531281
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of detecting and enumerating sulphate-reducing bacteria.
2. Description of Related Art
Sulphate-reducing bacteria (SRB) use sulphate as electron acceptor under anaerobic conditions, via the anaerobic respiration of sulphates (energy reduction), to produce sulphides while recovering, during this reduction, the energy necessary for their growth. This metabolic characteristic constitutes a common characteristic of these organisms, regardless of their phylogenetic position (Legall & Fauques, 1988).
These bacteria are recognized to be the principal microorganisms responsible for the biological formation of hydrogen sulphide (H
2
S). This H
2
S of biological origin in particular, and the metabolism of sulphate-reducing bacteria in general, cause many problems for industrialists such as the biological corrosion of steel, on the one hand, and the potential risk for staff, on the other (Postgate, 1979). In the petroleum industry, in addition to the abovementioned pernicious effects, the sulphate-reducing bacteria are also involved in impairing the quality of crude oil (Cord-Ruwich et al., 1987). The detection of these sulphurogenic bacteria therefore constitutes a major challenge for combating the production of H
2
S in a large number of industrial activities (Tatnall et al., 1988).
Microbial culture techniques applied to the detection and to the enumeration of these microorganisms have been developed (API, 1982; Magot et al., 1988; Scott & Davies, 1992). These methods require an incubation time of 10 to 21 days and are therefore poorly suited to the monitoring of contaminations of fluids in real time. Alternative methods allowing rapid measurement of the level of contamination have also been developed, such as for example the “Rapid Check™” from Conocco, based on the immunodetection of APS reductase (Horacek & Gawell, 1988, EP 272,916), or the “Hydrogenase test™” (Caproco), which detects the activity of hydrogenases, enzymes which are present in the SRBs but are not specific to these organisms (Scott & Davies, 1992). However, none of these methods is sufficiently sensitive or specific.
SUMMARY OF THE INVENTION
The authors of the present invention have developed a method of detecting and enumerating sulphate-reducing bacteria which combines these two advantages: sensitivity and specificity and which combines, in addition, speed with reliability. They therefore directed their attention to the sulphate energy reduction pathway and more specifically to that of APS reductase.
APS reductase (or Adenylylsulphate reductase) which allows the reduction of adenosine phosphosulphate (APS) (product of the activation of sulphate by ATP sulphurylase), is a cytoplasmic enzyme containing two subunits (&agr; and &bgr;) known to be involved only in the anaerobic respiration of sulphate (Legall & Fauques, 1988). This enzyme is not therefore present in non-sulphate-reducing organisms since it is not involved in the assimilatory reduction which allows the incorporation of sulphur into various molecules necessary for life, such as amino acids and vitamins.
On the basis of two sequences of the gene encoding this enzyme deposited in data banks, one derived from an organism in the domain of Bacteria (
Desulfovibrio vulgaris,
em_ba: 269372) and the other from the sector of Archaea (
Archaeoglobus fulgidus:
em_ba: X63434), the authors of the present invention were able to amplify and sequence various genes encoding APS reductase. Surprisingly, they observed that this gene is remarkably well conserved whereas the phylogenetic diversity of the organisms studied could not a priori suggest it. This result opened the perspective for using this gene as a target for the specific detection of sulphate-reducing bacteria, especially in the domain of Bacteria.
The subject of the present invention is therefore the use of at least one nucleotide sequence which hybridizes specifically with a fragment of the APS reductase gene or a fragment of the mRNA transcribed from the APS reductase gene to detect the presence of sulphate-reducing bacteria in a sample.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The subject of the present invention is more particularly a method for the specific, qualitative or quantitative detection of sulphate-reducing bacteria in a sample which is likely to contain them, the said method comprising the extraction of the DNA or of the RNA from the said sample and the detection of at least one fragment of the APS reductase gene or one fragment of the mRNA transcribed from the APS reductase gene, an indicator of the presence of sulphate-reducing bacteria in the said sample.
The extraction of the DNA or of the RNA from the said sample may be carried out by standard techniques which are well known to persons skilled in the art.
More particularly, the detection of at least one fragment of the APS reductase gene comprises the specific gene amplification of at least one fragment of the gene for the &agr; subunit of APS reductase. Advantageously, the gene amplification products may, in addition, be subjected to hybridization with a probe which is specific for the said fragment of the gene for the &agr; subunit of APS reductase, the said probe being labelled in a detectable manner.
According to another embodiment, the detection of at least one fragment of the APS reductase gene comprises the hybridization of the extracted DNA with a probe which is specific for the said fragment of the gene for the &agr; subunit of APS reductase, the said probe being labelled in a detectable manner.
According to another embodiment, the method of the invention comprises the extraction of the RNA from a sample which is likely to contain sulphate-reducing bacteria and the detection of at least one fragment of the mRNA which is transcribed from the APS reductase gene.
In this case, the detection may be carried out by direct hybridization of a specific nucleotide probe labelled in a detectable manner with the extracted mRNA, and/or by specific amplification of the mRNA encoding APS reductase, in particular by RT-PCR (reverse transcription followed by a polymerase chain reaction).
The subject of the present invention is also an oligonucleotide having a nucleotide sequence which is essentially identical to a sequence chosen from the sequences SEQ ID No. 1 to 25. Such an oligonucleotide is in particular useful as a primer for amplifying a fragment of the gene for the &agr; subunit of APS reductase, or as a probe which hybridizes with a fragment of the gene for the &agr; subunit of APS reductase or the product of amplification thereof.
Preferably, it is possible to use as a primer an oligonucleotide having a sequence which is essentially identical to one of the sequences SEQ ID No. 11 to 18, and as a probe an oligonucleotide having a sequence which is essentially identical to one of the sequences SEQ ID No. 19 to 25.
“Essentially identical” is understood to mean that the sequence of the oligonucleotide is identical to one of the sequences SEQ ID No. 1 to 25 or that it differs from one of these sequences without affecting the capacity of these sequences to hybridize with the gene for the &agr; subunit of APS reductase. A sequence which is “essentially identical” to one of the sequences SEQ ID No. 1 to 25 may in particular differ therefrom by a substitution of one or more bases or by deletion of one or more bases located at the ends of the oligonucleotide, or alternatively by addition of one or more bases at the ends of the oligonucleotide. Preferably, such an oligonucleotide has a minimum size of 10 nucleotides, preferably of at least 14 nucleotides.
According to a preferred embodiment of the invention, the method of detecting sulphate-reducing bacteria in a sample which is likely to contain them according to the invention advantageously comprises the steps consisting in:
extracting the DNA from the said sample;
bringing the DNA extracted in step i) into contact with at least one primer consisting of an oligonucleotide having a nucleotide s
Magot Michel
Ravot Gilles
Blank Rome LLP
Elf Exploration Production
Goldberg Jeanine
Jones W. Gary
LandOfFree
Method of detecting sulphate-reducing bacteria does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method of detecting sulphate-reducing bacteria, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method of detecting sulphate-reducing bacteria will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3003153