Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-05
2004-10-26
Sitton, Jehanne (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024320
Reexamination Certificate
active
06808879
ABSTRACT:
FIELD OF INVENTION
This invention may be generally described as a means of qualitative and quantitative analysis of microbial populations potentially present in a sample. More specifically, it relates to a means of qualitative and quantitative analysis using RNA targeted oligonucleotide probes.
BACKGROUND
The analysis of microbial populations potentially present is required for many types of solid and fluid samples. Some notable examples are those samples obtained from a natural or biological environment such as natural water or hot springs; samples taken from humans or animals such as blood, urine, vaginal and intestinal flora; and samples from urban, agricultural and industrial environments such as food products, industrial water, industrial effluents, municipal wastewater, industrial sludge, fermentation media, aerosols, filters or air from air conditioning systems.
Various laboratory techniques have been developed for the qualitative and quantitative analysis of microbial populations potentially present in a given sample.
One familiar technique involves a count of the microorganisms that develop after the sample (or an extract thereof) is cultured on various selective nutrient media under standard conditions. This technique is simple but entails significant risks of errors and artifacts (low specificity of morphological criteria, inability to detect viable but non-culturable microorganisms, inability to detect slow-growing microorganisms, need to maintain viability of bacteria between collection and enumeration). Moreover, this technique generally requires longer than 24 hours to yield results.
A second technique, which entails the measurement of the activity of one or more enzymes, allows a rapid quantification of populations of living microorganisms (culturable microorganisms and/or microorganisms in a viable but non-culturable form). This technique can be used, in particular, to monitor a set of populations, but does not achieve very high levels of specificity or sensitivity.
A third technique using immunological probes often requires a growth step and thus requires longer than 24 hours to yield results. Moreover, it frequently lacks both sensitivity and specificity (misidentification may occur due to cross-reactions).
The most recent techniques are based on the use of specific DNA probes, which are generally labeled to permit detection after hybridization with their targets. Two main categories of oligonucleotide probes have been developed: those that target DNA and those that target RNA (ribosomal RNA or messager RNA).
DNA-targeted probes, although potentially highly specific, have the drawback of low sensitivity due to the few copies of the target DNA genes in each microbial cell. Although the use of PCR (polymerase chain reaction) to amplify the target DNA sequences before detection can compensate for the lack of sensitivity of the DNA probes, it has several drawbacks of its own: for example, the presence of inhibitors can lead to false-negative reactions, while carry-over or similar contamination can lead to false-positive reactions. In contrast, the use of RNA-targeted probes prevents from such drawbacks. In particular, because of the large number of copies of rRNA that occur naturally in a microorganism (actively growing cells may contain 10
4
ribosomes, each a potential probe target), the use of rRNA-targeted probes does not require the amplification step, thereby overcoming the constraints and artifacts associated therewith. The advantage of targeting rRNA is that about 85-90 percent of the total RNA in a typical cell is rRNA.
The hybridization of RNA-targeted probes can be achieved either after cell lysis, extraction and purification of the total nucleic acids of the sample, or in situ on whole cells, generally after fixation (permeabilization) of the membrane (or wall) of the microorganisms potentially present in the sample.
However, cell lysis and the ensuing extraction and purification of the nucleic acids particularly total RNA, are delicate and time-consuming manipulations that require costly apparatus, trained personnel and strict experimental conditions, notably the prevention of contamination by nucleases during the procedure. This technique further implies the use of a solid support, such as a nylon membrane, onto which the purified nucleic acids are immobilized in such a way one can discriminate between them (e.g. dot-blot, slot-blot). It most generally also implies the use of radioactive probe labels, the handling of which requires special care. The cell lysis technique for RNA hybrididization is therefore ill-suited to use in routine analysis either in industry or in biological laboratories.
In situ hybridization in whole cells overcomes the need for preliminary extraction of the target nucleic acids by cellular lysis with all its associated disadvantages. The FISH (Fluorescent In Situ Hybridization) process, which employs fluorescence-labeled rRNA probes, is one existing in situ technique. This type of technique, generally involving fluorescence microscopy, provides a fast and sensitive qualitative analysis on many types of sample. Today, rRNA-targeted probes thus hybridized in situ with their target within whole cells can be quantified directly on the sample (flow cytometry, microscopy), although the method is not entirely satisfactory: quantification directly on the sample is technically costly, time-consuming, requires trained personnel and does not permit an accurate quantification of hybridized probes when the sample is complex and non-uniform (e.g. floc or aggregates formed by filamentous bacteria in sewage treatment sludge; samples containing naturally fluorescent microorganisms). As a result, the technique of in situ hybridization in whole cells using fluorescence-labeled oligonucleotide probes has, to date, remained an essentially qualitative technique that does not provide reliable quantitative results.
To meet the need for industrial-caliber performance on samples that can be complex and/or non-uniform, this invention provides a means for analyzing, both qualitatively and quantitatively, the microbial populations potentially present in a biological sample, said means overcoming the disadvantages of prior art techniques
SUMMARY OF THE INVENTION
The object of this invention is, therefore, a method of qualitative and quantitative analysis of the microbial population(s) potentially present in a sample, characterized in that it comprises:
contacting the microorganisms potentially present in said sample with at least one RNA-targeted oligonucleotide probe, hereafter called specific probe, able to target a desired microbiological population, under conditions favourable to in situ hybridization in whole cells,
extracting, by separation from their target and elution outside said cells, those probes which have become hybridized,
detecting the extracted probes and measuring the amount thereof or their respective amounts.
The present invention thus advantageously enables the extraction of said probes without destruction of said cells.
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Flax Jodi
Guillot Emmanuelle
Manem Jacques
Rittmann Bruce E.
Stahl David A.
Connolly Bove & Lodge & Hutz LLP
Sitton Jehanne
Suez Lyonnaise Des Eaux
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