Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-05-17
2002-10-22
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S007200, C435S091100, C435S091200, C536S023100, C536S024300, C536S024320, C536S026600
Reexamination Certificate
active
06468743
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel technology provided in the form of novel primers, probes, and beacons, and their use in a rapid and accurate assay and kit for assaying for the presence of a microorganism or virus, particularly a bacterium, in a sample. More specifically, this invention relates to an accurate method for assessing microbial, e. g. bacterial or viral, contamination of sterile substances, such as sterile foods, by microorganisms and/or viruses relying on PCR and fluorescent beacon technologies.
2. Description of the Background
Beacons are fluorescent probes which contain short complementary sequences of nucleotide, (arms) attached to the 5′ and 3′ ends of a probe of a sequence complementary to a target nucleic acid. In addition, the beacon comprises a fluor, fluorophore or fluorogen (fluorogenic agent) and a quencher attached, e. g. via linkers, to the ends of the stem or arms. In the absence of a target, the fluor and the quencher remain close to one another held in place by a hairpin loop stem formed by hybridization of the arms. In this conformation, the beacon does not fluoresce because of quenching. However, when the segment encompassed by the loop hybridizes to a complementary sequence, the hybridization of the arms is prevented, the fluor, fluorophore or fluorogenic agent and the quencher are held apart and fluorescence appears. Thus, the appearance of fluorescence is an indication of hybridization of the probe (DNA internal segment of the beacon) to a complementary nucleic acid sequence of a target. The annealing of a strand of nucleic acid to its complement, thus, may now be measured by following changes in the physical properties of the nucleic acids which occur upon hybridization.
Prior technology utilized, and immobilized the hybridized strands onto, a solid surface, removed unhybridized probes and then determined the number of probes attached to the solid phase. The need for removal of unhybridized probes precluded the application of solid phase hybridization to real time studies involving hybridization of nucleic acids. In addition, solid phase technology also has sensitivity limitations because the probes also bind non-specifically to the solid phase.
In the past the detection of microorganisms was made, generally, by phenotypic observation. Upon the realization that all living cells contain DNA and that DNA is responsible for the expression of phenotypic traits, novel detection methods relying on genetic parameters have been implemented. The polymerase chain reaction (PCR), a relatively recent technological development for amplification of DNA, provides significantly higher sensitivity and, thus, permits the detection of smaller quantities of nucleic acid by amplification and subsequent visualization, for instance, after electrophoretic separation and staining. Fluorescent dyes are also utilized by, for example, attachment to complementary oligomers (hybridization) that bind PCR-amplified DNA. These and other detection technologies eliminate the need for naked eye visualization of PCR products and, therefore, eliminate the need to electrophorese the nucleic acids, except for determining their molecular weight.
As is known in the art, the fidelity of specific DNA sequences produced by PCR is controlled by the primer DNA sequences used and by reaction conditions, such as thermocycling parameters, and the composition of the reaction mixture. DNA primers are generally designed to target specific sites vicinal to a desired sequence and applied to obtain DNA products of complementary sequences by the PCR, along with specifically tailored fluorescent molecular beacons designed to detect only the PCR product.
The detection of pathogens in edible products, cosmetics, medical fluids such as blood and IV solutions, and other products involved in commerce, is of great importance to avoid costly contamination, which may lead to outbreaks of disease and/or the need to discard large batches even after distribution. In most cases, even the etiological agent remains unidentified because of the sparcity of adequate testing technology to do this. Traditional technologies for recovering microorganisms from, for example, a foodstuff might include homogenization of the product in a buffered solution, inoculation of the homogenate in a selective enrichment medium, long hours of incubation at controlled temperatures, streaking of the broth onto a selective and/or differential agar medium, another incubation, isolation of colonies and finally a multiplicity of tests for biochemical or immunological characteristics and microscopic examination. Virulence is mostly tested on pure cultures which require several days of incubation. In addition, for sterility testing, the mere detection of biologically active DNA is critical.
Accordingly, there is a need for a rapid, simple, inexpensive and sensitive method for the general detection of microorganisms and/or viruses in commercial products as well as in pure cultures of microorganisms and viruses.
SUMMARY OF THE INVENTION
This invention relates to an in vitro method of detecting the presence of a microbe or a virus in a food sample, comprising the steps of:
(a) forming a polymerase chain reaction mixture by combining (1) a predetermined volume of a food sample to be tested for the presence of a nucleic acid sequence comprising a universal or specific nucleic acid sequence indicative of a microbe or a virus and sequences upstream and downstream of the universal or specific nucleic acid sequence, (2) known amounts of a first nucleic acid primer and a second nucleic acid primer for binding to the upstream sequence and the downstream sequence, respectively, and (3) polymerase chain reaction reagents;
(b) forming a polymerase chain reaction product by cycling the polymerase chain reaction mixture under conditions effective to amplify the universal or specific nucleic sequence, if present, to replicate and attain about 0.25 to about 10,000 &mgr;g nucleotide product/&mgr;l mixture; and
(c) determining whether or not the universal or specific nucleic acid sequence is present in the polymerase chain reaction product, the presence of the universal or specific nucleic acid sequence being indicative of the presence of a microbe or a virus in the food sample.
Any suitable method is used to determine the presence of the universal or specific nucleic acid sequence is present in the polymerase chain reaction product. Fluorescent intercalating reagents, such as ethidium bromide, can be used in conjunction with a fluorescence detection system. Alternatively, suitable oligonucleotide primers(s) that are hybridizably complementary to at least a portion of the reaction product, and bearing a fluorescent label and/or quencher can also be used in conjunction with the PCR amplification and/or fluorescence detection system as known in the art.
This invention also relates to an in vitro method of detecting the presence of a microbe or a virus in a food sample, comprising:
adding to a pre-determined volume of a sample suspected of comprising nucleic acid-containing microbe(s), known amounts of a pair of primers binding to sequences up-stream and down-stream to a universal or specific microbial and/or viral nucleic acid sequence and polymerase chain reaction (PCR) reagents to form a mixture;
cycling the mixture about under conditions effective to amplify the universal or specific microbial and/or viral nucleic acid sequence to attain about 0.25 to about 10,000 &mgr;g universal microbial nucleic acid/&mgr;l mixture;
adding a polynucleotide comprising a DNA internal segment that is hybridizably complementary to at least a portion of the universal or specific nucleic acid sequence; and a first and a second DNA arm segment adjoining the DNA internal segment, the first DNA arm segment ending in a 5′ terminus and the second DNA arm segment ending in a 3′ terminus, the arms segments comprising nucleotide sequences such that they are hybridizably complementary to one another. Opt
Fraser Mark S.
Romick Thomas L.
Christie Parker & Hale LLP
ConAgra Grocery Products Company
Forman B J
Jones W. Gary
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