PCR techniques for detecting microbial contaminants in foodstuffs
First Claim
1. An in vitro method of detecting the presence of a microbe in a food sample, comprising:
- (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 universal bacteria nucleic acid sequence comprising 5′
-CCACCGAATCTTCGTCGGTGG-3′
(SEQ. ID. NO.;
144) and sequences upstream and downstream of the universal bacteria 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, wherein a fluorogen is connected to one end of the first or second primer, and a (3) polynucleotide containing a single-stranded DNA probe that specifically hybridizes to SEQ. ID. NO.;
144 and wherein the polynucleotide assumes a stem-loop structure in the absence of the universal or bacterial nucleic acid sequence wherein the polynucleotide comprises a DNA internal segment being complementary to at least a portion of the universal bacterial nucleic acid sequence and a first and a second DNA arm segment adjoining the DNA internal segment, each of the arm segments comprising nucleotide sequences such that the arm segments are complementary to one another and (4) polymerase chain reaction reagents;
(b) forming a polymerase chain reaction product by cycling the polymerase chain reaction mixture under conditions whereby the universal bacterial nucleic acid sequence is replicated to from about 0.25 to about 10,000 μ
g nucleotide product/μ
l mixture;
(c) quenching any primer not bound to the upstream or the downstream sequences in the polymerase chain reaction product;
(d) hybridizing the DNA probe to the universal bacterial nucleic acid sequence, if present, and change the conformation of the stem-loop structure, the presence of a microbe in the sample is determined by detecting the conformational change in the stem-loop structure of the polynucleotide; and
(e) determining whether or not the universal bacterial nucleic acid sequence is present in the polymerase chain reaction product, the presence of the universal bacterial nucleic acid sequence detecting the presence of a microbe in the food sample.
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Accused Products
Abstract
A method or detecting the presence of living or dead microorganisms and viruses in a sample comprises adding to a pre-determined volume of a sample comprising nucleic acid-containing microbe (s) and/or virus (es), 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, cycling the mixture to amplify the universal or specific microbial and/or viral nucleic acid sequence; 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. Optionally, the terminus of one arm segment is operatively connected to a fluorogen and the terminus of the other arm segment is operatively connected to a quencher, such that the polynucleotide comprises a “fluorescent beacon.”
117 Citations
21 Claims
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1. An in vitro method of detecting the presence of a microbe in a food sample, comprising:
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(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 universal bacteria nucleic acid sequence comprising 5′
-CCACCGAATCTTCGTCGGTGG-3′
(SEQ. ID. NO.;
144) and sequences upstream and downstream of the universal bacteria 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, wherein a fluorogen is connected to one end of the first or second primer, and a (3) polynucleotide containing a single-stranded DNA probe that specifically hybridizes to SEQ. ID. NO.;
144 and wherein the polynucleotide assumes a stem-loop structure in the absence of the universal or bacterial nucleic acid sequence wherein the polynucleotide comprises a DNA internal segment being complementary to at least a portion of the universal bacterial nucleic acid sequence and a first and a second DNA arm segment adjoining the DNA internal segment, each of the arm segments comprising nucleotide sequences such that the arm segments are complementary to one another and (4) polymerase chain reaction reagents;
(b) forming a polymerase chain reaction product by cycling the polymerase chain reaction mixture under conditions whereby the universal bacterial nucleic acid sequence is replicated to from about 0.25 to about 10,000 μ
g nucleotide product/μ
l mixture;
(c) quenching any primer not bound to the upstream or the downstream sequences in the polymerase chain reaction product;
(d) hybridizing the DNA probe to the universal bacterial nucleic acid sequence, if present, and change the conformation of the stem-loop structure, the presence of a microbe in the sample is determined by detecting the conformational change in the stem-loop structure of the polynucleotide; and
(e) determining whether or not the universal bacterial nucleic acid sequence is present in the polymerase chain reaction product, the presence of the universal bacterial nucleic acid sequence detecting the presence of a microbe in the food sample. - View Dependent Claims (2)
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3. An in vitro method of detecting the presence of a microbe in a food sample, comprising the steps of:
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(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 bacteria nucleic acid sequence comprising 5′
-CCACCGAATCTTCGTCGGTGG-3′
(SEQ. ID. NO.;
144) of a microbe and sequences upstream and downstream of the universal bacterial 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, wherein a fluorogen is connected to one end of the first or second primer and (3) polymerase chain reaction reagents;
(b) forming a polymerase chain reaction product by cycling the polymerase chain reaction mixture under conditions whereby the universal bacterial nucleic acid sequence is replicated to from about 0.25 to about 10,000 μ
g nucleotide product/μ
l mixture;
(c) adding to the polymerase chain reaction product a polynucleotide containing a single-stranded DNA probe that specifically hybridizes to SEQ ID NO;
144 and wherein the polynucleotide assumes a stem-loop structure in the absence of the universal bacterial nucleic acid sequence wherein the polynucleotide comprises a DNA internal segment being complementary to at least a portion of the universal bacterial nucleic acid sequence and a first and a second DNA arm segment adjoining the DNA internal segment, each of the arm segments comprising nucleotide sequences such that the arm segments are complementary to one another;
(d) quenching any primer not bound to the upstream or the downstream sequences in the polymerase chain reaction product;
(e) determining whether or not the universal bacterial nucleic acid sequence is present in the polymerase chain reaction product, the presence of the universal bacterial nucleic acid sequence detecting the presence of a microbe in the food sample; and
(f) hybridizing the DNA probe to the universal bacterial nucleic acid sequence, if present, and change the conformation of the stem-loop structure, the presence of a microbe in the sample is determined by detecting the conformational change in the stem-loop structure of the polynucleotide. - View Dependent Claims (4)
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5. An in vitro method of detecting the presence of a bacterium in a food sample, comprising the steps of:
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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 nucleic acid sequence comprising 5′
-CCACCGAATCTTCGTCGGTGG-3′
(SEQ. ID. NO;
144) 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, wherein a fluorogen is connected to one end of the first or second primer, and (3) polymerase chain reaction reagents;
forming a polymerase chain reaction product by cycling the polymerase chain reaction mixture under conditions whereby the universal or specific nucleic acid sequence is replicated to from about 0.25 to about 10,000 μ
g nucleotide product/μ
l mixture;
adding to the polymerase chain reaction mixture or to the polymerase chain reaction product a polynucleotide comprising a DNA internal segment specifically hybridizes to SEQ ID NO;
144 and a first and a second DNA arm segment adjoining the DNA internal segment, each of the arm segments comprises nucleotide sequences such that the arm segments are complementary to one another, the polynucleotide assuming a stem-loop structure in the absence of the universal or specific nucleic acid sequence;
hybridizing the DNA probe to at the universal nucleic acid sequence, if present, and changing the conformation of the stem-loop structure;
quenching any primer not bound to the upstream or the downstream sequences in the polymerase chain reaction product; and
detecting the presence of a bacterium in the food sample by determining whether or not the universal or specific nucleic acid sequence is present in the polymerase chain reaction product by detecting whether there was a conformational change in the stem-loop structure of the polynucleotide. - View Dependent Claims (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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Specification