Composition for detecting microbial contamination comprising preparation for detecting nucleases, and use thereof
The present invention relates to a composition for detecting microbial contamination comprising a preparation for detecting nucleases, and a use thereof. A probe for measuring nucleic acid double-stranded nucleases, according to the present invention, may detect the comprehensive nucleic acid degradation capability of nucleases, whereby it is possible to quickly and precisely detect and quantify live microorganisms in a sample in a simple manner. Moreover, since the probe of the present invention is characterized in that signals of fluorescence consistently increase; is capable of measuring live microorganisms; and consists of a double-stranded nucleic acid, the probe is excellent for storage in a kit or a cartridge. As such, it is expected that the probe of the present invention may be used to easily and simply measure and compare the degree of contamination of microorganisms in an environment.
1. A composition for detecting microbial contamination, comprising:
an agent for detecting nuclease in microorganism,
wherein the agent is a double-stranded oligonucleotide;
wherein the double-stranded oligonucleotide is a double-stranded oligonucleotide consisting of SEQ ID NO: 1 and SEQ ID NO: 2 or a double-stranded oligonucleotide consisting of SEQ ID NO: 5 or SEQ ID NO: 6;
wherein one strand of the double-stranded oligonucleotide comprises a blunt end at the 5′-end or 3′-end; and the other strand of the double-stranded oligonucleotide comprises a sticky end on the opposite end of the blunt end;
wherein the double-stranded oligonucleotide has 1 to 10 nucleic acid mismatches between both ends; and wherein the oligonucleotide comprises a fluorescent material and a quencher, attached to 3′-end, 5′-end, both ends, or anywhere between both ends.
2. The composition according to claim 1 , wherein the sticky end comprises at least one mismatched base pair.
3. The composition according to claim 2 , wherein the nucleic acid mismatches in the sticky end are present at the 3′-end.
4. The composition according to claim 1 , wherein the fluorescent material is a luminous molecule, a metal ion, a complex compound, an organic dye, a conductor, a semiconductor, an insulator, a quantum dot, or a quantum wire.
5. A kit for detecting microbial contamination comprising the composition of claim 1 .
6. A method for detecting microbial contamination, comprising:
contacting a sample thought to be contaminated with microorganisms with a composition comprising an agent for detecting nuclease in microorganism, wherein the agent is a double-stranded oligonucleotide; wherein the double-stranded oligonucleotide is a double-stranded oligonucleotide consisting of SEQ ID NO: 1 and SEQ ID NO: 2 or a double-stranded oligonucleotide consisting of SEQ ID NO: 5 or SEQ ID NO: 6; wherein one strand of the double-stranded oligonucleotide comprises a blunt end at the 5′-end or 3′-end; and the other strand of the double-stranded oligonucleotide comprises a sticky end on the opposite end of the blunt end; wherein the double-stranded oligonucleotide has 1 to 10 nucleic acid mismatches between both ends; and wherein the oligonucleotide comprises a fluorescent material and a quencher, attached to 3′-end, 5′-end, both ends, or anywhere between both ends; and
identifying fluorescence development in the sample, the presence of fluorescence is indicative of microbial contamination in the sample.
7. The method according to claim 6 , wherein the method detects the presence of both Gram-negative and Gram-positive bacteria.
8. The method according to claim 6 , wherein the microbial contamination is quantified by determining the intensity of the fluorescence.
9. An oligonucleotide for measuring nuclease activity:
wherein the oligonucleotide is a double-stranded oligonucleotide consisting of SEQ ID NO: 1 and SEQ ID NO: 2 or a double-stranded oligonucleotide consisting of SEQ ID NO: 5 or SEQ ID NO: 6;
wherein one strand of the double-stranded oligonucleotide comprises a blunt end at the 5′-end or 3′-end; and the other strand of the double-stranded oligonucleotide comprises a sticky end on the opposite end of the blunt end;
wherein the double-stranded oligonucleotide comprises 1 to 10 mismatched base pairs between the blunt end and the sticky end; and wherein the oligonucleotide comprises a fluorescent material and a quencher, attached to 3′-end, 5′-end, both ends, or anywhere between both ends.