A kind of primer, probe, kit for detecting Ureaplasma urealyticum and its using method and application
A technology of Ureaplasma urealyticum and a kit, applied in the field of molecular biology, can solve the problems of long detection time, cumbersome use methods, and low detection sensitivity, and achieve the effects of shortened detection time, quick and easy use, and high sensitivity
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Embodiment 1
[0044] Embodiment 1, the detection of positive control substance and internal standard substance
[0045] The positive controls and internal standards are as follows:
[0046] The positive control substance is the pUC-57 plasmid containing the Ureaplasma urealyticum nucleic acid targeting sequence as shown in SEQ ID NO:4; the internal standard substance is the pUC-19 plasmid containing the nucleic acid sequence as shown in SEQ ID NO:8.
[0047] Dilute the positive control substance according to the following concentration gradient, the concentration is 5*10 4 copies / μl, 5*10 3 copies / μl, 5*10 2 copies / μl, 5*10copies / μl, 5copies / μl, spare.
[0048] Follow the steps below to check 1 # ~5 # Positive controls in centrifuge tubes:
[0049] Take six 1.5m1 centrifuge tubes, labelled 1 # ~6 # ,at 1 # ~6 # Add 2 μl of the sample to be tested, 2 μl of forward primer, 2 μl of reverse primer, 2 μl of the detection probe corresponding to the positive control substance (fluoresce...
Embodiment 2
[0051] Embodiment 2. Detection of different positive samples and internal standards
[0052] The different positive samples are as follows:
[0053] Positive sample 1 is Ureaplasma subtype 9 (UU, ATCC 33175) culture medium; positive sample 2 is Ureaplasma subtype 10 (UU, ATCC 33699) culture medium.
[0054] Follow the steps below to detect positive samples 1 to 2 in sequence:
[0055] Incubate the positive samples 1-2 at 99°C for 4min respectively;
[0056] Take four 1.5m1 centrifuge tubes, labelled 1 # ~4 # ,at 1 # ~4 # Add 2 μl of the sample to be tested, 2 μl of forward primer, 2 μl of reverse primer, 2 μl of the detection probe corresponding to the positive control substance (fluorescent reporter group is FAM-dT), 2 μl of the internal standard substance, and 2 μl of the corresponding internal standard substance into the centrifuge tube No. The detection probe (the fluorescent reporter group is HEX-dT) and 16 μl of the reaction solution were mixed evenly (among them, ...
Embodiment 3
[0058] Embodiment 3. Detection of different positive samples and different negative samples
[0059] The different positive samples are as follows:
[0060] Positive sample 1 is Ureaplasma subtype 9 (UU, ATCC 33175) culture medium; positive sample 2 is Ureaplasma subtype 10 (UU, ATCC 33699) culture medium; positive sample 3 is Ureaplasma subtype 11 (UU , ATCC 33695) culture medium; positive sample 4 is Ureaplasma subtype 12 (UU, ATCC 33696) culture medium; positive sample 5 is Ureaplasma subtype 13 (UU, ATCC 33698) culture medium.
[0061] The different negative samples are as follows:
[0062] Negative sample 1 is culture medium 1 that does not contain U. urealyticum nucleic acid; negative sample 2 is culture medium 2 that does not contain U. urealyticum nucleic acid; negative sample 3 is culture medium 3 that does not contain U. urealyticum nucleic acid.
[0063] Follow the steps below to detect positive samples 1 to 5 and negative samples 1 to 3 in sequence:
[0064] Inc...
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