Quintuple fluorescent PCR (polymerase chain reaction) quick and hypersensitive detection kit and application thereof
A kit and re-detection technology, applied in fluorescence/phosphorescence, recombinant DNA technology, microbial assay/inspection, etc., can solve the problems of no primer pairs and probe tips, no re-detection, and high homology
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Embodiment 1
[0089] The extraction of embodiment 1 nucleic acid
[0090] Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Ureaplasma urealyticum (UU), Mycoplasma hominis (MH) and Mycoplasma genitalium (MG) can be purchased from the Clinical Inspection Center of the Ministry of Health under license conditions, all of which are standard Products for nucleic acid extraction.
[0091] The extraction of nucleic acid is carried out according to the conventional magnetic bead extraction method. In order to adapt to the nucleic acid extraction of the above five microorganisms at the same time, the improvement is as follows: add 90uL nucleic acid extraction solution to 1uL standard (the formula and final concentration are: guanidine isothiocyanate 1.2M, Sodium edetate (pH8.0) 10mM, Tween-20 2% (W / W), sodium perchlorate 1M, ethanol 40% (V / V), Tris-HCl (pH8.0) 10mM) , incubate at 42°C for 10min, then add 10uL D-Beads DNA magnetic bead suspension (50mg / mL, can be purchased from Beijing Aibig...
Embodiment 2 5
[0093] Embodiment 2 Five-fold real-time PCR test
[0094] 1. Primer and probe sequences
[0095] Commission the synthesis of the following primer pairs and probes:
[0096] Primer pairs for detection of Neisseria gonorrhoeae:
[0097] NG-F:ACCGTAACGTCTCTAAGTC
[0098] NG-R: GCAAGATTTCCGATTTGGC
[0099] Probes for the detection of Neisseria gonorrhoeae:
[0100] NG-P: CAGACATCACGCACCGAAGCAT
[0101] Primer pairs for detection of Chlamydia trachomatis:
[0102] CT-F: TGTCCATATCTTTGATACGAT
[0103] CT-R:CTCTGATATTTTGAAGACTCTACA
[0104] Probes to detect Chlamydia trachomatis:
[0105] CT-P:CCAAGCCGAGTCTACAGTTATAGGTCA
[0106] Primer pairs for detection of Ureaplasma urealyticum:
[0107] UU-F: CTGCTCGTGAAGTATTACA
[0108] UU-R:GTACCATCTGGGAAAGTAC
[0109] Probes for the detection of Ureaplasma urealyticum:
[0110] UU-P:ACCAACCATTGTATCTACACCTTCCATA
[0111] Primer pairs for detection of Mycoplasma hominis:
[0112] MH-F:TGTGGTTTAATTTGAAGATACAT
[0113] MH-R:AGTCCT...
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