B19 (Human parvovirus B19), HTLV (Human T-cell lymphotropic virus) and HEV (Hepatitis E virus) quadruple fluorescent PCR (polymerase chain reaction) quick hypersensitivity detection kit and application thereof
A re-detection and kit technology, applied in the field of multiplex real-time PCR, can solve problems such as the inability to design quadruple non-interfering primer pairs and probes, mutual interference with fluorescence detection curves, and no prompts for primer pairs and probes.
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Embodiment 1
[0086] The extraction of embodiment 1 nucleic acid
[0087] Human parvovirus B19 (Human parvovirus B19), Human T-cell lymphotropic virus 1 (HTLV-I), Human T-cell lymphotropic virus 2 (HTLV-II), Hepatitis E virus (HEV) can be purchased from the Clinical Inspection Center of the Ministry of Health of the People's Republic of China under license conditions, and they are all standard products for nucleic acid extraction.
[0088] 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 four viruses 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 (5...
Embodiment 2 4
[0090] Embodiment 2 quadruple real-time PCR test
[0091] 1. Primer and probe sequences
[0092] Commission the synthesis of the following primer pairs and probes:
[0093] Primer pair for detection of human parvovirus B19 (Human parvovirus B19):
[0094] B-19-F: ACTTTTAGTGCCAACTCTG,
[0095] B-19-R: GACTAATGGTGCAAACCTT,
[0096] Probes for detecting Human parvovirus B19:
[0097] B-19-P: AAGTAGCTGCCACAATGCC,
[0098] Primer pair for detection of Human T-cell lymphotropic virus 1 (HTLV-I):
[0099] HTLV-I-F: CCTAGTAGCCTCCCCTCCATCACC,
[0100] HTLV-I-R: GCTGGTATTCTCGCCTCAATCCTT,
[0101] Probes for detecting human T-cell lymphotropic virus 1 (HTLV-I):
[0102] HTLV-I-P: CCCCTCCGTGTCCAAGCCAACA,
[0103] Primer pair for detection of Human T-cell lymphotropic virus 2 (HTLV-II):
[0104] HTLV-II-F: GATGACAGGCTACAACCCCAT,
[0105] HTLV-II-R: AGGTTCTGGTACTCCCGTCT,
[0106] Probes for detecting human T-cell lymphotropic virus 2 (HTLV-II):
[0107] HTLV-II-P: CCCCGCCCAGCAA...
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