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.

Active Publication Date: 2016-08-24
苏州华益美生物科技有限公司
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Problems solved by technology

[0011] Although human parvovirus B19 (Human parvovirus B19), human T-cell lymphotropic virus type I (Human T-cell lymphotropic virus 1, HTLV-I), human T-cell lymphotropic virus type II (Human T-cell lymphotropic virus 2, HTLV- II), hepatitis E virus (Hepatitis E virus, HEV) these four kinds of viruses, these viruses themselves all can use PCR method to carry out independent detection respectively, but their joint detection has not reported yet
At present, conventional real-time PCR detection, such as Chinese patents CN1768151A, CN101189505A, CN101273262A, CN101302473A, etc., are mostly only used to detect a single target nucleic acid
The Chinese patent CN101624629A of the inventor team discloses (in a single PCR reaction container) a real-time PCR method for multiple detection of target nucleic acids in samples, which is

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  • 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
  • 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
  • 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

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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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Abstract

The invention provides a 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 an application thereof, and relates to a real-time PCR method for quadruple detection of target nucleic acid in a nucleic acid extraction liquid in a single PCR reaction container. The method is used for detecting B19, HTLV-I, HTLV-2 and HEV.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection. Specifically, the invention relates to the ability to simultaneously detect human parvovirus B19 (Human parvovirus B19, B19), human T-cell lymphotropic virus type I (Human T-cell lymphotropic virus 1, HTLV-I), A multiplex real-time PCR method for detecting four pathogenic pathogens of human T-cell lymphotropic virus 2 (HTLV-II) and hepatitis E virus (HEV), which is fast and ultrasensitive And complete in one step. In addition, the present invention also relates to the reagents involved in the above PCR method, such as primers, probes, etc. that are irrelevant to each other, as well as the detection kit used in the above method and the preparation and application of the corresponding detection kit. Background technique [0002] Human parvovirus B19 (Human parvovirus B19) is a linear single-stranded DNA virus with a genome size of 5.6 kb. In 1975, when Cossart et al. detected the h...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12N15/10
CPCC12Q1/6806C12Q1/686C12Q1/701C12Q2537/143C12Q2561/113C12Q2563/107C12Q2531/113
Inventor 王川李振勇陈红干郭志武齐洁婷张必新
Owner 苏州华益美生物科技有限公司
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