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Reference-containing high-sensitivity fluorescent quantitative polymerase chain reaction (PCR) kit for Epstein-Barr virus

A fluorescence quantification and kit technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve problems such as affecting drug treatment, false negatives, and unprepared quantitative results of viral nucleic acid, so as to avoid false positives, Simple and fast operation, the effect of improving detection sensitivity

Active Publication Date: 2014-03-19
SHANGHAI XINGYAO MED TECH DEV CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the methods currently used for EBV fluorescence quantitative detection do not use internal references to monitor possible PCR inhibitors in nucleic acid extraction and amplification (Hill CE et al, Am J Clin Pathol, 2006, 125:665-67; ​​Liu Dixia et al., Modern Test Medical Journal, 2009, 24:93-95; patents CN201110165106, CN201110373390, CN201110177104), when PCR inhibitors in whole blood, plasma or nasopharyngeal swabs are brought into the amplification reaction for detection, it is easy to cause the quantitative results of viral nucleic acid to be unprepared Even "false negative" phenomenon occurs, which affects the drug treatment after viral infection

Method used

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  • Reference-containing high-sensitivity fluorescent quantitative polymerase chain reaction (PCR) kit for Epstein-Barr virus
  • Reference-containing high-sensitivity fluorescent quantitative polymerase chain reaction (PCR) kit for Epstein-Barr virus

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Embodiment 1: the design of EBV nucleic acid fluorescent PCR quantitative detection kit primer probe

[0030] According to the EBV and GAPDH gene sequences queried in the NCBI GenBank database, using Vector NTI, Oligo and other primer design software, the optimally obtained primer probe sequences are shown in Table 1, and the EBV primers amplify the 115bp on the BamHI-W gene of the virus Fragments and internal reference primers amplify a 144bp fragment on the human GAPDH gene.

[0031] Table 1 The designed kit EBV and internal reference detection primer probe

[0032]

[0033] The above primers and probes were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0034]

Embodiment 2

[0035] Embodiment 2: EBV nucleic acid fluorescent PCR quantitative detection kit preparation

[0036] The reaction buffer of the kit is self-prepared. According to the concentration and volume of each component in Table 2, the reaction buffer of the kit is prepared for 32 people. The prepared reaction buffer is divided into 20 μl for each reaction. After adding 10 μl of the template, the total reaction volume is 30 μl.

[0037]

[0038] Table 2 The volume of each component prepared by the reaction buffer of the kit

[0039] components The initial concentration Reaction final concentration (30μl) Volume for 32 servings (μl) Tris-HCl (pH8.3) 1000mM 10mM 9.6 KCl 500mM 50mM 96 dATP 100mM 0.2mM 1.92 dGTP 100mM 0.2mM 1.92 dCTP 100mM 0.2mM 1.92 dUTP 100mM 0.2mM 1.92 MgCl 2 50mM 3.5mM 67.2 EBV upstream primer 10μM 0.3μM 28.8 EBV downstream primers 10μM 0.3μM 28.8 EBV fl...

Embodiment 3

[0044] Embodiment 3: the mensuration of kit detection sensitivity

[0045] (1) EBV DNA extraction

[0046] EBV virus-positive plasma (5x10 4 copy / ml), using normal human negative plasma 10-fold serial dilution to 5x10 3 copy / ml, 5x10 2 copy / ml, 5x10 1 copy / ml, draw 400 μl each of the above-mentioned virus dilution, negative control of the kit, and 5 quantitative calibration products, and use the magnetic bead method virus DNA extraction reagent in Example 2 together to carry out virus DNA extraction on the nucleic acid extractor, and finally each 50 μl of each nucleic acid obtained from the sample.

[0047] (2) Fluorescent PCR detection

[0048] Take out the amplification part (reaction buffer) of the kit in Example 2 from the -20°C refrigerator, freeze-thaw, mix well, and centrifuge briefly, divide the reaction buffer into PCR reaction tubes at 20 μl / person, and then add 10 μl each of the negative control, quantitative calibrator, and template extracted from the v...

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Abstract

The invention relates to a kit for quantitative detection of an Epstein-Barr virus (EBV) nucleic acid by using a fluorescent polymerase chain reaction (PCR) technology. A pair of primers is used for amplifying an EBV specific nucleic acid sequence and EBVDNA is quantitatively detected through a fluorescence probe and meanwhile, an internal reference DNA is detected by using a human endogenous gene specificity primer and the fluorescence probe. By adopting the kit, existence of the EBV and internal reference nucleic acid is simultaneously detected by a single-tube double-wavelength fluorescent PCR technology, the EBVDNA in whole blood, plasma and a nasopharyngeal secretion sample can be quantitatively detected, and the whether a PCR inhibitor or template loss caused by a misoperation in detection exists in the sample is judged by the detection result of the internal reference nucleic acid. Thus, the kit is simple, convenient and fast in operation, can provide sensitive and accurate quantitative result, and can be widely applied to quantitative detection of clinical EBV virus infection.

Description

technical field [0001] The invention relates to a single-tube dual-wavelength fluorescent quantitative PCR detection kit for Epstein-Barr virus (EBV) containing an internal reference. Background technique [0002] Epstein-Barr virus (EBV) is a virus isolated from a Burkitt lymphoma cell line by British virologists in 1964. It is a DNA virus of the genus Lymphotropia in the family Herpesviridae. It is widely contagious in the crowd, and the transmission route is mainly through saliva, and it can also be transmitted through blood transfusion. The first infection of most people occurs in childhood, and most of them have no obvious symptoms after infection, or cause mild pharyngitis and upper respiratory tract infection. , but carry the virus for life, according to research, more than 95% of adults have the virus. This virus is the pathogen of infectious mononucleosis, especially because it is closely related to the occurrence of nasopharyngeal carcinoma, which has a high incid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/70C12Q2545/101C12Q2561/101C12Q2531/113
Inventor 吴大治夏懿吴梅
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD
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