Novel and highly-sensitive detection method for latent human cytomegalovirus (HCMV) in cell

A technology of human cytomegalovirus and cytomegalovirus, which is applied in the field of medical and microbiological detection, and can solve the problems of no research, analysis and expression

Inactive Publication Date: 2014-03-12
WENZHOU MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
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  • Novel and highly-sensitive detection method for latent human cytomegalovirus (HCMV) in cell
  • Novel and highly-sensitive detection method for latent human cytomegalovirus (HCMV) in cell
  • Novel and highly-sensitive detection method for latent human cytomegalovirus (HCMV) in cell

Examples

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example 1

[0033] Example 1: Establishment of UL138 testing standard curve

[0034] (1) The whole gene synthesis HCMV-UL138 is constructed on pcDNA3.1-zeo(+) plasmid to form a recombinant plasmid map (see image 3 A). Measure its concentration with a spectrophotometer: 490ng / ml, the base number of pcDNA3.1-zeo(+) / HCMV-UL138 recombinant plasmid is 5502bp, and the calculated copy number is 8×10 10 copies / μl, diluted to 8×10 as needed 9 , 8×10 8 , 8×10 7 , 8×10 6 , 8×10 5 , 8×10 4 , 8×10 3 , 8×10 2 , 8×10 1 , 8×10 0 copies / ul, according to the aforementioned nested PCR, the plasmids of different dilution concentrations were respectively subjected to the first round of PCR, and their corresponding first round PCR products were used as the standard products of Real-Time PCR in this experiment.

[0035] (2) Fluorescence quantitative PCR reaction solution composition: 2×SYBR Green Realtime PCR Master Mix 10μl, 10μmol / L inner upstream primer 0.8μl, 10μmol / L inner do...

example 2

[0040] Example 2 Using the present invention to detect HCMV latent infection in peripheral blood samples

[0041] Step 1: Method for separating white blood cells from peripheral blood

[0042] 1. Reagents and preparation

[0043] (1) 1× red blood cell lysate: weigh NH 4 CL 4.145g, KHCO 3 0.5g, Na 2 EDTA 18.6mg, add deionized water to make the volume to 500ml, store at room temperature after autoclaving.

[0044] (2) 10×PBS buffer: Weigh 8g NaCl, 0.2g KCl, Na 2 HPO 4 1.42g, KH 2 PO 4 0.27g, add 800ml deionized water, add dropwise concentrated hydrochloric acid to adjust the pH to 7.4, then add deionized water to make the volume to 1L, and store at room temperature after autoclaving. When washing white blood cells, it is necessary to dilute 10×PBS 10 times with deionized water into 1×PBS buffer.

[0045] Peripheral blood leukocyte separation steps

[0046] (1) Take 2 tubes of 2ml of EDTA anticoagulant blood from intravenous blood collection on the same day, and...

example 3

[0079] Example 3 Using the present invention to detect HCMV infection in peripheral blood samples

[0080] Step 1: DNA extraction method of peripheral blood leukocytes

[0081] 1. Reagents and preparation

[0082] (1) 50×TAE buffer for electrophoresis: Weigh 242g Tris, Na 2 EDTA·2H 2 Add about 800ml of deionized water to 37.2g of O, add 57.1ml of acetic acid, and dilute to 1L. For electrophoresis or agarose gel preparation, 50×TAE is diluted 50 times into 1×TAE buffer.

[0083] (2) Preparation of agarose gel

[0084] 1> Prepare an appropriate amount of 1×TAE buffer for electrophoresis and gel preparation.

[0085] Accurately weigh the agarose according to the amount of gel prepared and the concentration of the gel, add it to an appropriate conical flask, and then heat it in a microwave oven to melt the agarose. After the solution has cooled for a few minutes, add ethidium bromide solution (final concentration 0.5mg / ml) and mix well.

[0086] Pour the agarose solution into ...

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Abstract

The invention provides a novel and highly-sensitive detection method for human cytomegalovirus (HCMV). The method represents a nested quantitative PCR (polymerase chain reaction) technique which can detect both free HCMV and HCMV that infects a cell, and is characterized in that nucleic acid is extracted from human peripheral blood mononuclear cells (PBMCs), and a first round of PCR amplification is carried out through specific outer upstream and downstream primers; with the products of the first round of PCR amplification serving as a template, a second round of quantitative PCR amplification is carried out through specific inner upstream and downstream primers. By adopting the detection method provided by the invention, the HCMV detection sensitivity can reach 8 copies per microliter. Compared with the ELISA (enzyme-linked immunoabsorbent assay) method, compared with the ELISA method, the method adopting the PCR method for the detection of the latent HCMV infection has the advantages of rapidness, simplicity, high sensitivity, strong specificity, good stability and the like, and is a clinical method for early detection and diagnosis.

Description

technical field [0001] The invention relates to the field of medical and microbiological detection, in particular to a novel, highly sensitive intracellular latent human cytomegalovirus detection method, which can be applied in clinical testing and related research. Background technique [0002] Human cytomegalovirus (HCMV) is a double-stranded linear DNA virus belonging to the family Herpesviridae, which has a wide range of cell affinity and can infect epithelial cells, endothelial cells, fibroblasts, peripheral blood mononuclear cells and nerve cells, etc. . Crowd HCMV infection is very common in the population, more than 90% of adults are infected with HCMV, most of which are asymptomatic infections. However, neonatal intrauterine infection or perinatal infection often causes miscarriage, stillbirth, fetal malformation, developmental delay, hearing impairment, and nervous system developmental delay. Latent infection is an important biological characteristic of HCMV, whi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/70C12Q1/686C12Q2549/119C12Q2545/114C12Q2531/113
Inventor 薛向阳陈静张丽芳陈文静叶璐璐
Owner WENZHOU MEDICAL UNIV
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