Kit for detecting human cytomegalovirus nucleic acid

A human cytomegalovirus and kit technology, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of low detection sensitivity and detection lag, and achieve improved sensitivity and accuracy. Easy and fast effect of extraction and detection operation

Pending Publication Date: 2018-06-08
SHANGHAI XINGYAO MED TECH DEV CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Serological detection uses antigen-antibody reaction to detect virus-related proteins, which has the advantages of fast, simple, and easy-to-operate detection, but the detection sensitivity is not high, and there is still a certain lag in detection due to the "window period" of antibody production after infection

Method used

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  • Kit for detecting human cytomegalovirus nucleic acid
  • Kit for detecting human cytomegalovirus nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: Human cytomegalovirus nucleic acid detection kit primer probe design

[0028] According to the HCMV gene sequence inquired in the NCBI GenBank database, using molecular biology primer design software, the preferred primer probe sequence obtained is as shown in Table 1, and what HCMV primers amplified was the 139bp fragment on the IE2 gene of the virus.

[0029] Table 1 Design kit primer probe

[0030]

[0031] The above primers and probes were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

Embodiment 2

[0032] Example 2: Preparation of Human Cytomegalovirus Nucleic Acid Detection Kit

[0033] 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.

[0034] Table 2 The volume of each component prepared by the reaction buffer of the kit

[0035]

[0036] The negative control of the kit uses normal human negative plasma; the quantitative calibrator is the cloning plasmid containing the HCMV amplified target sequence fragment, which is diluted to 4 concentrations with TE buffer as the quantitative calibrator of the kit: 5.0x10 7 copies / ml, 5.0x10 6 copies / ml, 5.0x10 5 copies / ml, 5.0x10 4 copies / ml; the positive control is Escherichia coli containing the above cloned plasmid, diluted with human negative plasma ...

Embodiment 3

[0040] Embodiment 3: the mensuration of kit detection sensitivity

[0041] (1) Viral DNA extraction

[0042] Take HCMV-positive plasma with clinical identification and known concentration, and the concentration of HCMV in the sample is 2x10 5 copies / ml). The mixed sample was then serially diluted 10-fold to 2x10 using normal human negative plasma. 4 copies / ml, 2x10 3 copies / ml, 2x10 2 copies / ml, 2x10 1 copies / ml, draw 400 μl each of the above-mentioned virus dilution, the negative control of the kit, and the positive control, and use the magnetic bead method virus DNA extraction reagent in Example 2 to carry out virus DNA extraction on the nucleic acid extractor, and finally each sample obtained Nucleic acid each 50 μl.

[0043] (2) Fluorescent PCR detection

[0044] Take the reaction buffer of the kit in Example 2 out of the -20°C refrigerator, freeze-thaw, mix well, and centrifuge briefly, divide the reaction buffer into PCR reaction tubes at 20 μl / person, and the...

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Abstract

The invention relates to a kit for detecting human cytomegalovirus nucleic acid. The kit is characterized in that a virus-specific nucleic acid sequence is amplified by adopting a pair of primers; thehuman cytomegalovirus nucleic acid is detected by two fluorescent probes; the human cytomegalovirus nucleic acid in whole-blood, plasma, urea and milk samples can be qualitatively or quantitatively detected at high sensitivity. The kit has the advantages of simplicity, convenience and quickness in operation, and sensitive and accurate detection results, and can be used as an effective auxiliary detection means for human cytomegalovirus infection.

Description

technical field [0001] The invention relates to a fluorescent PCR detection kit for human cytomegalovirus nucleic acid, which belongs to the field of biotechnology. Background technique [0002] Human cytomegalovirus (Human Cytomegalic Virus, HCMV) belongs to the herpesviridae family and is the largest virus in the human herpesvirus group, with a diameter of 200nm and a core of double-stranded linear DNA, which has a typical herpesvirus structure. HCMV can only proliferate in human fibroblast culture, but not in other animal cells. The proliferation is very slow, and it takes more than one month for the initial isolation to appear cytopathic effect (CPE), which causes the cells to become round, swell, The cells and nuclei become enlarged, and large eosinophilic inclusion bodies appear around the nucleus, so it is also called cytomegalic inclusion disease (CID). Human infection with HCMV is a systemic infection syndrome. Clinically, multiple organs are involved, and the symp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/705C12Q2563/107
Inventor 吴大治夏懿吴梅
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD
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