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Reagent for rapidly detecting and quantifying subtypes of human herpes viruses and kit

A detection reagent and herpes virus technology, applied in the field of biochemistry and molecular biology, can solve the problems affecting HHV epidemiological investigation and early clinical diagnosis needs, and achieve the effect of high clinical diagnosis use value

Active Publication Date: 2017-06-13
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, up to now, there is no efficient and specific rapid detection and quantitative standard scheme for HHV subtypes, which greatly affects the epidemiological investigation and early clinical diagnosis of HHV.

Method used

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  • Reagent for rapidly detecting and quantifying subtypes of human herpes viruses and kit
  • Reagent for rapidly detecting and quantifying subtypes of human herpes viruses and kit
  • Reagent for rapidly detecting and quantifying subtypes of human herpes viruses and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1273

[0056] Example 1 Detection of 273 cases of skin tissue herpes virus subtype infection

[0057] Samples: 273 cases of skin tissue from Shanghai Dermatology Hospital patients, aged between 5 and 93 years old, male or female, collected different organs or tissues from each patient (choose lesion sites with typical characteristics, such as upper limbs, right feet, On the right, etc., the whole layer of skin was cut, including the epidermis and dermis), and the main pathological diagnosis was skin tumor, nevus cell nevus, seborrheic dermatitis or keratosis, eczema, etc.

[0058] 1. DNA extraction

[0059] The above-mentioned skin tissue samples were collected, placed in test tubes, and stored at -80°C. DNA was extracted using a DNA extraction kit (TissuegDNA kit, Biomiga), and the quality (OD260 / 280) and concentration of DNA extraction were initially detected.

[0060] The specific operation is as follows:

[0061] (1) Rinse the sample with PBS solution after taking it out, put ...

Embodiment 21

[0084] Example 2 Detection of herpes virus infection in 1 case of varicella patient (plasma)

[0085] Samples: From Huashan Hospital of Fudan University, samples were taken from female chickenpox patients.

[0086] 1. Using the HMW method to extract DNA from the patient's plasma

[0087] (1) Add 400ul HMW buffer to resuspend plasma (180ul), add 40ul 10% SDS at the same time, 20ul10mg / ul proteinase K, incubate in 60°C water bath for 5h;

[0088] The preparation of HMW buffer is as follows (mix well and store at 4°C):

[0089]

[0090] (2) Add an equal volume of phenol chloroform (1:1), 12000rpm / 5min at 4°C, collect the supernatant, and repeat the operation once;

[0091] (3) Add 1 / 10 volume of sodium acetate, 2 times the volume of ice ethanol, -80°C for 1h, 4°C at 12000rpm / 5min;

[0092] (4) Wash once with 70 ice ethanol, 4°C 12000rpm / 5min;

[0093] (5) Dry in air (20min), dissolve with ddH2O (20ul), and store at -20°C.

[0094] 2. PCR detection

[0095] PCR reaction s...

Embodiment 310

[0104] Example 3 Detection of herpes virus infection in 10 samples

[0105] Samples: From the Shanghai Center for Disease Control and Prevention, each sample was from a patient suspected of being infected with the measles virus.

[0106] 1. DNA extraction

[0107] Take an appropriate amount of sample at -80°C, use a DNA extraction kit (Tissue gDNA kit, Biomiga) to extract DNA, and initially detect the quality and concentration of the DNA extraction, the method is the same as in Example 1.

[0108] 2. PCR detection

[0109]Reaction system and reaction condition are the same as embodiment 1.

[0110] 3. Test results and analysis

[0111] Test results such as image 3 As shown, for HSV-1: 5 samples of simple infection with measles virus were all negative, and 5 samples of mixed infection were all positive; for HSV-2: 5 samples of simple infection with measles virus were all negative, and 5 samples of mixed infection were all negative. Infected samples were all positive; for ...

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Abstract

The invention discloses a reagent for rapidly detecting various subtypes of human herpes viruses. The reagent comprises forward and reverse primers specifically amplifying HSV-1 RL2, HSV-2 UL28, VZV ORF26, EBV EBNA1, HCMV IE, HHV-6A or HHV-6B U22, HHV-6A or HHV-6B IE, HHV-7 U95 and KSHV ORF72 and a corresponding internal reference standard. The invention also discloses a kit which contains the detection reagent and is used for rapidly detecting various subtypes of the human herpes viruses and also discloses a reagent which contains the detection reagent and is used for rapidly quantifying the various subtypes of the human herpes viruses as well as a detection quantification kit. The reagent disclosed by the invention can rapidly and effectively identify and diagnose the various subtypes on clinical samples in early stage of infection and with different sources, also can conveniently and accurately carry out virus copy number quantitative calculation and has high clinical diagnosis use value.

Description

technical field [0001] The invention belongs to the field of biochemistry and molecular biology, relates to a detection reagent and a kit, in particular to a reagent and a kit for rapid detection and quantification of various subtypes of human herpesviruses. Background technique [0002] Human herpesvirus (HHV) is a double-stranded DNA virus, which can be divided into three subfamilies α, β, and γ according to its conserved structural protein sequence, and can be divided into 8 subtypes according to its genome characteristics, namely, herpes simplex virus 1 , type 2 (HSV-1 / 2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), herpesvirus 6, 7 (HHV-6A / 6B / 7) and Kaposi's sarcoma virus (KSHV). It is known that human herpes virus has a high infection rate in the population, and can invade various tissue cells of the human body, thereby causing skin, mucous membrane, lymph, liver, lung, reproductive or nervous system and other organ infections...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q1/705C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 蔡启良丁玲朱青朱彩霞
Owner FUDAN UNIV
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