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EBER probe for detecting EBV infected tissue and detection kit

A technology for detection kits and probes, which is applied in the determination/inspection of microorganisms, recombinant DNA technology, and methods based on microorganisms. It can solve the problems of cumbersome processes, poor solubility of PNA, and high synthesis costs, and achieve accurate and good detection results. The effect of hybridization specificity

Pending Publication Date: 2021-07-23
GUANGZHOU LBP MEDICINE SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection process involves multi-step washing and incubation, and the process is cumbersome
In the detection of pathogenic microorganisms in FISH, the common application of PNA probes has ideal detection results, but the synthesis cost is high, the synthesis cycle is long, and the solubility of PNA in purified water is poor, requiring the help of heat or organic reagents Solubilization may affect the stability and hybridization uniformity of short fragment probes, greatly affecting the accuracy of detection results

Method used

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  • EBER probe for detecting EBV infected tissue and detection kit
  • EBER probe for detecting EBV infected tissue and detection kit
  • EBER probe for detecting EBV infected tissue and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] EBER probe design and synthesis

[0053] The EBER-1 and EBER-2 genes are EBV-encoded small RNAs that bind to host proteins and do not encode proteins, but are abundantly expressed in all EBV-positive cells, up to 10 in each cell 6 The copy can be used as the target of ISH to detect EBV. Retrieve the sequence (Human herpesvirus 4isolate SNU-1103EBER-1and EBER-2genes, completesequence, GenBank: EF187853.1), and analyze the sequence specificity, select the appropriate segment to design the EBER probe (see figure 1), to obtain the EBER-2 probe (5'-FAM-acttgaccgaagacggcagaaagcaga-3', SEQ ID NO: 1). At the same time, a control probe was synthesized with reference to the literature (Chinese patent application number CN201811313293.3), named EBER-1 probe (5'-FAM-ctcctccctagcaaaaccctcaggacggcg-3', SEQ ID NO: 2), used to compare new Set the performance of the probe.

Embodiment 2

[0055] Evaluation of two probes and two hybridization buffers

[0056] The two probes were formulated with two hybridization buffers to form a hybridization system, and the confirmed EBV-positive and negative nasopharyngeal carcinoma tissue samples FFPE sections were used for testing.

[0057] 1. System configuration

[0058] System 1 contained 10% dextran sulfate, 10mM NaCl, 30% formamide, 0.1% sodium pyrophosphate, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 50mM Tris-HCl (pH 7.5) and a final concentration of 5 μM fluorescently labeled probe needle (EBER-1 probe or EBER-2 probe).

[0059] System 2 contained 10% dextran sulfate, 50% formamide, 1% Triton X-100, 50mM Tris-HCl (pH 7.5) and a final concentration of 5 μM fluorescently labeled probe (EBER-1 probe or EBER-2 probe Needle).

[0060] 2. Detection method:

[0061] 1. Pre-treatment process:

[0062] Bake slices: bake slices at 65°C for 30 minutes.

[0063] Dewaxing: Environmentally friendly dewaxing agent twice for 10 ...

Embodiment 3

[0077] Method for detecting samples with FITC-labeled EBER-2 probe

[0078] 1. System preparation of probe hybridization solution

[0079] The system contained 10% dextran sulfate, 50% formamide, 1% Triton X-100, 50 mM Tris-HCl (pH 7.5) and a final concentration of 5 μM FITC-labeled EBER-2 probe.

[0080] 2. Detection method

[0081] The same method as in Example 2, wherein the sample numbers are 98172, K57084, K56035, K56302, K56926, K57085 and K55290 respectively.

[0082] 3. Test results

[0083] The results are shown in Figure 4.

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Abstract

The invention provides an EBER probe for detecting EBV infected tissues and a detection kit, and belongs to the technical field of virus detection. The EBER probe is a fluorescein-labeled EBER probe, and the nucleotide sequence of the EBER probe is shown in SEQ ID NO: 1. The probe hybridization solution is an aqueous solution containing 10-20 pmol / [mu]l of an EBER probe, 50% of formamide, 10% of dextran sulfate, 1.0% of Triton X-100 and 50 mmol / L of Tris-HCl. The invention also provides the detection kit which comprises the probe hybridization solution, a DAPI counterstaining agent and a quality control sheet group, can realize the detection of the EBER state in a tissue or cell sample, and assists in clinically judging whether EBV infection exists or not. Through verification, the kit is high in detection sensitivity and good in specificity, the detection method is simple and convenient, the defects of an existing product or method are overcome, and the kit has wide application prospects.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to an EBER probe and a detection kit for detecting EBV-infected tissues. Background technique [0002] Epstein-Barr Virus (EBV) is a common type 4 human herpes virus. Infection is common in the population, but most of them have no obvious symptoms, long-term incubation, and slow biological activities. However, in a few specific cases, such as low immune function or triggered by certain factors, Epstein-Barr virus can change from a latent state to a proliferative state, forming recurrent infections and causing clinical diseases. [0003] Diseases associated with EBV include: nasopharyngeal carcinoma, some gastric adenocarcinomas, infectious mononucleosis, chronic active EBV infection, EBV-associated hemophagocytosis syndrome, X-chromosome-associated lymphoproliferative syndrome, lymphoma Lymphoid granuloma, lymphomatoid papulosis, Burkitt's lymphoma, AIDS-relate...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6841C12N15/11C12R1/93
CPCC12Q1/705C12Q1/6841C12Q2527/125C12Q2563/173C12Q2563/107
Inventor 何瑰曾培源陈绍宇
Owner GUANGZHOU LBP MEDICINE SCI & TECH
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