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EB virus detection kit based on droplet digital pcr

A micro-droplet and kit technology, applied in the biological field, can solve the problems of detection sensitivity and accuracy limitations, and achieve the effect of high sensitivity, accurate results, and accurate detection of virus content

Active Publication Date: 2020-07-10
广东永诺医疗科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, under the conditions of low copy target molecules and slight differences in template concentration, the sensitivity and accuracy of its detection are limited.

Method used

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  • EB virus detection kit based on droplet digital pcr
  • EB virus detection kit based on droplet digital pcr
  • EB virus detection kit based on droplet digital pcr

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The Epstein-Barr virus detection kit based on droplet digital PCR of the present application includes:

[0044] (1) Reaction master mix for droplet digital PCR: including buffer (reaction buffer), dNTPs (deoxyribonucleoside triphosphate), taq enzyme (taq DNA polymerase), UDG enzyme (uracil-DNA glycosylation Enzymes); the droplet digital PCR reaction master mix should be compatible with the digital PCR equipment, and can be replaced with other digital PCR reaction master mixes, or can be prepared according to the digital PCR conditions.

[0045](2) Target fragment detection mixed solution and internal standard detection mixed solution: comprise the combination of primers and probes for detecting the target segment, and the combination of primers and probes for detecting the internal standard; the target segment is the DNA or DNA of Epstein-Barr virus in this embodiment Epstein-Barr virus DNA fragment (EBV DNA);

[0046] Wherein, the primer and probe combination of descr...

Embodiment 2

[0063] The use method of the Epstein-Barr virus detection kit based on droplet type digital PCR of the present application comprises:

[0064] (1). Sample collection: The applicable sample type is serum or plasma.

[0065] a. Serum Use a sterile syringe to draw 2ml of venous blood from the bend of the subject’s arm or the back of the hand. After the sample is separated from the serum, centrifuge at 1600rpm for 5 minutes at room temperature to separate the serum and transfer it to a 1.5ml sterilized centrifuge tube for later use.

[0066] b. Plasma Use a sterile syringe to draw 2ml of venous blood from the bend of the subject's arm or the back of the hand, inject it into a sterile collection tube containing EDTA, immediately mix it upside down gently, and centrifuge at 1600rpm at room temperature for 5 The plasma was separated in 1 minute and transferred to a 1.5ml sterilized centrifuge tube for later use.

[0067] Store samples immediately on ice or at 4°C after collection. ...

Embodiment 3

[0101] In this embodiment, the kit described in Embodiment 1 and the method described in Embodiment 2 are used to detect Epstein-Barr virus in the plasma samples of normal people and nasopharyngeal cancer patients. The specific detection methods are:

[0102](1). Sample extraction: MAGEN "HiPure AS Blood DNA Mini Kit" column extraction method was used to extract total DNA from plasma samples of 76 normal subjects and 268 newly diagnosed patients with nasopharyngeal carcinoma, positive control and negative control. According to the instructions of the nucleic acid extraction kit, 200ul samples were taken for extraction. When extracting, an internal standard was added to the samples, and the nucleic acid was extracted according to the operation instructions of the kit. The elution volume was 50µl.

[0103] (2). Reaction system preparation:

[0104] EBV DNA testing is carried out in batches, and each batch of testing needs to be tested in parallel with positive and negative contr...

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Abstract

The application discloses a detection kit for Epstein-Barr virus based on micro-droplet digital PCR. The kit includes: a reaction premix for micro-droplet digital PCR; a mixed solution for detection of target fragments, including amplification primers and probes for target fragments The sequence of the amplification primer of the target fragment is shown in SEQ ID No.1 and SEQ ID No.2, and the sequence of the probe of the target fragment is shown in SEQ ID No.3; internal standard and internal standard detection mixture, including As the exogenous recombinant plasmid of internal standard, and the amplification primer and probe of described internal standard, the sequence of the amplification primer of internal standard is as shown in SEQ ID No.4 and SEQ ID No.5, and the sequence of the probe of internal standard is as SEQ ID No.4 and SEQ ID No.5 ID No.6: Positive control master mix; Negative control master mix; This application optimizes the finished kit for absolute quantitative detection of Epstein-Barr virus by using digital PCR technology to provide accurate diagnostic basis for the diagnosis of Epstein-Barr virus-related diseases.

Description

technical field [0001] The application relates to the field of biotechnology, in particular to a droplet digital PCR-based detection kit for Epstein-Barr virus. Background technique [0002] Epstein-Barr virus (EBv), also known as human herpesvirus 4 (Human herpesvirus4 (HHV-4)). Epstein-Barr virus was the first time that Epstein and Barr successfully established Burkitt African children's lymphoma cells through in vitro suspension culture in 1964, and observed herpes virus particles in the cell smears of the established strain with electron microscopy, and believed that the virus was a variety of malignant One of the causes of tumors (such as Burkitt lymphoma, Hodgkin lymphoma, nasopharyngeal carcinoma, gastric cancer, etc.), it mainly infects human oropharyngeal epithelial cells and B lymphocytes. [0003] Nasopharyngeal carcinoma is a malignant tumor of the head and neck with a high incidence in southern and southeast coastal areas of my country. About 80% of nasopharyng...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12R1/93
CPCC12Q1/6851C12Q1/705C12Q2563/159
Inventor 罗景燕许少飞李伟琴赖炳权张钰莹曹红豆何继谦
Owner 广东永诺医疗科技有限公司
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