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Primer and crRNA (clustered regularly interspaced short palindromic repeats-derived ribonucleic acid) for DNA (deoxyribonucleic acid) detection on EB (Epstein-Barr) virus and application of primer and crRNA

A technology of Epstein-Barr virus and primer pair, which is applied in the field of tumor diagnosis and can solve problems such as complex instruments

Active Publication Date: 2020-05-15
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the instruments required for real-time fluorescent quantitative PCR technology are relatively complex, and it is difficult to promote them in remote areas.

Method used

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  • Primer and crRNA (clustered regularly interspaced short palindromic repeats-derived ribonucleic acid) for DNA (deoxyribonucleic acid) detection on EB (Epstein-Barr) virus and application of primer and crRNA
  • Primer and crRNA (clustered regularly interspaced short palindromic repeats-derived ribonucleic acid) for DNA (deoxyribonucleic acid) detection on EB (Epstein-Barr) virus and application of primer and crRNA
  • Primer and crRNA (clustered regularly interspaced short palindromic repeats-derived ribonucleic acid) for DNA (deoxyribonucleic acid) detection on EB (Epstein-Barr) virus and application of primer and crRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1LwCas13a protein expression and purification

[0045] Bacterial expression vectors were transformed into Rosetta™ 2(DE3)pLysS Singles competent cells (Millipore). Inoculate 16 mL of starter culture overnight in Terrific Broth 4 growth medium (12 g / L tryptone, 24 g / L yeast extract, 9.4 g / L K2HPO, 2.2 g / L KH2PO4, Sigma) (TB) Grow in 4L of TB at 37°C and 300RPM until the OD600 reaches 0.5. At this time, protein expression was induced at a final concentration of 500 μM by supplementing IPTG (Sigma) with IPTG, and the cells were cooled to 18° C. for 16 hours for protein expression. Cells were then centrifuged at 5200g for 15 minutes at 4°C. Cell pellets were collected and stored at -80 °C for further purification.

[0046]All subsequent steps of protein purification were performed at 4°C. The cell pellet was crushed and resuspended in lysis buffer (20mM Tris-HCl, 500mM NaCl, 1mM DTT, pH8.0), supplemented with protease inhibitors (Complete UltraEDTA-free table...

Embodiment 2

[0048] The design of embodiment 2 primers

[0049] Forward and reverse primer design

[0050] By testing multiple pairs of primers, the primers with the best effect are screened out. Recombinase polymerase amplification (RPA) forward primers were first screened (see Figure 4 ), it was found that F5 had the best effect in the forward primer; then use the forward primer F5 to screen the recombinase polymerase amplification technology (RPA) reverse primer (see Figure 5 ), it was found that the primer combination of F5 and R3 had the best effect.

[0051] Forward primer F5:

[0052] 5'-AATTCTAATACGACTCACTATAGGCCTAAGAAGGCACCGGTCGCCCAGTCCTACC-3' (SEQ ID NO: 1),

[0053] Reverse primer R3:

[0054] 5'-TGAACCGCTTACCACCTCCTCTTCTTGCTGGA-3' (SEQ ID NO: 2).

[0055] crRNA design

[0056] The crRNA sequence is:

[0057] 5'-GGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUCUACGGACUCGUCUGGGUUCUUGGCCC-3' (SEQ ID NO: 3).

[0058] crRNA preparation

[0059] The Epstein-Barr virus DNA standar...

Embodiment 3

[0060] Embodiment 3 A kind of quantitative detection method that carries out Epstein-Barr virus DNA with crRNA

[0061] (1) Extract the plasma of the patient to be tested, separate the total cell-free DNA in the plasma, and obtain the template;

[0062] (2) Carry out the corresponding reaction procedure of the obtained template DNA in the reaction system, first use the recombinase polymerase amplification technology and the primer pair described in SEQ ID NO: 1-2 to amplify the template DNA under the condition of 25-37°C reaction, and then transcribe the amplified product at 25-37°C to obtain the corresponding RNA, and use the crRNA and LwCas13a described in SEQ ID NO:3 to detect the fluorescent signal of the transcription product at 25-37°C. The fluorescent signal detection procedure is as follows: react in a fluorescent plate reader (BioTek) at 25-37° C. for 2-3 hours, and measure the fluorescent signal every 5 minutes. All the above-mentioned reactions can be integrated in...

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PUM

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Abstract

The invention discloses a specific primer for detecting DNA (deoxyribonucleic acid) of an EB (Epstein-Barr) virus, crRNA (clustered regularly interspaced short palindromic repeats-derived ribonucleicacid) for specific targeting detection on the EB virus, and a method for DNA detection on the EB virus of a patient suffering from nasopharynx cancer. By adopting the method disclosed by the invention, DNA of the EB virus can be detected without complex instruments such as a real-time fluorescence quantitative PCR (polymerase chain reaction) (qPCR) instrument, obtained results are generally accordant with detection results of a qPCR method, the clinical plasma sample detection sensitivity is 96%, and the specificity is 100%.

Description

technical field [0001] The present invention relates to the field of tumor diagnosis, in particular to molecular detection related to tumor diagnosis, more specifically to a specific detection primer for Epstein-Barr virus DNA and a specific crRNA targeting Epstein-Barr virus, and using the primer and crRNA to detect nasal Methods for quantitative detection of Epstein-Barr virus DNA in patients with pharyngeal carcinoma. Background technique [0002] Nasopharyngeal carcinoma (NPC) is a malignant tumor that occurs in the epithelial cells of the top and side walls of the nasopharyngeal cavity. Nasopharyngeal carcinoma is a common malignant tumor in southern China, and its incidence rate is as high as 20 / 100,000, so it is also called Guangdong cancer, ranking among the top ten in the incidence of all malignant tumors. [0003] Clinical studies have shown that plasma Epstein-Barr virus DNA levels are an independent biomarker for nasopharyngeal carcinoma. For the first time, Mu...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/705C12Q1/6851C12Q2521/507C12Q2522/101C12Q2531/119Y02A50/30
Inventor 曾木圣吴业涛刘尚鑫王芳
Owner SUN YAT SEN UNIV
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