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PCR (Polymerase Chain Reaction) detection kit and detection method for SRSF5 binding protein

A detection kit and protein-binding technology, which are used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., to achieve broad application prospects, accurate detection results, and not easy to mutate.

Pending Publication Date: 2022-04-12
成都纳比微特检测技术服务有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] At present, there is no real-time fluorescent quantitative PCR kit and detection method that can detect SRSF5 binding protein in host cells, and it is still in urgent need of development

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Primer Design and Synthesis

[0039] Using Primer premier 5.0 design software, primers (Gene ID: 6430) (Table 1) were designed according to the gene sequence of SRSF5 on GenBank, and the primers were synthesized by Suzhou Gemma Company. Wherein, the nucleotide sequence of the SRSF5 gene is shown in SEQ ID NO:3.

[0040] Examples The sequences of the primers designed are shown in Table 1. The present invention selects the primers in Table 1 mainly due to the failure to form a stable dimer or hairpin structure between the two primers, the internal stability of the double strand formed between the primer and the template is high, and it is not at the non-target site of the template. To initiate DNA polymerization, the GC content of primers and products is relatively high.

[0041] Table 1 Primer sequence information

[0042] name sequence (5'-3') SRSF5-F GGTGGTTGAGTTTGCCTCTTATG SRSF5-R GAGAACACCAGTCTCCAGTCGC

Embodiment 2

[0043] Embodiment 2 prepares the real-time fluorescent quantitative PCR detection kit of RNA binding protein SRSF5 of the present invention

[0044] A 20 μL reaction system was used to carry out related experiments with the obtained target gene as a template. The total volume of the reaction system for fluorescent quantitative PCR is 20 μL, including Green Realtime PCR Master Mix 10μL, upstream and downstream primers (10pmol / μL) each 0.8μL, template RNA 2μL, sterile water added to 20μL.

Embodiment 3

[0045] Embodiment 3 adopts detection kit of the present invention to carry out real-time fluorescent quantitative PCR detection

[0046] (1) Collect nucleic acid samples to be tested

[0047] a. Blood: Pipette 200 μL of blood into a centrifuge tube.

[0048] b. Tissue: Take an appropriate amount of tissue, grind it with liquid nitrogen, add an appropriate amount of normal saline, grind it evenly (in the form of tissue homogenate), centrifuge at 6000r / min for 3min, and take 200μL of the supernatant into a clean 1.5mL EP tube.

[0049] Add 500 μL RTL lysis buffer to each tube of the processed sample, place it on ice for 10 min and add 500 μL RNA binding buffer. Transfer the liquid to the upper layer of the column of the RNase-free collection tube, centrifuge at 12000rpm for 30s; discard the filtrate, add 500μL RW1 buffer to the upper layer, and centrifuge at 12000rpm for 30s; discard the filtrate, add 500μL RW2 buffer, centrifuge at 12000rpm for 30s, discard Repeat the washing...

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Abstract

The invention belongs to the technical field of influenza virus detection, and particularly relates to a PCR (Polymerase Chain Reaction) detection kit and a detection method for a key RNA (Ribonucleic Acid) binding protein SRSF5 interacting with virus RNA in an influenza virus life cycle. The invention provides a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for a key RNA (Ribonucleic Acid) binding protein SRSF5 in a life cycle of an influenza virus, which is used for detecting the influenza virus by using an existing real-time fluorescent quantitative PCR technology, the influenza virus is generally detected, related detection on an infected host is less, and a PCR technology for detecting the SRSF5 binding protein in a host cell does not exist yet, so that the invention provides the real-time fluorescent quantitative PCR detection kit for the key RNA binding protein SRSF5 in the life cycle of the influenza virus. The influenza infection condition in a body is judged by relatively quantitatively detecting SRSF5 binding protein in a host closely related to influenza virus. The detection kit disclosed by the invention is good in accuracy and sensitivity, is not easily influenced by influenza virus mutation, and is suitable for popularization and application.

Description

technical field [0001] The invention belongs to the technical field of influenza virus detection, and specifically relates to a PCR detection kit and a detection method for the key RNA binding protein SRSF5 interacting with viral RNA in the life cycle of influenza virus. Background technique [0002] Influenza virus is a highly contagious zoonotic pathogen with high morbidity and mortality worldwide, posing a serious threat to global public health security. At present, the only way to control seasonal influenza outbreaks around the world is vaccines, but due to the frequent mutation of influenza virus strains, the prediction of antigenic epitopes and the production of vaccines are relatively lagging behind, which affects the effective use of vaccines. After the host is infected with the virus, its innate immune system is activated to limit virus replication and eliminate pathogens, and then host acquired immunity participates in specific virus clearance. In addition, viruse...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
Inventor 余树芳袁旦一陈斌何谦辜良斌李晓琪张丹
Owner 成都纳比微特检测技术服务有限公司
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