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Circular RNA for diagnosing RSV infection and application thereof

A circular and versatile technology, applied in the field of circular RNA for the diagnosis of RSV infection, can solve the problems of unreported expression changes and achieve high sensitivity and specificity, good specificity, and high sensitivity

Inactive Publication Date: 2018-11-06
THE FIFTH AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the circular RNA produced by the human antiviral gene HERC5 has not been reported, nor has the expression change of the circular RNA caused by RSV virus infection been reported.

Method used

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  • Circular RNA for diagnosing RSV infection and application thereof
  • Circular RNA for diagnosing RSV infection and application thereof
  • Circular RNA for diagnosing RSV infection and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] This embodiment provides a kit for diagnosing RSV infection, the kit includes primers for detecting the expression level of circular RNA whose circBase ID is hsa_circ_0001426, and the primer sequences are such as SEQ ID NO.2 and SEQ ID NO.3 shown.

[0028] The primers are synthesized by conventional techniques in the art, prepared as a mother solution according to the instructions, and ddH can be used to 2 O was diluted to the working concentration.

[0029] The method for detecting the expression level of the circular RNA using the kit comprises the following steps:

[0030] (1) Total RNA extraction: total RNA was extracted by Trizol method.

[0031] (2) Reverse transcription: cDNA was synthesized using the reverse transcription kit PrimeScript RT reagent Kit with gDNA Eraser. The reaction system is 20ul (10ul of the reaction solution in step 1: 5×gDNA Eraser Buffer 2ul, gDNAEraser 1ul, RNA and water total 7ul; PrimeScript RT Enzyme Mix 1ul; RT Primer Mix 1ul; 5×Pri...

Embodiment 2

[0034] In this example, it is confirmed by high-throughput sequencing that the antiviral gene HERC5 produces circular RNA hsa_circ_0001426.

[0035] Methods: Total RNA was extracted from lung cancer cell A549 cells, ribosomal RNA was removed, and then RNase R enzyme was used to degrade the library to further construct the library. The constructed library was sequenced by RNA-Seq using Illumina HiSeq 2500 to analyze the circular RNA related to the antiviral gene HERC5.

[0036] Results: Through analysis, it was found that a circular RNA derived from the HERC5 gene was detected in A549 cells. Its sequence is shown in SEQ ID NO.1.

Embodiment 3

[0038] This example studies the features of hsa_circ_0001426.

[0039] (1) PCR amplification verification and sequencing verification of the reverse cleavage site of hsa_circ_0001426.

[0040] Method: According to the hsa_circ_0001426 sequence, a pair of bidirectional primers were designed to amplify the sequence containing the reverse cleavage site. The primer pair used included SEQ ID NO.2 and SEQ ID NO.3. Use the cDNA of the cell as a template for PCR amplification, and the reaction system is 20ul (including Takara LA Taq 0.2ul, 10×LA Taq Buffer 2ul, 25mM MgCl 2 2ul, dNTP Mixture 3.2ul, cDNA Template 1ul, forward and reverse primers 1.0ul each, ddH2O 9.6ul); the reaction program was: pre-denaturation at 94°C for 5min; denaturation at 98°C for 10s, annealing at 56°C for 30s, and extension at 72°C for 1min. 34 cycles; 72°C extension for 10 min. The PCR products were detected by 2.0% agarose gel electrophoresis, and sent to Guangzhou Aiji Biotechnology Co., Ltd. for sequenci...

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Abstract

The invention discloses a molecular marker for diagnosing RSV infection. The molecular marker is a circular RNA derived from an HERC5 gene, and circBase ID is hsa_circ_0001426. The invention also discloses application of the circular RNA in screening or preparing a reagent for diagnosing RSV infection and application of the reagent in preparing a kit for diagnosing RSV infection. The application finds that an expression quantity of the circular RNA in RSV-infected cells is significantly increased, the circular RNA can be used as a molecular indicator for detecting RSV infection, and has the advantages of simplicity, rapidity, high sensitivity, high specificity and the like.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a circular RNA for diagnosing RSV infection and its application. Background technique [0002] Respiratory syncytial virus (Respiratory Syncytial Virus, RSV) is one of the most important viral pathogens causing lower respiratory tract infections in infants, immunocompromised populations and the elderly. Young children die from RSV-associated lower respiratory tract infections at enormous medical cost. At present, there is no specific treatment plan for RSV disease except supportive treatment, and there is no RSV vaccine successfully developed to prevent the virus infection that can effectively reduce the incidence of the disease. [0003] With the rapid development of high-throughput sequencing technology, tens of thousands of non-coding transcripts have been discovered. Studies have shown that only 1% to 2% of human genome sequences have protein-coding...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2521/107
Inventor 姚文霞潘劲辉周新科
Owner THE FIFTH AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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