Method for detecting E6 or E7 nucleic acid of HPV16 or HPV18 virus based on RPA

A detection method and nucleic acid technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as physical trauma, uterine loss or bleeding, false negatives, etc., to achieve convenient use and detection. The effect of short time and mild detection conditions

Inactive Publication Date: 2019-04-16
郑筱玮
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0007] (1) The histopathological detection method in the prior art is sometimes difficult to accurately distinguish CIN from non-neoplastic lesions and different grades of CIN, and the treatment of different grades of CIN is different. In addition, histopathological detection requires biopsy , the puncture needle may cause uterine loss or bleeding during the puncture process, which will cause certain trauma to the body
[0008] (2) The repeatability of TCT in the prior art is not good, it will be affected by factors such as the environment and physician experience, and the consistency with biopsy is not high. In addition, the process of extracting cell samples is simple and the range is limited and it is easy to cause false negatives. The sensitivity of the test is only 40% to 60%, and some positive patients will be missed
[0009] (3) HPV nucleic acid amplification detection method in the prior art is a kind of detection method based on PCR, but needs complicated temperature control equipment, and can't directly detect the mRNA of HPV, and about 95% of high-risk HPV-DNA infected persons are not Can develop cervical cancer, the true carcinogenicity of the viral infection cannot be determined

Method used

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  • Method for detecting E6 or E7 nucleic acid of HPV16 or HPV18 virus based on RPA
  • Method for detecting E6 or E7 nucleic acid of HPV16 or HPV18 virus based on RPA
  • Method for detecting E6 or E7 nucleic acid of HPV16 or HPV18 virus based on RPA

Examples

Experimental program
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Effect test

Embodiment 1

[0075] This example is used to verify the RPA reaction amplification effect of the screened RPA primers directed against the E6 or E7 nucleic acid of HPV16 or HPV18 virus.

[0076] In this embodiment, a plasmid DNA containing E6 or E7 nucleic acid against HPV16 or HPV18 virus was selected as a template for detection. The specific sequence is as follows:

[0077] E6 of HPV16:

[0078]

[0079] E7 of HPV16:

[0080]

[0081] E6 of HPV18:

[0082]

[0083] E7 of HPV18:

[0084]

[0085] The sequence with a horizontal line is the screened RPA primer for E6 or E7 nucleic acid of HPV16 or HPV18 virus.

[0086] Select the corresponding primers to configure the RPA reaction system according to different target genes. RPA reaction system includes 2.4 μl pre-primer (10 μM), 2.4 μl post-primer (10 μM), 29.5 μl RPA buffer (from TwistDx company), 2 μl detection sample (containing 10 ng plasmid DNA), 11.2 μl double distilled water, 2.5 μl magnesium acetate (280mM); Once ...

Embodiment 2

[0095] This example is used to verify the specificity of the RPA primers screened against the E6 or E7 nucleic acid of HPV16 or HPV18 virus.

[0096] According to different target genes, select non-corresponding primers to configure the RPA reaction system. RPA reaction system includes 2.4 μl pre-primer (10 μM), 2.4 μl post-primer (10 μM), 29.5 μl RPA buffer (from TwistDx company), 2 μl detection sample (containing 10ng plasmid DNA or 1000ng human genomic DNA), 11.2 μl double distilled water , 2.5 μl of magnesium acetate (280 mM); once the magnesium acetate is added, the reaction starts timing.

[0097] The RPA reaction was carried out at 37°C for 30 minutes. After the reaction, it was observed by agarose gel electrophoresis. Such as image 3 As shown, the screened primers could not amplify non-corresponding HPV nucleic acid components and human genome, demonstrating the specificity of the method.

[0098] Such as image 3 As shown, the specificity verification of the RPA...

Embodiment 3

[0105] This embodiment is used for the sensitivity verification of the RPA primers screened against the E6 nucleic acid of HPV16.

[0106] The RPA reaction system includes 2.4 μl pre-primer (10 μM), 2.4 μl post-primer (10 μM), 29.5 μl RPA buffer (from TwistDx company), 2 μl detection sample (containing plasmid DNA with different copy numbers), 11.2 μl double-distilled water, 2.5 [mu]l magnesium acetate (280 mM); once the magnesium acetate was added, the reaction was timed. Both primers and templates are directed against E6 nucleic acid of HPV16.

[0107] The RPA reaction was carried out at 37°C for 30 minutes. After the reaction, it was observed by agarose gel electrophoresis. Such as Figure 4 As shown, the method can amplify plasmid DNA containing 10 copies of the target nucleic acid, demonstrating the high sensitivity of the method.

[0108] Such as Figure 4 As shown, the schematic diagram of the sensitivity verification of the screened RPA primers against the E6 nucl...

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Abstract

The invention belongs to the technical field of in vitro diagnosis, and discloses a method for detecting E6 or E7 nucleic acid of an HPV16 or HPV18 virus based on RPA. A primer for efficiently and specifically amplifying E6 or E7 nucleic acid of the HPV16 or HPV18 virus is designed and screened out; HPV16 or HPV18 virus is amplified through an RPA reaction, and DNA or mRNA is included. The specificity reaches 100%, and the sensitivity is 10 copies per reacton. The RPA isothermal amplification system is fast in reaction, no complex temperature control system is needed, effective amplification of target genes can be achieved at the temperature of 37 DEG C to 42 DEG C, and the method is suitable for field fast detection of E6 or E7 nucleic acid of the HPV16 or HPV18 virus. The RPA detecting method is utilized for detection, the detection condition is mild, the detection time is short, and use is more convenient.

Description

technical field [0001] The invention belongs to the technical field of in vitro diagnosis, and in particular relates to an RPA-based detection method for E6 or E7 nucleic acid of HPV16 or HPV18 virus. Background technique [0002] At present, the existing technologies commonly used in the industry are as follows: [0003] HPV virus is the abbreviation of human papillomavirus, which is a papillomavacuolating virus A genus of Papovaviridae, and is a sexually transmitted disease caused by spherical DNA virus infection. The main types are HPV1, 2, 6, 11, 16, 18, 31, 33 and 35, etc. Long-term infection of HPV16 and 18 may be related to female cervical cancer. [0004] HPV virus invades the basement membrane of the cervix, some of the infected cells spread laterally, and the other part migrates to the squamous epithelial cells, the virus integrates into the squamous epithelial cells, and then reproduces in large numbers, and the infected squamous epithelial cells gradually evolve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/708C12Q1/6844C12Q2521/507C12Q2522/101
Inventor 郑筱玮
Owner 郑筱玮
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