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Primer for specifically amplifying RNA as well as design method and application of primer

A specificity and primer length technology, applied in the field of molecular biology, can solve problems such as difficulty in ensuring RNA integrity and affecting the accuracy of RNA detection

Pending Publication Date: 2021-09-28
NINGBO HEALTH GENE TECHNOLOGIES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the DNA digestion process requires a certain amount of time (10-20 minutes) and temperature (37°C), even in a solution without ribonucleases, it is difficult to ensure the integrity of the RNA during the DNA digestion process, which will also occur in a certain amount of time. Affect the accuracy of RNA detection to a certain extent

Method used

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  • Primer for specifically amplifying RNA as well as design method and application of primer
  • Primer for specifically amplifying RNA as well as design method and application of primer
  • Primer for specifically amplifying RNA as well as design method and application of primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1: utilize designed primer to carry out one-step RT-PCR, distinguish human papillomavirus (HPV) type 16 DNA and RNA template

[0067] Persistent infection with high-risk human papillomavirus (HPV) type 16 is an important cause of cervical cancer. The E6 / E7 gene of HPV type 16 will be integrated into the human genome, resulting in a large amount of expression of E6 / E7 protein, promoting cell transformation, and finally inducing cancer. Therefore, the detection of E6 / E7 gene mRNA can better evaluate the severity of HPV infection in patients.

[0068] In this example, DNA and RNA were extracted from cervical exfoliated cells, and one-step RT-PCR was carried out under the same conditions, and finally the samples were separated by capillary electrophoresis to test the ability of the primers of the present invention to distinguish DNA and RNA templates. The specific steps as follows:

[0069] (1) According to Figure 4 Design the reverse transcription (reverse) ...

Embodiment 2

[0078] Embodiment 2: utilize designed primer to carry out one-step RT-PCR, distinguish human papillomavirus (HPV) type 18 DNA and RNA template

[0079] In this example, DNA and RNA were extracted from cervical exfoliated cells, and one-step RT-PCR was carried out under the same conditions, and finally the samples were separated by capillary electrophoresis to test the ability of the primers of the present invention to distinguish DNA and RNA templates. The specific steps as follows:

[0080] (1) According to figure 2 Design the reverse transcription (reverse) primer of HPV 18 type E6 / E7 gene according to the scheme in, and design the forward primer according to the conventional primer design scheme, and submit it to Shanghai Sangon Biological Co., Ltd. for primer synthesis, wherein the forward primer 5 ' end labeled FAM group. The specific forward primer sequence is 5'-TATGCTGCAACCGAGCACGA-3' (SEQ ID NO: 3), the reverse transcription (reverse) primer sequence is 5'-gatggacg...

Embodiment 3

[0089] Example 3: Using designed primers to carry out one-step RT-PCR to distinguish Enterococcus faecalis (Enterococcusfaecalis) cytolysin-encoding gene DNA and RNA templates

[0090] Alcoholic hepatitis is a serious life-threatening liver disease caused by excessive alcohol use. The latest research shows that the number of Enterococcus faecalis in the stool of patients with alcoholic hepatitis is much greater than that of patients with non-alcoholic hepatitis, and the cytolytic cells secreted by Enterococcus faecalis Lysin is positively correlated with the severity and mortality of alcoholic hepatitis. If the expression of Enterococcus faecalis cytolysin-encoding gene can be directly detected in stool samples, the severity of alcoholic hepatitis in patients can be better characterized.

[0091] In this example, DNA and RNA were extracted from feces samples, and one-step RT-PCR was carried out under the same conditions, and finally the samples were separated by capillary elect...

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PUM

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Abstract

The invention provides a reverse transcription primer. The reverse transcription primer is composed of an RNA template complementary part and an artificially introduced mismatched part. In a specific embodiment, the sequence of the reverse transcription primer is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, and / or SEQ ID NO: 14. The invention also provides a method for designing the reverse transcription primer. By using the primer in RT-PCR, RNA can be specifically amplified, and DNA of the same sequence cannot be amplified.

Description

technical field [0001] The present invention relates to the field of molecular biology. Specifically, the present invention relates to a reverse transcription primer and a design method thereof. By using such primers in RT-PCR, RNA can be specifically amplified without amplifying DNA of the same sequence. Background technique [0002] In the process of transcription, a strand of DNA is used as a template and a single strand of RNA is formed based on the principle of complementary base pairing. Its main function is to realize the expression of genetic information at the protein level, which is an important step in the process of transforming genetic information into phenotype. Bridge, so RNA detection is of great significance for gene expression and functional analysis. However, due to the formation mechanism of RNA, the sequence of the transcribed RNA must be completely identical (prokaryotes) or partially identical (eukaryotes) to a certain strand of DNA. Therefore, in n...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6806
CPCC12Q1/6806C12Q2521/107C12Q2531/113
Inventor 吴勇罗勇曾县平张迪骏张成
Owner NINGBO HEALTH GENE TECHNOLOGIES CO LTD
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