Primer for doubly detecting hepatitis e virus and hepatitis a virus through RT-RPA-lateral flow tomography, probe and kit

A technology of RT-RPA-, hepatitis E virus, applied in the field of biological nucleic acid molecular detection

Active Publication Date: 2018-11-20
JINZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports on the application of RPA

Method used

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  • Primer for doubly detecting hepatitis e virus and hepatitis a virus through RT-RPA-lateral flow tomography, probe and kit
  • Primer for doubly detecting hepatitis e virus and hepatitis a virus through RT-RPA-lateral flow tomography, probe and kit
  • Primer for doubly detecting hepatitis e virus and hepatitis a virus through RT-RPA-lateral flow tomography, probe and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Design and screening of embodiment 1 primers and probes

[0059] In order to achieve the above object, the present invention designs primers and probes for HEV ORF2 and HAV VP1. After comparing HEV ORF2 from DQ279091, AJ272108, AF060668, AF060669, D11092, M73218, GU206559, GU119961 in GenBank and KX088647, KX343015, KX343016, KX343P017, KX343V3 V018, the homologous comparison analysis of the two gene sequences was determined Conserve the core region, and design multiple sets of primers and probes for this region, perform RAP screening, and use deionized water as a template to amplify the designed primer pair and probe as a negative control, and finally select a pair of amplification products Can display clear detection bands and control bands in the colloidal carbon test card (such as image 3 ), and finally determine the following set of primer pairs and probe combinations as the universal gene detection target for detection of HEV and HAV.

[0060]

[0061] Note: ...

Embodiment 2

[0063] Embodiment 2: the establishment and inspection of the RT-RPA amplification detection method of HEV and HAV

[0064] 1. Establishment of RT-RPA amplification detection method for HEV and HAV

[0065] 1.1 Extraction of HEV and HAV viral RNA:

[0066] Take 200 μL of patient serum or saliva, and use the viral RNA magnetic bead method extraction kit to extract RNA. After washing three times, 100 mg of magnetic beads are obtained, and finally store in air-dried at room temperature for future use.

[0067] 1.2 RT-RPA amplification reaction

[0068] In this embodiment, the RT-PRA method is used to amplify HEV and HAV specific sequences, and the specific steps are:

[0069] (1) Add 20 μL of FastQant reverse transcription reaction solution to the spare magnetic bead tube obtained above and mix well, transfer to a 0.2ml reaction tube, put it in a constant temperature metal bath at 42°C, and treat for 10 minutes to obtain a cDNA template;

[0070] (2) Add each 2.1 μL (10 μmol / L) o...

Embodiment 3

[0088] Embodiment 3: clinical sample HEV and HAV detection

[0089] 1. Lateral flow chromatography RT-RPA detection of clinical samples

[0090] Take 3 clinical human serum samples that have been identified as HEV and HAV double-positive by fluorescent quantitative RT-PCR established in our laboratory, 7 single-positive samples, 4 double-negative samples, a total of 21 samples, according to the implementation The RT-RPA detection method described in steps 2.1 and 2.2 in Example 2 was used for RNA extraction and amplification. After RT-RPA amplification, the lateral flow chromatography test strips showed control bands and detection bands No. 1 and No. 2 in the reaction area of ​​the test strips of 3 samples, and control bands appeared in the reaction area of ​​the test strips of 7 samples And the No. 1 test strip, the control band appeared in the test strip reaction area of ​​7 samples, and the No. 2 test strip, and only the control band appeared in the test strip reaction are...

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Abstract

The invention discloses a RT-RPA-lateral flow tomography kit for doubly detecting hepatitis e virus and hepatitis a virus. The kit comprises an upstream primer, an intermediate probe and a downstreamprimer which are applicable to hepatitis e virus ORF2 gene sequence in the RT-RAP amplification technology, and/ or an upstream primer, an intermediate probe and a downstream primer which are applicable to hepatitis a virus VP1 gene sequence, general agents for recombinase polymerase amplification technology, and a lateral flow tomography test strip, wherein the lateral flow tomography test stripcomprises a sample adding pad, a control line, a #1 detecting line and/ or a #2 detecting line; the control line, the detecting lines and the primers are combined with a probe label. With the adoptionof the kit, the hepatitis e virus ORF2 gene and/ or hepatitis a virus VP1 gene in a sample can be rapidly, sensitively and specifically detected on site in a non-lab environment.

Description

technical field [0001] The invention belongs to the technical field of biological nucleic acid molecular detection, and in particular relates to a detection method using a reverse transcription recombinase-dependent amplification technology (Reverse Transcript Recombinase Ploymerase Amplification, RT-RPA) combined with a flow chromatography test strip detection method A method and a kit for realizing double detection of hepatitis E virus and hepatitis A virus. Background technique [0002] my country is a country with a large burden of liver disease. Hepatitis A and Hepatitis E are two important diseases that are transmitted through the digestive route with the highest incidence except hepatitis B. According to clinical and epidemiological observations, hepatitis A virus (HAV) mostly invades children and young people. The incidence rate decreases with age. The clinical manifestations usually start with fever, fatigue and loss of appetite, followed by hepatomegaly, tenderne...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844C12Q1/70C12N15/11
CPCC12Q1/6844C12Q1/706C12Q1/707C12Q2521/507C12Q2522/101C12Q2521/107C12Q2565/625Y02A50/30
Inventor 高慎阳李丹丹查恩辉张体银周铁忠岳喜庆
Owner JINZHOU MEDICAL UNIV
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