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RPA method for detecting 3 type human adenovirus, special primer, probe and application thereof

A technology of human adenovirus and probe, applied in the direction of microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as lack of understanding, poor awareness of protection, and increased morbidity

Inactive Publication Date: 2019-03-22
李越希
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The diseases caused by human adenovirus type 3 (HAdV3) mainly include acute respiratory diseases, infantile diarrhea, and pharyngeal conjunctival fever. Poor, it will lead to a rapid increase in the incidence of the disease in a short period of time

Method used

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  • RPA method for detecting 3 type human adenovirus, special primer, probe and application thereof
  • RPA method for detecting 3 type human adenovirus, special primer, probe and application thereof
  • RPA method for detecting 3 type human adenovirus, special primer, probe and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Design and screening of detection primers and probes for type 3 human adenovirus RPA

[0041] (1) Design of primers and probes

[0042] The inventor analyzed and determined through literature search that the specific sequence in the hexon gene of type 3 human adenovirus used in the present invention is the target gene. The known template gene sequence was obtained from the NCBI database, i.e. the nucleotide sequence shown in SEQ ID NO.1, and the above sequence was synthesized by Nanjing KingScript Biotechnology Co., Ltd. It can be used as a template in the process of needle screening, optimization of reaction system, etc. According to the principles of RPA primer and probe design, three pairs of primers and one probe were designed, as shown in Table 1.

[0043] Table 1 Primer and probe sequences

[0044]

[0045] (2) Primer screening

[0046] A positive plasmid containing the sequence shown in SEQ ID NO.1 of the type 3 human adenovirus hexon gene was a...

Embodiment 2

[0055] Example 2: Optimization of type 3 human adenovirus RPA reaction system, amplification and detection conditions

[0056] In the process of primer screening, there are still false positives in the detection of lateral flow nucleic acid detection test strips, so the RPA reaction system, amplification and detection conditions need to be optimized

[0057] (1) Primer probe concentration

[0058] Set the concentration gradient of the reverse primer as 10 μmol / L, 5 μmol / L, and 2.5 μmol / L, and the concentration of the probe as 10 μmol / L, 5 μmol / L, and 2.5 μmol / L. The primers were combined with the probes of three concentrations to form 9 combinations, and a set of negative controls was set for each combination. RPA amplification was carried out at 37°C, and the color development of the detection line of the lateral flow nucleic acid detection strip was used as an indicator for the completion of the amplification. The combination with the best amplification effect and no false...

Embodiment 3

[0067] Example 3: Evaluation of detection limit for type 3 human adenovirus RPA detection

[0068] Dilute the type 3 human adenovirus positive plasmid into 10 times 10 - 10 0 A series of different concentrations such as copies / μL, each taking 10 10 、10 9 、10 8 、10 4 、10 3 、10 2 、10 1 Copy / μL of positive plasmid 1 μL was added to the reaction system determined in Example 2, respectively, using the primer combination screened out, using the reaction conditions determined in Example 2 to perform RPA detection on the above templates with different copy numbers, and observe the detection of RPA detection limit.

[0069] see results Figure 4 , from 10 1 Copy / μL begins above sample all to be positive, and negative control is negative, illustrates that the detection limit of RPA detection method of the present invention is 10 1 copy / react.

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Abstract

The invention provides a RPA detection method for detecting 3 type human adenovirus, a special primer, a probe and an application thereof in detecting 3 type human adenovirus. The detection method, the special primer and the probe thereof are designed on the basis of 3 type human adenovirus hexon gene conserved sequence, with oligonucleotide sequences shown as SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4. According to the invention, a novel isothermal amplification technology RPA is firstly applied to the detection of 3 type human adenovirus. According to the method, an enzyme reaction process ofin vivo DNA replication is simulated; amplification of DNA template relies on a specific combination of enzyme and protein (recombinase, single-stranded binding protein and DNA polymerase); amplification of specific nucleotide sequence is realized under a constant temperature at 37 DEG C; amplified products can be visually identified through a lateral chromatography test strip. The detection method established by the invention has the advantages of high sensitivity, limit of detection reaching up to 101 copies / reaction, high specificity, no cross reaction with other pathogens, high detection speed, only 20min reaction time, simple and convenient operation, and suitability for onsite detection.

Description

technical field [0001] The invention relates to an RPA method for detecting type 3 human adenovirus, its special primers and probes, and uses thereof, which belong to the field of biotechnology, relate to the molecular biology of type 3 human adenovirus (human adenovirus, HAdV), and relate to a method for detecting type 3 The method and application thereof for human adenovirus, in particular to a method for rapidly detecting type 3 human adenovirus by using Recombinase Polymerase Amplification (RPA) technology, its special primers and probes and its application. Background technique [0002] The diseases caused by human adenovirus type 3 (HAdV3) mainly include acute respiratory diseases, infantile diarrhea, and pharyngeal conjunctival fever. Poor, the incidence of the disease will increase rapidly in a short period of time. Early detection of outbreaks is of great significance for disease prevention and control. [0003] In recent years, constant temperature nucleic acid a...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/701
Inventor 李越希齐永李晓玲李佳萌饶继先沈万鹏王长军曾雯雯刘苏云
Owner 李越希
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