RPA (recombinase polymerase amplification) method, PRA special primer and RPA probe for detecting universal human adenovirus, and application of RPA probe

A human adenovirus, general-purpose technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of non-specific clinical manifestations of infection, unsuitable for rapid diagnosis, mass infection, etc. , to shorten the detection time, save the detection time, and achieve the effect of strong specificity

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

Human adenovirus can cause regional epidemics or outbreaks, but because the clinical manifestations of its infection are usually non-specific, there is currently no specific treatment. Once it develops into severe pneumonia, the fatality rate is high, especially for types 3 and 7. Types, 11, 21, and 55 are highly contagious, and in places where people gather, such as schools and the mil

Method used

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  • RPA (recombinase polymerase amplification) method, PRA special primer and RPA probe for detecting universal human adenovirus, and application of RPA probe
  • RPA (recombinase polymerase amplification) method, PRA special primer and RPA probe for detecting universal human adenovirus, and application of RPA probe
  • RPA (recombinase polymerase amplification) method, PRA special primer and RPA probe for detecting universal human adenovirus, and application of RPA probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Design and screening of universal human adenovirus RPA detection primers and probes

[0044] (1) Design of primers and probes

[0045] The inventor analyzed and determined through literature search that the specific sequence in the hexon gene of the universal 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 and optimization of reaction system. According to the design principles of RPA primers and probes, two sets of primers and probes were designed, as shown in Table 1.

[0046] Table 1 Primer and probe sequences

[0047]

[0048] (2) Primer screening

[0049] Artificially synthesize a positive plasmid containing the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 of the...

Embodiment 2

[0058] Example 2: Optimization of Universal Human Adenovirus RPA Reaction System, Amplification and Detection Conditions

[0059] 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

[0060] (1) Primer probe concentration

[0061] 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 fa...

Embodiment 3

[0070] Example 3: Evaluation of the detection limit of universal human adenovirus RPA detection

[0071] Dilute the 3 / 7 type positive plasmid and the 11 / 21 / 55 type positive plasmid into 10-fold ratio 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.

[0072] see results Figure 4 , from 10 1 Copy / μL starts above two kinds of positive plasmids 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 an RPA (recombinase polymerase amplification) method, an PRA special primer and an RPA probe for detecting universal human adenovirus, and application of the PRA special primerand RPA probe in detection of the universal human adenovirus. The detection method, the special primer and the probe are designed based on a common conserved sequence of type 3, type 7, type 11, type21 and type 55 human adenovirus hexon genes, and have the oligonucleotide sequences shown as SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5. According to the method, the novel isothermal amplification technology is applied to detection of the universal human adenovirus for the first time; the enzymatic reaction process of in-vivo DNA replication is simulated, a specific enzyme and protein combination(recombinase, single-stranded binding protein and DNA polymerase) is used for DNA template amplification, amplification of a specific nucleotide sequence can be realized at a constant temperature of37 DEG C, and amplification products can be visually judged through a lateral chromatographic test strip. The detection method has high sensitivity, and the detection limit can reach 101 copies/reaction; the specificity is high, and no cross-reaction with other pathogens exists; the detection speed is high, and the method is simple and convenient to operate.

Description

technical field [0001] The invention relates to an RPA method for detecting a general-purpose human adenovirus, its special primers and probes, and uses thereof, which belong to the field of biotechnology, relate to the molecular biology of a general-purpose human adenovirus (human adenovirus, HAdV), and relate to a detection The method and its use of universal human adenovirus, especially a method for rapid detection of universal human adenovirus using recombinase polymerase constant temperature amplification (Recombinase Polymerase Amplification, RPA), its special primers and probes and its use . Background technique [0002] There are currently 57 serotypes of adenoviruses that are pathogenic to humans, which can be divided into 7 subgroups, A-G. This virus usually causes mild self-limiting respiratory diseases in healthy people, and in most cases it can be diagnosed clinically. Diagnosed, but sometimes adenoviruses can cause acute respiratory infections in humans, espec...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2563/107C12Q2565/625
Inventor 李越希李晓玲齐永李佳萌饶继先沈万鹏曾雯雯刘苏云郦钰超郑书龙
Owner 李越希
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