RPA method for detecting 21 type human adenovirus, special primer, probe and application thereof

A human adenovirus and probe technology, used in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of long detection time, reliance on expensive instruments, and inability to apply on-site detection.

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

At present, PCR and q-PCR are the main identification methods used in clinical practice, but both have shortcomings such as long detection time and dependence on expensive instruments, so they cannot be applied to on-site detection

Method used

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  • RPA method for detecting 21 type human adenovirus, special primer, probe and application thereof
  • RPA method for detecting 21 type human adenovirus, special primer, probe and application thereof
  • RPA method for detecting 21 type human adenovirus, special primer, probe and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Design and screening of detection primers and probes for type 21 human adenovirus RPA

[0042] (1) Design of primers and probes

[0043] The inventor analyzed and determined through literature search that the specific sequence in the hexon gene of type 21 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 principles of RPA primer and probe design, three pairs of primers and one probe were designed, as shown in Table 1.

[0044] Table 1 Primer and probe sequences

[0045]

[0046] (2) Primer screening

[0047] A positive plasmid containing the sequence shown in SEQ ID NO.1 of the 21-type human adenovirus hexon gene was ...

Embodiment 2

[0056] Example 2: Optimization of 21-type human adenovirus RPA reaction system, amplification and detection conditions

[0057] 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

[0058] (1) Primer probe concentration

[0059] 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 fals...

Embodiment 3

[0068] Example 3: Evaluation of the detection limit of type 21 human adenovirus RPA detection

[0069] Dilute the 21-type human adenovirus 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.

[0070] see results Figure 4 , from 10 2 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 2 copy / react.

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Abstract

The invention provides a RPA detection method for detecting 21 type human adenovirus, a special primer, a probe and an application thereof in detecting 21 type human adenovirus. The detection method,the special primer and the probe thereof are designed on the basis of 21 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 21 type human adenovirus. According to the method, an enzyme reaction processof in 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 102 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 discloses an RPA method for detecting type 21 human adenovirus, its special primers and probes and its use, which belong to the field of biotechnology, relate to the molecular biology of type 21 human adenovirus (human adenovirus, HAdV), and relate to a method for detecting type 21 The method and application thereof for human adenovirus, in particular to a method for rapidly detecting type 21 human adenovirus using recombinase polymerase constant temperature amplification (Recombinase Polymerase Amplification, RPA) technology, its special primers and probes and its application. Background technique [0002] In recent years, there have been occasional outbreaks of HAdV21 infection in densely populated units such as troops and schools. HAdV21 is a highly virulent HAdV, and its main symptom is febrile respiratory infection, which can cause severe pneumonia and even death in severe cases. At present, PCR and q-PCR are the main identification ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2521/507C12Q2522/101
Inventor 齐永李越希李晓玲李佳萌饶继先沈万鹏曾雯雯刘苏云郑书龙郦钰超
Owner 李越希
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