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Primers, probe, kit and detection method for human adenovirus quadruple real-time fluorescent PCR (Polymerase Chain Reaction) detection.

A detection kit and detection primer technology, applied in the field of human adenovirus detection, can solve the problems of not being able to meet the needs of HAdV detection work, unable to type, distinguish, etc., to achieve rapid detection and accurate detection, high sensitivity and good specificity Effect

Pending Publication Date: 2021-06-29
江西省疾病预防控制中心
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The real-time fluorescent PCR method is widely used in the early diagnosis of viral infectious diseases, but the current real-time fluorescent PCR method of HAdV is mostly aimed at the general type, which cannot be distinguished, and cannot meet the needs of HAdV detection.

Method used

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  • Primers, probe, kit and detection method for human adenovirus quadruple real-time fluorescent PCR (Polymerase Chain Reaction) detection.
  • Primers, probe, kit and detection method for human adenovirus quadruple real-time fluorescent PCR (Polymerase Chain Reaction) detection.
  • Primers, probe, kit and detection method for human adenovirus quadruple real-time fluorescent PCR (Polymerase Chain Reaction) detection.

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1: Human adenovirus B3 type, B7 type, B55 type and E4 type quadruple real-time fluorescent PCR detection method and use

[0064] (1) Detection primers and detection probes

[0065] The solid-phase phosphoramidite triester method is adopted to synthesize detection primers and detection probes, and the nucleotide sequences of the detection primers and detection probes are as follows:

[0066] F1: 5'-cttacaattcactcgctc-3';

[0067] R1: 5'-ggcttttcttctccttca-3';

[0068] F2: 5'-ggcacagcttacaattca-3';

[0069] R2: 5'-tctcccgttgtaactatc-3';

[0070] F3: 5'-tacggcttacaactctct-3';

[0071] R3: 5'-ctcctcaccattttttacac-3';

[0072] F4: 5'-gggaaaactcattgcaaac-3';

[0073] R4: 5'-ggaaatggtgttgttgga-3';

[0074] P1: 5'-cagtaactaccaccacaaacaca-3';

[0075] P2: 5'-tgattgtgtgcgtctgaggt-3';

[0076] P3: 5'-cgcgcctaacacatctca-3';

[0077] P4: 5'-aaaactgagaggggcaatgg-3';

[0078] Among them, F1~F4, R1~R4 are detection primers, F1 and R1, F2 and R2, F3 and R3, F4 and ...

Embodiment 2

[0107] Embodiment 2: Sensitivity experiment

[0108] (1) Positive standard

[0109] The target fragment with a length of 774bp was cloned into the pGEM®-T vector of Promega Company to prepare the recombinant plasmid as a positive control.

[0110] (2) Experimental steps

[0111] The positive plasmids of human adenovirus types B3, B7, B55 and E4 were purified, the concentration was measured by a BioDrop ultra-micro protein nucleic acid analyzer UV spectrophotometer, the copy number was calculated according to the Avogadro constant, and the recombinant plasmid were diluted to 1.0×10 7 copies / μL, 1.0×10 6 copies / μL, 1.0×10 5 copies / μL, 1.0×10 4 copies / μL, 1.0×10 3 copies / μL, 1.0×10 2 copies / μL, 1.0×10 1 copies / μL, 1.0×10 0 copies / μL, 1.0×10 -1copies / μ L, the fluorescent PCR detection method described in Example 1 is detected to the positive standard substance through gradient dilution, the sensitivity of verification established method, the result is as follows...

Embodiment 3

[0114] Embodiment 3: specificity experiment

[0115] (1) Test method

[0116]positive nucleic acid of human adenovirus type 1, type 2, type 5, type 6, type 21, type 37 and type 64, influenza A virus, rhinovirus, bocavirus, metapneumovirus and enterovirus As a template, a negative control and a positive control of human adenovirus B3, B7, B55 and E4 recombinant plasmids are set at the same time, and the fluorescent PCR detection method described in Example 1 is used for detection to verify the specificity of the established method. Experimental results such as Figure 9 shown.

[0117] (2) Experimental results

[0118] Depend on Figure 9 It can be seen that the positive controls of the positive plasmids of human adenovirus types B3, B7, B55, and E4 showed amplification curves, and no amplification curves were seen in the other 12 viral nucleic acid samples and negative controls, indicating that the detection method has good specificity.

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Abstract

The invention relates to primers, a probe, a kit and a detection method for human adenovirus quadruple real-time fluorescent PCR (Polymerase Chain Reaction) detection, the virus type in a sample is judged according to an amplification curve by performing fluorescent PCR amplification reaction, the operation is simple, the detection of four types of human adenovirus B3 type, B7 type, B55 type and E4 type can be realized only by one reaction, and the time consumed by the whole operation process is 1.5-2 hours, so that the primers, the probe, the kit and the detection method for human adenovirus quadruple real-time fluorescent PCR detection can be used for accurate, rapid and high-throughput analysis, and is beneficial to popularization and application in clinical practice.

Description

technical field [0001] The present application relates to the technical field of human adenovirus detection, in particular to a quadruple real-time fluorescent PCR detection primer, probe, kit and detection method. Background technique [0002] Human adenovirus (Human adenovirus, HAdV) belongs to the mammalian adenovirus genus. It is a non-enveloped double-stranded DNA virus and can be divided into seven subgenuses, A-G. The tissue tropism of the virus is different, thus causing different tissue and organ diseases. Among them, the human adenoviruses related to respiratory diseases are mainly B, C and E subgenus adenoviruses, and the etiological monitoring data show that B subgenus HAdV-7 and HAdV-3 are the main human adenoviruses causing respiratory infectious diseases in my country Types, HAdV-1 and HAdV-2 in group C are not uncommon, especially in southern China; they are gradually and widely popular in my country. [0003] HAdV infection can cause a variety of diseases, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143C12Q2563/107
Inventor 龚甜李健雄肖芳
Owner 江西省疾病预防控制中心
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