Loop-mediated isothermal amplification based human respiratory syncytial virus detection kit

A loop-mediated isothermal, syncytial virus technology, applied in the fields of molecular biology and nucleic acid detection, can solve problems such as difficult color change result determination

Inactive Publication Date: 2015-03-18
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Given that it is difficult to find suitable, broad-spectrum amplification primers, and it is difficult to judge results based on color changes, there is currently no RT-LAMP detection product for RSV at home and abroad.

Method used

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  • Loop-mediated isothermal amplification based human respiratory syncytial virus detection kit
  • Loop-mediated isothermal amplification based human respiratory syncytial virus detection kit
  • Loop-mediated isothermal amplification based human respiratory syncytial virus detection kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Embodiment 1, primer design

[0099] The whole genome sequence of 50 strains of HRSV (obtained from GenBank) was obtained, and multiple sequence alignment and sequence analysis were performed to find the conserved region. It was found that the difference between type A RSV and type B RSV was too large, and it was difficult to design general detection primers.

[0100] Further, for 50 strains of viruses, RSV type A and type B were separated and then compared, and it was found that type A was in the 1813-2030bp segment of the genome of the comparison result (ie: the 610-930th position of the N gene coding region of subtype A ), type B is highly conserved in the 12000-12600bp segment of the genome (namely: 2452-3052 in the coding region of the L gene of subtype B), which is suitable as a primer design region. For example, the 610-930th sequence of the coding region of the A subtype N gene includes any sequence of SEQ ID NO: 13-51; the 2452-3052nd sequence of the B subtype ...

Embodiment 2

[0115] Embodiment 2, system establishment and optimization

[0116] Taking the RT-LAMP system reported in the literature as the starting system (Ushio, M. and I. Yui, et al. (2005). J Med Virol77 (1): 121-7), the components are as follows:

[0117]

[0118] Three levels of memory orthogonal experiments were set up for the four factors in the above system.

[0119] Table 4. Factor level table

[0120]

[0121] Table 5. Table of experimental conditions

[0122]

[0123] RSV strains VR-1400 (type B) and VR-26 (type A) were used as templates to react with the designed primers and the combination of conditions listed in the experimental condition table, and the reaction process was monitored in real time with calcein.

[0124] The fluorescence quantitative detection results of the orthogonal experiment are as follows: figure 1 , the curve is the emission fluorescence of the reaction system in the range of 565-590nm over time, and the reaction curves 1-9 correspond to th...

Embodiment 3

[0131] Embodiment 3, visual and real-time monitoring of reaction process

[0132] Extract the RNA of RSV standard strains (A: VR-26, VR-1540; B: VR-1400, VR-955, VR-1580), use the mixed solution of RSV-A primer combination RSV-B primer set and so The extracted RNA was used as a template, and the following reaction system was used to react on a PCR instrument and a fluorescent quantitative PCR instrument to determine the effectiveness of the primer set.

[0133] Visual detection reaction system:

[0134]

[0135]

[0136] The reaction process is as follows:

[0137] Visual inspection was performed at 60°C for 60 minutes. The order of adding samples is shown in Table 7.

[0138] Table 7. Sampling order

[0139] 1

2

3

4

5

6

-

VR-26

VR-1540

VR-1580

VR-955

VR-1400

[0140] Fluorescence quantitative detection is:

[0141]

[0142] Fluorescence quantitative detection results such as figure 2 and Table 7. ...

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Abstract

The invention relates to a loop-mediated isothermal amplification based human respiratory syncytial virus detection kit. The inventor finds a loop-mediated isothermal amplification primer having excellent specificity to RSV after comparison and screening; the primer is prevented from specific amplification for nucleic acids other than nucleic acids containing the RSV. The loop-mediated isothermal amplification based human RSV detection kit can be well applied to identifying the ingredients of the RSV, and has excellent reproducibility and sensitivity.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and nucleic acid detection. Specifically, the present invention relates to a detection kit for human respiratory syncytial virus based on loop-mediated isothermal amplification. Background technique [0002] Respiratory syncytial virus (RSV) was first isolated from chimpanzees in 1955. And in 1955, a virus strain with the same antigenic characteristics as the chimpanzee strain was isolated from infants. In subsequent studies, it was found that the virus uses humans as its natural host and is an important pathogen that causes respiratory infections in humans. When the virus infects cells in vitro, it causes adjacent cells to form syncytia, resulting in blocky cytopathies, hence the name. RSV is a non-segmented negative-sense single-stranded RNA virus, belonging to Paramyxoviridae, Pneumoviridae, and Pneumovirus genus; it belongs to the same family as Parainfluenza virus, Measles virus,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/45C12Q1/70C12Q1/68C12N15/11
Inventor 蓝柯穆永林陈倩倩张驰宇
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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