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LAMP (loop-mediated isothermal amplification) primer combination for detecting six respiratory viruses, and application thereof

A primer combination and primer set technology, applied in the biological field, can solve problems such as expensive, relying on large-scale instrument prices, unfavorable rapid detection, etc.

Active Publication Date: 2017-06-23
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, molecular diagnostic methods such as polymerase chain reaction (PCR) have been used for the detection of respiratory viruses, which can accurately identify the target sequence, but it requires elements of thermal cycling system, which usually rely on large instruments and are expensive, Not conducive to rapid detection

Method used

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  • LAMP (loop-mediated isothermal amplification) primer combination for detecting six respiratory viruses, and application thereof
  • LAMP (loop-mediated isothermal amplification) primer combination for detecting six respiratory viruses, and application thereof
  • LAMP (loop-mediated isothermal amplification) primer combination for detecting six respiratory viruses, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Embodiment 1, the preparation that is used to detect the kit of 6 kinds of respiratory tract viruses

[0103] 1. Preparation of primer sets

[0104] 1. The primer set (hereinafter referred to as primer set I) for detecting influenza A H1N1 virus is as follows: outer primer F3 (sequence 1 in the sequence listing): 5'-AAGCTCAGCAATCCTACA-3'; outer primer B3 (sequence in the sequence listing 2): 5'-TCCCTCACTTTGGGTCTT-3'; Internal primer FIP (sequence 3 in the sequence listing): 5'-GACTTTGTTGGTCAGCACTAGTAGAAAAGGGAAAGAAGTCCTCG-3'; Internal primer BIP (sequence 4 in the sequence listing): 5'-TCTATCAGAATGCAGATGCATATGTTGCTATTTCCGGCTTGAA-3'; Loop Primer LF (sequence 5 in the sequence listing): 5'-GATGGTGAATGCCCCATAGC-3'; loop primer LB (sequence 6 in the sequence listing): 5'-TTTTGTGGGGTCATCAAGATACAG-3'.

[0105] 2. The primer set (hereinafter referred to as primer set II) for detecting influenza A H3N2 virus is as follows: outer primer F3 (sequence 7 in the sequence listing): 5...

Embodiment 2

[0115] Embodiment 2, specificity

[0116] Sample 1 to be tested: Influenza A H1N1 virus (recorded in the following documents: Wang Dayan, Gao Rongbao, Li Xiaodan, etc. Establishment of Rapid Nucleic Acid Detection Technology for Influenza A H1N1 Virus [J]. Acta Virus, 2009, 25 (B05): 1-3.).

[0117] Sample 2 to be tested: Influenza A H3N2 virus (recorded in the following documents: Qin Meng, Wang Dayan, Huang Fang, etc. Single-tube multiplex fluorescent quantitative PCR method for simultaneous detection of new A H1N1 and human seasonal H1N1 and H3N2 influenza viruses [J]. Acta Virus, 2010(2):97-102.).

[0118] Test sample 3: H5N1 avian influenza virus (recorded in the following literature: Imai M, Ninomiya A, MinekawaH, et al. Rapid diagnosis of H5N1avian influenza virus infection by newly developed influenza H5hemagglutinin gene-specific loop-mediated isothermalamplification method[J]. Journal of virological methods, 2007, 141(2):173-180.).

[0119] Test sample 4: H7N9 avi...

Embodiment 3

[0145] Embodiment 3, sensitivity

[0146] 1. Sensitivity of detecting influenza A (H1N1) virus

[0147] A. Kit detection

[0148] The experiment was repeated three times, and the steps for each repetition were as follows:

[0149] 1. Fixed primers

[0150] (1) Take the microfluidic chip, add 2 μL of the primer group I mixed solution prepared in step 2 of Example 1 to each well of reaction well #1 to reaction well #7, and add 2 μL of RNase-free water to reaction well #8 .

[0151] (2) After completing step (1), the microfluidic chip is placed in a clean ultra-clean bench to dry, and sealed with a sealing layer to obtain a microfluidic chip with fixed primer set I.

[0152] 2. Obtaining total RNA of influenza A (H1N1) virus

[0153] Refer to the operation steps of the Magnetic Viral DNA / RNA Kit to extract the total RNA of influenza A (H1N1) virus and name it RNA1-1. The concentration of RNA in RNA1-1 was 5 ng / μL.

[0154] Pipette 1mL RNA1-1 into a test tube filled with 9m...

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Abstract

The invention discloses LAMP (loop-mediated isothermal amplification) primer combination for detecting six respiratory viruses, and the application thereof. The primer combination provided by the invention consists of 36 DNA molecules as shown in the sequence 1 to the sequence 36. The primer combination can be used for detecting whether a to-be-detected virus is influenza A virus subtype H1N1, influenza A virus subtype H3N2, avian influenza virus subtype H5N1, avian influenza virus subtype H7N9, influenza B virus or human adenovirus, and can be used for detecting whether a to-be-detected sample contains influenza A virus subtype H1N1 and / or influenza A virus subtype H3N2 and / or avian influenza virus subtype H5N1 and / or avian influenza virus subtype H7N9 and / or influenza B virus and / or human adenovirus. The primer combination provided by the invention is used for detecting six respiratory viruses, has high specificity and high sensitivity, and can realize simple, convenient, rapid and accurate detection. A great promotional value is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a combination of LAMP primers for detecting six kinds of respiratory viruses and an application thereof. Background technique [0002] Respiratory tract infection is divided into upper respiratory tract infection and lower respiratory tract infection. About 90% of upper respiratory tract infections are caused by viruses, and bacterial infections are often secondary to viral infections. The disease can come on in all seasons and at any age, and it can be transmitted by droplets containing virus, droplets, or contaminated utensils. Whether it is a lower respiratory tract infection or an upper respiratory tract infection, most of them are caused by viruses, which are called viral respiratory tract infections. [0003] Influenza viruses belong to the Orthomyxoviridae family and are divided into three types: influenza A virus (such as influenza A H1N1 virus, influenza A H3N2...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/70C12Q1/68
CPCC12Q1/6844C12Q1/701C12Q2531/119
Inventor 宋宏彬王瑞丽郝荣章孔文李杨卢晓孙明璇赵荣涛
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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