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LAMP (loop-mediated isothermal amplification) primer group and kit for amplifying influenza A virus (IAV)

A technology of influenza A virus and primer set, which is applied in the biological field and can solve problems that have not been reported

Inactive Publication Date: 2018-01-16
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no report on the LAMP full typing detection technology of multiple conserved sites of IAV

Method used

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  • LAMP (loop-mediated isothermal amplification) primer group and kit for amplifying influenza A virus (IAV)
  • LAMP (loop-mediated isothermal amplification) primer group and kit for amplifying influenza A virus (IAV)
  • LAMP (loop-mediated isothermal amplification) primer group and kit for amplifying influenza A virus (IAV)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Example 1 Application of Eva Green to verify the amplification reaction to IAV-H1

[0115] Similar to SYBR Green I, Eva Green is a dye with a green excitation wavelength that binds to the minor groove region of all dsDNA double helices, and its inhibition on nucleic acid amplification reactions such as PCR is much smaller than that of the latter. In the free state, EvaGreen emits weak fluorescence, but once bound to double-stranded DNA, the fluorescence is greatly enhanced. Therefore, the fluorescence signal intensity of Eva Green is related to the quantity of double-stranded DNA, and the quantity of double-stranded DNA existing in the nucleic acid amplification system can be detected according to the fluorescence signal.

[0116] Reaction solution mix (25 μL)

[0117] 20mM Tris-HCl pH8.8

[0118] 10mM KCl

[0119] 10mM (NH 4 ) 2 SO 4

[0120] 14mM MgSO 4

[0121] 0.1% Triton X-100

[0122] 1M Betaine

[0123] 1.25mM dNTPs

[0124] 8U Bst DNA polymerase (NEW...

Embodiment 2

[0134] Example 2 Application of Eva Green to verify the amplification reaction to IAV-H2

[0135] Similar to SYBR Green I, Eva Green is a dye with a green excitation wavelength that binds to the minor groove region of all dsDNA double helices, and its inhibition on nucleic acid amplification reactions such as PCR is much smaller than that of the latter. In the free state, EvaGreen emits weak fluorescence, but once bound to double-stranded DNA, the fluorescence is greatly enhanced. Therefore, the fluorescence signal intensity of Eva Green is related to the quantity of double-stranded DNA, and the quantity of double-stranded DNA existing in the nucleic acid amplification system can be detected according to the fluorescence signal.

[0136] Reaction solution combination (25 μ L), with embodiment 1;

[0137] Primers:

[0138] 200nM H2-F3 / SEQ ID NO.5

[0139] 200nM H2-B3 / SEQ ID NO.6

[0140] 1600nM H2-FIP / SEQ ID NO.7

[0141] 1600nM H2-BIP / SEQ ID NO.8

[0142] Target: IAV-H2d...

Embodiment 3

[0145] Example 3 Application of Eva Green to verify the amplification reaction to IAV-H3

[0146] Similar to SYBR Green I, Eva Green is a dye with a green excitation wavelength that binds to the minor groove region of all dsDNA double helices, and its inhibition on nucleic acid amplification reactions such as PCR is much smaller than that of the latter. In the free state, EvaGreen emits weak fluorescence, but once bound to double-stranded DNA, the fluorescence is greatly enhanced. Therefore, the fluorescence signal intensity of Eva Green is related to the quantity of double-stranded DNA, and the quantity of double-stranded DNA existing in the nucleic acid amplification system can be detected according to the fluorescence signal.

[0147] Reaction solution combination (25 μ L), with embodiment 1;

[0148] Primers:

[0149] 200nM H3-F3 / SEQ ID NO.9

[0150] 200nM H3-B3 / SEQ ID NO.10

[0151] 1600nM H3-FIP / SEQ ID NO.11

[0152] 1600nM H3-BIP / SEQ ID NO.12

[0153] Target: IAV-...

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Abstract

The invention relates to the field of biotechnology and particularly relates to a LAMP (loop-mediated isothermal amplification) primer group and a kit for detecting influenza A virus (IAV). The primergroup comprises one or more of the following 29 primer groups: primer groups for amplifying segments of the regions H1-H18 of IVA and primer groups for amplifying segments of the regions N1-N11 of IVA. The invention also provides a kit for detecting IAV. The primer group provided by the invention has high sensitivity and high specificity; the kit prepared from the primer group can be used for quickly and accurately detecting the IAV contained in a to-be-detected sample and can be applied to the typing detection and identification of the IAV type in the to-be-detected sample. According to theprimer group and kit provided by the invention, the false positive and false negative phenomena are avoided in a process of adopting artificial RNA for control detection. The primer provided by the invention has high sensitivity and specificity; the provided visual kit brings great convenience to field detection; quick and accurate detection of IAV can be realized by preparing a kit from the primer.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a LAMP primer set and kit for amplifying influenza A virus (Influenza Avirus, IAV). Background technique [0002] Influenza viruses belong to the Orthomyxoviridae family and can be divided into types A, B, and C. Among them, seasonal influenza caused by influenza A and influenza B virus has high morbidity and mortality rate, constantly causing global health problems and causing huge economic losses. There are two surface fibers of hemagglutinin (hemagglutinin, HA) and neuraminidase (neuraminidase, NA) on the surface capsule of type A virus particles, which are two important glycoproteins and the main surface antigen of the virus. According to HA The difference from NA can be divided into different subtypes, such as H1N1 and H7N9, etc. Currently, there are 18 H subtypes (H1~H18) and 11 N subtypes (N1~N11). Types of viruses can almost all be isolated from aquatic birds, poultry and o...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
Inventor 杜昱光齐立斐毛瑞王倬
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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