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LAMP (loop-mediated isothermal amplification) primer group for amplifying MERS-CoV, and kit

A technology of primer sets and kits, which is applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems that have not been reported, achieve high sensitivity, rapid and accurate detection, and shorten the detection time Effect

Inactive Publication Date: 2017-06-20
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 detection technology of multiple conserved sites of MERS-CoV

Method used

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  • LAMP (loop-mediated isothermal amplification) primer group for amplifying MERS-CoV, and kit
  • LAMP (loop-mediated isothermal amplification) primer group for amplifying MERS-CoV, and kit
  • LAMP (loop-mediated isothermal amplification) primer group for amplifying MERS-CoV, and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Application of Eva Green to verify the amplification reaction to MERS-CoV orf1a

[0053] 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.

[0054] Reaction solution mix (25 μL)

[0055] 20mM Tris-HCl pH8.8

[0056] 10mM KCl

[0057] 10mM (NH 4 ) 2 SO 4

[0058] 14mM MgSO 4

[0059] 0.1% Triton X-100

[0060] 1M betaine

[0061] 1.25mM dNTPs

[0062] 8U Bst DNA polymer...

Embodiment 2

[0072] Example 2 Application of Eva Green to verify the amplification reaction to MERS-CoV orf1b

[0073] 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 EvaGreen 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 fluorescent signal.

[0074] Reaction solution mix (25 μL)

[0075] 20mM Tris-HCl pH8.8

[0076] 10mM KCl

[0077] 10mM (NH 4 ) 2 SO 4

[0078] 14mM MgSO 4

[0079] 0.1% Triton X-100

[0080] 1M betaine

[0081] 1.25mM dNTPs

[0082] 8U Bst DNA polymeras...

Embodiment 3

[0092] Example 3 Application of Eva Green to verify the amplification reaction to MERS-CoV N2

[0093] Similar to SYBR Green I, EvaGreen 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 EvaGreen 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 fluorescent signal.

[0094] Reaction solution mix (25 μL)

[0095] 20mM Tris-HCl pH8.8

[0096] 10mM KCl

[0097] 10mM (NH 4 ) 2 SO 4

[0098] 14mM MgSO 4

[0099] 0.1% Triton X-100

[0100] 1M betaine

[0101] 1.25mM dNTPs

[0102]8U Bst DNA polymerase (NE...

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Abstract

The invention relates to the field of biological technology, and in particular to an LAMP (loop-mediated isothermal amplification) primer group for amplifying MERS-CoV, and a kit. The primer group includes one or more of the following five primer groups: a primer group performing amplification for an orf1a region fragment of the MERS-CoV, a primer group performing amplification for an orf1b region fragment of the MERS-CoV, a primer group performing amplification for an N2 region fragment of the MERS-CoV, a primer group performing amplification for an N3 region fragment of the MERS-CoV and a primer group performing amplification for an upE region fragment of the MERS-CoV. The invention also provides the kit for detecting the MERS-CoV. According to the LAMP primer group and the kit provided by the invention, a false positive phenomenon is avoided in a process of using artificial RNA for control detection. The primer provided by the invention is high in both sensitivity and specificity; the provided visual kit can greatly facilitate on-site detection; and the prepared kit can achieve rapid and accurate detection of the MERS-CoV.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a LAMP primer set and kit for amplifying Middle East Respiratory Syndrome Virus (MERS-CoV). Background technique [0002] Middle East Respiratory Syndrome is a viral respiratory disease caused by a novel coronavirus (MERS-CoV), first identified in Saudi Arabia in 2012, and can cause a variety of illnesses ranging from the common cold to severe acute respiratory syndrome. Typical MERS symptoms include fever, cough and shortness of breath. At the same time, pneumonia is relatively common, and gastrointestinal symptoms including diarrhea have also been reported, but not in all cases. The case fatality rate of MERS reported cases is about 36%. In May 2016, the first imported case of MERS was confirmed in Huizhou, China, which aroused a certain degree of social concern. [0003] Because there is no vaccine and specific treatment drugs, it is extremely important for the prevention and c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119
Inventor 杜昱光毛瑞王倬
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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