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Reagent and method for detecting monkeypox virus through isothermal amplification

A monkeypox virus, isothermal amplification technology, applied in biochemical equipment and methods, microbial measurement/inspection, fluorescence/phosphorescence, etc., can solve problems such as hazards, and achieve specificity, high purity, and stable base difference Effect

Inactive Publication Date: 2014-04-02
浙江国际旅行卫生保健中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Smallpox vaccine has a certain resistance to monkeypox virus. At present, people under the age of 30 have not been vaccinated against smallpox vaccine. Once monkeypox virus is introduced, it will cause great harm

Method used

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  • Reagent and method for detecting monkeypox virus through isothermal amplification
  • Reagent and method for detecting monkeypox virus through isothermal amplification
  • Reagent and method for detecting monkeypox virus through isothermal amplification

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The preparation of embodiment 1 positive sample standard

[0034] According to the genbank database, the genome sequence of each subtype of monkeypox was analyzed, and after sequence comparison, the selected fragment was located between 48332-48780 bp in the genbank code HQ857563.1 of the monkeypox virus genome, a total of 448 bases. The base difference is stable, so that the designed primers can only target monkeypox virus and its subspecies, and will not amplify other poxviruses or other species. See SEQ ID NO: 1 and figure 1 . The target fragment is cloned into the pGSI plasmid and transformed into E. coli. When the foreign fragment is inserted into the multiple cloning site of the pGSI plasmid, the reading frame will be changed, the expressed protein will be inactivated, and the resulting amino acid fragment will lose α-complementation ability. Transformants containing recombinant plasmids can only form white colonies on the chromogenic induction medium. Transform...

Embodiment 2

[0036] Embodiment 2, the detection of monkeypox virus

[0037] 1. Primer Design

[0038] According to the monkeypox-specific sequence, a set of primers (F3, B3, FIP, BIP) were designed with Primer Ex plorerIV software, which are the front outer primer B3, the back outer primer F3, the front inner primer FIP, and the back inner primer BIP: among them FIP is composed of the complementary sequence of F1C and F2 sequence, and BIP is composed of the complementary sequence of B1C sequence and B2. The primer gene sequence is as follows:

[0039] Outer primer F3: 5'-CATTGATTTTTCGCGGGATA-3';

[0040] Outer primer B3: 5'-TCCATCTCCCTCCAGAATCTCC-3';

[0041] Inner primer FIP:

[0042] 5'-GTTGGTCTACGACAATGGATGCTCATCAGAATCTGTAGGCCGTGT-3';

[0043] Inner primer BIP:

[0044] 5'-TCCTTAGTCCAATGTTTTTAATAACCGAAAAATTTCTACGATCTATATTGACGAG-3'.

[0045] 2. The reaction system and reaction temperature of constant temperature amplification

[0046] The reaction system is 25 μl, the components ...

Embodiment 3

[0058] Embodiment 3, detection of monkeypox virus in human serum

[0059] Use 15 servings of serum from febrile patients who entered the airport port, and use the DNA extraction kit to extract DNA according to the instructions. The reaction system is 10×buffer2.5μl, and the primer mixture is 1μl (the final concentrations of the primers in the system are as follows: F3, B3, 40pM each, FIP, BIP each 800pM), template DNA 1μl, 8U BstDNA polymerase 0.8μl, betaine (8M) 2μl, dNTP (10mM) 2μl, MgSO 4 (100mM) 1.2ul, 1.25μl fluorescent dye 20×Eva Green, add ddH 2 Make up 25 μl of O; use sterilized double distilled water as the negative control, and the pGSI plasmid standard containing monkeypox specific gene sequence as the positive control. The reaction program was 63°C, 90 minutes. Observe the results after the reaction, the reaction results of the serum samples of 15 patients with fever were centrifuged at 6000rpm, no precipitation was seen for 3 minutes, no strong green fluorescenc...

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Abstract

The invention provides a reagent for detecting a monkeypox virus through isothermal amplification, which comprises a standard substance for detecting the monkeypox virus and primers, wherein the standard substance is a recombinant plasmid vector obtained by cloning an artificially synthesized gene segment into a pGSI plasmid; the base sequence of the gene segment is shown as SEQ ID NO:1; and the primers include an outer primer F3, an outer primer B3, an inner primer FIP and an inner primer BIP. The invention also provides a method for detecting a monkeypox virus through isothermal amplification, which can quickly and accurately detect the monkeypox virus by using the reagent.

Description

technical field [0001] The invention relates to a reagent and a method for detecting monkeypox virus, in particular to a reagent and a method for detecting monkeypox virus by using an isothermal amplification technique. Background technique [0002] Monkeypox virus is a zoonotic disease that occurs mainly in Central and West Africa. It was first discovered in monkeys in 1958, and it was first discovered in humans infected with monkeypox virus in the 1970s. It broke out in Wisconsin, USA in 2003, causing 82 people infected. Monkeypox virus is a double-stranded DNA virus, which belongs to the poxviridae family like smallpox virus. The symptoms of infection are similar to those of smallpox, such as fever, headache, swollen lymph nodes, cough and extreme pain all over the body. It is commonly known as "monkey smallpox". One of the dangerous viruses, which can be transmitted through direct contact with patients or infected animals, or through the body fluids of patients, and the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 吕沁风郑伟吴忠华徐琦罗鹏何蕾
Owner 浙江国际旅行卫生保健中心
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