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Zika virus detection kit based on loop-mediated isothermal amplification

A Zika virus and kit technology, applied in the fields of molecular biology and nucleic acid detection, can solve problems such as limiting the application of PCR-related technologies

Active Publication Date: 2021-08-06
中国科学院上海免疫与感染研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, PCR-based methods require thermal cyclers and other equipment, and there are also requirements for the detection site, and quantitative PCR requires special training, which limits the application of PCR-related technologies in rapid detection

Method used

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  • Zika virus detection kit based on loop-mediated isothermal amplification
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  • Zika virus detection kit based on loop-mediated isothermal amplification

Examples

Experimental program
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Effect test

Embodiment 6

[0141] Except for Example 6, the Zika virus used in other examples is the Zika virus cultured in vitro by Pasteur cells.

[0142] RT-LAMP reaction

[0143] The basic system of RT-LAMP reaction is shown in Table 1.

[0144]Table 1 The basic system of Zika RT-LAMP reaction

[0145]

[0146] The reaction steps are as follows:

[0147] (a) Visual inspection: directly observe the color change at 62°C for 60 minutes.

[0148] The dye is HNB or Calcein.

[0149] (b) Real-time real-time fluorescence quantitative detection is:

[0150] 62℃, 120s, 1 cycle,

[0151] 62°C, 60s, 60 cycles, collect fluorescence

[0152] The dye is SYTO 9, and the channel for collecting fluorescence is set as SYBR Green I channel.

[0153] II. Example

Embodiment 1

[0155] Primer design and screening

[0156] Obtain the whole genome sequence of all Zika viruses in GenBank, perform multiple sequence alignment and sequence analysis, and find the conserved regions. The 7621bp-7813bp segment of the Zika virus (taking KU740184.1 as an example) sequence is highly conserved and suitable for primer design area. The above regions were extracted from the comparison results, and primers were designed after re-alignment.

[0157] Screen the designed primers with the established RT-LAMP detection system to obtain primers that meet the requirements, and obtain 6 primers as shown in Table 2, and make these 6 primers into A, B, and C as shown in Table 3. Primer pair combinations at different final concentrations.

[0158] Table 2 Zika virus RT-LAMP specific primer sequence

[0159]

[0160] Table 3 Zika virus ABC3 primer combination

[0161]

[0162]

Embodiment 2

[0164] RNA in vitro transcription

[0165] In the example of in vitro transcription, the Zika virus plasmid template (pGH-new vector) was synthesized by Shanghai Jierui Bioengineering Co., Ltd., and the sequence was a segment of 7621bp-7813bp (taking KU740184.1 as an example). The template is amplified, and the amplified product is analyzed by electrophoresis on 1% agarose gel. After the correct size is determined, the target band is cut and recovered, and the purified DNA product can be used as a template for in vitro transcription.

[0166] Promega's "RiboMAX Large Scale RNA Production System-T7" kit was used for in vitro transcription, and the reaction process used the method provided by the reagent manufacturer. The concentration and purity of RNA products transcribed in vitro were measured using Nanodrop, and the unit was converted into copies / μL (copy / μL). To avoid repeated freezing and thawing of RNA, take an appropriate amount and dilute to 1×10 10 copies / μL were ali...

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Abstract

The invention provides a loop-mediated isothermal amplification (LAMP) primer and a related kit and a method for detecting Zika virus (also called Zika virus, Zika virus, ZIKV) using the primer. Using the primers can specifically loop-mediated isothermal amplification of Zika virus nucleic acid. The method of the invention has good specificity, high sensitivity, good repeatability, simple operation, convenience and quickness, can be well applied to identify Zika virus, and is suitable for clinical and laboratory detection.

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 Zika virus detection kit based on loop-mediated isothermal amplification. Background technique [0002] Zika virus (ZIKV) is an arbovirus transmitted by mosquitoes with an unknown host, mainly circulating in wild primates and tree-dwelling mosquitoes, such as Aedes africanus. The virus was first discovered by chance in rhesus monkeys in the Zika jungle of Uganda in 1947 through the Yellow Fever Surveillance Network, and subsequently in Ugandan and Tanzanian populations in 1952. The activity of the virus has been relatively hidden, and there are only sporadic cases of Zika virus infection in Africa, the Americas, Asia and the Pacific around the equator. The earliest outbreak occurred in Yapu Island in the Micronesian Islands in the Western Pacific in 2007, and a larger epidemic occurred in French Polynesia in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2527/127
Inventor 张驰宇李洋蓝柯胡轶红
Owner 中国科学院上海免疫与感染研究所
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