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Norovirus sequencing primer based on high-throughput sequencing, kit and detection method thereof

A detection method, a high-throughput technology, applied in the direction of microbial-based methods, biochemical equipment and methods, microbial measurement/inspection, etc., can solve the difficulty of norovirus and the inability to outline the complete type of norovirus in water Distribution and other issues, to achieve high accuracy, efficient typing and sequencing, and low false negative results

Active Publication Date: 2019-10-18
SHANGHAI BIOGERM MEDICAL TECH CO LTD
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Construction of a large number of clones still cannot outline the complete distribution of norovirus types in water bodies, and some norovirus types with low abundance are difficult or impossible to monitor through infectious cloning methods

Method used

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  • Norovirus sequencing primer based on high-throughput sequencing, kit and detection method thereof
  • Norovirus sequencing primer based on high-throughput sequencing, kit and detection method thereof
  • Norovirus sequencing primer based on high-throughput sequencing, kit and detection method thereof

Examples

Experimental program
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Embodiment 1

[0084] 1. Concentration and enrichment of viruses

[0085] Viruses in environmental water were concentrated and enriched by anionic membrane adsorption-ultrasonic oscillation method. The specific experimental steps are as follows:

[0086] (1) Centrifuge the collected sewage sample at 4°C and 3000r / min for 30 minutes, so that the suspended matter in the sample (including mud sand, clay, and water-insoluble inorganic matter, organic matter, microorganisms, etc.) settles at the bottom of the bottle;

[0087] (2) Take the above supernatant into a large sterile beaker equipped with a rotor, place it on a magnetic stirrer, and slowly add 2.5M MgCl while stirring 2 solution, so that the final concentration is 0.05M; then add 0.5M HCl to adjust the pH value to 3.5-4.0;

[0088] (3) Place the filter paper and the anion membrane in the installed filter successively, then transfer the above-mentioned pH-adjusted water sample to the filter, connect the filter to the positive pressure p...

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Abstract

The invention provides a Norovirus sequencing primer based on high-throughput sequencing, a kit and a detection method thereof. By designing the primer for constructing a Norovirus genotyping sequencing library, the amplified fragments can effectively represent the genotyping of Norovirus, the length of the amplified fragment of the primer is appropriate, and the double-end sequencing can be directly performed through a high-throughput sequencing platform without interrupting the gene fragment. Compared with a traditional Norovirus detection technology, the Norovirus genotyping detection method of the present invention no longer needs to use a specific primer to specifically detect a certain type one-to-one, an experiment is conducted to accurately distinguish and identify various Norovirus types in a water body, and the accuracy is higher and the repeatability is better; and high-depth sequencing is performed to ensure enough detection depth and lower false negatives.

Description

technical field [0001] The invention relates to the field of molecular biology technology, in particular to a norovirus sequencing primer, kit and detection method based on high-throughput sequencing. Background technique [0002] Norovirus (NoV), also known as Norwalk virus, belongs to the Norovirus genus of the Caliciviridae family and is the most important pathogen causing outbreaks of acute gastroenteritis. Due to its strong infectivity (approximately 18-1000 virus particles can be infected) and wide transmission route (through water, food, objects, air, etc.), norovirus spreads rapidly, so it is very easy to spread in crowded places such as schools and hospitals. and other places caused outbreaks. According to WHO's assessment of the global disease burden of foodborne diseases, there are more than 600 million cases of acute gastroenteritis caused by norovirus infection every year. Due to the lack of effective vaccines and drugs, virus infection causes 210,000 deaths e...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6806C12N15/11C12R1/93
CPCC12Q1/6806C12Q1/701C12Q2531/113C12Q2535/122C12Q2525/191
Inventor 陆靖李晖郑焕英易丽娜方苓夏昊强
Owner SHANGHAI BIOGERM MEDICAL TECH CO LTD
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