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Primer set, reagent, kit and detection method for detecting and/or assisting detection of GI type norovirus

An auxiliary detection and detection method technology, applied in the biological field, can solve problems such as unmatched detection conditions, limited application, product environmental pollution, etc., and achieve rapid on-site detection, high sensitivity, and good specificity

Inactive Publication Date: 2019-01-18
辽宁佰昊生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A major problem with LAMP technology at present is that its products seriously pollute the environment, and the reaction needs to be completed at a high temperature of 60-65°C, so its application is relatively limited
Moreover, the existing primer set for detecting GⅠ type norovirus is only suitable for specific methods (such as traditional PCR) and is not suitable for RPA amplification, so it cannot match the detection conditions to achieve the best detection effect

Method used

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  • Primer set, reagent, kit and detection method for detecting and/or assisting detection of GI type norovirus
  • Primer set, reagent, kit and detection method for detecting and/or assisting detection of GI type norovirus
  • Primer set, reagent, kit and detection method for detecting and/or assisting detection of GI type norovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Design of primer set and positive control sample

[0069] Aiming at the conserved sequence of GI norovirus, the RPA primer set for detecting GI norovirus was designed, and the product size was 112 bp. The positive control sample was designed for the primer set and amplified fragment as a DNA plasmid containing positive control. The sequence of the positive control contained in the specific primer set and the positive control sample is as follows:

[0070] Table 1 Sequences of primer sets and positive controls

[0071]

Embodiment 2

[0072] Example 2 Kit for detection and / or auxiliary detection of type GI norovirus

[0073] The kit includes the upstream primer RPA-NGI-F in Example 1, the downstream primer RPA-NGI-R, GI Norovirus positive control sample (0.2ng / μL), a tube containing lyophilized enzyme powder, rehydration Buffer (Rehydration Buffer), magnesium acetate solution (280mmol / L). The above tubes containing lyophilized enzyme powder, rehydration buffer (Rehydration Buffer) and magnesium acetate solution (280mmol / L) are all from RPA amplification kit TwistAmp Basic kits.

Embodiment 3

[0074] Embodiment 3 Identify the detection method of GI type norovirus

[0075] (a) RPA amplification

[0076] Using the cDNA of the sample to be tested as a template, using the kit in Example 2, and using the RPA-NGI-F and RPA-NGI-R primer sets to perform RPA amplification, the RPA amplification product was obtained. At the same time, a blank control was set (DNA template was nuclease-free water).

[0077] The preparation method of the RPA amplification system is as follows: add 29.5 μL of Rehydration Buffer, 12.5 μL of deionized water, and 2.4 μL of upstream and downstream primers to a 0.2 mL TwistAmp reaction tube containing lyophilized enzyme powder (primers). The final concentration is 0.48 μmol / L), template DNA or positive control sample is 1 μL, and finally 2.5 μL of magnesium acetate solution (280 mmol / L) is added.

[0078] RPA amplification reaction conditions: fully mix the above RPA amplification system, and place it in a metal bath or water bath at 37°C for 40 mi...

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Abstract

The invention relates to the field of biotechnology, in particular to a primer set, a reagent, a kit and a detection method for detecting and / or assisting detection of GI type norovirus. A primer setfor detecting and / or assisting in the detection of a type GI norovirus is provided, comprising a single-stranded DNA molecule as shown in SEQ ID NO. 1 and a single-stranded DNA molecule as shown in SEQ ID NO. 2. The experiment proves that the primer set of the invention has good specificity, high sensitivity, short detection time, no special instrument is needed, the experiment time cost, the simplicity of operation and the detection cost are obviously reduced. The reagent or kit containing the primer set can be used for specific detection and identification of Norovirus type GI, and the RPA detection method of Norovirus type GI based on the primer set is sensitive, accurate, simple and rapid, which can realize the screening and rapid diagnosis of Norovirus type GI.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a primer set, a reagent, a kit and a detection method for detecting and / or assisting the detection of type GI norovirus. Background technique [0002] Norovirus is the most common cause of epidemiological and sporadic cases of acute gastroenteritis worldwide and the leading cause of foodborne gastroenteritis. Acute gastroenteritis caused by norovirus is on the rise in my country, especially in schools, kindergartens, nursing homes and other places where people with low resistance gather, which are prone to collective outbreaks. Norovirus is highly contagious and can be transmitted through fecal-oral transmission, human-to-human transmission, and virus-contaminated water and food. It is estimated that outbreaks of nonbacterial diarrhea caused by norovirus account for about 50%-80% of the total. Outbreaks are usually associated with shellfish or freshly prepared vegetable and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2522/101C12Q2531/119
Inventor 董进法姚雪春卢星忠梁耀极那永东林春美周雪洁孙晓丽郭微
Owner 辽宁佰昊生物科技有限公司
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