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Primer set, kit and method for detecting norovirus type gi and gii constant temperature amplification

A constant temperature amplification and primer set technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., to achieve the effects of avoiding time loss, simple operation, and strong specificity

Active Publication Date: 2022-06-14
DALIAN NATIONALITIES UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional PCR and nested PCR are cumbersome to operate, and the amplified product needs to be detected by electrophoresis to observe the amplification result, which takes a long time to detect and has poor sensitivity
Real-time fluorescence quantitative PCR has high experience requirements for operators and strict partition requirements for the experimental environment, which is not conducive to the promotion and use at the grassroots level
The detection cost of gene chip technology is expensive and the stability is poor, so it cannot be popularized and applied at the grassroots level

Method used

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  • Primer set, kit and method for detecting norovirus type gi and gii constant temperature amplification
  • Primer set, kit and method for detecting norovirus type gi and gii constant temperature amplification
  • Primer set, kit and method for detecting norovirus type gi and gii constant temperature amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1 Design and Screening of Primer Sets for Norovirus Type GI and Type GII Real-time Fluorescence Constant Temperature Amplification

[0076] 1. Primer set design

[0077]The present invention screens norovirus type GI genome-specific sequences from the aspects of intra-species consistency and stability, inter-species specificity, copy number, etc., and screens norovirus type GI specific sequences that do not exist in other viruses or Gene fragments with low homology were used as detection molecules. The design of primers needs to consider whether it is prone to mismatch, the length of the amplified fragment, and the reaction temperature. The norovirus genome has three open reading frames (ORFs), ORF1 encodes a polymeric protein, which can produce six non-structural proteins with different functions, including RNA polymerase, under the action of 3C protease. According to the order of N-C terminal: protein 48 (p48), NTPase (NTPase), protein 22 (p22 / p20), viral ge...

Embodiment 2

[0100] The specific amplification verification of embodiment 2 primer set

[0101] 1. Preparation of positive control substance

[0102] According to the best Norovirus type GI and GII type-specific constant temperature amplified gene sequences obtained in Example 1, select the positively amplified sequence containing Norovirus type GI and GII types as shown in SEQ ID NO.13 The sequence construction plasmid was used as a positive control, in which 1 restriction site base sequence was added to the upstream and downstream of the positive control gene sequences of type GI and type GII, and 2 restriction sites were added in the middle of type GI and type GII In the base sequence, two restriction site base sequences are added after the GII type. Norovirus type GI and GII positive controls such as SEQ ID NO.13 were artificially synthesized to prepare plasmids, and RNA was transcribed in vitro as a positive control (entrusted to Bao Biological Engineering (Dalian) Co., Ltd. to compl...

Embodiment 3

[0130] Example 3 Real-time fluorescence constant temperature amplification detection sensitivity test

[0131] 1. Sensitivity test method

[0132] 1.1 Optimization of primer set, 2× RNA constant temperature amplification reaction buffer and SYBR Green I dye concentration

[0133] The screened inner primers are incremented from 1.5 μM to 1.7 μM at a distance of 0.1 μM; the outer primers are incremented at a distance of 0.1 μM from 0.1 μM to 0.3 μM; the loop primers are incremented at a distance of 0.1 μM from 0.7 μM to 0.9 μM; 2 ×RNA constant temperature amplification reaction buffer increases from 0.75× to 1.25× with a step of 0.25×; SYBR Green I dye concentration increases from 1.5× to 2.5× with a step of 0.5×. The Taguchi method was used to explore the optimal combination of primer concentrations.

[0134] 1.2 Screening and optimization of enzyme reaction solution

[0135] The enzyme reaction solution is Bst DNA polymerase. In order to determine the optimal dosage and rat...

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Abstract

The invention belongs to the field of biological detection, and in particular relates to a primer set, a kit and a method for detecting norovirus type GI and GII constant temperature amplification, and the primer set includes a norovirus type GI primer set and a norovirus type GII primer set , Norovirus type GI primer set includes outer primer GI‑OF, outer primer GI‑OB, inner primer GI‑IF, inner primer GI‑IB, loop primer GI‑LF and loop primer GI‑LB, norovirus type GII The primer set includes outer primer GII-OF, outer primer GII-OB, inner primer GII-IF, inner primer GII-IB, loop primer GII-LF, and loop primer GII-LB. The primer set can specifically and sensitively amplify norovirus types GI and GII. The detection method is simple, fast, accurate, high-sensitivity, and low in cost. It has low requirements for experimental operators and equipment, and is suitable for large-scale promotion. and apply.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a primer set, a kit and a method for detecting norovirus type GI and GII constant temperature amplification. Background technique [0002] Norovirus (Norwalk Viruses) is a non-enveloped single-stranded positive-sense RNA virus belonging to the Caliciviridae family and is the main cause of nonbacterial gastroenteritis. Noroviruses can be divided into five gene groups G I to G V, among which the GI and GII groups mainly cause human infection. Of the reported foodborne illnesses caused by pathogenic bacteria, more than 58% of the cases were caused by norovirus. According to statistics, in the United States, norovirus causes 570 to 800 deaths, 56,000 to 71,000 hospitalizations, 400,000 emergency visits, and 1.7 million to 1.9 million outpatient visits each year. The main features of norovirus disease are projectile vomiting, non-bloody diarrhea, nausea, abdominal cra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/166C12Q2531/119C12Q2563/107C12Q2545/113C12Q2521/107C12Q2537/1376
Inventor 曹际娟郑秋月杨莉莉朴永哲胡冰
Owner DALIAN NATIONALITIES UNIVERSITY