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Kit for precisely and quantitatively detecting norovirus type I and detection method

A technology of quantitative detection and detection method, which is applied in the field of molecular biology, can solve the problems of quantitative detection of Norovirus by RT-ddPCR, etc., and achieve the effects of high tolerance, reduced detection deviation, and easy standardization

Active Publication Date: 2016-11-16
BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no relevant reports on the quantitative detection of Norovirus type Ⅰ by RT-ddPCR at home and abroad.

Method used

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  • Kit for precisely and quantitatively detecting norovirus type I and detection method
  • Kit for precisely and quantitatively detecting norovirus type I and detection method
  • Kit for precisely and quantitatively detecting norovirus type I and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Design of RT-ddPCR primers and probes

[0033] In order to realize the specific detection and absolute quantitative analysis of Norovirus type I, we selected the Norovirus typing gene fragment RdRp-VP1, and used the NCBI online tool for sequence analysis and comparison, using Prime Express software V4.0 (ABI , Foster City, CA, USA) designed more than 10 pairs of primer and probe combinations. After screening, one set of primer / probe combinations with strong specificity and suitable for RT-ddPCR was finally obtained. The sequence is shown in Table 1. The 5'end of the probe is labeled with FAM, and the 3'end is labeled with BHQ. The primers / probes were synthesized by Beijing Liuhetong Economic and Trade Co., Ltd.

Embodiment 2

[0034] Example 2 Establishment of detection method

[0035] (1) RNA extraction: use a commercial kit for extraction; or use the traditional Trizol method to extract RNA, the specific operation is as follows: ①Add 1mL Trizol to 100μL sample, shake for 30s, then leave at room temperature for 5min; ②Add 250μL chloroform, shake vigorously 30s, placed at room temperature for 5min; 4℃, 12000g, centrifuged for 5min; ③Transfer the supernatant to a new centrifuge tube, add 500μL of isopropanol, shake vigorously for 30s, leave at room temperature for 5min; ④4℃, 5000g, centrifuge for 5min; ⑤Carefully remove the supernatant, wash the precipitate with 1mL 70% ethanol and centrifuge at 12000g at 4℃ for 5min (aspirate the supernatant as much as possible, and place the centrifuge tube on the ultraclean table to blow dry the precipitate); ⑥Add 50μL RNase-free water Dissolve RNA (in order to better dissolve viral RNA, heat at 60°C for 10 minutes). The extracted RNA is stored at -80°C for later us...

Embodiment 3

[0045] Example 3 Kit composition

[0046] 1. The composition of the kit (stored at -20℃)

[0047] (1) The primers and probes SEQ ID No.1-3 designed in Example 1 for detecting Norovirus type I were synthesized by Beijing Liuhetong Economic and Trade Co., Ltd.;

[0048] (2) One-step RT-ddPCR supermix, 25mM magnesium acetate: purchased from BioRad in the United States, article number 186-3021;

[0049] (3) Microdroplet generating oil: purchased from BioRad in the United States, article number 186-3030;

[0050] (4) Droplet generation card: purchased from BioRad in the United States, article number 186-4007;

[0051] (5) Aluminum foil heat-sealing film: purchased from BioRad, the United States, article number 181-4000;

[0052] (6) Twin Tec Semi-Skirted 96-well plate: purchased from Eppendorf, Germany, catalog number 0030128605;

[0053] (7) Negative control: DEPC water; purchased from Shanghai Shenggong, item number: D1005;

[0054] (8) Positive control: Norovirus type I RNA standard material;...

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Abstract

The invention discloses a kit for precisely and quantitatively detecting norovirus type I and a detection method, and in particular relates to a primer and a probe for detecting norovirus type I, a kit comprising the primer and the probe, and a detection method of the kit, wherein the kit has nucleotide sequences as shown from SEQ ID No.1 to SEQ ID No.3. The invention further provides a detection method for precisely and quantitatively detecting norovirus type I by using a microdroplet digital PCR technique, and the detection method does not depend on certified reference materials or other standard products, and is relatively high in precision, good in sensitivity and repeatability and easy to standardize.

Description

Technical field [0001] The present invention belongs to the field of molecular biology, and specifically relates to a kit and oligonucleotides for detecting Norovirus type I, more specifically, a reverse transcription-microdroplet for detecting Norovirus type I Digital polymerase chain reaction (droplet digital reverse transcriptional polymerase chain reaction, RT-ddPCR) rapid quantitative detection kit. Background technique [0002] Norovirus (Norovirus) belongs to the Caliciviridae family. It is a non-enveloped single-stranded positive-stranded RNA virus with a full-length genome of 7.5-7.7kp, including 3 open reading frames (ORF), of which ORF1 encodes the nuclear Non-structural proteins such as triphosphatase, protease and RNA-dependent RNA polymerase (RdRp), ORF2 encodes the major structural protein VP1, ORF3 encodes the minor structural protein. Based on the difference of RdRP and VP1 gene sequence information, Norovirus can be divided into 5 genotypes GⅠ-GⅤ. Among them, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2563/159
Inventor 徐蕾蕊曾静赵晓娟马丹
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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