A kit and detection method for accurate and quantitative detection of Norovirus type Ⅱ

A quantitative detection and kit technology, applied in the field of molecular biology, can solve the problems of quantitative detection of norovirus type Ⅱ by RT-ddPCR, achieve high accuracy, high tolerance, and reduce the effect of detection deviation

Active Publication Date: 2019-11-26
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 Ⅱ using RT-ddPCR at home and abroad.

Method used

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  • A kit and detection method for accurate and quantitative detection of Norovirus type Ⅱ
  • A kit and detection method for accurate and quantitative detection of Norovirus type Ⅱ
  • A kit and detection method for accurate and quantitative detection of Norovirus type Ⅱ

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 RT-ddPCR primer, probe design

[0033] In order to realize the specific detection and absolute quantitative analysis of Norovirus type Ⅱ, we selected the RdRp-VP1 fragment, which is a norovirus genotyping fragment, located at 4000bp-6500bp of the whole genome of Norovirus, through the NCBI online tool For sequence analysis and comparison, more than 10 pairs of primers and probe combinations were designed using Prime Express software V4.0 (ABI, Foster City, CA, USA). One set of each probe combination, 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. Primers / probes were synthesized by Beijing Liuhetong Economic and Trade Co., Ltd.

Embodiment 2

[0034] The establishment of embodiment 2 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, and leave at room temperature for 5min; ②Add 250μL chloroform, shake vigorously 30s, place at room temperature for 5min; 4°C, 12000g, centrifuge for 5min; ③ Transfer the supernatant into a new centrifuge tube, add 500μL of isopropanol and shake vigorously for 30s, place at room temperature for 5min; ④ 4°C, 5000g, centrifuge for 5min; ⑤Remove the supernatant carefully, wash the precipitate with 1mL 70% ethanol, then centrifuge at 12000g at 4°C for 5min (absorb the supernatant as much as possible, and place the centrifuge tube on an ultra-clean bench to dry the precipitate); ⑥Add 50μL RNase-free water Dissolve RNA (in order to dissolve virus RNA better, heat at 60°C for 10min). The extracted RNA was stored at -80°C for ...

Embodiment 3

[0045] Embodiment 3 kit composition

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

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

[0048] (2) one-step RT-ddPCR supermix, 25mM magnesium acetate: purchased from BioRad, USA, Cat. No. 186-3021;

[0049] (3) Droplet generating oil: purchased from BioRad, USA, product number 186-3030;

[0050] (4) Droplet generation card: purchased from BioRad, USA, product number 186-4007;

[0051] (5) Aluminum foil heat-sealing film: purchased from BioRad, USA, item number 181-4000;

[0052] (6) Twin Tec Semi-Skirted 96-well plate: purchased from Eppendorf, Germany, Cat. No. 0030128605;

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

[0054] (8) Positive control: norovirus type Ⅱ RNA standard substance;

[0055] (9) DEPC water: purchased from Shanghai Sangong,...

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Abstract

The invention discloses a reagent kit and a method for accurately and quantitatively detecting type-II norovirus, and particularly relates to a group of primers and probes for detecting type-II norovirus, a reagent kit with the primers and the probes and a method implemented by the aid of the reagent kit. The primers and the probes are provided with nucleotide sequences shown from SEQ ID No.1 to SEQ ID No.3 in a sequence table. The reagent kit and the method have the advantages that the method can be used for accurately and quantitatively detecting the type-II norovirus by the aid of microdroplet digital PCR (polymerase chain reaction) technologies and is high in sensitivity, accuracy and repeatability and easy to standardize.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a kit and oligonucleotide for detecting Norovirus type II, more precisely, a reverse transcription-microdroplet for detecting Norovirus type II Digital reverse transcriptional polymerase chain reaction (droplet digital reverse transcriptional polymerase chain reaction, RT-ddPCR)) rapid quantitative detection kit. Background technique [0002] Norovirus (Norovirus) belongs to the family Caliciviridae. It is a non-enveloped single-stranded positive-sense RNA virus with a genome length of 7400-7700bp, including 3 open reading frames (Open Reading Frame, ORF), of which ORF1 encodes nucleosides ORF2 encodes the major structural protein VP1, and ORF3 encodes the minor structural protein. Based on the difference in RdRP and VP1 gene sequence information, NoV can be divided into five genotypes GⅠ-GV, of which GⅠ and GⅡ are the most common and can infect humans. [0003] Sin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2563/159C12Q2563/107C12Q2545/114
Inventor 曾静徐蕾蕊魏海燕赵晓娟
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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