Kit and detection method for accurate quantitative detection of group A rotaviruses

A technology for quantitative detection of rotavirus, applied in the field of molecular biology, can solve the problems of quantitative detection of rotavirus in the RT-ddPCRA group, achieve high accuracy, reduce detection deviation, and be easy to standardize

Active Publication Date: 2016-12-14
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 group A rotavirus by RT-ddPCR at home and abroad

Method used

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  • Kit and detection method for accurate quantitative detection of group A rotaviruses
  • Kit and detection method for accurate quantitative detection of group A rotaviruses
  • Kit and detection method for accurate quantitative detection of group A rotaviruses

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 group A rotavirus, we selected group A rotavirus typing gene fragment VP6, and performed sequence analysis and comparison through NCBI online tool, using Prime Express software V4.0 (ABI , Foster City, CA, USA) designed more than 10 pairs of primer and probe combinations, and finally obtained 1 set of primer / probe combinations with strong specificity and suitable for RT-ddPCR after screening. The sequences are 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 to 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 fu...

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 for detecting group A rotavirus designed in Example 1 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: Group A rotavirus RNA standard substance;

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

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Abstract

The invention discloses a kit and detection method for accurate quantitative detection of group A rotaviruses, and particularly relates to a set of primers and probes having nucleotide sequences shown in sequence tables of SEQ ID No.1 to SEQ ID No.3 used for detecting the group A rotaviruses, and the kit containing the primers and probes and the detection method for the group A rotaviruses. The detection method for the accurate quantitative detection of the group A rotaviruses by using a droplet digital PCR (Polymerase Chain Reaction) technology provided by the invention has higher sensitivity, accuracy and repeatability, and is easily standardized.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a kit and oligonucleotides for detecting group A rotaviruses, more precisely, a reverse transcription-droplet method for detecting group A rotavirus types Digital polymerase chain reaction (droplet digital reverse transcriptional polymerase chain reaction, RT-ddPCR) rapid quantitative detection kit. Background technique [0002] Rotavirus (Rotavirus) belongs to the family Reoviridae and the genus Rotavirus. Spherical, 60-80nm in diameter, double-layered capsid, no envelope, observed under the electron microscope, the shape of the virus is wheel-shaped, hence the name rotavirus. Rotavirus is a double-stranded RNA virus, composed of 11 gene segments, each segment contains an open reading frame, each gene segment size is between 667-3302bg, encoding 6 structural proteins (VP1, VP2, VP3 , VP4, VP6, VP7) and 5 nonstructural proteins (NSP1-NSP5). According to the antigen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2563/159C12Q2545/113
Inventor 徐蕾蕊马丹魏咏新赵晓娟
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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