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Kit and detection method for accurately and quantitatively detecting astrovirus

A technique for quantitative detection of astroviruses, applied in the field of molecular biology, can solve the problems of quantitative detection of astroviruses that have not been seen by RT-ddPCR, achieve high tolerance, high accuracy, and reduce detection deviations

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 astrovirus by RT-ddPCR at home and abroad

Method used

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  • Kit and detection method for accurately and quantitatively detecting astrovirus
  • Kit and detection method for accurately and quantitatively detecting astrovirus
  • Kit and detection method for accurately and quantitatively detecting astrovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0033] In order to realize the specific detection and absolute quantitative analysis of astrovirus, we selected the target sequence of astrovirus open reading frame 2 (Open Reading Frame, ORF2) encoding caspid protein, and performed sequence analysis and alignment through NCBI online tools. Prime Express software V4.0 (ABI, Foster City, CA, USA) designed more than 10 pairs of primers and probe combinations, and finally obtained 1 set of primers / probe combinations with strong specificity and suitable for RT-ddPCR after screening. See 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 it 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, and 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...

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 astroviruses 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: Astrovirus RNA standard substance;

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

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Abstract

The invention discloses a kit and a detection method for accurately and quantitatively detecting astrovirus and particularly relates to a group of primers and probes for detecting the astrovirus as well as the kit containing the primers and the probes and a detection method of the astrovirus, wherein the primers and the probes have nucleotide sequences shown in SEQ ID No. 1 to SEQ ID No. 3 in a sequence table. The invention provides the detection method for accurately and quantitatively detecting the astrovirus by using a droplet digital PCR (Polymerase Chain Reaction) technology; the method does not need to rely on certified reference materials or other standard substances, and has higher accuracy, sensitivity and repeatability, and is easy to standardize.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a test kit and oligonucleotide for detecting astrovirus, more precisely, a reverse transcription-droplet digital polymerase chain method for detecting astrovirus Reaction (dropletdigital reverse transcriptional polymerase chain reaction, RT-ddPCR)) rapid quantitative detection kit. Background technique [0002] Appleton et al. first discovered Human Astrovirus (HAstV) in the feces of patients with gastroenteritis in 1975, and there are outbreaks of Astrovirus infection in various countries every year. Astrovirus is a single-stranded positive-strand RNA virus without a capsid, with a diameter of 28-35nm and 5-6 small horns, making the entire virus particle a star-shaped structure. The viral gene is 6.8kb in length, including a 5' non-coding region (NCR), three open reading frames (ORFs) ORF1a, ORF1b and ORF2, and 80 nucleotides of 3 'noncoding regions and a polyA tai...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/701C12Q2563/159C12Q2563/107C12Q2545/114
Inventor 魏海燕马丹徐蕾蕊李丹
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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