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Group-A rotavirus rapid real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) detection kit

A group A rotavirus, RT-PCR technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, microbial measurement / inspection, etc., can solve the problems of acute onset, lack of sensitivity, difficult to promote, etc., to improve reliability and accuracy, fast detection speed, avoiding false negatives and false positives

Inactive Publication Date: 2017-12-22
NANTONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF THE PEOPLES REPUBLIC OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 1. Electron microscope observation: Sometimes immunoelectron microscopy and ultrathin section electron microscopy are also used. The role of this method in diagnosis is highly appraised, but due to equipment limitations, it is difficult to popularize
[0007] 2. Enzyme immunosorbent assay: the double sandwich method is the most commonly used detection method at present. WHO lists this method as the standard method for diagnosing RV. Related products are already on the market at home and abroad, but this rapid detection The sensitivity of the method has a relatively large deficiency
At present, the kit developed by this method needs at least 2 hours from reverse transcription to complete PCR detection, but the patients with group A rotavirus infection are mainly outpatients, and the onset is urgent, so it is urgent to develop a method that can quickly detect A in samples. Group of Rotavirus Nucleic Acid Products

Method used

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  • Group-A rotavirus rapid real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) detection kit
  • Group-A rotavirus rapid real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) detection kit
  • Group-A rotavirus rapid real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Preparation and optimization of the rapid real-time fluorescent RT-PCR detection kit for group A rotavirus according to the present invention

[0054] 1. Design of primers and probes: through sequence comparison and analysis of the nucleic acid sequences of the reported group A rotaviruses, a segment with no secondary structure and a high degree of conservation was selected. According to the basic principles of primer and probe design, the software And artificially design multiple pairs of primers and probes.

[0055] 2. Selection of clinical samples: According to relevant domestic and foreign literature reports, select stool samples.

[0056] 3. Establishment and optimization of the reaction system

[0057] Screening of primer probes: Use the multiple sets of primer probes designed in 1 above to detect the RNA of the above-mentioned positive quality control products and negative quality control products. Primer Probe Assemblies. (The forward primer sequen...

Embodiment 2

[0065] Embodiment 2: use the detection method of group A rotavirus rapid real-time fluorescent RT-PCR detection kit of the present invention

[0066] 1. Specimen collection, transportation and storage

[0067] Take 500-1000μl of the sample to be tested and the watery diarrhea, put it into a sterile 1.5ml centrifuge tube, and send it directly for testing or store it at -20°C, and the storage period should not exceed six months. Ship sealed in dry ice to avoid freeze-thaw.

[0068] 2. Nucleic acid extraction

[0069] Take 200ul water sample diarrhea and add it to a 1.5ml centrifuge tube, vortex fully, centrifuge at 5000rpm for 30s, take 50ul supernatant and add 5ul lysate, which consists of 0.5% (w / v) sodium lauroyl sarcosinate, 200mmol / L dithiothreitol, TE (pH7.4), incubate at 95°C for 2min, centrifuge at 12,000rpm for 3min, collect the supernatant, which can be directly used for PCR detection or temporarily stored at -20°C. If stored for more than one month, it can be store...

Embodiment 3

[0084] Embodiment 3: the use of the quality control product in the rapid real-time fluorescent RT-PCR detection kit of group A rotavirus of the present invention

[0085] The quality control substances in the rotavirus rapid PCR kit include positive quality control substances and negative quality control substances, which are used for quality control in clinical trials. The operation method is the same as that of the samples to be tested, see Example 2.

[0086] The result is as follows:

[0087] The amplification curve of the positive quality control product is S-shaped and the Ct value is ≤33, see the attached figure 2 , the amplification curve shown in the figure is an S-shaped curve, indicating that the detection system effectively amplifies the group A rotavirus nucleic acid.

[0088] The amplification curve of the negative quality control product is not S-shaped, see the attached image 3 , the graph shows that the amplification curve is a relatively straight broken l...

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Abstract

The invention discloses a group-A rotavirus rapid real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) detection kit which comprises an amplification primer, a forward primer 5'-gttgatgctcaagatggagt-3', a reverse primer 5'-acttcattgtaatcatattgaatacc-3' and a specific fluorescent probe 5'-X-cagcaacaactgcagcttcaaaagaagtgt-Y-3'. The kit can meet the requirement of timeliness for group-A rotavirus detection, group-A rotaviruses are rapidly and quantitatively detected in real time according to one-step fluorescent RT-PCR technology by the design of the primers, the probe and reaction conditions in a reaction system and heat-resisting DNA (deoxyribonucleic acid) rapid polymerase, and sensitivity can reach 10<2> PFU / ml.

Description

technical field [0001] The invention relates to the technical field of molecular diagnosis, in particular to a rapid real-time fluorescent RT-PCR detection kit for group A rotavirus. Background technique [0002] Rotavirus is the most common pathogen in viral gastroenteritis. In 1973, Australian scholar Bishop et al first discovered it in ultra-thin sections of the duodenal mucosa of children with acute non-bacterial gastroenteritis, and named it rotavirus in 1974. (White GB, Ashton CI, Roberts C, Parry HE.1974.Letter: "Rotavirus" inastroenteritis.Lancet.2(7882):726; Bryden AS, Davies HA, Hadley RE, FlewettTH..1975.Rotavirus enteritis in the West Midlands during 1974.Lancet.2(7928):241-3.) This pathogen can cause respiratory infections in vaccinated animals and can be isolated from infected animals. [0003] Rotaviruses belong to the Reoviridae family and are spherical, double-stranded RNA viruses with a wide shell cover, short and thin edges. The average diameter is abou...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/701C12Q2563/107C12Q2545/113C12Q2521/107
Inventor 陈峰陈琳徐聪灵蔡丽萍唐晓宇吴尔翔
Owner NANTONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF THE PEOPLES REPUBLIC OF CHINA
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