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Primer sets, probes and kits for Kawasaki disease detection

A Kawasaki disease and reagent technology, applied in the field of primer sets, probes and kits for Kawasaki disease detection, can solve problems such as affecting the specificity of the detection results, and achieve the effect of accurate results

Active Publication Date: 2020-07-28
湖南赛哲智造科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The principle of fluorescent quantitative PCR using ordinary dye method utilizes the characteristics that nucleic acid double-stranded DNA molecules can combine with multi-molecular dyes and excite fluorescence under specific conditions. Its detection sensitivity is extremely high, but primer dimers, single-stranded secondary structures and non-specific Amplified products may affect the specificity of the test results

Method used

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  • Primer sets, probes and kits for Kawasaki disease detection
  • Primer sets, probes and kits for Kawasaki disease detection
  • Primer sets, probes and kits for Kawasaki disease detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1 detects the reverse transcription primer, amplification primer and probe sequence of Kawasaki disease

[0065] The detection markers of Kawasaki disease are: hsa-miR-197, hsa-miR-671, hsa-miR-1246 and hsa-miR-4436.

[0066] The reverse transcription primer sequence is:

[0067] RT-miR-197:

[0068] GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCTGGG;

[0069] RT-miR-671:

[0070] GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTTTTTTTTTTTTCCAGCC;

[0071] RT-miR-1246:

[0072] GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCTGCT;

[0073] RT-miR-4436:

[0074] GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGCAGGGC;

[0075] The amplification primer sequences are:

[0076]Universal reverse primer R-KD: TCGTATCCAGTGCAGGG;

[0077] F-miR-197: TTCACCACCTTTCTCCACC;

[0078] F-miR-671:GAGAGGAAGCCCTGGAG;

[0079] F-miR-1246: GCCGAATGGATTTTTGGAG;

[0080] F-miR-4436: TCCTGTCCACTTCTGCCT;

[0081] The probe sequence is: CCGAGGTATTCGCACTGGAT.

[0082] Among the a...

Embodiment 2

[0083] The detection method of embodiment 2 Kawasaki disease

[0084] (1) Extraction of serum exosome miRNA The operation of extracting serum exosome miRNA includes the following steps:

[0085] 1) Place 250 μl of serum on ice to dissolve naturally, then add 60 μl of exosome extraction reagent, gently blow and mix with a pipette, then let it stand on ice for 30 minutes; centrifuge at 1500g at 4°C for 10 minutes; remove as much as possible with a pipette All supernatants and precipitated parts are exosomes;

[0086] 2) Add 1ml Trizol to the exosome extracted above to fully lyse (mixed by ultrasonic oscillation), and let it stand for 5 minutes;

[0087] 3) Add 200 μl chloroform, shake and mix well for about 30 s, make the aqueous phase and organic phase fully contact, and let stand at room temperature for about 10 min;

[0088] 4) Centrifuge at 14000g for 15min at 4°C, transfer the RNA in the upper aqueous phase to another new RNase-free EP tube;

[0089] 5) Add an equal volu...

Embodiment 3

[0122] Embodiment 3 specificity evaluation experiment

[0123] There are currently 8 Kawasaki disease-specific miRNAs: miR-4739, miR-16, miR-483, miR-21, miR-19, miR-22, miR-1260, and miR-134 in serum exosomes of patients with Kawasaki disease and healthy people. The mean difference is significant, which may affect the detection specificity of the primers and probes of the present invention.

[0124] Using miR-4739, miR-16, miR-483, miR-21, miR-19, miR-22, miR-1260, miR-13410nM standard cDNA after reverse transcription as a template, using the above-mentioned Example 1 and The primers, probes and methods described in 2 were tested to verify 8 Kawasaki disease-specific miRNAs: miR-4739, miR-16, miR-483, miR-21, miR-19, miR-22, miR-1260, Whether miR-134 can be amplified.

[0125] Experimental results such as figure 1 shown. figure 1 Among them, the curves A~H are respectively the cDNA Q- PCR reaction results; from the amplification curve, it can be seen that miR-4739, miR-1...

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Abstract

Enclosed in the present invention is a primer set, a probe, and a kit for Kawasaki disease detection. A nucleic acid sequence combination comprises reverse transcription primers, amplification primers, and a probe sequence, and can qualitatively / quantitatively detect hsa-miR-197, hsa-miR-671, hsa-miR1246, and hsa-miR4436 in human serum effectively.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a primer set, a probe and a kit for Kawasaki disease detection. Background technique [0002] Kawasaki disease (Kawasaki disease KD) is an acute febrile systemic vasculitis syndrome of unknown etiology. The disease was first discovered in Japan by Japanese scholar Tomisaku Kawasaki in 1961 and first reported in 1967. Since 1970, KD has been reported successively in most countries or regions in the world, and the incidence rate of Asians is the highest. In recent years, KD has become one of the common diseases in children. KD mainly affects infants under 5 years old. The clinical features are fever, mucositis, rash, cervical lymph node enlargement, and extremity changes. The pathological changes are mainly systemic small and medium arterial vasculitis, especially It is easy to cause coronary artery inflammatory injury and the resulting thrombotic infarction, stenosis, dilation and aneu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/178
Inventor 曾宏彬陈杰罗宝花余旻斐
Owner 湖南赛哲智造科技有限公司