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Kit based on serum miRNA as well as use method and application of kit

A kit and serum technology, applied in the field of serum miRNA-based kits, can solve problems such as difficulty in laboratory development, poor sensitivity, and dependence on viremia

Inactive Publication Date: 2014-09-24
JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Virus isolation is time-consuming and laborious, and the biosafety requirements of the test operation are high, which is difficult for general laboratories to carry out
Serological methods for detecting specific IgG antibodies are suitable for retrospective diagnosis and epidemiological investigations, while methods for detecting early antibody IgM are less sensitive
PCR-based viral nucleic acid detection is fast, specific, and sensitive, but depends on the presence of viremia

Method used

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  • Kit based on serum miRNA as well as use method and application of kit
  • Kit based on serum miRNA as well as use method and application of kit
  • Kit based on serum miRNA as well as use method and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1: serum total RNA is extracted

[0016] Collect 6 serum samples from patients with acute SFTS and 6 healthy normal serum samples, mix the two groups of serum (200μL each), and extract total RNA using the phenol-chloroform method. Specific steps: 1. Take 100ul serum samples and add 300ul DEPC water 1.5ml EP, then add 1ul10f / ul cel-miR-238 for parameter calibration, mix well, add 200ul water-saturated phenol, shake vigorously, let stand for 3min, add 200ul chloroform, shake well again and mix 16000g Centrifuge for 15 minutes at room temperature. 2. Aspirate the supernatant, add isopropanol twice the volume of the supernatant, then add 1 / 10 of the volume of the supernatant 3M sodium acetate about 40ul, mix well, let stand at -20°C for 2h, and then store at 4°C, 16000g , Centrifuge for 20min. 3. Discard the supernatant, add 1ml of 75% ethanol, centrifuge at 16000g at 4°C for 20min. 4. Discard the supernatant, dry at room temperature, add 20ul DEPC water to di...

Embodiment 2

[0017] Example 2: Detection of differential expression of serum miRNA in SFTS acute infection

[0018] TaqMan Array Human v3.0 chip (Applied Biosystems, USA) was used for detection. For each group of samples, 212 miRNAs were detected on the Array A plate, and U6snRNA was selected as the internal reference. 1. Reverse transcription using Taqman MiRNA Reverse Transcription Kit (Applied Biosystems, USA): add 50ng total RNA to 4.5ul Megaplex pool reverse transcription mixture, including Megaplex RT Primers (10×) 0.8μl, dNTPs 0.2μl, MultiScribe Reverse Transcriptase (50U / μl) 1.5μl, RT Buffer (10×) 0.8μl, MgCl2 (25mM) 0.9μl, RNase inhibitor (20U / μl) 0.1μl, Nuclease-free water 0.2μl. 2. Pre-amplification: TaqMan PreAmp Mastermix (2×) 12.5 μl and Megaplex PreAmp Primer (10×) 2.5 μl, reverse transcription product 2.5 μl, Nuclease-free water 7.5 μl. 3. The qRT-PCR reaction was carried out on a 7900HT instrument (Applied Biosystems): dilute the pre-amplification reaction solution 4 tim...

Embodiment 3

[0020] Example 3: Verification of serum differentially expressed miRNA

[0021] The 30 miRNAs specifically expressed in the sera of SFTS acutely infected patients obtained in Example 2 were verified by qRT-PCR. 58 sera were selected for the verification experiment, 29 of which were from the acute phase of SFTS infection, and 29 were from healthy people. 30 miRNA-specific primers and probes were designed and synthesized by Applied Biosystems in the United States, and artificially synthesized single-stranded miR-16 was used as an internal reference gene standard. Each sample of 22 miRNAs was detected in triplicate, and the differential expression level was calculated by 2-(ΔCtmiRNA-ΔCtcontrol). In this experiment, SPSS18.0 software was used for statistical analysis, and the data representation method was median ± SD. The comparison between groups was performed by t test, and P<0.05 was considered significant difference.

[0022] Table 1 Comparison of serum miRNA expression dif...

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Abstract

The invention belongs to the technical field of biology and in particular relates to a kit based on serum miRNA as well as a use method of the kit and application of the kit to early infection detection of fever with thrombocytopenia syndrome viruses. MiRNA molecules comprise hsa-miR-188-3p, hsa-miR-517a-3p, hsa-miR-518f-5p and hsa-miR-511-3p. The expression of the four miRNAs in early attack of the fever with thrombocytopenia syndrome viruses is specifically and remarkably increased through identification by adopting a Taqman low-density chip and a qRT-PCR (quantitative reverse transcription polymerase chain reaction) method. The four serum miRNAs are used as molecular targets, and a quick, sensitive and specific detection method for acute infection of the fever with thrombocytopenia syndrome viruses is built. The hsa-miR-188-3p, the hsa-miR-517a-3p, the hsa-miR-518f-5p and the hsa-miR-511-3p are applied to early diagnosis of fever with thrombocytopenia syndrome virus infection, and the kit is used for detecting the hsa-miR-188-3p, the hsa-miR-517a-3p, the hsa-miR-518f-5p and the hsa-miR-511-3p.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit based on serum miRNA, a use method thereof and an application in detection of early infection of fever with thrombocytopenia syndrome virus. Background technique [0002] Fever with thrombocytopenia syndrome (Severe fever with thrombocytopenia syndrome, SFTS) is a newly discovered hemorrhagic disease caused by bunya virus in my country in recent years, and the pathogen is called SFTS virus. The disease is mainly distributed in rural areas in mountainous and hilly areas, and is sporadic, and the crowd is generally susceptible. The clinical manifestations are fever, with body temperature above 38°C, accompanied by fatigue, nausea, and vomiting. Some cases have headache, muscle aches, and diarrhea. The vast majority of patients have a good prognosis, but some cases are critically ill, with disturbance of consciousness, skin ecchymosis, gastrointestinal bleeding, pulmo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6809C12Q1/6883C12Q2600/158C12Q2600/178C12Q2531/113C12Q2561/101C12Q2525/207
Inventor 祁贤汤奋扬鲍倡俊崔仑标宋勇春周明浩
Owner JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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