Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Application of HSP70 as molecular marker to detection of thalassemia and preparation of diagnostic kit

A thalassemia and molecular marker technology, applied in the application field of preparing diagnostic kits, can solve the problems of cumbersome operation steps, inability to make accurate judgments, long diagnosis time, etc., and achieve good specificity, diversified detection techniques, and convenient detection. Effect

Active Publication Date: 2021-04-27
CENT SOUTH UNIV
View PDF7 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Blood routine diagnosis cannot accurately determine whether it is thalassemia, and it needs to be combined with genetic diagnosis for further judgment; diagnosis based on globin gene DNA sequence requires high laboratory equipment and technical personnel, such as DNA chip and high-throughput DNA sequencing, the operation steps are relatively cumbersome , the diagnosis time is longer and the cost is higher

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Application of HSP70 as molecular marker to detection of thalassemia and preparation of diagnostic kit
  • Application of HSP70 as molecular marker to detection of thalassemia and preparation of diagnostic kit
  • Application of HSP70 as molecular marker to detection of thalassemia and preparation of diagnostic kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: In this example, qRT-PCR technology was used to detect the mRNA level of HSP70 in peripheral blood of mice.

[0021] Experimental steps:

[0022] 1) Collect peripheral blood of mice with anticoagulant EP tube;

[0023] 2) Add 1ml RNA extraction reagent (Trizol) solution to lyse red blood cells and extract total RNA; μ

[0024] 3) Detect the concentration of total RNA and reverse transcribe it into cDNA;

[0025] 4) Configure 10 μL of qPCR reaction system, as follows:

[0026] cDNA (30ng, 2μL); SYBR Green fluorescent dye (5μL); double distilled water (1μL); HSP70 or internal reference gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) primer (10mM, 2μL). The HSP70 forward primer sequence is 5'-CCATCGAGGAGGTGGATTAGA-3', as shown in SEQ ID NO.1; the HSP70 reverse primer sequence is 5'-AGTGCTGCTCCCAACATTAC-3', as shown in SEQ ID NO.2; GAPDH forward The primer sequence is 5'-ATCATCCCTGCATCCACT-3', as shown in SEQ ID NO.3; the GAPDH reverse primer sequence i...

Embodiment 2

[0029] Example 2: In this example, immunofluorescence was used to detect the expression level of HSP70 in peripheral blood of mice.

[0030] Experimental steps:

[0031] 1) Collect peripheral blood of mice with anticoagulant EP tube;

[0032] 2) Take 10 μl of anticoagulated blood and slowly add 1ml of ice-methanol for internal fixation for 20 minutes;

[0033] 3) After the fixation, evenly smear the blood cells on the glass slide;

[0034] 4) Permeabilize the membrane with 0.1% Triton X-100 by volume for 10 minutes, and wash 3 times with pH7.2-7.4 phosphate buffer saline (PBS);

[0035] 5) blocking with bovine serum albumin at a mass concentration of 1% for 30 minutes;

[0036] 6) HSP70 primary antibody (Abcam, USA) was incubated overnight at 4°C;

[0037] 7) Recover the antibody, wash it with PBS for 3 times, add the antibody of erythroid cell surface marker glycoprotein Ter119 (Biolegend, USA), and the goat anti-rabbit secondary antibody labeled with fluorescein isothioc...

Embodiment 3

[0041] Example 3: In this example, flow cytometry was used to detect the expression level of HSP70 in peripheral blood of mice.

[0042] Experimental steps:

[0043] 1) Collect peripheral blood of mice with anticoagulant EP tube;

[0044] 2) Take 10 μl of anticoagulated blood and slowly add 1ml of ice-methanol for internal fixation for 20 minutes;

[0045] 3) Wash the cells once, and permeabilize the membrane with 0.1% Triton X-100 for 10 minutes;

[0046] 4) After waiting for natural sedimentation, discard the supernatant, then add HSP70, Ter119, Hoechst 33342 flow-type antibodies and incubate in the dark for 20 minutes;

[0047] 5) After the incubation, the cells were washed and detected by a flow cytometer.

[0048] Experimental results such as image 3 Shown: the proportion of HSP70 positive cells detected by flow cytometry in peripheral blood cells of wild-type mice (Ter119 positive and Hoechst3342 negative) was 0.42 ± 0.09% ( image 3 Middle A), while thalassemia mi...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses an application of HSP70 as a molecular marker to detection of thalassemia and preparation of a diagnostic kit. mRNA and protein levels of peripheral blood HSP70 can be used as specific markers for diagnosis and treatment of thalassemia, expression level of the HSP70 in thalassemia is very high, whether the thalassemia exists or not can be accurately identified, and along with alleviation of thalassemia symptoms, the expression of the HSP70 is reduced. Therefore, the HSP70 not only can be used as the molecular marker for thalassemia diagnosis, but also can be used as a basis for evaluating a thalassemia treatment effect.

Description

technical field [0001] The invention relates to HSP70 as a molecular marker for detecting thalassemia and its application in preparing a diagnostic kit. The invention belongs to the technical field of thalassemia molecular markers. Background technique [0002] Thalassemia (referred to as "thalassemia") is a fatal and disabling genetic hemolytic anemia that seriously threatens human health. Streptoglobin thalassemia (beta thalassemia for short). Thalassemia is mainly caused by the imbalance of globin chains in red blood cells. According to the degree of imbalance in the number of α / β chains, thalassemia can be divided into mild, intermediate and severe thalassemia. Existing diagnostic techniques for thalassemia mainly include blood routine diagnosis, globin gene diagnosis, and hemoglobin analysis. Among them, blood routine diagnosis cannot determine whether it is thalassemia, and gene analysis is generally required to detect it; globin gene diagnosis has high requirements...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883G01N33/68
CPCC12Q1/6883G01N33/6893C12Q2600/158G01N2800/22
Inventor 刘静陈慧勇彭元亮张海航梁龙
Owner CENT SOUTH UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com