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Method for detecting haptoglobin (Hp) classified type

A technology of haptoglobin and polymerase, which is applied in the field of biomedicine and can solve problems such as low accuracy rate and complex typing methods for detecting haptoglobin

Active Publication Date: 2017-11-28
深圳中科唯新生物技术有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The present invention provides a method for detecting haptoglobin typing, to at least solve the technical problems of complex and low accuracy in the prior art for detecting haptoglobin typing

Method used

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  • Method for detecting haptoglobin (Hp) classified type
  • Method for detecting haptoglobin (Hp) classified type
  • Method for detecting haptoglobin (Hp) classified type

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Step 1: Saliva sample collection and processing

[0053] Rinse your mouth 30 minutes before sampling to remove food residues and residual microorganisms. Do not eat, drink, brush your teeth, smoke or chew gum within 30 minutes after rinsing your mouth; relax your cheeks and rub gently for 15-30 seconds to produce saliva, collect 2ml of saliva into a sterile collector.

[0054] Step 2: DNA (deoxyribonucleic acid) extraction from saliva samples

[0055] (1) Take 500ul (microliter) saliva sample, add 1ml (milliliter) buffer (100mmol / L (mmol / liter) Tris-HCl (trishydroxymethylaminomethane-hydrochloric acid buffer), pH8.0,0.5 %SDS (sodium dodecyl sulfonate), 10mmol / LEDTA (ethylenediaminetetraacetic acid)) and 6ul 20mg / ul (mg / microliter) of proteinase K, shake and mix on a vortex instrument;

[0056] (2) Treat the saliva treated in step (1) in a water bath at 55°C for 20 minutes;

[0057] (3) Add 600ul of phenol: chloroform: isoamyl alcohol (25:24:1), mix by inverting, cent...

Embodiment 2

[0078] Step 1: Peripheral blood sample processing and DNA extraction

[0079] (1) Take 2ml of peripheral blood containing EDTA anticoagulant, put it in a 10ml sterilized centrifuge tube, add 6mL 0.1mM EDTA, treat for 15 minutes to break the red blood cells, centrifuge at 3000rpm for 10min, discard the supernatant, and collect the precipitate;

[0080] (2) Add 1ml 0.1mM EDTA to the precipitate, mix well, transfer to a 2ml sterilized centrifuge tube, centrifuge at 5400rpm for 5min, and discard the supernatant;

[0081] (3) Add 1ml of buffer solution (100mmol / LTris-HCl, pH8.0, 0.5% SDS, 10mmol / LEDTA) and 6ul of 20mg / ul proteinase K to the precipitate, shake and mix on a vortex instrument, and then treat in a water bath at 55°C 20min;

[0082] (4) Add 600ul of phenol: chloroform: isoamyl alcohol (25:24:1), mix by inverting, centrifuge at 10000rpm for 5min, and take the supernatant;

[0083] (5) Add 600ul of chloroform:isoamyl alcohol (24:1) to the supernatant of step (4), centri...

Embodiment 3

[0103] Step 1: Same as Step 1 of Example 2.

[0104] Step 2: Gene PCR (polymerase chain reaction) amplification and product identification

[0105] (1) PCR amplification:

[0106] Use 10X Mix buffer, its composition is to contain Tris-HCl (final concentration: 12mmol / L), pH is 8.6, potassium chloride (final concentration: 40mmol / L), magnesium chloride (final concentration 1.8mmol / L), Taq enzyme (final concentration: 0.8U), a mixed solution of dNTP (final concentration: 250umol / L), and the DNA extracted from the saliva sample were used as templates for PCR amplification. Amplification primers are as follows:

[0107] F: 5'-ATAATACAGTTCGCGAGCTTCT-3';

[0108] R: 5'-GGGCAAGATACTCAACCTGTC-3'.

[0109] The PCR reaction procedure is as follows:

[0110] 92°C for 3min; 92°C for 48s, 55°C for 40s, 74°C for 45s, 26 cycles; 70°C for 10min; 8°C∝.

[0111] That is, pre-denaturation at a temperature of 92°C for 3 minutes; denaturation at a temperature of 92°C for 48 seconds, anneal...

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PUM

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Abstract

The invention provides a method for detecting the haptoglobin (Hp) type. According to the method, by designing specific primers, a to-be-detected sample containing Hp genes is subjected to gene amplification, and thus amplified products are obtained, and by conducting electrophoresis detection on the amplified products, the gene type of Hp of the to-be-detected sample can be determined. The method that type classifying is conducted on the Hp from the perspective of genes is built, and operation of the method is easy, convenient, fast and accurate so that the method can be applied to clinical practice.

Description

technical field [0001] The invention relates to the field of biomedicine, more specifically, to a method for detecting haptoglobin typing. Background technique [0002] At present, the population of diabetic patients is increasing day by day, and they are gradually becoming younger. These patients have a high risk of concurrent cardiovascular disease. According to rough statistics, about 75% of diabetic patients die of cardiovascular disease. In order to alleviate the symptoms of hyperglycemia in patients, reduce the occurrence of microvascular and macrovascular complications, and enable patients to live a normal life, it is particularly important to use appropriate drugs to prevent cardiovascular complications. Since cardiovascular disease in diabetic patients has a certain correlation with oxidative stress in the body, the American College of Cardiology and the World Health Organization (WHO) recommend the use of vitamin E and other antioxidants to prevent cardiovascular c...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 王天泽李敏
Owner 深圳中科唯新生物技术有限公司
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