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A gene detection kit free from nucleic acid purification

A detection kit and sodium chloride technology, applied in the fields of molecular biology and gene diagnosis, can solve the problems of unsolved sample DNA extraction, cumbersome sample DNA extraction steps, concentration and cross-contamination of open tubes, etc., and shorten the detection cycle. , saving detection cost, low cost effect

Active Publication Date: 2021-08-27
众福健康科技(杭州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even if high-temperature pretreatment steps are used to release DNA in blood samples without extracting nucleic acids, there are still steps that may easily cause cross-contamination, such as concentration and tube opening
The extraction steps of sample DNA are cumbersome, the operation time is long and it is easy to cause pollution. The existing technology does not solve the problem of sample DNA extraction

Method used

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  • A gene detection kit free from nucleic acid purification
  • A gene detection kit free from nucleic acid purification
  • A gene detection kit free from nucleic acid purification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Kit preparation:

[0040] Sample Diluent 1:

[0041] 0.1M sodium hydroxide, 100mM sodium chloride

[0042] PCR reaction solution 1 is shown in Table 1:

[0043] Table 1 PCR reaction solution component 1

[0044]

[0045]

[0046] When the kit was used for B5801 gene detection, the primers and probe sequences used were as shown in Table 2:

[0047] Table 2 Primers and probes for B5801 gene detection

[0048]

Embodiment 2

[0050] Sample Diluent 2:

[0051] 0.04M NaOH 200mM NaCl

[0052] PCR reaction solution 2 is shown in Table 3 or Table 4:

[0053] Table 3 PCR reaction solution components 2-1

[0054]

[0055]

[0056] Table 4 PCR reaction solution components 2-2

[0057] components (μl) 10×PCRBuffer 2 2mMdNTPs 1 10 μM Primer 1 1 10 μM Primer 2 1 10 μM fluorescent probe 2 1 Internal reference gene primer 1 1 Internal reference gene primer 2 1 Internal reference gene fluorescent probe 1 DMSO (dimethyl sulfoxide) 1 Tween-20 1 Sorbitol 2 anti-jamming polymerase 0.6

[0058] When the kit was used for ALDH2 gene detection, the primers and probe sequences used were as shown in Table 5:

[0059] Table 5 Primers and probes for ALDH2 gene detection

[0060]

[0061]

Embodiment 3

[0063] The nucleic acid amplification carried out by the kit of the present invention mainly includes the following steps:

[0064] Collection of human peripheral blood samples

[0065] Use a disposable sterile syringe to extract 1-2 ml of venous blood from the subject, inject it into a blood collection tube containing EDTA anticoagulant, and immediately invert the blood collection tube gently for 5 to 10 times to mix the anticoagulant and venous blood thoroughly .

[0066] sample dilution

[0067] Sample diluent:

[0068] 0.1M sodium hydroxide, 100mM sodium chloride

[0069] Take 10 μL of the collected human peripheral blood sample and add it to 290 μL of the sample diluent, shake and mix well, and centrifuge briefly to collect the liquid to the bottom of the tube.

[0070] Reaction solution preparation

[0071] The anti-interference PCR reaction solution, primer-probe mixture, positive control and negative control were mixed to prepare a fluorescent PCR reaction solutio...

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Abstract

The invention relates to a gene detection kit free from nucleic acid purification, belonging to the technical fields of molecular biology and gene diagnosis. The kit provided by the present invention includes: sample diluent, anti-interference PCR reaction solution, anti-interference polymerase, primer probe mixture, positive control substance and negative control substance; the sample diluent is composed of sodium hydroxide and chloride Sodium aqueous solution; the anti-interference PCR reaction solution includes fluorescent PCR buffer, dNTPs and anti-interference agent. Using the kit of the invention to detect genes does not require nucleic acid purification of samples, the detection is fast and simple, the detection cycle is greatly shortened, and the detection efficiency and detection throughput are effectively improved.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and gene diagnosis, in particular to a gene detection kit free from nucleic acid purification. Background technique [0002] Polymerase chain reaction (PCR) was first conceived by Mullis of the United States in 1983. In 1985, he invented the polymerase chain reaction, that is, a simple DNA amplification method, which means the real birth of PCR technology. PCR is a process in which a specific nucleic acid molecule is used as a template to repeatedly synthesize double-stranded DNA through primer extension. It can be regarded as a special DNA replication in vitro. The biggest feature of PCR is that it can greatly increase a small amount of DNA. PCR technology is very sensitive, theoretically only need a nucleic acid molecule can be amplified. PCR (Polymerase Chain Reaction) uses DNA denaturation in vitro at a high temperature of 95°C to become a single strand. At a low temperature (often...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/686
CPCC12Q1/686C12Q2563/107
Inventor 倪小龙李亚波欧阳云
Owner 众福健康科技(杭州)有限公司
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