Method for co-extracting DNA/RNA (Deoxyribonucleic Acid/Ribonucleic Acid) virus nucleic acid

A RNA virus and co-extraction technology, which is applied in the field of rapid co-extraction of nucleic acid from respiratory specimens based on magnetic beads, can solve the problems of cumbersome steps, great harm to human body, and reduced amount of nucleic acid, and achieve good application prospects and accurate and reliable results

Inactive Publication Date: 2016-09-07
HANGZHOU FIRST PEOPLES HOSPITAL
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Problems solved by technology

[0003] Although the current nucleic acid separation technology is developing rapidly, there are many shortcomings. For example, the viral nucleic acid co-extraction kits on the market are mainly used to detect viruses in blood, such as the TIANGEN kit, and are generally based on the media adsorption method. Transf

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  • Method for co-extracting DNA/RNA (Deoxyribonucleic Acid/Ribonucleic Acid) virus nucleic acid
  • Method for co-extracting DNA/RNA (Deoxyribonucleic Acid/Ribonucleic Acid) virus nucleic acid
  • Method for co-extracting DNA/RNA (Deoxyribonucleic Acid/Ribonucleic Acid) virus nucleic acid

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Embodiment 2

[0044] Example 2 Detection of housekeeping genes GAPDH and HTN3

[0045] The GAPDH gene is an essential gene to maintain the minimum function of human cells, that is, the housekeeping gene. The amount of DNA in the nucleic acid extracted from the throat swab can be measured by detecting the amount of the GAPDH gene, and the HTN3 gene (Histatin 3) is the saliva The specific marker of mRNA can measure the amount of RNA in the nucleic acid extracted from the throat swab by detecting the amount of the HTN3 gene. The gene sequence is shown in Table 3.

[0046] (1) Co-extraction of housekeeping gene nucleic acid from throat swab specimens

[0047] (1) Sample preparation: Collect 5 throat swab specimens from healthy people, add 1ml of sterilized physiological saline to a sterile tube, shake and mix well, squeeze the cotton swab dry, and immediately transfer all the liquid to a 1.5ml centrifuge tube, Take 200μl and add it to another 1.5ml centrifuge tube, centrifuge for 5min, and disca...

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Abstract

The invention provides a method for co-extracting DNA/RNA (Deoxyribonucleic Acid/Ribonucleic Acid) virus nucleic acid. The method comprises the following steps: cracking a sample, which is diluted with saline, with a guanidine isothiocyanate cracking solution containing an RNA precipitating aid agent; releasing and dissociating DNA or RNA of different nucleic acid viruses in the sample into a cracking solution; adding magnetic beads to form a magnetic bead-nucleic acid compound; washing to obtain a nucleic acid eluting solution. The novel method for co-extruding the DNA and the RNA from a respiratory tract sample is established based on the nano magnetic beads; in a whole process, toxic reagents including phenol, chloroform, beta-mercaptoethanol and the like are not used, and time and labor are saved. The method is used for commonly extracting the DNA and the RNA from the respiratory tract sample and is also applicable to nucleic acid co-extraction of other samples with different types, such as urine and blood; impurities including protein and the like are efficiently removed and the degradation of the nucleic acid is reduced; the aim of detecting DNA viruses and RNA viruses in the same tube at the same time is realized. The method is simple, convenient, rapid and safe, and is particularly suitable for fluorescent quantitative PCR (Polymerase Chain Reaction) detection and the like.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a method for rapidly co-extracting nucleic acids from respiratory specimens based on magnetic beads, including DNA viruses and RNA viruses. Background technique [0002] Multiplex RT-qPCR (Reverse transcription quantitative PCR, RT-qPCR) technology has become the main method for detecting viruses in clinical laboratories due to its advantages of simple operation, high sensitivity, strong specificity, and relatively low cost. Currently, multiplex PCR technology is mostly used for Detect the same type of viral nucleic acid, but in clinical practice, common respiratory viruses are divided into DNA viruses and RNA viruses, such as adenovirus (Adenovirus, ADV) is a DNA virus, and respiratory syncytial virus (Respiratory syncytial virus, RSV) is For RNA viruses, both are equally important, and the simultaneous detection of DNA and RNA viruses by multiplex quantitative PCR has...

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
CPCC12N15/1013C12Q1/6806C12Q2563/143C12Q2563/149C12Q2523/308
Inventor 余道军何慧陈懿
Owner HANGZHOU FIRST PEOPLES HOSPITAL
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