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Rapid nucleic acid extraction method for fluorescent quantitative PCR (polymerase chain reaction) detection

A technology of fluorescence quantification and extraction method, applied in the fields of molecular biology and medical detection, can solve the problems of cumbersome operation and time-consuming, and achieve the effect of avoiding cross-contamination, shortening time, and improving amplification efficiency.

Inactive Publication Date: 2017-07-28
江苏睿玻生物科技有限公司
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Problems solved by technology

Although the purity of DNA extracted by this method is high, the operation is cumbersome and time-consuming

Method used

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  • Rapid nucleic acid extraction method for fluorescent quantitative PCR (polymerase chain reaction) detection

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Embodiment Construction

[0035] 1. Use two methods to extract DNA in urethral secretions, and use these two extraction methods to extract 3 samples at the same time, and compare them in parallel.

[0036] 1.1 Use Tiangen Oral Swab Genomic DNA Extraction Kit (DP322) to extract DNA from urethral secretions.

[0037] 1.1.1 Take 200μl of cell preservation solution containing urethral secretions and add 400μl of buffer GA.

[0038] 1.1.2 Add 20 μl of Proteinase K solution, vortex for 10 sec to mix, place at 56°C for 60 min, and vortex for several times every 15 min.

[0039] 1.1.3 Add 400μl buffer GB, mix thoroughly by inversion, and place at 70°C for 10min. At this point the solution should be clear and centrifuged briefly to remove droplets from the inner wall of the cap.

[0040] 1.1.4 Add 200μl absolute ethanol, mix thoroughly by inversion, and briefly centrifuge to remove the liquid droplets on the inner wall of the tube cap.

[0041] 1.1.5 Put the solution and flocculent precipitate obtained in th...

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Abstract

The invention relates to a rapid nucleic acid extraction method for fluorescent quantitative PCR (polymerase chain reaction) detection. The method is applicable to extraction of DNA (deoxyribonucleic acid) of pathogenic microorganisms such as mycoplasmas, Gram negative bacteria and viruses, and the extracted product DNA can be used for fluorescent quantitative PCR detection. Samples of the pathogenic microorganisms such as the mycoplasmas, the Gram negative bacteria and the viruses are washed once by normal saline, solution containing 5% of Chelex-100, 0.5% of NP-40, 0.5% of Triton X-100 and 1mM of EDTA (ethylene diamine tetraacetic acid) is added in precipitation, the pH (potential of hydrogen) of the solution ranges from 11 to 12, the solution is re-suspended, heated for 10 minutes at the temperature of 80-90 DEG C and centrifuged for 5 minutes at the speed of 12000rpm, and supernatant is used for fluorescent quantitative PCR detection. Compared with an existing nucleic acid extraction method, the rapid nucleic acid extraction method has the advantages that operation is simple and rapid, cross infection among the samples is avoided, the DNA yield of the samples is increased, and fluorescent quantitative PCR amplification efficiency is improved.

Description

technical field [0001] The invention belongs to the fields of molecular biology and medical detection, and relates to a rapid nucleic acid extraction method for fluorescent quantitative PCR detection, specifically referring to the simple and reliable method for extracting clinical sample DNA, and then using the fluorescent quantitative PCR method to extract DNA nucleic acid detection. Background technique [0002] Nucleic acid is the carrier of genetic information and the material basis of gene expression. Whether it is for nucleic acid structure or function research, it is first necessary to quickly and effectively separate and extract the required genomic nucleic acid from complex and diverse biological samples, and the quality and integrity of the extracted nucleic acid will directly affect the subsequent experimental results. At present, the traditional solution-based extraction methods commonly used for nucleic acid extraction include: alkaline extraction, guanidine is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 卿志荣田青
Owner 江苏睿玻生物科技有限公司
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