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RNA one-step-lysis and one-tube RT-PCR detection method

A technology of RT-PCR and detection method, which is applied in the field of RNA one-step cleavage and one-tube RT-PCR detection, which can solve problems such as difficult control of condition optimization, high complexity and difficulty, non-specific amplification and false positives, and achieve Improve reaction efficiency and success rate, reduce non-specific reactions, and reduce the effect of occurrence probability

Active Publication Date: 2013-10-02
ZHEJIANG JFK BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the optimal reaction conditions of RT reaction and PCR reaction are inconsistent, it is difficult to control the optimization of the conditions of the one-tube RT-PCR on the market, which often causes serious non-specific amplification, and the cost / selling price is high. Compared with the traditional two-pipe method, there is a great improvement
[0004] Although RT-PCR has a high degree of popularization in the field of scientific research, there is still a lot of room for improvement in clinical testing applications; the main problems include: the steps of RNA extraction and purification are cumbersome, and generally require additional DNase enzyme digestion to remove traces. The steps of quantitative genomic DNA; the two kinds of enzymatic reactions (RT and PCR) have different optimal reaction conditions, are easily affected by various inhibitory substances, and are also easily troubled by non-specific amplification and false positives; therefore, the overall The complexity and difficulty of online operations are high, which is not conducive to the promotion and application of clinical testing

Method used

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  • RNA one-step-lysis and one-tube RT-PCR detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Washing to remove genomic DNA in the supernatant of cell lysate

[0042] Primer A for detecting hTERT mRNA is Oligo(dT) 29 , PCR primer sequences are: 5'-AAGTCCTACGTCCAGTGCCAGGGGA-3' and 5'-GCTTGTTCTCCATGTCGCCGTAGC-3'.

[0043] Human lung cancer cell line A549 cells were cultured in a 24-well plate, 1000 cells / well, after overnight culture, the culture medium was aspirated, + 200ul lysate, repeatedly pipetted, transferred to a 1.5ml centrifuge tube, and shaken at room temperature for 10min , centrifuged at 4°C and 15000rpm for 20min, and the supernatant was taken to obtain the cell lysate supernatant; Mercaptoethanol, 0.1mg / ml protamine DNA (Sigma Company), the solvent is Tris-HCl with 10mmol / L and pH7.5.

[0044] Take 20ul of the supernatant of the above-mentioned cell lysate, add it to a 0.2ml PCR thin-walled tube, incubate at 50°C for 30min, then blot up the liquid in the tube, and directly add 20ul of the PCR reaction solution (containing 20ul of PCR bu...

Embodiment 2

[0049] Example 2: Immobilization of Primer A by Magnetic Beads

[0050] Primer A is Oligo(dT) 29 . PCR primers are hTERT-specific primers:

[0051] 5'-AAGTCCTACGTCCAGTGCCAGGGGA-3' and

[0052] 5'-GCTTGTTTCTCCATGTCGCCGTAGC-3'. TBST buffer 20ul, containing 10ug M-280 streptavidin magnetic beads and 2pmol biotinylated primer A (biotinylated primer A is directly modified and added to the 5' end during synthesis), put into a 0.2ml PCR thin-walled tube, room temperature for 1hr , absorb the magnetic beads in the tube wall with a magnet, blot the liquid in the tube, add 50ul TBST buffer solution, mix well, blot dry, wash repeatedly in this way for a total of 6 times, and finally blot dry; add 50ul TE buffer solution, set aside.

[0053] Cell lysis: Cell culture in 24-well plate, absorb culture medium, + 200ul lysate (same as Example 1), pipette repeatedly, transfer to 1.5ml centrifuge tube, shake at room temperature for 10min, centrifuge at 4°C and 15000rpm for 20min, take to ...

Embodiment 3

[0057] Example 3: Coupling and immobilizing primer A in direct PCR tube

[0058] TBST buffer 50ul, containing 5pmol biotinylated Oligo(dT) 29 (See Example 2), put it into a streptavidin-coated 0.2ml PCR thin-wall tube, room temperature for 1hr, blot the liquid in the tube, add 100ul TBST buffer, mix well, blot dry, and wash repeatedly for a total of 3 Add 100ul TE buffer solution, then, blot the liquid in the tube, add 50ul cell lysate supernatant (same as Example 1), incubate at 50°C for 30min, blot the liquid in the tube, wash once with TBST buffer , add 50ul RT reaction solution (containing 50ul RT buffer, 0.5mmol / LdNTP, 10U MMLV RT enzyme), incubate at 42°C for 30min, blot the liquid in the tube, add 50ul PCR reaction solution (SYBR green I fluorescence quantification), and carry out the PCR program (Pre-denaturation at 95°C for 2min, followed by 40 cycles of 95°C for 3s and 66°C for 30s, two-step method), SYBR green I fluorescence quantitative analysis. With this method...

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Abstract

The invention provides a RNA one-step-lysis and one-tube RT-PCR (one-Step-lysis and One-tube RT-PCR, SORT-PCR) detection method. Compared to the conventional RT-PCR method, the RNA one-step-lysis and one-tube RT-PCR method does not need to purify and extract RNA, RNA is hybridly combined to a fixed specific primer or Oligo primer after cell lysis, other components in the cell lysis solution supernatant are washed away, then the RNA is added into a RT reaction system to perform RT reactions. After being dried again, the RNA is added into a PCR reaction system to perform PCR reactions, and the result can be detected by the fluorescence real-time quantitative analyzing method. The RNA one-step-lysis and one-tube RT-PCR detection method shortens the RNA detection process of more than 3 hours to 2 hours, and has the advantages of simple operation, high detection sensitivity and specificity, and suitability for detecting RNA from various sources of sample lysis.

Description

(1) Technical field [0001] The invention relates to a one-step cleavage-tube RT-PCR detection method for RNA. (2) Background technology [0002] RNA is the carrier of genetic information transmission. RT-PCR (reverse transcription–polymerase chain reaction) refers to a method that combines RT (Reverse Transcription, reverse transcription) and PCR (Polymerase Chain Reaction, polymerase chain reaction) reactions. RT-PCR uses RNA as a template cDNA synthesis, or RT, combined with PCR provides a fast and sensitive method for analyzing gene expression. RT-PCR detection or quantitative analysis of RNA is more sensitive and easier to operate than other RNA analysis techniques including Northern blotting, RNase protection analysis, in situ hybridization and S1 nuclease analysis. It is a powerful means for detection and analysis, detection and analysis of pathogens, etc. [0003] The template for RT-PCR can be total RNA or poly(A)+RNA. RT requires RT enzymes (such as MMLV, AMV, e...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 陈燃金晓铮
Owner ZHEJIANG JFK BIOLOGICAL TECH
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