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Top-speed RNA library building method and kit

A kit and extremely fast technology, applied in the biological field, can solve the problems of low detection rate of low-copy pathogens, low success rate of library construction, and low concentration, and achieve the effect of simple operation, wide application, and short process

Pending Publication Date: 2021-11-30
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for the clinical diagnosis industry, the concentration of RNA extraction from pathogenic samples is low, generally around 5-10 ng / μL, and then the host rRNA is removed, and the success rate of library construction is low.
Therefore, pathogen detection generally uses the method of not removing host rRNA for RNA library construction, but in the extracted total RNA, since host rRNA accounts for 90%, the detection rate of low-copy pathogens will decrease or even miss detection. The probability

Method used

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  • Top-speed RNA library building method and kit
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  • Top-speed RNA library building method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: A method for extremely fast RNA library construction

[0033] In this example, using the total RNA of 293 Cells, an extremely fast RNA library construction process was established. See the schematic diagram for the process and principle. figure 1 . The specific method is as follows:

[0034] 1) Host rRNA removal:

[0035] The Random primer used is NNNNNN (synthetic), 5.8S / 18S / 28S rRNA probe sequence reference CN202110257924 .X).

[0036] Table 1

[0037] components Dosage 293 Total RNA 10~500ng 4 μM 5.8S / 18S / 28S rRNA probe mix (CN202110257924 .X) 1 μL 1 mM Random Primer 1 μL Add DEPC water to 15 μL

[0038] 5 min at 85°C, 2 min at 72°C, 10 min at 60°C, and store at 4°C.

[0039] 2) Synthesis of 1st cDNA:

[0040] 1st Reaction buffer: 200 mM Tris-HCl, 250 mM KCl, 10 mM MgCl2, 5 mM dNTP, 20 mM DTT, pH 8.3.

[0041] Table 2

[0042] components Dosage The above reaction system 15μL 1st Re...

Embodiment 2

[0061] Example 2: Comparative analysis of an extremely fast RNA library construction method and traditional library construction methods

[0062] In this example, we compared the extremely fast RNA library construction method of the present invention with the traditional RNA library construction method. The traditional RNA library construction method uses NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina for library construction, the input amount of RNA is 500 ng, and the library construction process is carried out according to the product manual. In this extremely fast RNA library construction method, we have carried out library construction with RNA inputs of 500ng, 100ng, and 50ng, and the library construction process refers to Example 1.

[0063] Qubit was used to quantify the constructed library, and Agilent 2100 Bioanalyzer was used to verify the library size distribution. The constructed library was sequenced and analyzed on the Illumina NovaSeq 6000 pla...

Embodiment 3

[0067] Example 3: Application test of a kit for rapid RNA library construction in pseudovirus detection

[0068] In this example, we will COVID-19-pseudovirus (2019-nCOV pseudovirus) (Yeasen, 11900) according to 10 4 -10 7 Add 2.5 μL to 50 ng of 293 Total RNA at the ratio of cells / mL, use an extremely fast RNA library building kit to build a library and send it for sequencing.

[0069] COVID-19-pseudovirus (2019-nCOV pseudovirus) (Yeasen, 11900) retroviral vector, the vector is loaded with the partial sequence of the COVID-19 virus ORF1 a / b gene, the entire sequence of the E gene and the N gene coding region, where N The gene sequence is shown in SEQ ID No.1. According to the N gene sequence, we used QRT-PCR to detect the primers ORF1ab-F: 5´-GTGARATGGTCATGTGTGGCGG-3´, ORF1ab-R: 5´-CARATGTTAAASACACTATTAGCATA-3´, and detected the RNA of COVID-19-pseudovirus after 1st cDNA synthesis 1st cDNA synthesis efficiency. Quantitative PCR analysis was performed on the 100-fold dilute...

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Abstract

The invention provides a top-speed RNA library building method. The method comprises the following steps: adding a reverse transcription hindering probe 5.8S / 18S / 28S rRNA probe mix and a random primer into total RNA, conducting mixing, and then performing annealing; adding reverse transcriptase, and synthesizing 1st cDNA; synthesizing 2nd cDNA by using polymerase, and recovering the 2nd cDNA by using magnetic beads to obtain ds-cDNA; carrying out fragmentation, terminal repairing and linker connection on the ds-cDNA; and performing library amplification and recovery. The invention further discloses a corresponding top-speed RNA library building kit. According to the invention, rapid library building of RNA can be completed within 3 hours, and the kit can be widely applied to the field of pathogen diagnosis.

Description

technical field [0001] The patent of the present invention relates to an extremely fast RNA library construction method and a kit, which belong to the field of biotechnology. Background technique [0002] RNA-seq is a technique for analyzing the genome by examining gene expression levels. Transcriptome research can study gene function and gene structure from the overall level, reveal biological development and disease occurrence process, and has been widely used in basic research, clinical diagnosis and drug research and other fields. RNA-seq technology can provide more transcription information of organisms more quickly and accurately, and is widely used in the field of biomedicine. In the direction of clinical diagnosis, through RNA-seq technology, the detection of bacteria, fungi, RNA viruses, parasites and other pathogens can be completed at one time, with high detection rate and high sensitivity. Especially in the era of the new coronavirus epidemic from the end of 20...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06
CPCC40B50/06
Inventor 刘娜江翱罗元廷曹振宋东亮
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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