Construction method of novel coronavirus whole-genome high-throughput sequencing library, and kit for library construction

A coronavirus and whole genome technology, applied in the biological field, can solve problems such as slow timeliness, high requirements, and low data volume requirements

Active Publication Date: 2020-06-26
福州福瑞医学检验实验室有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sequencing of the whole virus genome by targeting the liquid-phase hybridization capture system has strong specificity and low data volume requirements; the disadvantage is that the requirement for the initial target cDNA is high, and there is a risk that the virus genome cannot be captured. The design cost is high, the timeliness is slow (more than 12 hours in total for hybridization and capture), and the adaptability to clinical transformation is not high; through literature search, the whole genome of RNA virus is high-throughput by using Targeted Multiplexing-PCR Sequencing (TMS, Targeted Multiplexing-PCR Sequencing) technology Quantitative sequencing studies are rarely reported

Method used

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  • Construction method of novel coronavirus whole-genome high-throughput sequencing library, and kit for library construction
  • Construction method of novel coronavirus whole-genome high-throughput sequencing library, and kit for library construction
  • Construction method of novel coronavirus whole-genome high-throughput sequencing library, and kit for library construction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] The viral RNA used in this example was extracted from the alveolar lavage fluid of patients with new coronary pneumonia by the magnetic bead method, a total of two cases; Safety level 3 (P3) laboratory completed.

[0087] The method provided in this example can be used to detect virus species in alveolar lavage fluid or to detect virus genome mutations from patients with confirmed new coronary pneumonia; the virus copy number is measured by the new coronavirus nucleic acid standard substance (high concentration) GBW (E) 091089 (China The N gene copy number of the Institute of Metrology and Science) was used as a reference, and the virus concentration (Copies / μL) was determined by absolute quantitative qRT-PCR (Table 3).

[0088] Table 3 RNA virus copy number and clinical information in alveolar lavage fluid

[0089]

[0090] The specific experimental method is as follows:

[0091] The viral single-stranded RNA extracted from the alveolar lavage fluid was reverse-tr...

Embodiment 2

[0114] The viral RNA samples 46d1 and 50d1 used in this embodiment are the same as those in Embodiment 1.

[0115] The specific experimental method is as follows:

[0116] Viral single-stranded RNA extracted from alveolar lavage fluid used 34 gene-specific reverse primers (COV-1-R, COV-8-R, COV-12-R, COV-20-R, COV-30- R, COV-38-R, COV-47-R, COV-54-R, COV-62-R, COV-71-R, COV-80-R, COV-86-R, COV-94-R, COV-102-R, COV-111-R, COV-119-R, COV-125-R, COV-132-R, COV-141-R, COV-146-R, COV-155-R, COV- 162-R, COV-172-R, COV-179-R, COV-187-R, COV-195-R, COV-202-R, COV-210-R, COV-220-R, COV-228- R, COV-233-R, COV-239-R, COV-247-R, COV-252-R) mixed (genome direction 3'-5'), reverse transcribed into a strand of cDNA (1st cDNA), The 1st cDNA synthesis kit is selected as: TAKARA PrimeScript 1 st strand cDNA Synthesis Kit (TAKARA, CatNo.6110A); 1st cDNA was purified for subsequent amplification.

[0117] Use the anchored multiple amplification primer set 1 to carry out PCR reaction. The PC...

Embodiment 3

[0140] The viral RNA used in this example was extracted from throat swab samples of patients with new coronary pneumonia by magnetic bead method; the extraction and quality inspection of RNA was conducted by Chinese Academy of Medical Sciences / Peking Union Medical College Hospital Institute of Pathogen Biology Biosafety Level 3 ( P3) The laboratory is completed.

[0141] The method provided in this example can be used to detect virus species in throat swab samples or to detect viral genome mutations from patients with confirmed or suspected novel coronavirus pneumonia; The copy numbers of N gene and E gene (National Institute of Metrology, China) were used as reference, and the virus concentration (Copies / μL) was determined by absolute quantitative qRT-PCR (Table 8). .

[0142] Table 8 Pharyngeal swab RNA virus copy number, clinical information

[0143]

[0144] The specific experimental method is as follows:

[0145] The viral single-stranded RNA extracted by the magnet...

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Abstract

The invention provides a construction method of a novel coronavirus whole-genome high-throughput sequencing library, and a kit for library construction. The method comprises the following steps: 1) carrying out reverse transcription on virus RNA; (2) carrying out a first round of PCR reaction by utilizing multiple amplification primers of an anchoring part Illumina linker sequence; and 3) carryingout a second round of PCR reaction by using a labeled Illumina library amplification primer, and purifying the amplification product to obtain the high-throughput sequencing library. The anchoring multiplex amplification primer combination provided by the invention is utilized, so the efficient targeted enrichment can be performed on the genome of the novel coronavirus COVID-19, the influence that an existing method is low in targeting, low in experimental timeliness and prone to being brought into host background pollution is overcome, COVID-19 virus whole genome sequencing can be completedwithin a short time with the small sequencing data volume and cost, so differential diagnosis and virus mutation identification of the COVID-19 virus are achieved.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a method for constructing a novel coronavirus whole genome high-throughput sequencing library and a kit for library construction. Background technique [0002] The new coronavirus COVID-19 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) belongs to the Betacoronavirus genus, similar to other coronavirus genomes that have been discovered, the COVID-19 genome contains 6 main open reading frames (ORF , Open Reading Frame), respectively orf1ab, ORF3a, ORF6, ORF7a, ORF8 and ORF10, and some other accessory genes (S gene, E gene, M gene and N gene). High-depth sequencing of the whole genome of the virus is currently one of the most sensitive and specific methods to obtain the overall nucleic acid mutation, virus typing, and evolutionary relationship of the virus genome; however, due to host nucleic acid background interference and other factors, the current mainstream ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/08C12Q1/70C12Q1/6869G16B20/20G16B20/30C12R1/93
CPCC40B50/08C12Q1/701C12Q1/6869G16B20/20G16B20/30C12Q2535/122Y02A50/30
Inventor 王洋李杰王辰高汉林郭超王健伟任丽丽杨明刘静赵晔
Owner 福州福瑞医学检验实验室有限公司
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