Bronchoalveolar lavage fluid sample library building method based on nanopore sequencing platform, identification method and kit

A technology of bronchoalveolar lavage fluid and kit, applied in the field of metagenomic identification, can solve the problems of increasing the difficulty and time of biosignal splicing, imbalance of bacterial abundance ratio, serious identification problems, etc., and achieves great commercial promotion value and rapid detection. The effect of meeting the demand and shortening the time of building the library

Active Publication Date: 2020-04-07
BEIJING SIMCERE MEDICAL LAB CO LTD +2
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AI Technical Summary

Problems solved by technology

There are many problems in the identification of next-generation sequencing, such as: the identification of next-generation sequencing usually takes a long time from sample to result (>72h); Multi-sample combined sequencing to reduce costs; third-generation sequencing such as nanopore sequencing, with its excellent features including ultra-long sequence read length, real-time data generation, bioinformatics analysis, and compact and portable equipment, seems to be a relatively fast and convenient metagenomic research at present. method
[0007] First of all, there are some necessary purification steps in the whole wet experiment process, so the overall process is not as presented in the article and can be completed in 4 hours. According to the actual test, the whole process needs to be completed in 5.5 hours; secondly, because the host DNA step is removed as much as possible Most of the host DNA in the sample is removed, resulting in the extraction concentration of the sample is often extremely low
For example, in the process of building a sample library, due to the failure of PCR, it is impossible to continue building the library; more importantly, due to the PCR step in the library building process, the amplification bias is inevitably introduced, resulting in some bacteria with high GC content. There are serious problems in the identification of bacteria, and the proportion of bacterial species abundance is seriously unbalanced

Method used

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  • Bronchoalveolar lavage fluid sample library building method based on nanopore sequencing platform, identification method and kit
  • Bronchoalveolar lavage fluid sample library building method based on nanopore sequencing platform, identification method and kit
  • Bronchoalveolar lavage fluid sample library building method based on nanopore sequencing platform, identification method and kit

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Effect test

Embodiment 1

[0130] Embodiment 1 Removal of host reaction system optimization

[0131] The present invention is by setting different saponin concentration (5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%...) and the dosage of HL-SAN enzyme (10 μ L, 5 μ L, 3 μ L, 1 μ L …) carried out host removal experiments on several clinical samples, and found that although the heterogeneity of the samples was very large, the effect of saponin concentration above 1% was similar to that of the 5% saponin used by Professor Grady. Significantly increase the amount of final extracted nucleic acid. Finally, in order to meet the requirements of library construction, we determined the treatment conditions of sputum and alveolar lavage fluid respectively by comprehensively considering various factors such as the effect of host removal and the balance of bacterial loss, and further used randomly collected clinical samples. After pre-processing, the library construction kit was used to build the library on the machine, an...

Embodiment 2

[0132] The identification result of embodiment 2 clinical positive pathogen sample

[0133] The present invention aims at 30 samples each of bacterial infection sputum and alveolar lavage fluid samples collected at random from clinical culture positive, carries out metagenome database construction and sequencing identification according to the above method, and uses clinical culture results as a control (the clinical culture results derived from microbial culture and mass spectrometric identification). Results All the databases were successfully built and put on the computer. Table 1 below shows the comparison results of the detailed identification results and the culture gold standard.

[0134] Table 1. Detailed comparison table between the pathogen detection results of the process of the present invention and the culture gold standard

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[0141] The results showed that 20 cases of alveolar lavage fluid an...

Embodiment 3

[0144] Embodiment 3 and the comparison of Grady flow process

[0145] The present invention further randomly collected 27 cases of clinically positive bacterial infection sputum samples and 23 cases of alveolar lavage fluid samples, and carried out pathogen identification strictly according to Grady's development ( figure 1 ), the results showed that there were 1 case of sputum sample and 12 cases of alveolar lavage fluid samples, all of which failed to build the database (because the nucleic acid concentration after PCR was <4ng / μg, they could not be sequenced on the machine). The following table 3 is the comparison result of the detailed identification results of the samples successfully constructed by Grady and the culture gold standard.

[0146] Table 3. The detailed comparison table of the successful results of the Grady process pathogen database construction and the culture gold standard

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[0150]Table 4. Successful results and positiv...

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Abstract

The invention relates to a bronchoalveolar lavage fluid sample library building method based on a nanopore sequencing platform, an identification method and a kit. According to the method, based on the combination of the nanopore sequencing platform and incomplete host removal, host removal pretreatment and sample library building are carried out on metagenome samples of human bodies and animal bodies, and then used for sequencing identification of metagenomes. Compared with the traditional process, the method provided by the invention is simpler and faster, meets the reasonable detection requirements, omits the processes of library building PCR and the like in the identification process, eliminates the bias caused by PCR amplification, and can more truly and accurately identify the metagenome microorganisms.

Description

[0001] This divisional application is based on the Chinese patent application with the application number 201811528417.X, the application date is December 13, 2018, and the title of the invention is "Metagenomic sample library construction method, identification method and kit based on nanopore sequencing platform" divisional application. technical field [0002] The invention relates to the field of metagenomic identification, in particular to a method for building a library of alveolar lavage fluid samples based on a nanopore sequencing platform, an identification method and a kit. Background technique [0003] Compared with the traditional culture identification method, metagenomic sequencing identification has the advantages of short identification period and low requirements on the technical level of operators and identification personnel. Moreover, metagenomic sequencing overcomes the disadvantages of identifying many microorganisms that cannot be cultivated, and is in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C40B50/06
CPCC12Q1/6806C12Q1/6869C40B50/06C12Q2535/122C12Q2565/631C12Q2527/125
Inventor 张烨程晨周水莲梁晓雪胡龙李杜衡涂浩波任用
Owner BEIJING SIMCERE MEDICAL LAB CO LTD
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