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Method and reagent for enriching intracellular microorganisms

A technology of microorganisms and cells, which is applied in the measurement/testing of microorganisms, biochemical equipment and methods, and the separation of microorganisms. It can solve the problems of improving sequencing depth and increasing sequencing costs, and achieve the effect of increasing the sequence ratio

Active Publication Date: 2021-02-23
广州微远医疗器械有限公司 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If whole blood is used for nucleic acid extraction and library construction, the release of a large number of human host genomes in the cells will cover up very small amounts of pathogenic microorganism information, making it necessary to increase the sequencing depth to ensure that trace amounts of microbial information are detected. Increased sequencing costs to a certain extent

Method used

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  • Method and reagent for enriching intracellular microorganisms
  • Method and reagent for enriching intracellular microorganisms
  • Method and reagent for enriching intracellular microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] A method for enriching intracellular microorganisms, comprising the following steps:

[0047] 1. Collect cells

[0048] Cellular components of the sample are collected.

[0049] 1.1 Whole blood sample: Place the blood collection tube (containing whole blood, about 5-10ml) in a centrifuge, centrifuge at 1600g for 10min, transfer the upper layer of plasma, and use a pipette to absorb the middle layer of white blood cells.

[0050] 1.2 Other clinical infection samples containing cells: take 500-1000 μl of samples, centrifuge at 10,000-12,000 rpm for 5 minutes, and collect cells.

[0051] 2. Release microorganisms

[0052] Use cell treatment agents to make cell membranes more permeable and release intracellular microorganisms, specifically:

[0053] Take the cell fraction collected in step 1, and use 400-600 μl cell treatment agent to treat the cells (about 5x10 6 ), incubate on ice for 5-20 min, and shake and mix continuously during the period to increase the permeabil...

Embodiment 2

[0066] In this example, the method in Example 1 is used to detect whole blood samples.

[0067] 1. Sample preparation.

[0068] Divide the 5ml whole blood sample into 2 parts, one of which is centrifuged at 1600g for 10min, collect the upper layer plasma and the middle layer white blood cell samples into 2.0ml EP tubes, respectively marked as A and B, and the other whole blood sample Marked as C.

[0069] 2. Sample pretreatment and preparation.

[0070] Sample B (white blood cells) was treated with reference to steps 2-6 in Example 1; after sample A was directly extracted with the Micro DNA Extraction Kit (DP316) of Tiangen Biochemical Technology (Beijing) Co., Ltd., refer to Example 1 Steps 5-6 for processing; Sample C is processed with reference to steps 4-6 in Example 1. All three samples were constructed as qualified high-throughput (NGS) sequencing libraries.

[0071] 3. Detection

[0072] The A, B and C samples in the above step 2 were sequenced on the machine at th...

Embodiment 3

[0081] In this example, the method in Example 1 is used to detect the lower respiratory tract sample (alveolar lavage fluid).

[0082] 1. Sample preparation.

[0083] Divide 2ml of alveolar lavage fluid sample into 2 parts, one of which takes a certain volume (500-1000μl) sample, centrifuges at 10,000-12,000rpm for 5min, collects cells, and marks it as D; the other takes the same volume as sample D The original sample, labeled E.

[0084] 2. Sample pretreatment and preparation.

[0085] Sample D was processed with reference to steps 2-6 in Example 1; sample E was processed with reference to steps 4-6 in Example 1. Both samples were constructed into qualified high-throughput (NGS) sequencing libraries.

[0086] 3. Detection

[0087] The D and E samples in the above step 2 were sequenced on the machine at the same time, and the same sequencing depth was adopted, and the results were as follows:

[0088] Table 2 The sequencing results of the above D and E samples

[0089] ...

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Abstract

The invention relates to a method and a reagent for enriching intracellular microorganisms, and belongs to the technical field of microorganism gene detection. The method for enriching intracellular microorganisms comprises the following steps: collecting cells, namely obtaining a to-be-detected sample, and collecting host cells from the to-be-detected sample; carrying out microorganism release, namely treating the collected host cells by using a cell treatment agent so as to have membrane of the host cells ruptured with nucleus membrane of the host cells kept intact, thereby allowing releaseof intracellular microorganisms; and then, enriching microorganisms, namely removing host cell residues in the sample so as to obtain the product. According to the method for enriching intracellular microorganisms, cell membrane permeability of the cells contained in the sample is increased by using the cell treatment agent so as to have the intracellular microorganisms released; and then, the cell residues without microorganisms are removed so as to have the part containing microorganisms reserved; and thus, intracellular pathogenic microorganisms are released with host nucleic acid removed.Therefore, the purposes of separating and enriching intracellular pathogenic microorganisms are achieved.

Description

technical field [0001] The invention relates to the technical field of microbial gene detection, in particular to a method and reagent for enriching intracellular microorganisms. Background technique [0002] Clinical methods for pathogen diagnosis include morphological detection, antigen-antibody detection, in vitro culture, serological detection, and molecular biology methods such as PCR (Polymerase chain reaction), all of which have their own advantages and disadvantages. [0003] Traditional pathogen detection methods are mainly limited by low throughput, and each detection can only identify one or several pathogens. Usually, clinicians need to make judgments based on experience and guess the possible pathogens. According to the existing detection methods of these pathogens, the detection is carried out separately; on the other hand, under the current technical conditions, only 0.1% to 1% of the microorganisms are cultivable, and even if they are cultivable, they face th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/02C12Q1/6806C12Q1/04
CPCC12N1/02C12Q1/6806C12Q2527/125
Inventor 许腾曾伟奇吴婉婷李永军王小锐苏杭
Owner 广州微远医疗器械有限公司
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