Method for selectively removing host nucleic acid in liquid biological sample

A biological sample and selective technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of affecting the efficiency of nucleic acid extraction of pathogenic microorganisms, reducing detection sensitivity and accuracy, crowding out the amount of sequencing data, etc.

Pending Publication Date: 2021-05-18
INQUIRE LIFE DIAGNOSTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] First, the high concentration of host nucleic acid released when the host cells in the sample are lysed will compete with the nucleic acid capture material, affecting the extraction efficiency of nucleic acid from pathogenic microorganisms
[0006] Second, high-concentration host nucleic acid will compete with reagents such as primers and enzymes during the amplification reaction (PCR reaction) of the nucleic acid sequence of the target pathogenic microorganism, which may lead to the generation of a large number of non-specific amplification products and inhibit the nucleic acid of the target pathogenic microorganism. sequence amplification
[0007] Third, wh...

Method used

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  • Method for selectively removing host nucleic acid in liquid biological sample
  • Method for selectively removing host nucleic acid in liquid biological sample
  • Method for selectively removing host nucleic acid in liquid biological sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] A method for selectively removing host nucleic acid from a liquid biological sample, the method comprising the steps of:

[0069] (1) Mix the liquid biological sample and the saponin solution evenly, and let stand at room temperature for 5 minutes to specifically lyse the cells of non-target components such as host cells to obtain a mixture, the final concentration of saponin in the mixture is 1.7%;

[0070] (2) The mixture is centrifuged at high speed, and the supernatant is discarded after centrifugation to obtain the first precipitate, the centrifugation speed is 4000rpm, and the time is 30 minutes;

[0071] (3) Add nuclease reaction solution and nuclease to the first precipitation, nuclease contains 10 enzyme units, the purpose is to degrade the non-target components in the sample, mainly host nucleic acid; after step (3), enrich the target microorganism 1 , rinse target microorganism 1 with water and / or biological buffer, the biological buffer is PBS; repeat step (...

Embodiment 2

[0077] A method for selectively removing host nucleic acid from a liquid biological sample, the method comprising the steps of:

[0078] (1) Mix the liquid biological sample and the saponin solution evenly, and let stand at room temperature for 10 minutes to specifically lyse the cells of non-target components such as host cells to obtain a mixture, the final concentration of saponin in the mixture is 0.04%;

[0079] (2) The mixture is centrifuged at high speed, and the supernatant is discarded after centrifugation to obtain the first precipitate, the centrifugation speed is 4200rpm, and the time is 28 minutes;

[0080] (3) Add nuclease reaction solution and nuclease to the first precipitation, nuclease contains 50 enzyme units, the purpose is to degrade the non-target components in the sample, mainly host nucleic acid; after step (3), enrich the target microorganism 1 , use water and / or biological buffer to wash target microorganism 1, the biological buffer is PBS; repeat ste...

Embodiment 3

[0086] A method for selectively removing host nucleic acid from a liquid biological sample, the method comprising the steps of:

[0087] (1) Mix the liquid biological sample and the saponin solution evenly, and let stand at room temperature for 15 minutes to specifically lyse the cells of non-target components such as host cells to obtain a mixture. The final concentration of saponin in the mixture is 0.85%;

[0088] (2) Centrifuge the mixture at high speed, pour off the supernatant after centrifugation, and obtain the first precipitate, the centrifugation speed is 4500rpm, and the time is 26 minutes;

[0089] (3) Add nuclease reaction solution and nuclease to the first precipitation, nuclease contains 100 enzyme units, the purpose is to degrade the non-target components in the sample, mainly host nucleic acid; after step (3), enrich the target microorganism 1 , use water and / or biological buffer to wash target microorganism 1, the biological buffer is PBS; repeat step (3) 2 t...

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Abstract

The invention provides a method for selectively removing host nucleic acid in a liquid biological sample, and belongs to the technical field of molecular biology and in-vitro molecular diagnose.The method comprises the following steps: (1), mixing the liquid biological sample and a saponin solution uniformly, leaving the mixture to stand for 5-30 minutes at the room temperature to enable cells of non-target components such as host cells to be subjected to specific lysis to obtain a mixture, wherein the final concentration of the saponin in the mixture is 0.02-1.7%; (2) carrying out high-speed centrifugation on the mixture, and pouring out a supernate after centrifugation to obtain a first precipitate; (3) adding a nuclease reaction liquid and nuclease to the first precipitate, wherein the nuclease contains 10-200 enzyme units and aims at degrading non-target components in the sample, which mainly comprises host nucleic acid; (4) inactivating nuclease; and (5) enriching a target microorganism 2. Pathogenic bacteria with the concentration as low as about 2.0 CFU/mL can be detected, and the clinical diagnosis requirements on infectious diseases such as blood flow infection and the like are met. The method has the advantages of few used reagents and equipment, low cost, simple process and wide application range.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and in vitro molecular diagnosis, in particular to a method for selectively removing host nucleic acid from liquid biological samples. Background technique [0002] Pathogenic microorganisms such as bacteria, mycoplasma, chlamydia, rickettsia and spirochetes are the main pathogenic factors that cause infection and infectious diseases. They enter the human body through contact or other media, causing diseases. During the development of infectious diseases, some pathogens can enter the patient's blood circulation through various channels, or appear in different types of body fluids such as cerebrospinal fluid, fluid in internal organs, pus, etc., or appear in patients deliberately obtained through medical means. organ lavage fluid such as bronchoalveolar lavage fluid. Therefore, liquid biological samples obtained from these various sources become an effective way to diagnose infectious p...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/04
CPCC12Q1/04C12Q1/6806C12Q2521/327
Inventor 江山庞白冰
Owner INQUIRE LIFE DIAGNOSTICS INC
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