Method for selectively removing host nucleic acid in liquid biological sample

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...

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, when using high-throughput sequencing (NGS) methods to detect target pathogenic microorganisms, the high concentration of host nucleic acid will cause the host nucleic acid sequence to occupy most of the sequencing data, thereby crowding out and compressing the amount of sequencing data for the nucleic acid sequence of the target pathogenic microorganism, Reduce the sensitivity and accuracy of detection

Due to the above reasons, most of the currently available methods and kits for isolating and detecting target microorganisms in biological samples are not suitable for liquid biological samples. There is a strong demand for methods and kits for pathogenic microorganisms

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