Evaluation of host nucleic acid removal and pathogen enrichment methods in animal tissue samples
10.3969/j.issn.1002-2694.2025.00.091
- VernacularTitle:动物组织样本宿主核酸去除与病原体富集方法评估研究
- Author:
Xuezhi HUANG
1
;
Zuming ZHANG
;
Hao ZHOU
;
Ting ZHAO
;
Zirui XIONG
;
Guangqian PEI
;
Yunfei WANG
;
Mengnan CUI
;
Yan GUO
;
Haifeng PAN
;
Yujun CUI
;
Hang FAN
Author Information
1. 安徽医科大学公共卫生学院流行病学与卫生统计学系,合肥 230032;军事科学院军事医学研究院,病原微生物生物安全全国重点实验室,北京 100071
- Publication Type:Journal Article
- Keywords:
infected tissue samples;
pathogen identification;
host nucleic acid removal;
nonspecific amplification;
metagenome;
high-throughput sequencing
- From:
Chinese Journal of Zoonoses
2025;41(7):682-690
- CountryChina
- Language:Chinese
-
Abstract:
This study was aimed at investigating the effectiveness of various host nucleic acid removal and non-specific amplifica-tion techniques in animal tissue samples,to increase the accuracy of pathogen identification in tissue samples.Simulated samples were prepared with a mixture of mouse lung tissue homogenates and Klebsiella pneumoniae fluids,and processed with six host nucleic acid removal kits and three non-specific amplification techniques.The effectiveness of each method in removing host DNA and enriching nucleic acids of pathogenic microorganisms was evaluated through real-time fluorescence quantitative PCR and high-throughput se-quencing.For host nucleic acid removal techniques,the method of selective cleavage and quantitative degradation of host DNA(Com-plete5 kit)effectively decreased the host nucleic acid content in tissue samples and increased the relative abundance of pathogen nucleic acids.In contrast,the magnetic bead method for host DNA removal(Next microbiome DNA enrichment Kit kit)was less effec-tive.At lower pathogen concentrations(77 CFU/mL),the Vazyme kit was more effective than the other kits in removing host nucleic acids.Non-specific amplification techniques(MALBAC whole genome amplification,MDA isothermal amplification,and random primer amplification)were not applicable to tissue samples and were not effective in increasing the relative abundance of pathogen nucleic acids.Selective lysis and quantitative degradation of host DNA were suitable for processing tissue samples with high host back-ground and low pathogenic microorganism levels,whereas non-specific amplification methods were not applicable to tissue samples for pre-processing of macro-genome high-throughput sequencing.
