Development and performance verification of a high throughput sequencing method for pathogenic microbial metagenomics
10.13602/j.cnki.jcls.2025.01.01
- VernacularTitle:一种病原宏基因高通量测序方法的开发与性能确认
- Author:
Haiyun CHAI
1
;
Yuemei HUANG
1
;
Haiyan TAN
1
;
Wei WANG
1
Author Information
1. 广东芯海医学检测实验室有限公司,广东 佛山 528200
- Publication Type:Journal Article
- Keywords:
pathogenic microorganism;
high throughput sequencing;
performance verification
- From:
Chinese Journal of Clinical Laboratory Science
2025;43(1):1-6
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a high throughput sequencing method for pathogenic microbial metagenomics and verify its detection performance.Methods First,the library construction conditions with different input levels of nucleic acid were studied.Three differ-ent input levels of nucleic acid such as 5 ng,50 ng,and 100 ng,three fragmentation times such as 20 min,25 min,and 30 min,and three amplification cycles such as 7,5,and 3 cycles were tested.The optimal library construction conditions were determined by the li-brary concentration and fragment size.The representative bacteria covering fungi and gram-positive/negative bacteria,including Candi-da albicans,Staphylococcus aureus,Staphylococcus epidermidis,Escherichia coli and Cryptococcus Gattii,were selected.Three different concentrations of mixed bacterial suspensions were prepared at a 1∶1 ratio.Then,106/mL of T-cell suspensions were added to prepare reference samples.The performance of the self-established method(Method 1)was verified by the reference samples,and its consis-tency with Method 2(DNA sample library construction kit combined with probe anchored polymerase chain reaction sequencing method)was compared.Results The optimal library construction conditions of the sequencing method for pathogenic microbial metagenomics were determined as follows:a nucleic acid input of 50 ng,a nucleic acid fragmentation time of 20 min,and 5 amplification cycles for PCR.The positive coincidence rate(accuracy)and negative coincidence rate(specificity)of Method 1 were 100%,and its detection limit was 500 CFU/mL.The correlation coefficients(r)of the reads per million(RPM)in linear samples of various microorganisms were greater than 0.9.The CV of bacterial detection were less than 10%,while those of fungal detection were around 20%.The consis-tency between Method 1 and Method 2 was 100%.Conclusion A sequencing method for pathogenic microbial metagenomics is estab-lished successfully,which can acquire accurate,stable,and reliable results and is suitable for the pathogen detection of alveolar lavage fluid samples.
