Establishment of genomic detection system for Alzheimer′s disease risk based on time-of-flight mass spectrometry
10.3760/cma.j.cn114452-20250209-00068
- VernacularTitle:基于飞行时间质谱建立阿尔茨海默病风险基因组检测体系
- Author:
Yuyan KUANG
1
;
Ting ZHANG
;
Wenyan GE
;
Huimin GUO
;
Qingmin RAO
;
Yongyin HE
;
Qiang WANG
;
Xiaomei ZHONG
;
Yuping NING
;
Yulong LIN
;
Haiying LIU
Author Information
1. 广州医科大学附属脑科医院检验科，广州 510370
- Publication Type:Journal Article
- Keywords:
Alzheimer′s disease;
Mass spectrometry;
Genetic testing
- From:
Chinese Journal of Laboratory Medicine
2025;48(12):1571-1580
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a genomic nucleic acid mass spectrometry detection platform for allelic risk associated with Alzheimer's disease.Methods:Whole blood samples of 61 patients diagnosed as Alzheimer's disease in the Affiliated Brain Hospital of Guangzhou Medical University from December 28th, 2023 to 31st, March 2024 were collected and deoxynucleic acid (DNA) was extracted, including 22 males and 39 females, aged (67.36 ± 8.18) years old. After screening out 17 risk gene loci in Chinese population, multiplex polymerase chain reaction primers, single-base extension primers and Sanger sequencing primers were designed. Ten samples were used for primer optimization and debugging through Sanger sequencing and time-of-flight mass spectrometry to establish a detection system. The remaining samples were genotyped using a time-of-flight mass spectrometer and verified by Sanger sequencing for accuracy evaluation. Five samples were selected for gradient dilution and then subjected to time-of-flight mass spectrometry detection to evaluate the detection limit. Three clinical samples, one case of Escherichia coli and one case of Staphylococcus aureus genomic DNA samples were selected for cross-reaction research. The anti-interference ability of the detection system was evaluated against hemolysis, chylous substances and conventional anticoagulants in the samples. Two samples, one wild and one homozygous mutation sample with representative peak shapes, were selected to evaluate the anti-interference ability. Four samples containing the common genotypes of all gene loci in the system were selected and repeated 10 times to evaluate the precision.Results:The minimum intensity of single-base extension primers on mass spectrometry is greater than half of the maximum intensity. All 17 risk gene loci screened were successfully typed. The time-of-flight mass spectrometry detection results of 1,037 loci from 61 samples showed that the genotyping detection rate was 100%. The genotypes of the 20 DNA samples were completely consistent with the results of Sanger sequencing, with an accuracy rate of 100%. The mass spectrometry detection results of five samples after gradient dilution indicated that the low detection limit was 5 ng of DNA. The reaction system has a strong anti-interference ability against hemolysis of samples, chylous substances, conventional anticoagulants and DNA cross-contamination. Homologous allele interference and no cross-reaction between the bacterial genome and 17 gene loci do not affect the risk genome detection results. The results of 10 repeated mass spectrometry tests on 4 samples showed that the precision was 100%.Conclusion:The genomic detection system of Alzheimer's disease risk has been successfully established to provide an auxiliary mean for disease diagnosis and risk assessment.
