A real-time fluorescence quantitative PCR method was established for the detection and identification of Ochrobactrum anthropi and Brucella
10.3760/cma.j.cn231583-20231117-00108
- VernacularTitle:建立一种实时荧光定量PCR方法检测与鉴定人苍白杆菌及布鲁氏菌
- Author:
Yihan WU
1
;
Liping WANG
Author Information
1. 内蒙古自治区疾病预防控制中心（内蒙古自治区预防医学科学院）微生物实验室，呼和浩特 010030
- Keywords:
Ochrobactrum anthropi;
Brucella;
Real-time fluorescence quantitative PCR
- From:
Chinese Journal of Endemiology
2024;43(5):416-420
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a real-time fluorescence quantitative PCR method for detection and identification of Ochrobactrum anthropi ( O.anthropi) and Brucella, and to apply the method in the detection of clinical samples. Methods:The gene nucleotide sequences of 16S ribosomal RNA (16S rRNA) of Ochrobactrum spp. and strains which were adjacent to Ochrobactrum spp. were downloaded from the GenBank database of the National Center for Biotechnology Information (NCBI) website in the United States. A phylogenetic tree was constructed by using MEGA 5.1 software. The primers and probes for the real-time fluorescence quantitative PCR assay, which were specific to O.anthropi were designed using DNAstar (V7.1) software, the sensitivity and specificity of the method was verified. And the method was applied to detect O.anthropi infection in blood samples of clinical patients in pastoral areas. Results:A total of 37 strains of 16S rRNA gene nucleotide sequences from 17 species of Ochrobactrum spp. and 15 strains from 8 species of Brucella spp. were downloaded from the NCBI website. The phylogenetic tree of 16S rRNA gene revealed a high degree of homology (similarity of 100.0%) between O.anthropi, O.cytisi, O.lupini and O.tritici in Ochrobactrum spp. The similarity was 99.1% between O.anthropi and 8 species of Brucella spp. The similarities was 84.5% - 98.6% between O.anthropi and other 13 species of Ochrobactrum spp. The real-time fluorescence quantitative PCR method (nested PCR) was successfully established. For the detection of O.anthropi standard strain DNA, the minimum detection limit of single real-time fluorescence quantitative PCR was 3.67 fg nucleic acid DNA, with a cycle threshold (CT) of 36.40, and a nested PCR detection CT of 14.67, Brucella spp. and other non- Ochrobactrum spp. strains were not detected. Totally 126 blood samples from clinical patients were collected and tested, and the detection rate of O.anthropi was 17.46% (22/126). Conclusion:A real-time fluorescence quantitative PCR method has been established to detect O.anthropi, which has high sensitivity and specificity and can be used for the identification of O.anthropi and Brucella, as well as clinical sample detection.
