Simultaneous detection of 7 important Rickettsiales pathogens by TaqMan-probe quantitative real-time PCR.
10.3760/cma.j.cn112338-20221011-00875
- Author:
Xiao Jing JIN
1
;
Zhong Qiu TENG
2
;
Pei Xing XU
2
;
Xiang Rong SUN
3
;
Wen WANG
2
;
Xin Cheng QIN
2
;
Tian QIN
4
Author Information
1. Department of Epidemiology, School of Public Health, Shanxi Medical University, Taiyuan 030001, China.
2. National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention/State Key Laboratory of Infectious Disease Prevention and Control, Beijing 102206, China.
3. Nanchang Center for Disease Control and Prevention, Nanchang 330038, China.
4. Department of Epidemiology, School of Public Health, Shanxi Medical University, Taiyuan 030001, China National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention/State Key Laboratory of Infectious Disease Prevention and Control, Beijing 102206, China.
- Publication Type:Journal Article
- MeSH:
Humans;
Rickettsiales;
Real-Time Polymerase Chain Reaction;
RNA, Ribosomal, 16S;
Reproducibility of Results;
Orientia tsutsugamushi;
Spotted Fever Group Rickettsiosis
- From:Chinese Journal of Epidemiology
2023;44(5):816-822
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To establish and optimize a TaqMan-probe quantitative real-time PCR (qPCR) assay for the detection of 7 important Rickettsiales pathogens and simultaneous identification of the infection types. Methods: Based on the ompB gene of Rickettsia prowazekii, Rickettsia mooseri and spotted fever group rickettsiae, the groEL gene of Orientia tsutsugamushi, the 16S rRNA of Ehrlichia chaffeensis, the gltA gene of Anaplasma phagocytophilum and the com1 gene of Coxiella burnetii, we synthesized primers and TaqMan-probes and optimized the reaction system and reaction process to same solution. The sensitivity, specificity and reproducibility of this assay were evaluated and the assay was used for the detection of simulated and actual samples. Results: The Ct value of the standard curves of the 7 pathogens showed a good linear relationship with the number of DNA copies (all R2 >0.990 0), the minimum detection limit was 10 copies/μl, showing good specificity. In the 96 tick nucleic acid extracts, Coxiella burnetii was detected in 1 sampleand spotted fever group Rickettsiae was detected in 3 samples. In the 80 blood samples from patients with undefined febrile illness, Orientia tsutsugamushi was detected in 1 sample and spotted fever group rickettsiae was detected in 2 samples. Conclusions: In this study, based on the established TaqMan-probe qPCR assay, the reaction system and reaction condition of the 7 important pathogens of Rickettsiales were optimized to the same solution. This method overcomes the shortcomings of using different reaction systems and reaction conditions for different pathogens, which can precisely identify the species of 7 important pathogens of Rickettsiales in clinical sample detections and is important for the infection type identification and laboratory detection time reduction to facilitate precise treatment of the patients.
