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Published on: February 4, 2018
A highly sensitive one-tube nested quantitative real-time PCR assay for specific detection of Bordetella pertussis
Rui-Qing Zhang1, Zheng Li2, Gui-Xia Li3
1Hebei Medical University, Shijiazhuang, 050031, Hebei, China; NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing 102206, China; Hebei General Hospital, Shijiazhuang, 050051, Hebei, China.
Objectives:
Bordetella pertussis is a highly contagious respiratory agent and is the causative pathogen of pertussis, which primarily affects children. Current diagnostic techniques for this pathogen have a variety of limitations including a long culture time, low bacterial load, and lack of specificity.
Methods:
This article reports the development of a one-tube nested quantitative real-time PCR assay using the locked nucleic acid (LNA) technique (LNA-OTN-q-PCR), targeting the BP485 gene and using a simple inexpensive extraction method. A total of 130 clinical samples from patients with clinically suspected pertussis, collected from the Children's Hospital of Hebei, China, were tested by LNA-OTN-q-PCR assay. RT-PCR and two-step semi-nested PCR assays were performed in parallel for comparison.
Results:
Only strains of B. pertussis were identified as positive, whereas all of the remaining strains were appropriately identified as negative by the LNA-OTN-q-PCR assay. A single copy per reaction can be detected by the LNA-OTN-q-PCR assay. Additionally, the sensitivity of this method was 100 times that of the RT-PCR assay (100 copies per reaction). Sixty-three of the 130 clinical samples were detected positive by LNA-OTN-q-PCR assay; in contrast, RT-PCR was able to detect only 41 positive samples. Following this, all 63 samples were positively identified by two-step semi-nested PCR. Compared with the two-step semi-nested PCR assay, both the specificity and sensitivity of the LNA-OTN-q-PCR assay using purified DNA and crude extract were 100%.
Conclusions:
This assay was able to detect B. pertussis infection with high sensitivity and specificity. This test shows great potential as a promising technique to detect B. pertussis in both clinical laboratories and public health settings.
Insights
A new one-tube nested quantitative real-time PCR assay using locked nucleic acid (LNA-OTN-q-PCR) accurately detects Bordetella pertussis. This highly sensitive and specific method offers a promising advancement for pertussis diagnostics in clinical and public health settings.
Area of Science:
- Microbiology
- Molecular Biology
- Infectious Diseases
Background:
- Bordetella pertussis causes highly contagious pertussis, primarily in children.
- Current diagnostic methods for B. pertussis suffer from limitations like long turnaround times, low sensitivity, and poor specificity.
Purpose of the Study:
- To develop and evaluate a novel one-tube nested quantitative real-time PCR (LNA-OTN-q-PCR) assay for detecting B. pertussis.
- To assess the sensitivity and specificity of the LNA-OTN-q-PCR assay compared to existing PCR methods.
Main Methods:
- Development of a one-tube nested quantitative real-time PCR assay utilizing locked nucleic acid (LNA) technology targeting the BP485 gene.
- Application of a simple and inexpensive DNA extraction method.
- Testing of 130 clinical samples from suspected pertussis cases using LNA-OTN-q-PCR, RT-PCR, and two-step semi-nested PCR.
Main Results:
- The LNA-OTN-q-PCR assay demonstrated 100% specificity, correctly identifying B. pertussis strains and negative for others.
- The assay detected as little as a single copy of the target gene per reaction, showing 100 times greater sensitivity than RT-PCR.
- Out of 130 samples, LNA-OTN-q-PCR identified 63 positive cases, compared to 41 by RT-PCR, with full agreement from two-step semi-nested PCR.
Conclusions:
- The developed LNA-OTN-q-PCR assay provides a highly sensitive and specific method for detecting Bordetella pertussis infection.
- This advanced diagnostic technique holds significant promise for routine use in clinical laboratories and public health surveillance for pertussis.
