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Updated: Jun 27, 2026
Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV
Published on: November 10, 2023
Establishment and Preliminary Application of a Multiplex TaqMan Real-Time Fluorescence Quantitative PCR Assay for the
Qiuyang Sun1, Yuanzhi Xie1, Yufang Feng1
1Institute of Laboratory Animal Resources (National Rodent Experimental Animal Resource Bank), National Institutes for Food and Drug Control, Beijing 102629, China.
Abstract:
Pneumocystis is an opportunistic fungal pathogen that causes severe Pneumocystis pneumonia (PCP) in immunocompromised individuals and laboratory animals. Three host-specific species-Pneumocystis murina (P. murina), Pneumocystis carinii (P. carinii), and Pneumocystis jirovecii (P. jirovecii)-are closely associated with infections in humans and laboratory animals. However, the conventional method, microscopic staining, suffers from low sensitivity, operator-dependent subjectivity, and inability to differentiate species, highlighting the urgent need for a multiplex qPCR assay. In this study, we established a multiplex qPCR method targeting the mtLSUrRNA gene of P. murina, the TS gene of P. carinii, and the mtSSUrRNA gene of P. jirovecii. Primers and probes were designed and optimized using a matrix approach. The method was systematically evaluated for sensitivity, specificity, and reproducibility using recombinant plasmid standards and laboratory animal samples. Validation was performed on 260 mouse lung samples, 30 P. murina-positive samples, 25 rat lung samples, 6 rat bronchoalveolar lavage fluid (BALF) samples, and 8 P. carinii-positive samples. Results were compared with single-plex qPCR and staining microscopy (performed on 68 mouse lung samples, 38 Pneumocystis-positive samples). The limits of detection (LOD) were 5 copies/μL for P. murina, 6 copies/μL for P. carinii, and 8 copies/μL for P. jirovecii. Standard curves showed excellent linearity (R2 ≥ 0.999) with amplification efficiencies of 90-110%. No non-specific reactions were observed with 22 common pathogens, and intra-/inter-group coefficients of variation (CV%) were below 1%. Moreover, interference testing revealed minimal matrix effects on the amplification system and no mutual interference among the primers and probes. The multiplex qPCR detected all 38 positive samples (100%), showing 100% concordance with single-plex qPCR, whereas Giemsa staining detected none (0%) and toluidine blue staining only 60% (3/5) of the tested positives, suggesting that the multiplex qPCR achieved higher detection rates than staining microscopy. In conclusion, this novel multiplex qPCR method offers high sensitivity, specificity, and reproducibility, providing a sensitive and specific tool for laboratory animal health monitoring and epidemiological surveillance. Its clinical application for human PCP diagnosis requires further validation with authentic human specimens.
Insights
A new multiplex qPCR assay accurately detects three species of Pneumocystis, offering a sensitive tool for laboratory animal health monitoring. This method surpasses traditional staining techniques in detecting Pneumocystis pneumonia (PCP).
Area of Science:
- Mycology and Infectious Diseases
- Molecular Diagnostics
- Veterinary Medicine
Background:
- Pneumocystis is an opportunistic fungal pathogen causing severe Pneumocystis pneumonia (PCP) in immunocompromised hosts.
- Three species, Pneumocystis murina, Pneumocystis carinii, and Pneumocystis jirovecii, infect humans and laboratory animals.
- Conventional microscopic staining for Pneumocystis lacks sensitivity and species differentiation, necessitating advanced diagnostic tools.
Purpose of the Study:
- To establish and validate a multiplex quantitative PCR (qPCR) assay for the simultaneous detection of P. murina, P. carinii, and P. jirovecii.
- To evaluate the sensitivity, specificity, and reproducibility of the developed multiplex qPCR method.
- To compare the diagnostic performance of the multiplex qPCR assay against conventional staining methods and single-plex qPCR.
Main Methods:
- Development of a multiplex qPCR assay targeting the mtLSUrRNA, TS, and mtSSUrRNA genes specific to P. murina, P. carinii, and P. jirovecii, respectively.
- Optimization of primers and probes using a matrix approach.
- Systematic evaluation using recombinant plasmid standards and validation on 260 mouse lung samples, rat samples, and known positive samples, compared with microscopy.
Main Results:
- The multiplex qPCR assay demonstrated high sensitivity with limits of detection as low as 5 copies/μL for P. murina.
- Excellent linearity (R² ≥ 0.999) and amplification efficiencies (90-110%) were achieved, with no cross-reactivity against 22 common pathogens.
- The assay detected 100% of positive samples, showing 100% concordance with single-plex qPCR, significantly outperforming Giemsa and toluidine blue staining.
Conclusions:
- The novel multiplex qPCR method provides a highly sensitive, specific, and reproducible tool for detecting Pneumocystis species in laboratory animals.
- This assay significantly improves upon traditional staining methods for P. murina and P. carinii detection and monitoring.
- Further validation with human specimens is required for clinical application in diagnosing human PCP.
