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Updated: Apr 10, 2026
Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1
Published on: March 13, 2018
A streamlined integrated system integrating lysate release, freeze-dried reagents for multiplex polymerase chain
Miao Fu1, Siying He1, Yingchao Wu2
1Department of Clinical Laboratory, Affiliated Jinhua Hospital, Zhejiang University School of Medicine, Jinhua, Zhejiang, China.
Introduction:
Current Toxoplasma gondii (TOX), Epstein-Barr virus (EBV), rubella virus, human cytomegalovirus (HCMV), and herpes simplex virus types I and II (HSV I and II) (TORCHes) diagnostic methods are limited by challenges, such as multi-step workflows and cold-chain dependency. This study aimed to develop an integrated "sample-in-result-out" system combining rapid nucleic acid release, room-temperature-stable multiplex polymerase chain reaction (PCR), and automated interpretation to improve diagnostic accuracy.
Methods:
We utilized a self-prepared lysis buffer for fast nucleic acid extraction from diverse sample types. Primers, probes, and reaction components for six TORCHes pathogens were fabricated into freeze-dried microspheres via vacuum freeze-drying, enabling single-tube multicolor melting curve analysis (MMCA). The companion software, TORCHes-MCAv1.0, was used to automate pathogen typing and result reporting. Evaluation metrics encompassed sensitivity, specificity, precision, stability, and anti-interference ability. The system was validated using 210 clinical samples compared to commercial single-target qPCR, with Sanger sequencing resolving any discrepancies.
Results:
Nucleic acid extraction and release were completed within 1 min. Freeze-dried microspheres demonstrated exceptional repeatability, with T m intra- and inter-batch CVs of 0.03-0.18%. Limits of detection (LoD) were 200 copies/mL for HSV-I/II, HCMV, and TOX, and 500 copies/mL for EBV and RV. Specificity analysis showed no cross-reactivity with non-target pathogens. Stability testing confirmed the microspheres could be stored stably at room temperature for 1 year. Clinical validation showed near-perfect agreement with qPCR (Kappa = 0.965-1.000), with all seven discrepant results confirmed correct via sequencing. The TORCHes-MCAv1.0 software achieved 100% concordance with manual interpretation.
Conclusion:
We developed a robust, streamlined integrated TORCHes detection system. Its simplicity, stability, and high-throughput nature make it a valuable complementary tool for confirming active infection and differentiating pathogen subtypes in complex clinical scenarios.
Insights
This study introduces a rapid, room-temperature-stable multiplex PCR system for diagnosing TORCHes infections, improving accuracy and efficiency in clinical settings. The integrated approach simplifies workflows, offering a valuable tool for identifying and differentiating common pathogens.
Area of Science:
- Clinical diagnostics
- Molecular biology
- Infectious disease detection
Background:
- Current TORCHes diagnostics face challenges like complex workflows and cold-chain requirements.
- There is a need for streamlined, accurate, and stable methods for detecting Toxoplasma gondii (TOX), Epstein-Barr virus (EBV), rubella virus (RV), human cytomegalovirus (HCMV), and herpes simplex virus (HSV I and II).
Purpose of the Study:
- To develop an integrated "sample-in-result-out" system for TORCHes detection.
- To combine rapid nucleic acid release, room-temperature-stable multiplex PCR, and automated interpretation for improved diagnostic accuracy.
Main Methods:
- Developed a self-prepared lysis buffer for rapid nucleic acid extraction.
- Created freeze-dried microspheres containing primers, probes, and reaction components for six TORCHes pathogens.
- Utilized single-tube multicolor melting curve analysis (MMCA) and companion software (TORCHes-MCAv1.0) for automated interpretation.
Main Results:
- Achieved nucleic acid extraction and release within 1 minute.
- Demonstrated high repeatability (Tm CVs 0.03-0.18%) and stability (room temperature for 1 year).
- Clinical validation showed near-perfect agreement with qPCR (Kappa 0.965-1.000), with high sensitivity and specificity.
Conclusions:
- Developed a robust and streamlined integrated TORCHes detection system.
- The system offers simplicity, stability, and high-throughput capabilities.
- This method serves as a valuable tool for confirming active infections and differentiating pathogen subtypes.
