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Published on: March 13, 2018
Development and validation of a new and rapid molecular diagnostic tool based on RT-LAMP for Hepatitis C virus
Sonia Arca-Lafuente1, Cristina Yépez-Notario2, Pablo Cea-Callejo3
1Viral Hepatitis Reference and Research Laboratory, National Center of Microbiology (CNM), Carlos III Health Institute (ISCIII), Madrid, Majadahonda, Spain.
Insights
A new Hepatitis C virus (HCV) diagnostic tool uses Loop-Mediated Isothermal Amplification (LAMP) for rapid, affordable point-of-care detection. This pangenotypic system simplifies diagnosis for hard-to-reach populations.
Area of Science:
- Molecular Diagnostics
- Virology
- Point-of-Care Testing
Background:
- Hepatitis C virus (HCV) affects millions globally, with underdiagnosis in remote populations hindering control efforts.
- Current diagnostic methods can be complex and inaccessible for point-of-care (POC) applications.
- Development of rapid, user-friendly molecular diagnostic systems is crucial for effective HCV screening.
Purpose of the Study:
- To develop a pangenotypic molecular diagnostic tool for Hepatitis C virus (HCV) detection.
- To create a rapid and easy-to-use system for point-of-care (POC) molecular detection of HCV.
- To enable HCV diagnosis in hard-to-reach populations through a simplified assay.
Main Methods:
- A novel pangenotypic diagnostic tool based on Loop-Mediated Isothermal Amplification (LAMP) was developed.
- The system incorporates a direct sample lysis procedure, eliminating the need for RNA purification.
- Validation involved 129 HCV-positive samples, 27 HCV-negative/HIV-positive samples, and 11 healthy donors, using parallel RT-qPCR for comparison.
Main Results:
- The developed RT-LAMP assay detected HCV in 109/116 positive serum samples and 11/13 positive blood samples within 40 minutes.
- The assay demonstrated high accuracy with 94% sensitivity and 100% specificity compared to RT-qPCR.
- A limit of detection of 3.26 log10 IU/mL (10-20 copies per reaction) was achieved.
Conclusions:
- An accurate, affordable, and rapid molecular diagnostic system for Hepatitis C virus (HCV) has been successfully developed.
- The system's ability to use direct sample lysis from capillary blood makes it ideal for point-of-care (POC) use.
- This RT-LAMP assay is recommended as a valuable tool for molecular detection of HCV, particularly in resource-limited settings.
Purpose:
Globally, it is estimated that 1.0 million individuals are newly infected by Hepatitis C virus (HCV) every year, and nearly 50 million people live with a chronic infection, according to World Health Organization. To overcome underdiagnosis of HCV infection among hard-to-reach populations, it is essential to develop new rapid and easy-to-use molecular diagnostic systems. In this work, we have developed a pangenotypic diagnostic tool based on Loop-Mediated Isothermal Amplification (LAMP), coupled to a direct sample lysis procedure for molecular detection of HCV at point-of-care (POC).
Methods:
Procedure validation was performed using 129 different samples from HCV infected patients (116 serum samples, and 13 fresh blood samples), 27 individuals who tested negative for HCV but positive for HIV, and 11 healthy donors. Serum was collected, lysed for 10 min at room temperature, and assayed by RT-LAMP. To achieve this, a set of 9 LAMP-primers was used for the first time. Parallel RT-qPCR assays were conducted for HCV to both validate the procedure and quantify viral loads.
Results:
HCV was detected by RT-LAMP in 109/116 HCV positive serum samples, and in 11/13 positive blood samples in less than 40 min. Compared to RT-qPCR results, our RT-LAMP procedure showed a sensitivity of 94 %, 100 % specificity, and a limit of detection of 3.26 log10 IU/mL (10-20 copies per reaction).
Conclusions:
We have developed an accurate system, more affordable than the current available rapid tests for HCV. Since no prior RNA purification step from capillary blood is required, we strongly recommend our RT-LAMP system as a valuable and rapid tool for the molecular detection of HCV at POC.
