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Updated: Jan 6, 2026
Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification
Published on: March 31, 2021
Validation of a dual-purpose conventional PCR strategy for SARS-CoV-2 detection and lineage screening in
Liliana Iñiguez-Gutiérrez1, María Teresa Magaña-Torres2, José Daniel Ruiz-Carrillo3
1Departamento Disciplinas Especializantes I, Decanato de Ciencias de la Salud, Universidad Autónoma de Guadalajara, Jalisco, 45110, Zapopan, Mexico.
Purpose:
Affordable molecular tools for SARS-CoV-2 surveillance are crucial, particularly in settings where access to advanced technologies is limited. This study aimed to validate a dual-purpose strategy based on conventional PCR and polyacrylamide gel electrophoresis for both diagnosis and lineage screening.
Methods:
Fifty-five nasopharyngeal swab samples were analyzed through RNA extraction, reverse transcription, and conventional PCR targeting the SARS-CoV-2 spike (S) gene. Amplicons were visualized on 6% polyacrylamide gels, and the diagnostic results were compared with those of RT‒qPCR. Fragment size shifts via electrophoresis were used for lineage inference and confirmed via Sanger sequencing.
Results:
The method demonstrated 100% concordance with RT‒qPCR for both diagnostic detection and accurate lineage classification in all analyzed samples. Characteristic gel migration patterns allowed identification of Omicron sublineages, and two samples presented unique mutational profiles not previously recorded in databases at the time of sampling. One of them matched the BA.2.86 lineage later reported in July 2024, indicating the method's potential for early variant detection. The approach also enabled multiplex PCR and internal control verification using a human LDLR gene fragment.
Conclusion:
This dual-purpose PCR strategy offers a cost-effective, scalable, and technically accessible alternative for SARS-CoV-2 surveillance in low-resource settings. Its high diagnostic reliability and ability to detect emerging variants through amplicon profiling support its implementation as a screening tool, pending broader validation across diverse populations.
