Optimization of a Semi-nested PCR Protocol for Amplifying the Entire Spike Protein Region to Identify SARS-CoV-2 Variants in Wastewater

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• Amplifying the spike protein region enhances the detection of SARS-CoV-2 variants in wastewater systems.
• The optimized semi-nested PCR protocol improves amplification efficiency and specificity for variant identification.
• Assessment of wastewater analysis across multiple sites shows increased sensitivity in detecting variants of SARS-CoV-2.
• Findings emphasize the method's role in public health monitoring and potential outbreak response strategies.