Enhanced Two-Step LAMP-CRISPR Assay with an Engineered Zst Polymerase for Contamination-Free and Ultrasensitive DNA...

Area of Science:

• Molecular Diagnostics
• Biotechnology
• Isothermal Amplification

Background:

• Current loop-mediated isothermal amplification (LAMP)-coupled clustered regularly interspaced short palindromic repeats (LAMP-CRISPR) assays face challenges with aerosol contamination in two-step formats and compromised performance in one-step formats.
• Next-generation molecular diagnostics require sensitive, specific, and contamination-free detection methods.

Purpose of the Study:

• To develop an enhanced two-step LAMP-CRISPR (ETL-CRISPR) assay for ultrasensitive DNA detection.
• To eliminate aerosol contamination issues inherent in current LAMP-CRISPR methods.
• To achieve highly specific and sensitive detection of human papillomaviruses (HPV).

Main Methods:

• Engineered Zst polymerase capable of polymerizing dATP, dCTP, dGTP, and dUTP (without dTTP) for contamination-free amplification.
• Targeting the L1 gene of human papillomaviruses (HPV) 16 and the E7 gene of HPV18.
• Validation using 85 HPV clinical swab samples.

Main Results:

• The ETL-CRISPR assay demonstrated single-copy level sensitivity and high specificity within 1 hour.
• Ultrasensitive DNA detection was achieved without aerosol contamination.
• Clinical sample validation showed a 98.8% accuracy for HPV detection, comparable to real-time quantitative polymerase chain reaction.

Conclusions:

• ETL-CRISPR provides a contamination-free and ultrasensitive method for molecular diagnosis.
• The engineered Zst polymerase is key to overcoming limitations of previous LAMP-CRISPR assays.
• This assay offers a straightforward strategy for point-of-care diagnosis of clinical pathogens.