An ERA-CRISPR/Cas12a Method for Highly Sensitive Detection of Human Adenovirus Type 55

TL;DR

This paper presents a fast and sensitive method for detecting human adenovirus type 55 using a combination of ERA and CRISPR-Cas12a technologies.

Contribution

The study introduces a novel ERA-CRISPR/Cas12a method for detecting HAdV55 with high sensitivity and specificity.

Findings

The detection limit was as low as 9 copies/reaction for standard plasmids and 2.5 copies/reaction for cultured HAdV55 strains.

The method showed no cross-reactivity with other common respiratory pathogens or adenovirus subtypes.

The detection process was completed within 30 minutes at a constant temperature of 42 °C.

Abstract

Background/Objectives: Human adenovirus 55 (HAdV55) is a notable pathogen causing community-acquired pneumonia; outbreaks occur frequently in military camps, hospitals, and schools, thereby posing a threat to public health security. This study aimed to develop a method for detecting HAdV55 nucleic acid by targeting the conserved region of the Hexon gene. The sequence was amplified using enzymatic recombination isothermal amplification (ERA) technology, in conjunction with CRISPR-Cas12a technology, to enhance the amplification signal. Methods: Optimized primer and crRNA sequences were selected through ERA isothermal amplification testing. The ERA-CRISPR/Cas12a detection method was completed within 30 min at a constant temperature of 42 °C. Results: Sensitivity was assessed by detecting standard plasmids and live strains at various dilution concentrations. The detection limits were determined to be 9 copies/reaction for standard plasmids and 2.5 copies/reaction for cultured HAdV55 strains. Specificity tests were conducted on positive samples for five common respiratory pathogens and five other adenovirus subtypes, all of which showed no cross-reactivity. Conclusions: A rapid ERA-CRISPR/Cas12a nucleic acid detection method for HAdV55 has been successfully developed, demonstrating high sensitivity and specificity without the need for expensive or complex instruments. This method holds promise for on-site pathogen screening and detection.

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