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Published on: December 23, 2022
A highly specific and ultrasensitive approach to detect Prymnesium parvum based on RPA-CRISPR-LbaCas12a-LFD system
Hai-Long Huang1, Ning-Jian Luo1, Wei-Zhong Chen1
1Key Laboratory of Marine Biotechnology of Zhejiang Province, School of Marine Science, Ningbo University, Ningbo, Zhejiang, 315211, China.
Background:
Harmful algal blooms (HABs), caused by the rapid proliferation or aggregation of microorganisms, are catastrophic for the environment. The Prymnesium parvum is a haptophyte algal species that is found worldwide and is responsible for extensive blooms and death of larval amphibians and bivalves, causing serious negative impacts on the ecological environment. For the prevention and management of environmental pollution, it is crucial to explore and develop early detection strategies for HABs on-site using simple methods. The major challenge related to early detection is the accurate and sensitive detection of algae present in low abundance.
Results:
Herein, recombinase polymerase amplification (RPA) was combined with clustered regularly interspaced short palindromic repeats and Cas12a protein (CRISPR-LbaCas12a) systems, and the lateral flow dipstick (LFD) was used for the first time for early detection of P. parvum. The internal transcribed spacer (ITS) of P. parvum was selected as the target sequence, and the concentration of single-strand DNA reporters, buffer liquid system, reaction time, and amount of gold particles were optimized. The RPA-CRISPR-LbaCas12a-LFD approach demonstrated highly specificity during experimental testing, with no cross-reaction against different microalgae used as controls. In addition, the lowest detection limit was 10,000 times better than the lowest detection limit of the standalone RPA approach. The feasibility and robustness of this approach were further verified by using the different environmental samples. It also observed that P. parvum are widely distributed in Chinese Sea, but the cell density of P. parvum is relatively low (<0.1 cells/mL).
Significance:
The developed approach has an excellent specificity and offers 10,000 times better sensitivity than the standalone RPA approach. These advantages make this approach suitable for early warning detection and prevention of HAB events in environmental water. Also, the outcomes of this study could promote a shift from traditional laboratory-based detection to on-site monitoring, facilitating early warning against HABs.
Insights
A new method combining recombinase polymerase amplification (RPA) with CRISPR-LbaCas12a and lateral flow dipstick (LFD) enables highly sensitive and specific early detection of Prymnesium parvum harmful algal blooms (HABs). This approach significantly improves detection limits for environmental monitoring.
Area of Science:
- Environmental Science
- Molecular Biology
- Biotechnology
Background:
- Harmful algal blooms (HABs) pose significant environmental threats, necessitating effective early detection strategies.
- Prymnesium parvum blooms cause ecological damage, including the death of aquatic organisms.
- Accurate and sensitive detection of low-abundance algae is critical for HAB management.
Purpose of the Study:
- To develop a simple, on-site, and highly sensitive method for the early detection of Prymnesium parvum.
- To improve upon existing detection methods for harmful algal blooms.
Main Methods:
- Development of a novel detection system integrating recombinase polymerase amplification (RPA) with clustered regularly interspaced short palindromic repeats and Cas12a protein (CRISPR-LbaCas12a).
- Utilized a lateral flow dipstick (LFD) for rapid, visual detection.
- Optimized key reaction parameters including DNA reporter concentration, buffer system, reaction time, and gold particle quantity.
Main Results:
- The developed RPA-CRISPR-LbaCas12a-LFD assay demonstrated high specificity, with no cross-reactivity against other microalgae.
- Achieved a 10,000-fold improvement in detection limit compared to standalone RPA.
- Successfully validated the method's feasibility and robustness using diverse environmental samples, detecting low P. parvum cell densities.
Conclusions:
- The novel RPA-CRISPR-LbaCas12a-LFD approach offers superior specificity and sensitivity for early HAB detection.
- This method facilitates a transition from laboratory-based analysis to on-site environmental monitoring.
- The developed assay is suitable for early warning systems to prevent and manage harmful algal bloom events.
