Development of a rapid screening test for Karenia mikimotoi by using loop-mediated isothermal amplification and lateral flow dipstick

Wang, Liang; Zhang, Chunyun; Chen, Guofu; Wang, Yuanyuan; Fu, Mengqi

doi:10.1007/s10811-020-02203-3

Cited by 5 publications

(4 citation statements)

References 60 publications

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“…Commonly used target genes for discriminating microalgae species include small subunit rDNA (Zhen et al, 2008 , 2009 ), large subunit rDNA (Guillou et al, 2002 ; Haywood et al, 2007 ; Bowers et al, 2017 ; Zhang et al, 2018 ), and ITS (Huang et al, 2017a , b , 2020 ; Engesmo et al, 2018 ; Zhang et al, 2019 ; Wang et al, 2020 ; Han et al, 2022 ). The ITS regions have been widely used for species discrimination in microalgae (Connell, 2002 ), including K. veneficum (Huang et al, 2017a ; Fu et al, 2019 ) and K. mikimotoi (Yuan et al, 2012 ; Zhang et al, 2019 ; Huang et al, 2020 ; Wang et al, 2020 ; Han et al, 2022 ). For most dinoflagellate genera, the ITS region is considered an effective species identifier (Stern et al, 2012 ).…”

Section: Discussionmentioning

confidence: 99%

“…Selecting the target gene for probing or priming is a crucial step in the development of a molecular detection assay. Commonly used target genes for discriminating microalgae species include small subunit rDNA (Zhen et al, 2008(Zhen et al, , 2009, large subunit rDNA (Guillou et al, 2002;Haywood et al, 2007;Bowers et al, 2017;Zhang et al, 2018), and ITS (Huang et al, 2017a(Huang et al, ,b, 2020Engesmo et al, 2018;Zhang et al, 2019;Wang et al, 2020;Han et al, 2022). The ITS regions have been widely used for species discrimination in microalgae (Connell, 2002), including K. veneficum (Huang et al, 2017a;Fu et al, 2019) and K. mikimotoi (Yuan et al, 2012;Zhang et al, 2019;Huang et al, 2020;Wang et al, 2020;Han et al, 2022).…”

Section: Discussionmentioning

confidence: 99%

“…To overcome this limitation, isothermal amplification techniques that do not require thermocyclers have shown promise for on-site application, including recombinase polymerase amplification (RPA) (Piepenburg et al, 2006 ), loop-mediated isothermal amplification (LAMP) (Notomi et al, 2000 ), and hyperbranched rolling circle amplification (Dean et al, 2001 ). Following pre-amplification, lateral flow dipstick (LFD) detection technology has been frequently used in conjunction with these isothermal amplification techniques for readout of the results, as demonstrated in methods developed for monitoring K. mikimotoi , such as hyperbranched rolling circle amplification coupled with LFD (Zhang et al, 2019 ) and LAMP-LFD (Huang et al, 2020 ; Wang et al, 2020 ; Han et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

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Development of a rapid detection method for Karenia mikimotoi by using CRISPR-Cas12a

Wang,

Chen,

Pan

et al. 2023

Front. Microbiol.

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Harmful algal blooms (HABs), mainly formed by dinoflagellates, have detrimental effects on marine ecosystems and public health. Therefore, detecting HABs is crucial for early warning and prevention of HABs as well as the mitigation of their adverse effects. Although various methods, such as light microscopy, electron microscopy, real-time PCR, and microarrays, have already been established for the detection of HABs, they are still cumbersome to be exploited in the field. Therefore, rapid nucleic detection methods such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP)-lateral flow dipstick (LFD) have been developed for monitoring bloom-forming algae. However, the CRISPR/Cas-based detection of HABs has yet to be applied to this field. In this study, we developed a method for detecting Karenia mikimotoi (K. mikimotoi), a typical ichthyotoxic dinoflagellate responsible for global blooms. Our method utilized Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) to target and cleave the internal transcribed spacer (ITS) of K. mikimotoi, guided by RNA. We leveraged the target-activated non-specific single-stranded deoxyribonuclease cleavage activity of LbCas12a to generate signals that can be detected using fluorescence-read machines or LFDs. By combining RPA and LbCas12a with reporters, we significantly enhanced the sensitivity, enabling the detection of ITS-harboring plasmids at concentrations as low as 9.8 aM and genomic DNA of K. mikimotoi at levels as low as 3.6 × 10−5 ng/μl. Moreover, we simplified the genomic DNA extraction method using cellulose filter paper (CFP) by directly eluting the DNA into RPA reactions, reducing the extraction time to < 30 s. The entire process, from genomic DNA extraction to result reporting, takes less than an hour, enabling the identification of nearly a single cell. In conclusion, our method provided an easy, specific, and sensitive approach for detecting K. mikimotoi, offering the potential for efficient monitoring and management of K. mikimotoi blooms.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Development of a rapid detection method for Karenia mikimotoi by using CRISPR-Cas12a

Wang,

Chen,

Pan

et al. 2023

Front. Microbiol.

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“…To date, molecular identification and detection methods for HABs have largely focused on species-specific molecular assays (Gray et al, 2003;Zhang et al, 2008;Al-Kandari et al, 2011;Toldrà et al, 2018;Huang et al, 2020;Wang et al, 2020) or universal eukaryotic molecular markers (Scorzetti et al, 2009;Zheng et al, 2009a;Marie et al, 2010;Yuan et al, 2012;Wu et al, 2015;. Kareniaceae species are generally present at a low cell density before a bloom occurs, which is usually undetectable or underestimated using universal eukaryotic primers (Gentien et al, 2005;Liu et al, 2020).…”

Section: Feasibility Of the Specific Barcodes For Kareniaceae Species...mentioning

confidence: 99%

Development of Specific DNA Barcodes for the Dinophyceae Family Kareniaceae and Their Application in the South China Sea

Zhang

Smith

et al. 2022

Front. Mar. Sci.

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Species from the family Kareniaceae (Dinophyceae) frequently cause harmful algal blooms (HABs), with serious ecological impacts and risks to human safety and aquaculture activities in coastal waters worldwide. However, due to their small size, lack of morphological divergence, and low abundance during non-bloom periods, the diversity within this family is not well understood. By comparing the commonly used molecular markers, the Internal Transcribed Spacer (ITS) region was found to have an appropriate mutation rate to distinguish three of the most common genera (Karenia, Karlodinium, and Takayama) within the Kareniaceae family and different geographical strains of Kareniaceae. Specific primers targeting the ITS region of Karenia, and the other primers specific to the genera Karlodinium and Takayama, were designed. Specificity of the primers was tested using 17 strains of Kareniaceae species and 15 non-target species. Representative Kareniaceae species could be successfully detected even at low concentrations of target DNA template with a limit of detection of 3.2 pg. The primers were also assessed using high-throughput sequencing with two environmental samples from the South China Sea (SCS). Analysis of the reads identified as Kareniaceae species revealed a high diversity and the existence of unreported Kareniaceae species in the SCS. In conclusion, the newly developed molecular barcodes specifically detected Kareniaceae species in the field and will provide technical support for the effective warning and monitoring of Kareniaceae HABs.

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Advances in multiplex molecular detection technologies for harmful algae

Zhang

Wang

et al. 2022

Environ Sci Pollut Res

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Development of a rapid screening test for Karenia mikimotoi by using loop-mediated isothermal amplification and lateral flow dipstick

Cited by 5 publications

References 60 publications

Development of a rapid detection method for Karenia mikimotoi by using CRISPR-Cas12a

Development of a rapid detection method for Karenia mikimotoi by using CRISPR-Cas12a

Development of Specific DNA Barcodes for the Dinophyceae Family Kareniaceae and Their Application in the South China Sea

Advances in multiplex molecular detection technologies for harmful algae

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