Development of a rapid screening test for Karenia mikimotoi by using loop-mediated isothermal amplification and lateral
- PDF / 1,101,313 Bytes
- 13 Pages / 595.276 x 790.866 pts Page_size
- 16 Downloads / 214 Views
Development of a rapid screening test for Karenia mikimotoi by using loop-mediated isothermal amplification and lateral flow dipstick Liang Wang 1,2 & Chunyun Zhang 1,2 & Guofu Chen 1 & Yuanyuan Wang 1 & Mengqi Fu 1,2 Received: 17 March 2020 / Revised and accepted: 3 July 2020 # Springer Nature B.V. 2020
Abstract As a bloom-forming dinoflagellate, Karenia mikimotoi is toxic and globally distributed. Frequent outbreaks of K. mikimotoi blooms usually lead to the mass mortality of pelagic and benthic marine life, resulting in large economic losses. Therefore, the simple, rapid, and highly effective monitoring of K. mikimotoi is crucial to minimize the damage caused by this alga. This study aimed to establish a convenient, cost-effective, and efficient molecular detection technique through loop-mediated isothermal amplification (LAMP) combined with chromatographic lateral flow dipstick (LFD) for the daily monitoring and real-time detection of K. mikimotoi. The internal transcribed spacer (ITS) and large subunit (LSU) rDNA sequences of K. mikimotoi were used to design and screen an optimal primer (KmLF3) and a detection probe (KmLF3HP). The optimized LAMP conditions were as follows: dNTP concentration, 1.2 mM; betaine concentration, 1 M; ratio of the inner primer to the outer primer, 8:1; magnesium ion concentration, 8 mM; amplification temperature, 59 °C; and amplification time, 60 min. Cross-reactivity tests confirmed the specificity of LAMP–LFD. The detection limit of LAMP–LFD for K. mikimotoi genomic DNA was 1.70 × 10−4 ng μL−1, which is 100 times lower than that of conventional PCR. The detection limit of LAMP–LFD for the recombinant plasmid containing the LSU rDNA of K. mikimotoi was 6.21 × 103 copies μL−1, which was 10 times more sensitive than that of LAMP followed by agarose gel electrophoresis and SYBR Green I dye staining and 100 times lower than that of conventional PCR. The practicability of LAMP–LFD was validated by testing with simulated field-water samples, displaying a detection limit of 10−1 cells mL−1. In conclusion, the developed LAMP–LFD is a specific, sensitive, and accurate detection method and may be competent for the field detection of K. mikimotoi. Keywords Karenia mikimotoi . Dinoflagellate . Loop-mediated isothermal amplification . Chromatographic lateral flow dipstick . Detection
Introduction The unarmored dinoflagellate Karenia mikimotoi, also recorded as Gymnodinium mikimotoi, Gyrodinium aureolum, and
* Chunyun Zhang [email protected] * Guofu Chen [email protected] 1
School of Marine Science and Technology, Harbin Institute of Technology (Weihai), Weihai 264209, PR China
2
School of Enviroment, Harbin Institute of Technology, Harbin 150000, PR China
Gymnodinium nagasakiense (Ottway et al. 1979; Hansen et al. 2000), is a notorious harmful algal bloom (HAB)-forming microalga that has a worldwide distribution (Brand et al. 2012; O'Boyle et al. 2016). The HABs caused by K. mikimotoi that was first reported in 1935 in Gokasho Bay, Japan, are frequently associated with mortal
Data Loading...
