Establishment of double probes rolling circle amplification combined with lateral flow dipstick for rapid detection of Chattonella marina

Highlights

• A novel method referred to dpRCA-LFD was developed for the detection of Chattonella marina.

• The dpRCA-LFD conditions were optimized.

• The developed dpRCA-LFD is specific for Chattonella marina.

• Background DNA did not affect the performance of dpRCA-LFD.

• The dpRCA-LFD is competent for convenient and accurate analysis of field samples.

Abstract

Chattonella marina is one of the main algae that could cause harmful algal blooms. It has killed a large number of cultured fish in coastal areas of many countries, causing serious economic losses. Therefore, it is necessary to establish a method that can specifically detect C. marina at pre-bloom abundance, so that timely measures can be taken before this alga causes harm. In this study, a long probe, a short probe and a pair of amplification primers were first designed by using the internal transcribed spacer (ITS) sequence of C. marina as the target gene and using the CD74 gene of a distant species Gallus gallus as the base sequence. The double probes rolling circle amplification (dpRCA) system was then established with the designed probes and amplification primers. A novel detection protocol referred to as dpRCA-LFD by combining the dpRCA products and lateral flow dipstick (LFD) was finally established, which can make the detection results visible to the naked eyes. The reaction conditions of dpRCA were optimized and the optimal conditions were as follows: cycle number of ligation reaction, 12; ligation temperature, 58 °C; amplification temperature, 60 °C; and amplification time, 60 min. The specificity test that was performed using the optimized dpRCA conditions indicated that dpRCA-LFD was exclusively specific for the target alga. The tests with the genomic DNA of C. marina and the recombinant plasmid containing the ITS sequence of C. marina showed that the sensitivity of dpRCA-LFD was 100 times higher than that of conventional PCR. The detection limit (DL) for the genomic DNA was 8.3 × 10⁻³ ng µL⁻¹ (8.3 × 10⁻³ ng per reaction), and the DL for the recombinant plasmid DNA was 7.8 copies µL⁻¹ (7.8 copies per reaction). The practicality of the developed dpRCA-LFD was further validated by test with the spiked samples containing C. marina and field samples. The simulative test showed that the dpRCA-LFD has a DL of 10 cells mL⁻¹. The dpRCA-LFD could successfully recognize the target cells from the field samples. In summary, the dpRCA-LFD established in this study has advantages of good specificity, high sensitivity, and easily visible detection results, and therefore is promising for the analysis of C. marina in field samples.

Introduction

In recent years, the occurrence frequency of harmful algal blooms (HABs) has increased due to the intensified human activities and deteriorating natural environmental conditions (Gobler et al., 2017). HABs have caused serious negative impacts on marine biological resources, ecosystem, and economic development, which has attracted attention from most coastal countries. To reduce the negative impacts caused by HABs, some countries have already enacted relevant laws and regulations (Inaba et al., 2019), and actively explored effective monitoring methods (Seltenrich, 2014; Zohdi and Abbaspour, 2019) and control strategies for HABs (Beaulieu et al., 2005; Jin et al., 2019).

It is an important strategy to monitor the growth status of harmful algae in real time and to take measures in time for particular situations to reduce the negative effects of HABs (Smith, 2019). Monitoring harmful algal species in marine environment can be mainly conducted through chemical and biological methods. Between them, the chemical detection methods mainly rely on determining the content of nitrogen, phosphorus (Ergül et al., 2018), lipid (Park et al., 2016) or lipophilic marine toxins (Orellana et al., 2015) in the algal cells using chemical instruments, or detecting algal cells using an electrochemical luminescence detection system (Zhu et al., 2012). The biological detection methods mainly include detection of living algal cells using quartz crystal microbalance (Sousa et al., 2014), microscopic examine based on morphological observation, molecular detection (Zhen et al., 2009), and so on. Harmful algae may produce toxic and harmful substances, or cause anoxia of water body, which usually leads to the death of marine organisms. Therefore, accurate recognition and identification of algal species is essential to take corresponding measures to reduce the harm caused by them (Suh et al., 2016). Molecular biological techniques can play an important role in this field. At present, several molecular detection methods have been developed, including quantitative PCR (He et al., 2007; Zhang and Li, 2012), sandwich hybridization integrated with nuclease protection assay (Zhen et al., 2008), reverse dot blot assay combined with DNA array (Zhang et al., 2015), fluorescence in situ hybridization (Zhang et al., 2010; Liu et al., 2019a), and multiplex PCR (Sun et al., 2019). In particular, the isothermal amplification techniques with high sensitivity have recently been applied to the detection of harmful algae, including loop-mediated isothermal amplification (LAMP) (Qin et al., 2019; Wang et al., 2019), rolling circle amplification (RCA) (Nie et al., 2017; Liu et al., 2019b), recombinase polymerase amplification (RPA) (Toldrà et al., 2018; Fu et al., 2019) and nucleic acid sequence-based amplification (NASBA) (Ulrich et al., 2010).

Double probes rolling circle amplification (dpRCA) is a novel isothermal amplification technique that is developed on the basis of RCA (Wang, 2013). Unlike the traditional RCA that only requires single padlock probe, dpRCA mainly relies on a short probe (PS, approximately 18 nt) and a long probe (PL, approximately 58 nt), both of which are phosphorylated at the 5′ end. The schematic representation of dpRCA probes is shown in Fig. 1. The PL is composed of T1, P2c, and T2. The sequences of T1, PS, and T2 are reverse complementary to the target gene, and their binding regions are continuous in the template. P2c is the reverse complementary sequence of amplification primer P2, which is designed with a gene sequence from a species that is distantly related to the target species. dpRCA is performed using the cyclization products of the PL and PS as the template, with the sequence same as the PS as the forward amplification primer (P1), and P2 as the reverse amplification primer. dpRCA can be divided into three steps. First, T1, T2, and PS complementarily bind with the template sequence. The phosphate and hydroxyl groups that are respectively labeled at the 5′ end and 3′ end of the probes form phosphodiester with Taq DNA ligase, producing the cycled ligation products that are served as template for the subsequent amplification reaction. Next, exonuclease is added to the ligation products, which can catalyze the hydrolysis of single nucleotides from the 3′ end of the unlinked probes. After removing the free probes, the amplification primer P2 and P1 are finally added to the digested products for exponential RCA. First, P2 reversely binds with the circular template, and RCA occurs with DNA polymerase that has strand displacement function. Then, P1 begins to extend by using the newly synthesized strand as template. The exponential RCA is accomplished by repeating the same process as mentioned above. dpRCA combined with lateral flow dipstick (dpRCA-LFD) can detect dpRCA products simply and quickly. In the LFD assay, the probe labeled with carboxyfluorescein (FAM) combines with probe labeled with biotin, and the resultant hybrid then binds to the colloidal gold-labeled anti-FAM antibody to form a ternary complex. The mixture is bound to the biotin antibody on the detection line, so the detection line turns red. In the meantime, the free FAM-labeled probe binds to the colloidal gold-labeled anti-FAM antibody to form a binary complex that can pass through the detection line by chromatography and binds with the quality control line, making it appear red. The LFD assay is simple, safe, and rapid. The whole procedure of LFD assay only takes 5–10 min and can be completed without special instruments. The analytic results can be determined by visually observing whether the colored detection line is formed.

Chattonella marina is one of the typical harmful microalgae with highly adaptive capability, which can grow in the temperature at 10–30 °C and salinity ranges of 15–50 psu respectively (Tiffany et al., 2001). It is one of the most common HABs-forming species that can cause mass death of cultured fish in coastal areas (Shen et al., 2010). Since it was reported for the first time in 1968, C. marina has been recorded to cause huge economic losses to many coastal areas (Dorantes-Aranda et al., 2013). For example, C. marina caused economic losses of 7.1 billion yen for Japan in 1972 (Marshall and Hallegraeff, 1999). In 1996, C. marina was responsible for the death of a large number of southern bluefin tuna Thunnus maccoyii in Australia, resulting in economic losses of $40 million (Masuda et al., 2011). In 2009 and 2010, the economic losses caused by C. marina in the Yatsushiro Sea in Japan exceeded 3 billion yen and 5 billion yen, respectively (Mukai et al., 2019). Therefore, the accurate and rapid identification and detection of C. marina is particularly important to reduce the possible losses for cultured fish.

In this study, the internal transcribed spacer (ITS) sequence of C. marina was cloned and sequenced to design probes and primers for dpRCA. Followed by establishing and optimizing the reaction conditions of dpRCA, dpRCA was combined with LFD to establish a novel detection method for C. marina, namely, dpRCA-LFD. The specificity, sensitivity and practicability of dpRCA-LFD were tested to confirm if the developed detection protocol could meet the requirements for field analysis.