Loop-mediated isothermal amplification: rapid detection of Angiostrongylus cantonensis infection in Pomacea canaliculata

Background Angiostrongylus cantonensis is a zoonotic parasite that causes eosinophilic meningitis in humans. The most common source of infection with A. cantonensis is the consumption of raw or undercooked mollusks (e.g., snails and slugs) harbouring infectious third-stage larvae (L3). However, the parasite is difficult to identify in snails. The purpose of this study was to develop a quick, simple molecular method to survey for A. cantonensis in intermediate host snails. Findings We used a loop-mediated isothermal amplification (LAMP) assay, which was performed using Bst DNA polymerase. Reactions amplified the A. cantonensis 18S rRNA gene and demonstrated high sensitivity; as little as 1 fg of DNA was detected in the samples. Furthermore, no cross-reactivity was found with other parasites such as Toxoplasma gondii, Plasmodium falciparum, Schistosoma japonicum, Clonorchis sinensis, Paragonimus westermani and Anisakis. Pomacea canaliculata snails were exposed to A. cantonensis first-stage larvae (L1) in the laboratory, and L3 were observed in the snails thirty-five days after infection. All nine samples were positive as determined by the LAMP assay for A. cantonensis, which was identified as positive by using PCR and microscopy, this demonstrates that LAMP is sensitive and effective for diagnosis. Conclusions LAMP is an appropriate diagnostic method for the routine identification of A. cantonensis within its intermediate host snail P. canaliculata because of its simplicity, sensitivity, and specificity. It holds great promise as a useful monitoring tool for A. cantonensis in endemic regions.


Findings
Angiostrongylus cantonensis can be found in the lungs and arteries of insectivores, rodents, canines and felines [1][2][3][4][5][6]. They are prevalent in the Pacific islands and Southeast Asia, and are the most common cause of eosinophilic meningitis in humans in areas where the parasite is endemic [7]. The definitive hosts of A. cantonensis are various species of rats. Modes of transmission of this parasite include ingestion of raw or undercooked snails and fresh leafy vegetables contaminated by infective third-stage larvae (L 3 ) [8]. First-stage larvae (L 1 ) of A. cantonensis grow to infective L 3 [9], and the results showed that the endemicity of A. cantonensis is primarily attributable to P. canaliculata and Achatina fulica, which were imported into P.R.China in 1981 [10,11] and 1931 [12,13], respectively, and have rapidly extended their geographic ranges. Indeed, these two snails are now listed as invasive species by the Chinese government. P. canaliculata played an important role in a recent angiostrongyliasis outbreak in P. R.China [14][15][16][17].
Current surveying of A. cantonensis intermediate host snails depends mainly on microscopic examination. However, the parasite is difficult to identify and it is often overlooked if the infective load is low. Furthermore, microscopy is laborious and time-consuming and is not suitable for large-scale surveys. Application of polymerase chain reaction (PCR) is considered a more accurate and practical diagnostic method [18]; however, despite its excellent sensitivity and specificity, a PCR approach requires special equipment and is not suitable for field application. With this in mind, we propose a novel detection method based on loop-mediated isothermal amplification (LAMP).
LAMP is a novel method for the rapid amplification of DNA. Its advantages include rapidity and minimal equipment requirements. Bst DNA polymerase can synthesize a new strand of DNA, while simultaneously displacing the complementary strand, thereby enabling DNA amplification at a single temperature with a single enzyme. Four primers are required for the LAMP reaction: FIP, BIP, F3 and B3. F3 and B3 contribute to the formation of a stem-loop structure, while the other two primers, FIP and BIP, which are complementary to the inner sequence of the stem-loop structure, are used to amplify the target sequence. Thus, using four amplification primers provides higher specificity to the reaction than conventional PCR method. Another advantage of LAMP is that amplification from stem-loop structures leads to the accumulation of a large amount of products of varying lengths, which make detection of amplified DNA much easier, the result can easily be seen by the naked eye.
Recently, the applicability of LAMP for the detection of parasites such as Trypanosoma, Babesia and Plasmodium has been demonstrated [19][20][21][22][23][24][25][26]. However, a LAMP application for survey of the intermediate host snails of A. cantonensis has not been developed. This report describes the establishment of a LAMP assay for the sensitive and specific detection of A. cantonensis in snails.

Ethics statement
The institutional ethics committee of Zhejiang Academy of Medical Sciences in Hangzhou approved this study. Animal experiments were carried out in adherence to institutional guidelines for animal husbandry.

Parasite preparation and experimental infection
An A. cantonensis animal infection model had been established in the lab [27,28]. A total of five Sprague-Dawley (SD) rats were infected by intragastric injection of 50-80 L 3 . Thirty-five days post-infection, fresh faeces (i.e., not older than 12 h) were collected from infected rats and the L 1 larvae were isolated by the Baermann technique [5]. Eggs of P. canaliculata were cultured in the laboratory to make certain that developing snails remained uninfected throughout the study. A total of nine adult snails were fasted for 48 h, and then exposed to a 200-ml suspension of 40,000 L 1 in a dish (radius = 8.5 cm, height = 6.5 cm) for 12 h at room temperature. Subsequently, the snails were transferred to aquariums with clean water maintained at a temperature of 21 ± 1°C and were fed a mixed diet consisting of vegetables and dried fish food. For the examination of P. canaliculata, a recently developed method relying on specific lung tissue features of this species was employed [28,29]. In brief, the lungs were separated from the snail body and opened. The nodules containing A. cantonensis larvae were then directly observed under a microscope. The above establishment of the infection model and experimental snail infection was conducted at the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention in Shanghai.

DNA extraction
Genomic DNA from A. cantonensis larvae, T. gondii, P. falciparum, adult worms of S. japonicum, C. sinensis, P. westermani and Anisakis larvae was extracted by using a QIAamp DNA mini kit (QIAGEN Inc; Valencia, CA, USA) and the concentration of DNA was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific; Wilmington, DE, USA). The lungs of nine infected and nine uninfected P. canaliculata snails were separated from the snail body and DNA was extracted for the LAMP and PCR assay. DNA derived from T. gondii, P. falciparum, S. japonicum, C. sinensis, P. westermani and Anisakis was used to demonstrate the specificity of the LAMP assay. The DNA was finally eluted in a 60-μl volume of elution buffer and stored at -20°C until use.

LAMP reaction
The LAMP reaction was conducted following methods described by Notomi, with minor modifications [30]. In brief, LAMP primers for the specific amplification of the A. cantonensis 18S rRNA gene were designed according to sequence data in GenBank (accession number AY295804.1) using the Primer Explorer programme. The F3 and B3 primers were located outside of the two primers (FIP and BIP) in the A. cantonensis 18S rRNA gene region. The sequences of the primers are shown in  The LAMP reaction mixture was incubated at a range of temperatures (61, 63, 65, 67 and 69°C) for different times (30,40, 50 and 60 min) to optimize the reaction conditions. The reaction was terminated by incubation at 95°C for 2 min.

LAMP product detection
As part of the assay development, the LAMP products were initially detected by several methods, including electrophoresis in 1.5% agarose gels that were stained with GelRed™ (Biotium Inc.; Hayward, CA, USA) and photographed using a BIORAD gel system (Bio-Rad Laboratories; Hercules, CA, USA). A DL 2,000™ DNA Marker (Takara; Dalian, P.R.China) was used in electrophoresis to determine molecular weights. In addition, the products were visually detected by a colour change after the addition of SYBR green I dye to the tubes. One microliter of SYBR green I dye (100×) (Cambrex BioSciences Inc.; Rockland, ME, USA) was added to each tube containing LAMP products.

Sensitivity and specificity of LAMP
The sensitivity of the LAMP assay was determined using A. cantonensis DNA samples and nine infected snail samples. A serial dilution of A. cantonensis genomic DNA was used at concentrations from 0.01 fg to 100 pg. The purified DNA was dissolved in 60 μl of double-distilled water, and 1 μl of the solution was used as the template for LAMP. The specificity of LAMP assay was tested using DNA samples from T. gondii, P. falciparum, S. japonicum, C. sinensis, P. westermani and Anisakis.

PCR reaction
To compare and evaluate the sensitivity and efficiency of the LAMP protocol with the classical PCR technique, a simple PCR was conducted targeting the same gene using the same samples as for LAMP assay. The PCR reaction was performed following methods described by Qvarnstrom Y, with minor modifications [18]. A 405-bp DNA fragment of the A. cantonensis 18S rRNA gene was amplified with sense primer (F, 5'-TTCGAGTATCCAGTG-GAG GG-3') and antisense primer (R, 5'-GCAAATGC TTTCGCTTTAGG -3'). The PCR products were purified and sequenced.

Development of an 18s rRNA-based LAMP assay
Agarose gel electrophoresis of all positive LAMP reactions revealed a ladder pattern with many bands of different sizes (Figure 2A), indicating that stem-loop DNA with inverted repeats of the target sequence was produced. In addition to gel electrophoresis, we used other methods to detect a positive reaction. Addition of SYBR green I dye to tubes after the LAMP reaction changed the colour of a positive reaction to yellowish green ( Figure 2B, tube 1-7), whereas the negative reactions remained reddish orange ( Figure 2B, tube N). All samples that were positive as determined by gel electrophoresis were also positive as determined by visual detection of colour change. The effect of LAMP amplification with different temperatures and periods of time was observed by gel electrophoresis to determine the optimum reaction condition. The best results were obtained when the reaction was incubated at a temperature of 65°C for 60 min (Data not shown).

Sensitivity and specificity
The sensitivity of the LAMP and PCR reaction was determined using 10× serial dilutions of A. cantonensis genomic DNA. The PCR detection limit was 100 pg (Figure 3), similar to the paper published before [12,31,32], while the LAMP detection limit was 1 fg (Figure 2), which shows that the LAMP assay is more sensitive than the PCR method. There is no cross-reaction with T. gondii, P. falciparum, S. japonicum, C. sinensis, P. westermani and Anisakis genomic DNA (Figure 4).

LAMP and PCR assay of infected snails
Nine samples from artificially infected snails were verified using microscopic examination, then analyzed using the LAMP and PCR method. The sequence results of PCR products demonstrate specific A. cantonensis gene was amplified. All these samples were positive for A. cantonensis, while the samples from uninfected snails remained negative ( Figure 5), which shows that LAMP is sensitive and effective for diagnosis of A. cantonensis.
In conclusion, the detection limit of the LAMP assay was 1 fg of A. cantonensis genomic DNA, while the detection limit of the PCR is 100 pg, which shows that LAMP assay is more sensitive than PCR method. The specificity of the LAMP assay was confirmed using DNA extracted from T. gondii, P. falciparum, S. japonicum, C. sinensis, P. westermani and Anisakis, and no crossreactivity was found. Nine samples from artificially infected snails determined by the microscopic examination were analyzed using the LAMP and PCR method. All these samples were positive for A. cantonensis, while samples from uninfected snails remained negative, which shows that LAMP is sensitive and effective for diagnosis. Thus, it was demonstrated that the LAMP technique could be used to amplify A. cantonensis DNA with high specificity and efficiency.
In recent years, the detection of snails by PCR has become an important tool for surveillance of A. cantonensis. Despite the clinical utility of PCR-based techniques, they have the inherent disadvantage of being timeconsuming as well as requiring special equipment which is not suitable for field application. We demonstrate in this study that the LAMP technique can be used to amplify A. cantonensis DNA under isothermal conditions with high specificity and efficiency. It has a minimal equipment requirement and can be accomplished in one   Positive reactions turned green after the addition of SYBR green I. Tube N, negative control; tube 1, A. cantonensis; tube 2, T. gondii; tube 3, P. falciparum; tube 4, S. japonicum; tube 5, C. sinensis; tube 6, P. westermani; tube 7, Anisakis hour or less. The specificity and sensitivity of the LAMP assay shows that LAMP is effective for diagnosis of A. cantonensis and is suitable for large-scale field surveys.