Loop-mediated isothermal amplification: rapid detection of Angiostrongylus cantonensis infection in Pomacea canaliculata
- Rui Chen†1,
- QunBo Tong†1,
- Yi Zhang2,
- Di Lou1,
- QingMing Kong1,
- Shan Lv2,
- MingMing Zhuo1,
- LiYong Wen1 and
- ShaoHong Lu1Email author
© Chen et al; licensee BioMed Central Ltd. 2011
Received: 23 March 2011
Accepted: 25 October 2011
Published: 25 October 2011
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.
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.
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.
Angiostrongylus cantonensis can be found in the lungs and arteries of insectivores, rodents, canines and felines [1–6]. 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 . 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 (L3) . First-stage larvae (L1) of A. cantonensis grow to infective L3 in intermediate host snails. This disease is difficult to detect because of the long incubation period in patients and few diagnostic symptoms. Thus, in order to control A. cantonensis, efforts should be directed towards building a surveillance system for the intermediate host snails of this parasite.
Many species of snails can serve as intermediate hosts for A. cantonensis. The first national survey pertaining to A. cantonensis and its definitive and intermediate hosts in the People's Republic of China (P.R.China) was implemented in 2006-2007 , 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 [14–17].
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 ; 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 [19–26]. 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.
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 L3. Thirty-five days post-infection, fresh faeces (i.e., not older than 12 h) were collected from infected rats and the L1 larvae were isolated by the Baermann technique . 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 L1 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.
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 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.
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 . A 405-bp DNA fragment of the A. cantonensis 18S rRNA gene was amplified with sense primer (F, 5'-TTCGAGTATCCAGTGGAG GG-3') and antisense primer (R, 5'-GCAAATGCTTTCGCTTTAGG -3'). The PCR products were purified and sequenced.
Development of an 18s rRNA-based LAMP assay
Sensitivity and specificity
LAMP and PCR assay of infected snails
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 cross-reactivity 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 time-consuming 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 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.
This work is supported by the National S & T Major Program (Grant No. 2008ZX10004-011), Zhejiang Science and Technology Project (2007F30016, 2009F20036), the Projects of Zhejiang Health Department (XKQ-009-003, XKQ-010-001), and Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents.
- Alicata JE: The discovery of Angiostrongylus cantonensis as a cause of human eosinophilic meningitis. Parasitol Today. 1991, 7: 151-153. 10.1016/0169-4758(91)90285-V.View ArticlePubMedGoogle Scholar
- Koo J, Pien F, Kliks MM: Angiostrongylus (Parastrongylus) eosinophilic meningitis. Rev Infect Dis. 1988, 10: 1155-1162. 10.1093/clinids/10.6.1155.View ArticlePubMedGoogle Scholar
- Rosen L, Loison G, Laigret J, Wallace GD: Epidemiologic and clinical observations on Pacific islands and the possible etiologic role of Angiostrongylus cantonensis. Am J Epidemiol. 1967, 85: 17-44.PubMedGoogle Scholar
- Tsai HC, Liu YC, Kunin CM, Lai PH, Lee SS, Chen YS, Wann SR, Lin WR, Huang CK, Ger LP, Lin HH, Yen MY: Eosinophilic meningitis caused by Angiostrongylus cantonensis associated with eating raw snails: correlation of brain magnetic resonance imaging scans with clinical findings. Am J Trop Med Hyg. 2003, 68: 281-285.PubMedGoogle Scholar
- Barçante JM, Barçante TA, Dias SR, Vieira LQ, Lima WS, Negrão-Corrêa D: A method to obtain axenic Angiostrongylus vasorum first stage larvae from dog feces. Parasitol Res. 2003, 89: 89-93. 10.1007/s00436-002-0719-z.View ArticlePubMedGoogle Scholar
- Anderson RC: Keys to the genera of the superfamily Metastrongyloidea. Edited by: Anderson RC, Chabaud AG, Willmont S. Commonwealth Agricultural Bureaux, Farnham Royal, Bucks, 40-C.I.H. Keys to the Nematode Parasites of Vertebrates, No. 5, 1978
- Alicata JE: Angiostrongyliasis cantonensis (eosinophilic meningitis): historical events in its recognition as a new parasitic disease of man. J Wash Acad Sci. 1988, 78: 38-46.Google Scholar
- Alto W: Human infections with Angiostrongylus cantonensis. Pac Health Dialog. 2001, 8: 176-182.PubMedGoogle Scholar
- Lv S, Zhang Y, Liu HX, Hu L, Yang K, Steinmann P, Chen Z, Wang LY, Utzinger J, Zhou XN: Invasive snails and an emerging infectious disease: results from the first national survey on Angiostrongylus cantonensis in China. PLoS Negl Trop Dis. 2009, 3: e368-10.1371/journal.pntd.0000368.PubMed CentralView ArticlePubMedGoogle Scholar
- Lv S, Zhang Y, Steinmann P, Zhou XN: Emerging angiostrongyliasis in Mainland China. Emerg Infect Dis. 2008, 14: 161-164. 10.3201/eid1401.061529.PubMed CentralView ArticlePubMedGoogle Scholar
- Joshi RC, Sebastian LS: Global advances in ecology and management of golden apple snails. Nueva Ecija: PhilRice. 2006, 588-Google Scholar
- Mead AR: The giant African snail: a problem in economic malacology. 1961, Chicago: University of Chicago Press, 257-Google Scholar
- Jarreit VHC: The spread of the snail Achatina fulica to south China. Hong Kong Nat. 1931, 2: 262-264.Google Scholar
- Zheng RY, Jin R, Lin BC, Pan CW, Xue DY: Probing and demonstrating etiological factor for outbreak of angiostrongyliasis cantonensis in Wenzhou. Sh J Prev Med. 2001, 13: 105-107.Google Scholar
- He ZY, Jia L, Huang F, Liu GR, Li J, Dou XF, Wang QY, He X, Gao ZY, Yang P, Wu J: Investigation on outbreak of angiostrongyliasis cantonensis in Beijing. Chin J Public Health. 2007, 23: 1241-1242.Google Scholar
- Deng ZH, Cai JS, Lin RX, Pei FQ, Cui HE, Qu Y, Gao XX, Wang K, Zhou SQ, Xie YM: The first local outbreak of Angiostrongylus cantonensis infection in Guangdong province. South China J Prev Med. 2007, 33: 17-20.Google Scholar
- Lv S, Zhang Y, Chen SR, Wang LB, Fang W, Chen F, Jiang JY, Li YL, Du ZW, Zhou XN: Human angiostrongyliasis outbreak in Dali, China. PLoS Negl Trop Dis. 2009, 3: e520-10.1371/journal.pntd.0000520.PubMed CentralView ArticlePubMedGoogle Scholar
- Qvarnstrom Y, Sullivan JJ, Bishop HS, Hollingsworth R, da Silva AJ: PCR-based detection of Angiostrongylus cantonensis in tissue and mucus secretions from molluscan hosts. Appl Environ Microb. 2007, 73 (5): 1415-1419. 10.1128/AEM.01968-06.View ArticleGoogle Scholar
- Mori Y, Nagamine K, Tomita N, Notomi T: Detection of loopmediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun. 2001, 289: 150-154. 10.1006/bbrc.2001.5921.View ArticlePubMedGoogle Scholar
- Aonuma H, Suzuki M, Iseki H, Perera N, Nelson B, Igarashi I, Yagi T, Kanuka H, Fukumoto S: Rapid identification of Plasmodium-carrying mosquitoes using loop-mediated isothermal amplification. Biochem Biophys Res Commun. 2008, 376: 671-676. 10.1016/j.bbrc.2008.09.061.View ArticlePubMedGoogle Scholar
- Ikadai H, Tanaka H, Shibahara N, Matsuu A, Uechi M, Itoh N, Oshiro S, Kudo N, Igarashi I, Oyamada T: Molecular evidence of infections with Babesia gibsoni parasites in Japan and evaluation of the diagnostic potential of a loop-mediated isothermal amplification method. J Clin Microbiol. 2004, 42: 2465-2469. 10.1128/JCM.42.6.2465-2469.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Kuboki N, Inoue N, Sakurai T, Di Cello F, Grab DJ, Suzuki H, Sugimoto C, Igarashi I: Loop-mediated isothermal amplification for detection of African trypanosomes. J Clin Microbiol. 2003, 41: 5517-5524. 10.1128/JCM.41.12.5517-5524.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Poon LL, Wong BW, Ma EH, Chan KH, Chow LM, Abeyewickreme W, Tangpukdee N, Yuen KY, Guan Y, Looareesuwan S, Peiris JS: Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat treated blood by loop-mediated isothermal amplification. Clin Chem. 2006, 52: 303-306.View ArticlePubMedGoogle Scholar
- Han ET, Watanabe R, Sattabongkot J, Khuntirat B, Sirichaisinthop J, Iriko H, Jin L, Takeo S, Tsuboi T: Detection of four Plasmodium species by genus- and species-specific loop-mediated isothermal amplification for clinical diagnosis. J Clin Microbiol. 2007, 45: 2521-2528. 10.1128/JCM.02117-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Iseki H, Alhassan A, Ohta N, Thekisoe OM, Yokoyama N, Inoue N, Nambota A, Yasuda J, Igarashi I: Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites. J Microbiol Methods. 2007, 71: 281-287. 10.1016/j.mimet.2007.09.019.View ArticlePubMedGoogle Scholar
- Aonuma H, Yoshimura A, Perera N, Shinzawa N, Bando H, Oshiro S, Nelson B, Fukumoto S, Kanuka H: Loop-mediated isothermal amplification applied to filarial parasites detection in the mosquito vectors: Dirofilaria immitis as a study model. Parasit Vectors. 2009, 2: 15-10.1186/1756-3305-2-15.PubMed CentralView ArticlePubMedGoogle Scholar
- Lv S, Zhang Y, Liu HX, Zhang CW, Steinmann P, Zhou XN, Utzinger J: Angiostrongylus cantonensis: morphological and behavioral investigation within the freshwater snail Pomacea canaliculata. Parasitol Res. 2009, 104: 1351-1359. 10.1007/s00436-009-1334-z.View ArticlePubMedGoogle Scholar
- Lv S, Zhou XN, Zhang Y, Liu HX, Zhu D, Yin WG, Steinmann P, Wang XH, Jia TW: The effect of temperature on the development of Angiostrongylus cantonensis (Chen 1935) in Pomacea canaliculata (Lamarck 1822). Parasit Res. 2006, 99: 583-587. 10.1007/s00436-006-0198-8.View ArticleGoogle Scholar
- Liu HX, Zhang Y, Lv S, Zhu D, Ang XH, Hu L, Zhou XN: A comparative study of three methods in detecting Angiostrongylus cantonensis larvae in lung tissue of Pomacea canaliculata. Chin J Parasitol Parasit Dis. 2007, 25: 53-56.Google Scholar
- Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T: Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000, 28: E63-10.1093/nar/28.12.e63.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang Y, Zhou XN, Liu HX, Lv S, Li LS, Lin JX, Li YS: Development of PCR assay for detection of Angiostrongylus cantonensis in Pomacea canaliculata. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2006, 24: 353-355.PubMedGoogle Scholar
- Qvarnstrom Y, da Silva AC, Teem JL, Hollingsworth R, Bishop H, Graeff-Teixeira C, da Silva AJ: Improved molecular detection of Angiostrongylus cantonensis in mollusks and other environmental samples with a species-specific internal transcribed spacer 1-based TaqMan assay. Appl Environ Microbiol. 2010, 76: 5287-5289. 10.1128/AEM.00546-10.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.