Hostname: page-component-cd9895bd7-p9bg8 Total loading time: 0 Render date: 2024-12-25T09:22:03.041Z Has data issue: false hasContentIssue false
  • English
  • Français

Development and evaluation of loop-mediated isothermal amplification (LAMP) for rapid detection of Clonorchis sinensis from its first intermediate hosts, freshwater snails

Published online by Cambridge University Press:  22 July 2013

Y. CHEN ,
T. WEN ,
D.-H. LAI ,
Y.-Z. WEN ,
Z.-D. WU ,
T.-B. YANG ,
X.-B. YU ,
G. HIDE  and
Z.-R. LUN
Y. CHEN
Affiliation:
Centre for Parasitic Organisms, State Key Laboratory of Biocontrol and Key Laboratory of Gene Engineering (Sun Yat-Sen University), Ministry of Education, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, P.R. China
T. WEN
Affiliation:
Centre for Parasitic Organisms, State Key Laboratory of Biocontrol and Key Laboratory of Gene Engineering (Sun Yat-Sen University), Ministry of Education, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, P.R. China
D.-H. LAI
Affiliation:
Centre for Parasitic Organisms, State Key Laboratory of Biocontrol and Key Laboratory of Gene Engineering (Sun Yat-Sen University), Ministry of Education, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, P.R. China
Y.-Z. WEN
Affiliation:
Centre for Parasitic Organisms, State Key Laboratory of Biocontrol and Key Laboratory of Gene Engineering (Sun Yat-Sen University), Ministry of Education, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, P.R. China
Z.-D. WU
Affiliation:
Key Laboratory of Tropical Disease and Control (Sun Yat-Sen University), Ministry of Education, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, P.R. China
T.-B. YANG
Affiliation:
Centre for Parasitic Organisms, State Key Laboratory of Biocontrol and Key Laboratory of Gene Engineering (Sun Yat-Sen University), Ministry of Education, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, P.R. China
X.-B. YU
Affiliation:
Key Laboratory of Tropical Disease and Control (Sun Yat-Sen University), Ministry of Education, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, P.R. China
G. HIDE
Affiliation:
Ecosystems and Environment Research Centre and Biomedical Research Centre, School of Environment and Life Sciences, University of Salford, Salford, M5 4WT, UK
Z.-R. LUN*
Affiliation:
Centre for Parasitic Organisms, State Key Laboratory of Biocontrol and Key Laboratory of Gene Engineering (Sun Yat-Sen University), Ministry of Education, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, P.R. China Key Laboratory of Tropical Disease and Control (Sun Yat-Sen University), Ministry of Education, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, P.R. China Ecosystems and Environment Research Centre and Biomedical Research Centre, School of Environment and Life Sciences, University of Salford, Salford, M5 4WT, UK
*
*Corresponding author: School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, P.R. China. E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Summary

Clonorchiasis, caused by Clonorchis sinensis, is a key foodborne zoonosis, which is mainly found in China, Korea and Vietnam. Detection of this parasite from the second intermediate host, the freshwater fish is the common method for epidemiological surveys of this parasite, but is time consuming, labour intensive and easily leads to misdiagnosis. In this study, we have developed a rapid, sensitive and reliable molecular method for the diagnosis of C. sinensis from its first intermediate hosts, freshwater snails, based on a loop-mediated isothermal amplification (LAMP) method. The specific amplified fragment from genomic DNA of C. sinensis did not cross-react with those from other relevant trematodes and a range of hosts (freshwater fish, shrimps and snails) of C. sinensis living in similar environments. The detection limit of the LAMP method was as low as 10 fg which was 1000 times more sensitive than conventional PCR, which was also demonstrated by successful application to field samples. These results show that the LAMP method is a more sensitive tool than conventional PCR for the detection of C. sinensis infection in the first intermediate hosts and, due to a simpler protocol, is an ideal molecular method for field-based epidemiological surveys of this parasite.

Keywords

Clonorchis sinensisloop mediated isothermal amplification (LAMP)first intermediate hostITS2field samplesrapid detection
Type
Research Article
Information
Parasitology , Volume 140 , Issue 11 , September 2013 , pp. 1377 - 1383
Copyright
Copyright © Cambridge University Press 2013 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Ai, L., Dong, S. J., Zhang, W. Y., Elsheikha, H. M., Mahmmod, Y. S. and Lin, R. Q. (2010). Specific PCR-based assays for the identification of Fasciola species: their development, evaluation and potential usefulness in prevalence surveys. Annals of Tropical Medicine and Parasitology 104, 65–72.CrossRefGoogle ScholarPubMed
Chen, R., Tong, Q., Zhang, Y., Lou, D., Kong, Q., Lv, S., Zhuo, M., Wen, L. and Lu, S. (2011). Loop-mediated isothermal amplification: rapid detection of Angiostrongylus cantonensis infection in Pomacea canaliculata. Parasites and Vectors 4, 204.CrossRefGoogle ScholarPubMed
Chen, Y. D., Zhou, C. H. and Xu, L. Q. (2012). Analysis of the results of two nationwide surveys on Clonorchis sinensis infection in China. Biomedical and Environmental Sciences 25, 163–166.Google ScholarPubMed
Coordinating Office of the National Survey on the Important Human Parasitic Diseases (2005). A report of the national survey on the current status of the important human parasitic diseases. Chinese Journal of Parasitology and Parasitic Diseases 23, 332–334 (in Chinese).Google Scholar
Cox, A., Tilley, A., McOdimba, F., Fyfe, J., Hide, G. and Welburn, S. C. (2005). A PCR based assay for detection and differentiation of African trypanosome species in blood. Experimental Parasitology 111, 24–29.CrossRefGoogle ScholarPubMed
Cox, A., Tosas, O., Tilley, A., Picozzi, K., Coleman, P. G., Hide, G. and Welburn, S. C. (2010). Constraints to estimating the prevalence of trypanosome infections in East African Zebu Cattle. Parasites and Vectors 3, 82.CrossRefGoogle ScholarPubMed
Deng, C. H., Liu, Z. Q., Zeng, W., Huang, L. Y., Chen, Y., Guo, L. L., Chen, X. Y., Shen, H. X., Li, X. M., Ma, C. L. and Chen, D. X. (2003). Investigation on the freshwater fish infection with encysted metacercaria of Clonorchis sinensis in some areas of Guangdong. Chinese Journal of Misdiagnostics 3, 343–344 (in Chinese).Google Scholar
Fang, Y. Y. (2007). Prevention and control of clonorchiosis. South China Journal of Preventive Medicine 33, 70–72 (in Chinese).Google Scholar
Fürst, T., Keiser, J. and Utzinger, J. (2012). The global burden of human food-borne trematodiasis: first estimates derived from a systematic review and meta-analysis. Lancet Infectious Diseases 12, 210–221.CrossRefGoogle Scholar
Huang, X. H., Zhong, W. Z., Chi, W. J., Liu, D. X., Zhang, X. J., Deng, F. T. and Huang, A. (2006). The prevalent characteristics of Clonorchis sinensis in limestone mountainous area and control measures. China Tropical Medicine 6, 408–409 (in Chinese).Google Scholar
International Agency for Research on Cancer (2012). Opisthorchis viverrini and Clonorchis sinensis. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans 100B, 341–370.Google Scholar
Kaewkes, S. (2003). Taxonomy and biology of liver flukes. Acta Tropica 88, 177–186.CrossRefGoogle ScholarPubMed
Kim, T. S., Cho, S. H., Huh, S., Kong, Y., Sohn, W. M., Hwang, S. S., Chai, J. Y., Lee, S. H., Park, Y. K., Oh, D. K. and Lee, J. K. (2009). A nationwide survey on the prevalence of intestinal parasitic infections in the Republic of Korea, 2004. Korean Journal of Parasitology 47, 37–47.CrossRefGoogle ScholarPubMed
Lai, D. H., Wang, Q. P., Chen, W., Cai, L. S., Wu, Z. D., Zhu, X. Q. and Lun, Z. R. (2008). Molecular genetic profiles among individual Clonorchis sinensis adults collected from cats in two geographic regions of China revealed by RAPD and MGE-PCR methods. Acta Tropica 107, 213–216.CrossRefGoogle ScholarPubMed
Li, F. L., Lin, R. X., Huang, B. M., Zhou, Y. L., Ou, B. W., Luo, C. H., Tan, Y. F. and Chen, X. J. (2005). Jiangmen city clonorchiosis epidemiological survey and treatment measures exploration. Chinese Journal of Parasitology and Parasitic Diseases 18, 214–215 (in Chinese).Google Scholar
Lim, M. K., Ju, Y. H., Franceschi, S., Oh, J. K., Kong, H. K., Hwang, S. S., Park, S. K., Cho, S. I., Sohn, W. M., Kim, D. I., Yoo, K. Y., Hong, S. T. and Shin, H. R. (2006). Clonorchis sinensis infection and increasing risk of cholangiocarcinoma in the Republic of Korea. American Journal of Tropical Medicine and Hygiene 75, 93–96.CrossRefGoogle ScholarPubMed
Lin, R. Q., Ai, L., Zou, F. C., Verweij, J. J., Jiang, Q. and Li, M. W. (2008). A multiplex PCR tool for the specific identification of Oesophagostomum spp. from pigs. Parasitology Research 103, 993–997.CrossRefGoogle ScholarPubMed
Lin, R. Q., Tang, J. D., Zhou, D. H., Song, H. Q., Huang, S. Y., Chen, J. X., Chen, M. X., Zhang, H., Zhu, X. Q. and Zhou, X. N. (2011). Prevalence of Clonorchis sinensis infection in dogs and cats in subtropical southern China. Parasites and Vectors 4, 180.CrossRefGoogle ScholarPubMed
Liu, Y. S. and Chen, M. (1998). Biology of Clonorchis sinensis and Control of Clonorchiasis. Science Press, Beijing (in Chinese).Google Scholar
Lun, Z. R., Gasser, R. B., Lai, D. H., Li, A. X., Zhu, X. Q., Yu, X. B. and Fang, Y. Y. (2005). Clonorchiasis: a key foodborne zoonosis in China. Lancet Infectious Diseases 5, 31–41.CrossRefGoogle ScholarPubMed
Mori, Y., Nagamine, K., Tomita, N. and Notomi, T. (2001). Detection of loop-mediated isothermal amplification by turbidity derived from magnesium pyrophosphate formation. Biochemical and Biophysical Research Communications 289, 150–154.CrossRefGoogle ScholarPubMed
Müller, B., Schmidt, J. and Mehlhorn, H. (2007). Sensitive and species-specific detection of Clonorchis sinensis by PCR in infected snails and fishes. Parasitology Research 100, 911–914.CrossRefGoogle ScholarPubMed
Nagamine, K., Hase, T. and Notomi, T. (2002). Accelerated reaction by loop-mediated isothermal amplification using loop primers. Molecular and Cellular Probes 16, 223–229.CrossRefGoogle ScholarPubMed
Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Wananabe, K., Amino, N. and Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research 28, e63.CrossRefGoogle ScholarPubMed
Parvathi, A., Karunasagar, I., Sanath Kumar, H., Kenchanna Prakasha, B., Lu, J., Xu, X., Hu, W. and Feng, Z. (2007). Clonorchis sinensis: development and evaluation of a nested polymerase chain reaction (PCR) assay. Experimental Parasitology 115, 291–295.CrossRefGoogle ScholarPubMed
Rim, H. J. (1986). The current pathobiology and chemotherapy of clonorchiasis. Korean Journal of Parasitology 24 (Suppl.), 1–141.CrossRefGoogle ScholarPubMed
Sato, M., Thaenkham, U., Dekumyoy, P. and Waikagul, J. (2009). Discrimination of O. viverrini, C. sinensis, H. pumilio and H. taichui using nuclear DNA-based PCR targeting ribosomal DNA ITS regions. Acta Tropica 109, 81–83.CrossRefGoogle Scholar
Tang, H. J., Lan, Y. G., Wen, Y. Z., Zhang, X. C., Desquesnes, M., Yang, T. B., Hide, G. and Lun, Z. R. (2012). Detection of Trypanosoma lewisi from wild rats in Southern China and its genetic diversity based on the ITS1 and ITS2 sequences. Infection Genetics and Evolution 12, 1046–1051.CrossRefGoogle ScholarPubMed
Tian, L. G., Chen, J. X., Wang, T. P., Cheng, G. J., Steinmann, P., Wang, F. F., Cai, Y. C., Yin, X. M., Guo, J., Zhou, L. and Zhou, X. N. (2012). Co-infection of HIV and intestinal parasites in rural area of China. Parasite and Vectors 5, 36.CrossRefGoogle ScholarPubMed
Xu, Z. M., Zhou, Z. Z., Wu, X. Y., Tao, Y. P., Li, M. H., Le, Q., Hua, Y., Chen, Q. and Wang, B. (2007). The changes on the infection rate of Clonorchis sinensis cyst in second intermediate host and ecological environment analysis. Journal of Pathogen Biology 2, 61–66 (in Chinese).Google Scholar
Zhang, X. C., Pei, F. Q., Zhang, Q. M., Lin, R. X., Huang, S. Y., Wang, J. L., Cen, Y. Z., Lu, W. C. and Chao, B. (2010). Current status of environmental sanitation and Clonorchis sinensis intermediate host infection of freshwater aquaculture in partial areas of Guangdong Province. South China Journal of Preventive Medicine 36, 9–13 (in Chinese).Google Scholar
Zhu, X. Q., D'Amelio, S., Gasser, R. B., Yang, T. B., Paggi, L. and He, F. (2007). Practical PCR tools for the delineation of Contracaecum rudolphii A and Contracaecum rudolphii B (Ascaridoidea: Anisakidae) using genetic markers in nuclear ribosomal DNA. Molecular and Cellular Probes 21, 97–102.CrossRefGoogle ScholarPubMed
Zou, X. H., Chen, L. G., He, L. J., Luo, J. P., Pan, D. and Cui, W. J. (2008). A survey of Clonorchis sinensis infection in Wujiang district of Shaoguan city. Journal of Tropical Medicine 8, 1285–1293 (in Chinese).Google Scholar
Supplementary material: Image

CHEN et al. supplementary material

Supplementary figure

Download CHEN et al. supplementary material(Image)
Image 86.2 KB
Supplementary material: Image

CHEN et al. supplementary material

Supplementary figure

Download CHEN et al. supplementary material(Image)
Image 2.9 MB