Loop-mediated isothermal amplification: rapid detection of Angiostrongylus cantonensis infection in Pomacea canaliculata - PubMed (original) (raw)

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

Rui Chen et al. Parasit Vectors. 2011.

Abstract

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.

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Figures

Figure 1

Figure 1

Specific primers used in this study. A: Position of primer sets in A. cantonensis 18S rRNA gene for LAMP and PCR assay. B: Specific primers for the LAMP and PCR assay.

Figure 2

Figure 2

Sensitivity of the LAMP assay for A. cantonensis detection. LAMP reactions were performed with various concentrations of A. cantonensis DNA. lane 1, 1000 pg; lane 2, 100 pg; lane 3, 10 pg; lane 4, 1 pg; lane 5, 100 fg; lane 6, 10 fg; lane 7, 1 fg; lane 8, 0.1 fg; lane 9, 0.01 fg; lane M, DL2000 DNA Marker; lane N, negative control.

Figure 3

Figure 3

Sensitivity of PCR for A. cantonensis detection. PCR reactions were performed with various concentrations of A. cantonensis DNA. lane 1, 1000 pg; lane 2, 100 pg; lane 3, 10 pg; lane 4, 1 pg; lane 5, 100 fg; lane 6, 10 fg; lane 7, 1 fg; lane 8, 0.1 fg; lane 9, 0.01 fg; lane M, DL2000 DNA Marker; lane N, negative control.

Figure 4

Figure 4

Specificity of the LAMP assay for A. cantonensis detection. Detection of DNA from A. cantonensis and other parasites by LAMP. LAMP reaction tubes were inspected visually. 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

Figure 5

Figure 5

PCR and LAMP amplification results from artificially infected snails. A: Detection of A. cantonensis in snail samples by PCR. B: Detection of A. cantonensis in snail samples by LAMP. lane M, DL2000 DNA Marker; lane P, positive control; lane N, negative control; lanes 1-9, infected snails; lanes 10-18, uninfected snails.

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