Fig 1.
Primer sets used for amplification of the Clonorchis sinensis cytochrome c oxidase subunit 1 (cox1) gene by the loop-mediated isothermal amplification (LAMP) technique.
(A) Locations of the primer sequences. (B) Names, length and sequences of six primers. F3 and B3 represent forward and backward external primers, respectively; FIP and BIP represent forward and backward internal primers, respectively; and LF and LB represent forward and backward loop primers, respectively. Primer FIP consists of F1 complementary sequence and F2 direct sequence. Primer BIP consists of B1 direct sequence and B2 complementary sequence.
Fig 2.
Sensitivity of the loop-mediated isothermal amplification (LAMP) assay and PCR for the detection of Clonorchis sinensis genomic DNA.
Ten-fold serial dilutions starting from 1 ng of genomic DNA (lane 1) down to 1 fg (lane 7) were tested. (A) Naked eye detection of LAMP products using SYBR Green I. A green color indicates a positive reaction, and an orange color indicates a negative reaction. (B) Fluorescence of LAMP products after using SYBR Green I followed by detection under UV light. (C) Agarose gel electrophoresis of LAMP products followed by ethidium bromide staining and detection under UV light. (D) PCR with outer primers F3 and B3. Values in the left are in base pairs. Lane 8, non-template control (NTC); lane M, molecular marker.
Fig 3.
Sensitivity of the loop-mediated isothermal amplification (LAMP) assay for the detection of Clonorchis sinensis eggs in feces experimentally spiked with a known number of eggs in ten-fold serial dilutions from 10,000 eggs (lane 1) to 1 egg (lane 5).
(A) Naked eye detection of LAMP products using SYBR Green I. A green color indicates a positive reaction, and an orange color indicates a negative reaction. (B) Fluorescence of LAMP products after using SYBR Green I followed by detection under UV light. (C) Agarose gel electrophoresis of LAMP products followed by ethidium bromide staining and detection under UV light. Values in the left are in base pairs. Lane 6, negative stool DNA; lane M, molecular marker.
Fig 4.
Specificity of the loop-mediated isothermal amplification (LAMP) assay for the detection of Clonorchis sinensis genomic DNA.
(A) Naked eye detection of LAMP products using SYBR Green I. A green color indicates a positive reaction, and an orange color indicates a negative reaction. (B) Fluorescence of LAMP products after using SYBR Green I followed by detection under UV light. (C) Agarose gel electrophoresis of LAMP products followed by ethidium bromide staining and detection under UV light. Values in the left are in base pairs. Lane 1, Clonorchis sinensis; lane 2, Metagonimus yokogawai; lane 3, Opisthorchis viverrini; lane 4, Fasciola gigantica; lane 5, Spirometra erinacei; lane 6, Diphyllobothrium latum; lane 7, Ascaris lumbricoides; lane 8, Ascaris suum; lane 9, Necator americanus; lane 10, Trichuris trichiura; lane 11, Cryptosporidium parvum; lane 12, Entamoeba histolytica; lane 13, Giardia lamblia; lane 14, Escherichia coli; lane 15, non-template control; M, molecular marker.
Table 1.
Results of the loop-mediated isothermal amplification (LAMP) assay for the detection of Clonorchis sinensis DNA in 120 stool samples compared with the results diagnosed by Kato-Katz (KK) and real-time PCR.
Table 2.
Amplification of Clonorchis sinensis DNA in 120 stool samples by loop-mediated isothermal amplification (LAMP) according to the infection intensity presented as eggs per gram of feces (EPG).