Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay

Abstract

Abstract. For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical.

Detection of helminths by loop-mediated isothermal amplification assay: a review of updated technology and future outlook

“…The impressive progress currently made in the LAMP assay for helminths includes trematodes of Clonorchis sinensis [12, 26, 60], Opisthorchis viverrini [14, 61, 62], Amphimerus spp. [63, 64], Paragonimus westermani [15], Fasciola hepatica [65–67], F. gigantica [65], Schistosoma japonicum [16, 27, 68–70], S. mansoni [13, 71–77], S. haematobium [51, 71, 72, 76]; nematodes of Necator americanus [78, 79], Ascaris lumbricoides [17, 79] , Trichuris trichiura [79], Toxocara canis [80] and T. cati [81], Strongyloides stercoralis [52, 82], Onchocerca volvulus [83–86], Wuchereria bancrofti [86, 87], Brugia malayi [86, 88], B. tomori [88], Loa loa [89–91], Dirofilaria repens [92], Angiostrongylus cantonensis [93, 94], Trichinella spiralis [95, 96], Bursaphelenchus xylophilus [97], and Haemonchus contortus [98, 99]; cestodes of T. solium [44, 100–103] , T. saginata [44, 100–103], T. asiatica [44, 100–103], T. hydatigena [104], T. multiceps [104], T. pisiformis [104] and T. crassiceps [104], Echinococcus granulosus [104–106], E. multilocularis [104, 107], E. equinus [108], E. canadensis [108], E. felidi [108], E. ortleppi [108, 109] and E. shiquicus [104] , have been covered in this review for further insight into its adoption for clinical diagnosis, field surveys and surveillance of helminths. The sensitivity and specificity of...…”

DNA-Detection Based Diagnostics for Taenia solium Cysticercosis in Porcine

“…Loop-mediated isothermal amplification (LAMP) is a DNA based isothermal amplification technique first described by Notomi et al [15] and has a wide application as a diagnostic tool for variety of helminthic and protozoan infections for both humans and [27] Cysticerci COX1, internal transcribed spacer 1 [32] Nested PCR Proglottids Tsol31 [10,26] Serum COX1 [14,33] Multiplex PCR cysticerci, proglottids COX1 [14] animals [36][37][38][39][40][41]. As shown in (Table 3), only few studies have utilized LAMP as a diagnostic tool for the detection of T. solium infections [12,13,42]. e LAMP method targeted the mitochondrial cytochrome c oxidase subunit 1 (cox1) which exists in a cell in large amounts.…”

“…e LAMP method targeted the mitochondrial cytochrome c oxidase subunit 1 (cox1) which exists in a cell in large amounts. Nkouawa et al [12] developed a multiplex LAMP assay in combination with a dot enzymelinked immunosorbent assay as a fast field-based test for the differentiation of human Taenia species. e LAMP assay was found to be more sensitive than the multiplex PCR technique for the differential detection of Taenia species in stool and cysticercus samples [43].…”

“…Because of the low sensitivity and specificity of the conventional diagnosis based on lingual and serological examination, molecular tools are more reliable for differential diagnosis of porcine cysticercosis. Several molecular diagnostic tools for detection of porcine cysticercosis have been developed elsewhere [11][12][13][14]; a good example is PCR based techniques that have been developed and adopted for molecular identification of porcine cysticercosis and for screening and authentication of meat [11]. Although PCR provides sensitive and reliable diagnostic results, it is laboratory based and expensive thus unsuitable for most low-income countries and inapplicable in the low resource areas where porcine cysticercosis infections are endemic.…”

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

“…One of the example is combining mLAMP with dot enzyme-linked immunosorbent assay (dot-ELISA) for differential identification of Taenia solium, Taenia saginata and Taenia asiatica, where cytochrome c oxidase subunit 1 gene was chosen as the target (Nkouawa et al 2016). In this study, a single tube of mLAMP reaction was performed with FIP primers labelled with fluorescein isothiocyanate (FITC), digoxigenin (DIG) and tetramethylrhodamine (TAMRA), and BIP primers labelled with biotin.…”