Development of LAMP assays for the molecular detection of taeniid infection in canine in Tibetan rural area

Abstract

For field-identification of taeniid cestodes in canine animals in Tibetan area, loop-mediated isothermal amplification (LAMP) assays for Echinococcus multilocularis, E. shiquicus, Taenia hydatigena, T. multiceps, T. pisiformis and T. crassiceps were developed and evaluated along with the reported assay for E. granulosus. The LAMP assays showed specific reaction with their corresponding target species DNA with the detection limit of 1 to 10 pg. Moreover, the assays for E. granulosus, E. multilocularis, T. hydatigena and T. multiceps could detect DNA extracted from 3 or more eggs of their corresponding target species. Then, the LAMP assays were applied on samples containing 3 to 35 taeniid eggs obtained from 61 field-collected canine feces in Qinghai, and the result was compared with a reported multiplex PCR and sequence analysis. The LAMP assays and the PCR detected single species DNA of E. granulosus, E. shiquicus, T. hydatigena and T. multiceps in 5, 2, 44 and 2 samples, respectively. In the rest 8 samples, DNA of both E. granulosus and T. hydatigena were detected by the PCR but the LAMP assays detected those DNAs in 2 samples and only T. hydatigena DNA in 6 samples. It was assumed that less than 3 E. granulosus eggs were mixed in the samples although the samples contained 21 to 27 eggs in total. In conclusion, the LAMP assays were less sensitive than the multiplex PCR, but would have adequate sensitivity for field use in Tibetan area.

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...…”

“…with 10 000 protoscoleces–Egg and larvaStoolDog10 pg of DNA/rxn100%[104] E. granulosus sensu strictoProtoscolex––Nad1AF2976171/10 or 1/50 of one proscolex E. granulosus G1, E. granulosus G3 positive;[108]100% E. equinus Protoscolex––Nad1AF3464031/10 or 1/50 of one proscolex100%[108] E. Canadensis Protoscolex––Nad1AB2080631/10 or 1/50 of one proscolex E. Canadensis G6, E. Canadensis G7, E. Canadensis G8, E. Canadensis G10 positive;[108]100% E. felidi Egg––Nad1EF5583571/10 and 1/50 egg100%[105] E. ortleppi Protoscolex––Nad1AB2358461/10 or 1/50 of one proscolex100%[105]Protoscolex and associated germinal layerHydatid cystCamel and humanNad1JN63717710 pg of DNA/rxn100%[109] E. multilocularis ProtoscolexMultilocular cystic massesMouseNad5AB0313511 pg of DNA/rxn100%[107]EggStoolDogpositive from 12 days p.i. with 10 000 protoscoleces100%Larva–HumanCox1AB461411 pg of DNA/rxn100%[104]EggStoolDog5 egg of DNA extraction100% E. shiquicus Adult–FoxCox1JF9061310 pg of DNA/rxn100%[104] T. hydatigena …”

DNA Footprints: Using Parasites to Detect Elusive Animals, Proof of Principle in Hedgehogs

“…Further improvements could be made through development as a loop-mediated isothermal amplification (LAMP-PCR), using similar methodology to that previously described [ 59 , 60 ]. This has potential for a test which could be used in a field setting: Feng et al [ 59 ] found the LAMP-PCR method had lower, but adequate sensitivity for the specific detection of cestode DNA as compared to multiplex PCR, while Abbasi et al [ 60 ] demonstrated 10-fold increased sensitivity over PCR for the detection of Schistosoma spp. in infected snails.…”

Coenurosis of Yak, Bos grunniens, caused by Taenia multiceps: A Case Report with Molecular Identification in Qinghai Tibetan Plateau Area, China

“…The yak case pathogen was identified as the larval stage of T. multiceps by clinical anatomy and molecular biology methods, and epidemiologically, there were no cases of human infections in this pastoral area. This case may be caused by wild canids (definitive hosts) because Li et al [9] reported T. multiceps in Tibetan sand foxes in Qinghai Province of China with a 6% prevalence; on the other hand, Feng et al [10] isolated T. multiceps eggs from the faces of farm dogs also in Qinghai Province of China. In the same environment and area, there were many wild foxes and domestic dogs (definitive hosts) and sheep, goat and yaks (intermediate hosts) even wandered around people’s residences in some situations, threatening human health.…”