Genetic variability and discrimination of low doses of Toxocara spp. from public areas soil inferred by loop-mediated isothermal amplification assay as a field-friendly molecular tool

• Published in: Veterinary World 2016-12, Vol.9 (12), p.1471-1477
• Main Authors: Ozlati, Maryam, Spotin, Adel, Shahbazi, Abbas, Mahami-Oskouei, Mahmoud, Hazratian, Teimour, Adibpor, Mohammad, Ahmadpour, Ehsan, Dolatkhah, Afsaneh, Khoshakhlagh, Paria
• Format: Article
• Language: English
• Subjects: Ascaridida; gene flow; Genetic aspects; Genetic variation; Identification and classification; Iran; loop-mediated isothermal amplification; Medical research; polymerase chain reaction; soil; Soil microbiology; Toxocara spp
• ISSN: 0972-8988; 2231-0916
• DOI: 10.14202/vetworld.2016.1471-1477

Aim: One of the main diagnostic problems of conventional polymerase chain reaction (PCR) is indiscrimination of low parasitic loads in soil samples. The aim of this study is to determine the genetic diversity and identification of spp. from public areas soil inferred by loop-mediated isothermal amplification (LAMP) assay. A total of 180 soil samples were collected from various streets and public parks of northwest Iran. The DNA of recovered eggs were extracted and amplified by PCR and LAMP following ZnSO flotation technique. The amplicons of internal transcribed spacer-2 gene were sequenced to reveal the heterogeneity traits of spp. In addition, sequences of southwest Iran were directly retrieved to compare gene flow between two distinct populations. spp. eggs were found in 57, 14 and 77 of soil samples using the microscopy, PCR and LAMP (detection limit 1-3 eggs/200 g soil), respectively. 7.7% of isolates were identified as by PCR method, while LAMP was able to detect 27.2%, 15.5% and 12.2% as , and mixed infections, respectively. The kappa coefficient between LAMP and microscopy indicated a strong agreement (0.765) but indicated a faint agreement among LAMP-PCR (0.203) and PCR-microscopy (0.308) methods. A pairwise fixation index ( st) as a degree of gene flow was generally low (0.02156) among populations of northwest and southwest Iran. The statistically significant st value indicates that the populations are not genetically well differentiated between northwest and southwest Iran. This shows that here is possibly an epidemiological drift due to the transfer of alleles. The LAMP assay because of its shorter reaction time, more sensitivity, and simultaneous detection of environmental contamination to be appears as valuable field diagnosis compared to PCR. Therefore, the detection of low spp. loads from public area soils will help to expand epidemiological understanding of toxocariasis and establishing preventive strategies in resource-limited endemic of Iran.