Open Access
Research
(Published
online: 27-12-2016)
23.
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 -
Maryam Ozlati, Adel Spotin, Abbas Shahbazi, Mahmoud Mahami-Oskouei,
Teimour Hazratian, Mohammad Adibpor, Ehsan Ahmadpour, Afsaneh
Dolatkhah and Paria Khoshakhlagh
Veterinary World, 9(12): 1471-1477
doi:
10.14202/vetworld.2016.1471-1477
Maryam Ozlati:
Infectious and Tropical Diseases Research Center, Tabriz
University of Medical Sciences, Tabriz, Iran; Department of
Parasitology and Mycology, Faculty of Medicine, Tabriz
University of Medical Sciences, Tabriz, Iran; maryamomedicals@gmail.com
Adel Spotin:
Infectious and Tropical Diseases Research Center, Tabriz
University of Medical Sciences, Tabriz, Iran; Department of
Parasitology and Mycology, Faculty of Medicine, Tabriz University
of Medical Sciences, Tabriz, Iran; adelespotin@gmail.com
Abbas Shahbazi:
Department of Parasitology and Mycology, Faculty of Medicine,
Tabriz University of Medical Sciences, Tabriz, Iran; shahbazy42@yahoo.com
Mahmoud Mahami-Oskouei:
Department of Parasitology and Mycology, Faculty of Medicine,
Tabriz University of Medical Sciences, Tabriz, Iran; mmahami@gmail.com
Teimour Hazratian:
Department of Parasitology and Mycology, Faculty of Medicine,
Tabriz University of Medical Sciences, Tabriz, Iran;
hazratian2222@yahoo.com
Mohammad Adibpor:
Department of Parasitology and Mycology, Faculty of Medicine,
Tabriz University of Medical Sciences, Tabriz, Iran; adibpourm@yahoo.com
Ehsan Ahmadpour:
Drug Applied Research Center, Tabriz University of Medical
Sciences, Tabriz, Iran; ehsanahmadpour@gmail.com
Afsaneh Dolatkhah:
Department of Parasitology and Mycology, Faculty of Medicine,
Tabriz University of Medical Sciences, Tabriz, Iran;
afsaneh.dolatkhah@gmail.com
Paria Khoshakhlagh:
Department of Parasitology and Mycology, Faculty of Medicine,
Tabriz University of Medical Sciences, Tabriz, Iran;
paria.khoshakhlagh@gmail.com
Received: 31-08-2016, Accepted: 24-11-2016, Published online:
27-12-2016
Corresponding author:
Adel Spotin, e-mail: adelespotin@gmail.com
Citation:
Ozlati M, Spotin A, Shahbazi A, Mahami-Oskouei M, Hazratian T,
Adibpor M, Ahmadpour E, Dolatkhah A, Khoshakhlagh P (2016) 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,
Veterinary World, 9(12):
1471-1477.
Abstract
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
Toxocara
spp. from public areas soil inferred by loop-mediated isothermal
amplification (LAMP) assay.
Materials and Methods:
A
total of 180 soil samples were collected from various streets and
public parks of northwest Iran. The DNA of recovered
Toxocara
eggs were extracted and amplified by PCR and LAMP following ZnSO4
flotation technique. The amplicons of internal transcribed
spacer-2 gene were sequenced to reveal the heterogeneity traits of
Toxocara
spp. In addition,
Toxocara canis
sequences of southwest Iran were directly retrieved to compare
gene flow between two distinct populations.
Results:
Toxocara
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
T. canis
by PCR method, while LAMP was able to detect 27.2%, 15.5% and
12.2% as
Toxocara cati,
T. canis
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 (Fst)
as a degree of gene flow was generally low (0.02156) among
Toxocara
populations of northwest and southwest Iran.
Conclusions:
The statistically significant
Fst
value indicates that the
T. canis
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
Toxocara
spp. loads from public area soils will help to expand
epidemiological understanding of toxocariasis and establishing
preventive strategies in resource-limited endemic of Iran.
Keywords:
gene flow, Iran, loop-mediated isothermal amplification,
polymerase chain reaction, soil,
Toxocara
spp.
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