Development of Mitochondrial Loop-Mediated Isothermal Amplification for Detection of the Small Liver Fluke Opisthorchis viverrini (Opisthorchiidae; Trematoda; Platyhelminthes)

Tháng Mười 11, 2017

Development of Mitochondrial Loop-Mediated Isothermal
Amplification for Detection of the Small Liver Fluke
Opisthorchis
viverrini
(Opisthorchiidae; Trematoda; Platyhelminthes)

Thanh Hoa Le,a Nga Thi Bich Nguyen,a Nam Hai Truong,b and Nguyen Van Dec
Department of Immunology, Institute of Biotechnology, Vietnam Academy of Science and Technology (VAST), Cau Giay District, Hanoi, Vietnama; Department of Genetic
Engineering, Institute of Biotechnology, VAST, Cau Giay District, Hanoi, Vietnam
b; and Department of Parasitology, Hanoi Medical University, Dong Da District, Hanoi,Vietnam.


Mitochondrial DNA sequences offer major advantages over the more usual nuclear targets for loop-mediated isothermal amplification approaches (mito-LAMP) because multiple copies occur in every cell. Four LAMP primers [F3, FIP(F1cF2),
BIP(B1c
B2), and B3] were designed based on the mitochondrial nad1 sequence of Opisthorchis viverrini and used for a highly
specific assay (mito-OvLAMP) to distinguish DNA of
O. viverrini from that of another opisthorchiid (Clonorchis sinensis) and
other trematodes (
Haplorchis pumilio, Haplorchis taichui, Fasciola hepatica, and Fasciola gigantica). Conventional PCR was
applied using F3/B3 primer pairs to verify the specificity of the primers for
O. viverrini DNA templates. All LAMP-positive samples could be detected with the naked eye in sunlight, by gel electrophoresis (stained with ethidium bromide), and by addition of
SYBR green I to the product in sunlight or under UV light. Only DNA from
O. viverrini yielded amplification products by LAMP
(and by PCR verification), and the LAMP limit of detection was as little as 100 fg (10
4 ng DNA), indicating that this assay is 10
to 100 times more sensitive than PCR. Field testing was done using representative egg and metacercarial samples collected from
localities where the fluke is endemic. With the advantages of simplicity, rapidity, sensitivity, and cost effectiveness, mito-OvLAMP is a good tool for molecular detection and epidemiology studies in regions or countries where
O. viverrini is endemic,
which can lead to more effective control of opisthorchiasis and trematodiasis.

https://drive.google.com/file/d/0BwAVbkCHjoJ8eHdPSkwxSTcwTzQ/view?usp=sharing