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A rapid and inexpensive method for the isolation and identification of Giardia.

ABSTRACT: This work details a protocol for the isolation of Giardia species from their host animals and their identification based on their morphological and molecular characteristics. Giardia are intestinal protozoan parasites found in almost all vertebrates and the epidemiology of Giardia has attracted the attention of scientists due to their harm to humans and live stocks worldwide. Giardia trophozoites adhere to the surface of host's intestines using their ventral disc, and they also adhere the culture tube wall during in vitro culturing. We developed a method of isolating Giardia trophozoites according to this phenomenon, and a method of isolating Giardia cysts according to their special density as well. We validated the protocol by isolating and identifying Giardia species from their host animals, and all the results support that this methodology has a certain validity. It could help the further epidemiological researches and other researches requiring relatively pure living organism as materials of this harmful parasite.•An isolation method with high efficiency which remains the physiological activity of isolated Giardia.•An identification method with high accuracy which avoids the influences from other organisms.•Low-cost, fast and convenient methodology.

SUBMITTER: Lyu Z

PROVIDER: S-EPMC7408348 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A rapid and inexpensive method for the isolation and identification of Giardia.

Lyu Zhangxia Z Wen Jianfan J

MethodsX 20200723

This work details a protocol for the isolation of Giardia species from their host animals and their identification based on their morphological and molecular characteristics. Giardia are intestinal protozoan parasites found in almost all vertebrates and the epidemiology of Giardia has attracted the attention of scientists due to their harm to humans and live stocks worldwide. Giardia trophozoites adhere to the surface of host's intestines using their ventral disc, and ...[more]

PMID: 32793426

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Project description:BackgroundKoi Herpesvirus (KHV) affects both juvenile and adult common carp and koi, and is especially lethal to fry. The high mortalities caused by the disease have had a negative impact on the international koi trade. Different diagnostic techniques have been used to detect KHV, including: isolation of the virus in cell culture, electron microscopy, several PCR tests, ELISA and in situ hybridisation. All of these methods are time consuming, laborious and require specialised equipment.ResultsA rapid field diagnosis of KHV in common and koi carp was developed using loop-mediated isothermal amplification (LAMP). The LAMP reaction rapidly amplified nucleic acid with high specificity and efficiency under isothermal conditions using a simple water bath. Two methods of extracting DNA from host tissue were compared: extraction by boiling and by using a commercial extraction kit. A set of six primers--two inner primers, two outer primers and two loop primers--was designed from a KHV amplicon. The reaction conditions were optimised for detection of KHV in 60 min at 65 degrees C using Bst (Bacillus stearothermophilus) DNA polymerase. When visualised by gel electrophoresis, the products of the KHV LAMP assay appeared as a ladder pattern, with many bands of different sizes from 50 base-pairs (bp) up to the loading well. The KHV LAMP product could also be simply detected visually by adding SYBR Green I to the reaction tube and observing a colour change from orange to green. All samples positive for KHV by visual detection were confirmed positive by gel electrophoresis. The KHV LAMP had the same sensitivity as a standard PCR assay for the detection of KHV.ConclusionThis paper describes an accelerated LAMP assay for diagnosis of KHV. The entire procedure took only 90 minutes to produce a result: 15 minutes for DNA extraction; 60 min for the LAMP reaction; 2 min for visual detection using SYBR Green I. The test can be used under field conditions because the only equipment it requires is a water bath.

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A rapid and inexpensive method for the isolation and identification of Giardia.

Publications

A rapid and inexpensive method for the isolation and identification of <i>Giardia</i>.

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