Development of an in-house PCR for the detection of giardia lamblia in cat and dog stool samples

Giardiasis is disease caused by a parasitic protozoan that can be transmitted through faecal-oral routes and carries a zoonotic transmission risk. Household pets, such as cats and dogs, can serve as carriers of this parasite, posing a transmission risk to vulnerable groups like children and the elde...

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Main Author:	Hussein, Ikran Ahmed
Format:	Thesis
Language:	English
Published:	2023
Subjects:	R Medicine RC Internal medicine
Online Access:	http://eprints.usm.my/60256/1/Ikran%20Ahmed%20Hussein-E.pdf
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id	my-usm-ep.60256
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spelling	my-usm-ep.602562024-04-16T01:57:27Z Development of an in-house PCR for the detection of giardia lamblia in cat and dog stool samples 2023-08 Hussein, Ikran Ahmed R Medicine RC Internal medicine Giardiasis is disease caused by a parasitic protozoan that can be transmitted through faecal-oral routes and carries a zoonotic transmission risk. Household pets, such as cats and dogs, can serve as carriers of this parasite, posing a transmission risk to vulnerable groups like children and the elderly. Although chronic and intermittent Giardia infections rarely lead to fatal outcomes, they can result in malnutrition or stunted growth in children and the elderly. Scheduling regular screenings of cat and dog stools is crucial to minimize the zoonotic risk of giardiasis. Stool microscopy is a robust and cost-effective method for detecting the parasite; however, its low sensitivity can lead to false negatives. On the other hand, PCR has higher sensitivity and can reduce false negatives due to the intermittent release of the parasite in stool. Thus, our study aimed to develop an in-house PCR method for detecting Giardia DNA in dog and cat stool samples. We established an axenic Giardia trophozoite culture and extracted Giardia DNA from these cells for use as an assay template. An in-house PCR was developed using Gia2029 and Gia2150c primers targeting a 250 bp amplicon. Additionally, we set up an internal control PCR (IC-PCR) using T7-promoter and T7-terminator primers targeting the Entamoeba histolytica SREHP gene (800 bp) in the pET14b-SREHP plasmid. The optimized annealing temperature for both Giardia-PCR and IC-PCR was 58 °C. The analytical limit of detection was approximately 20 pg of genomic DNA, equivalent to around 1000 trophozoites. In our preliminary PCR screening of Giardia in environmental stool samples from cats (n = 7) and dogs (n = 3) that tested negative by microscopy, we found unspecific amplicons in cat stool samples (6/7), but none in dog samples (0/3). All stool specimens showed positive PCR amplicons for the internal control gene, ruling out interference by PCR inhibitors. In conclusion, the PCR method based on Gia2029 and Gia2150c primers appears suitable for dog samples, while further evaluation via sequencing is required for cat samples. 2023-08 Thesis http://eprints.usm.my/60256/ http://eprints.usm.my/60256/1/Ikran%20Ahmed%20Hussein-E.pdf application/pdf en public masters Universiti Sains Malaysia Pusat Pengajian Sains Perubatan
institution	Universiti Sains Malaysia
collection	USM Institutional Repository
language	English
topic	R Medicine RC Internal medicine
spellingShingle	R Medicine RC Internal medicine Hussein, Ikran Ahmed Development of an in-house PCR for the detection of giardia lamblia in cat and dog stool samples
description	Giardiasis is disease caused by a parasitic protozoan that can be transmitted through faecal-oral routes and carries a zoonotic transmission risk. Household pets, such as cats and dogs, can serve as carriers of this parasite, posing a transmission risk to vulnerable groups like children and the elderly. Although chronic and intermittent Giardia infections rarely lead to fatal outcomes, they can result in malnutrition or stunted growth in children and the elderly. Scheduling regular screenings of cat and dog stools is crucial to minimize the zoonotic risk of giardiasis. Stool microscopy is a robust and cost-effective method for detecting the parasite; however, its low sensitivity can lead to false negatives. On the other hand, PCR has higher sensitivity and can reduce false negatives due to the intermittent release of the parasite in stool. Thus, our study aimed to develop an in-house PCR method for detecting Giardia DNA in dog and cat stool samples. We established an axenic Giardia trophozoite culture and extracted Giardia DNA from these cells for use as an assay template. An in-house PCR was developed using Gia2029 and Gia2150c primers targeting a 250 bp amplicon. Additionally, we set up an internal control PCR (IC-PCR) using T7-promoter and T7-terminator primers targeting the Entamoeba histolytica SREHP gene (800 bp) in the pET14b-SREHP plasmid. The optimized annealing temperature for both Giardia-PCR and IC-PCR was 58 °C. The analytical limit of detection was approximately 20 pg of genomic DNA, equivalent to around 1000 trophozoites. In our preliminary PCR screening of Giardia in environmental stool samples from cats (n = 7) and dogs (n = 3) that tested negative by microscopy, we found unspecific amplicons in cat stool samples (6/7), but none in dog samples (0/3). All stool specimens showed positive PCR amplicons for the internal control gene, ruling out interference by PCR inhibitors. In conclusion, the PCR method based on Gia2029 and Gia2150c primers appears suitable for dog samples, while further evaluation via sequencing is required for cat samples.
format	Thesis
qualification_level	Master's degree
author	Hussein, Ikran Ahmed
author_facet	Hussein, Ikran Ahmed
author_sort	Hussein, Ikran Ahmed
title	Development of an in-house PCR for the detection of giardia lamblia in cat and dog stool samples
title_short	Development of an in-house PCR for the detection of giardia lamblia in cat and dog stool samples
title_full	Development of an in-house PCR for the detection of giardia lamblia in cat and dog stool samples
title_fullStr	Development of an in-house PCR for the detection of giardia lamblia in cat and dog stool samples
title_full_unstemmed	Development of an in-house PCR for the detection of giardia lamblia in cat and dog stool samples
title_sort	development of an in-house pcr for the detection of giardia lamblia in cat and dog stool samples
granting_institution	Universiti Sains Malaysia
granting_department	Pusat Pengajian Sains Perubatan
publishDate	2023
url	http://eprints.usm.my/60256/1/Ikran%20Ahmed%20Hussein-E.pdf
_version_	1804888899682566144

Development of an in-house PCR for the detection of giardia lamblia in cat and dog stool samples

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