Optimization and evaluation of sarcocystosis thermostable PCR detection kit / Nurul Fathiyah Zaipul Anuar

Sarcocystis spp. is a parasite that can cause sarcocystosis in human and animals. They are intracellular protozoan parasite that can be found mostly in cattle, sheep and goats. Diagnosis of Sarcocystis spp. is mainly carried by histological investigation or serological methods. Histological exami...

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Main Author:	Zaipul Anuar, Nurul Fathiyah
Format:	Thesis
Language:	English
Published:	2020
Subjects:	Infectious and parasitic diseases
Online Access:	https://ir.uitm.edu.my/id/eprint/60062/1/60062.pdf
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spelling	my-uitm-ir.600622022-05-21T02:39:06Z Optimization and evaluation of sarcocystosis thermostable PCR detection kit / Nurul Fathiyah Zaipul Anuar 2020-04 Zaipul Anuar, Nurul Fathiyah Infectious and parasitic diseases Sarcocystis spp. is a parasite that can cause sarcocystosis in human and animals. They are intracellular protozoan parasite that can be found mostly in cattle, sheep and goats. Diagnosis of Sarcocystis spp. is mainly carried by histological investigation or serological methods. Histological examination has limitations, as it depends on the site of collection and load of parasitic infection. On the other hand, serological diagnosis of active infection is also unreliable because activation is not always accompanied by changes in antibody levels. Molecular-based techniques are very efficient, as detection of large sample size can be carried out with enhanced specificity and sensitivity. Hence the most reliable method would be PCR technique. However, the conventional PCR requires trained personnel and cold chain transportation/storage. With the knowledge of the disease caused by the organism, its prevalence and geographical distribution, there is a need to develop and improvise the existing molecular tools which can be handy for early detection of Sarcocystis in rural areas, where facilities and molecular biologist might not be available, whilst reducing sample collection to processing time. Thermostable premix to detect Sarcocystis spp. was developed to make conventional PCR more user-friendly. The aim of this study is to develop a sarcocystosis thermostable PCR detection kit and evaluate this kit in terms of the sensitivity and specificity, stability, limit of detection (LOD), and repeatability. PCR amplification was carried out to detect the D2 region in conserved regions of 18S rRNA in Sarcocystis spp. A pair of primers to detect KMT1 gene in Pasteurella multocida was used as internal control. Sarcocystis capranis was used as positive control for this kit. The premix contents were lyophilized in the presence of enzyme stabilizer to ensure the Taq polymerase work efficiently at room temperature. A total number of 48 samples were used to test the specificity and sensitivity while the stability of this PCR kit was evaluated using Q10 accelerated aging method. The sensitivity and the specificity of the developed thermostable PCR kit was 100%. The Q10 accelerated ageing method at 37℃ was found to be about 2.4 month. The limit of detection (LOD) was evaluated using two-fold dilution DNA of Sarcocystis capranis. The limit of detection is 0.547 ng/μL. In the repeatability test, different samples with various concentrations of DNA samples were used. The repeatability study was used to observe the consistency of the PCR results from thermostable sarcocystosis premix based on the intensity of the bands. The percentage of coefficient of variance (COV) ranged from 1.00 % to 3.22 %. From this result, it is evident that this thermostable PCR kit has a good range of repeatability. The developed sarcocystosis thermostable PCR detection kit is very efficient in detection of Sarcocystis spp. with high sensitivity and specificity with an advantage of being a cold chain free PCR system. 2020-04 Thesis https://ir.uitm.edu.my/id/eprint/60062/ https://ir.uitm.edu.my/id/eprint/60062/1/60062.pdf text en public masters Universiti Teknologi MARA Faculty of Medicine Hussaini, Jamal (Associate Professor Dr.)
institution	Universiti Teknologi MARA
collection	UiTM Institutional Repository
language	English
advisor	Hussaini, Jamal (Associate Professor Dr.)
topic	Infectious and parasitic diseases
spellingShingle	Infectious and parasitic diseases Zaipul Anuar, Nurul Fathiyah Optimization and evaluation of sarcocystosis thermostable PCR detection kit / Nurul Fathiyah Zaipul Anuar
description	Sarcocystis spp. is a parasite that can cause sarcocystosis in human and animals. They are intracellular protozoan parasite that can be found mostly in cattle, sheep and goats. Diagnosis of Sarcocystis spp. is mainly carried by histological investigation or serological methods. Histological examination has limitations, as it depends on the site of collection and load of parasitic infection. On the other hand, serological diagnosis of active infection is also unreliable because activation is not always accompanied by changes in antibody levels. Molecular-based techniques are very efficient, as detection of large sample size can be carried out with enhanced specificity and sensitivity. Hence the most reliable method would be PCR technique. However, the conventional PCR requires trained personnel and cold chain transportation/storage. With the knowledge of the disease caused by the organism, its prevalence and geographical distribution, there is a need to develop and improvise the existing molecular tools which can be handy for early detection of Sarcocystis in rural areas, where facilities and molecular biologist might not be available, whilst reducing sample collection to processing time. Thermostable premix to detect Sarcocystis spp. was developed to make conventional PCR more user-friendly. The aim of this study is to develop a sarcocystosis thermostable PCR detection kit and evaluate this kit in terms of the sensitivity and specificity, stability, limit of detection (LOD), and repeatability. PCR amplification was carried out to detect the D2 region in conserved regions of 18S rRNA in Sarcocystis spp. A pair of primers to detect KMT1 gene in Pasteurella multocida was used as internal control. Sarcocystis capranis was used as positive control for this kit. The premix contents were lyophilized in the presence of enzyme stabilizer to ensure the Taq polymerase work efficiently at room temperature. A total number of 48 samples were used to test the specificity and sensitivity while the stability of this PCR kit was evaluated using Q10 accelerated aging method. The sensitivity and the specificity of the developed thermostable PCR kit was 100%. The Q10 accelerated ageing method at 37℃ was found to be about 2.4 month. The limit of detection (LOD) was evaluated using two-fold dilution DNA of Sarcocystis capranis. The limit of detection is 0.547 ng/μL. In the repeatability test, different samples with various concentrations of DNA samples were used. The repeatability study was used to observe the consistency of the PCR results from thermostable sarcocystosis premix based on the intensity of the bands. The percentage of coefficient of variance (COV) ranged from 1.00 % to 3.22 %. From this result, it is evident that this thermostable PCR kit has a good range of repeatability. The developed sarcocystosis thermostable PCR detection kit is very efficient in detection of Sarcocystis spp. with high sensitivity and specificity with an advantage of being a cold chain free PCR system.
format	Thesis
qualification_level	Master's degree
author	Zaipul Anuar, Nurul Fathiyah
author_facet	Zaipul Anuar, Nurul Fathiyah
author_sort	Zaipul Anuar, Nurul Fathiyah
title	Optimization and evaluation of sarcocystosis thermostable PCR detection kit / Nurul Fathiyah Zaipul Anuar
title_short	Optimization and evaluation of sarcocystosis thermostable PCR detection kit / Nurul Fathiyah Zaipul Anuar
title_full	Optimization and evaluation of sarcocystosis thermostable PCR detection kit / Nurul Fathiyah Zaipul Anuar
title_fullStr	Optimization and evaluation of sarcocystosis thermostable PCR detection kit / Nurul Fathiyah Zaipul Anuar
title_full_unstemmed	Optimization and evaluation of sarcocystosis thermostable PCR detection kit / Nurul Fathiyah Zaipul Anuar
title_sort	optimization and evaluation of sarcocystosis thermostable pcr detection kit / nurul fathiyah zaipul anuar
granting_institution	Universiti Teknologi MARA
granting_department	Faculty of Medicine
publishDate	2020
url	https://ir.uitm.edu.my/id/eprint/60062/1/60062.pdf
_version_	1783735079042809856

Optimization and evaluation of sarcocystosis thermostable PCR detection kit / Nurul Fathiyah Zaipul Anuar

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