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 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.