1.A quadriplex PCR assay for rapid detection of diarrhoeacausing parasitic protozoa from spiked stool samples
Al-Talib, H. ; Julia Ashazila, M.J. ; Hussaini, J. ; Wang, S.M. ; Mohd Shah, N.A. ; Al-Khateeb, A. ; Chandrika, M.
Tropical Biomedicine 2019;36(2):348-356
Diarrhoea is a leading killer of children, accounting for 9% of all deaths among
children under age 5 worldwide and 3% in Malaysia in 2015. A large proportion of diarrhoea
illnesses among children in developing countries are ascribed to an unknown etiology
because microscopic examination was the only available technique which has low detection
limits. The proposed study aimed to evaluate a new quadriplex PCR assay to detect
parasitic pathogens namely E. histolytica, G. lamblia and C. parvum which considered
responsible for the majority of human infections. Three set of specific primer pairs were
designed for detection of parasitic pathogens. Quadriplex PCR assay was optimized and an
internal amplification control was incorporated to check for PCR inhibitors in samples.
The PCR assay was evaluated using spiked stool samples. Specific primer pairs were
successfully designed and simultaneously amplified the targeted genes. The analytical
sensitivity of the quadriplex PCR at the DNA level was found to be 50 ng DNA. The
analytical specificity was evaluated with 11 reference protozoal and bacterial strains and
was found to be 100%. We concluded that the developed quadriplex PCR assay was rapid
and gave results within 5 hours which is essential for the identification of parasitic pathogen
and might be useful as an additional diagnostic tool whenever time is important in the
diagnosis of parasite that cause diarrhoea.