Malaysian Journal of Analytical Sciences Vol 23 No 3 (2019): 401 - 406

CHEMICAL COMPONENTS OF POLYMERASE CHAIN REACTION IN 18S rRNA FOR DETECTION OF Cryptosporidium FROM RIVER WATER SAMPLES

(Komponen Kimia Tindak Balas Berantai Polimerase di dalam 18S rRNA untuk Pengesanan Cryptosporidium dari Sampel Air Sungai)

Mohd Aiman Barudin¹, Mohammad Lokman Md Isa^2,3, Afzan Mat Yusof^2,3*

¹Department of Biomedical Science, Kulliyyah of Allied Health Sciences

²Department of Basic Medical Sciences, Kulliyyah of Nursing

³Integrated Cellular and Molecular Biology Cluster (iMolec)

International Islamic University Malaysia, Jalan Sultan Ahmad Shah, Bandar Indera Mahkota,

25200 Kuantan, Pahang, Malaysia.

*Corresponding author: afzan@iium.edu.my

Received: 19 August 2018; Accepted: 18 May 2019

Abstract

The gene of 18S ribosomal RNA or 18S rRNA is the universal gene function as a general genetic marker for species identification of microorganisms including parasites. Cryptosporidium has distinct 18S rRNA genes along different species within the same genus. In this study, polymerase chain reaction or PCR was used to study chemical components of PCR setup in amplification of 18S rRNA gene of this parasite. Cryptosporidium was collected from river water samples and its presence was confirmed using specific immunofluorescence detection of this parasite. Isolated water containing Cryptosporidium was then subjected to genomic DNA extraction before PCR step. The chemical components of PCR consisting of MgCI₂, deoxynucleotide triphosphate (DNTPs), Polymerases, free DNase-water, universal primers and PCR buffer were studied in different volume and concentration. Each chemical component of PCR was optimized differently in yielding the same final volume of 20 µL per each reaction. The value range of chemical components of PCR consisted of MgCI₂ (0.1 µM-0.5 µM), dNTPs (50-250 mM), free DNase water (5-10 µL), polymerases (0.2-0.5 U) and universal primers (2-20 µM). The result indicated that 0.2 µM of MgCI₂, 100 mM of dNTPs, less than 10 µL of free DNase water, 0.5 U of polymerases and 10 mM of universal primers were the best combination to get better result for molecular identification of 18S rRNA Cryptosporidium. As a conclusion, accurate and proper concentration or volume of each PCR chemical components is essential for molecular identification of 18S rRNA Cryptosporidium gene. In future studies, study on gradient temperature parameters of PCR run can be included to study the chemical nature of amplified genes either in denaturation, annealing or extension steps.

Keywords: chemical, Cryptosporidium, polymerase chain reaction, 18S ribosomal RNA gene

Abstrak

Gen 18S ribosomal RNA adalah gen universal yang berfungsi sebagai penanda genetik umum untuk pengenalpastian spesies mikroorganisma termasuk parasit. Cryptosporidium mempunyai gen 18S rRNA yang berlainan daripada spesies berbeza dalam genus yang sama. Dalam kajian ini, tindak balas berantai polymerase atau PCR digunakan untuk mengkaji komponen kimia susun atur PCR dalam amplifikasi gen 18S rRNA bagi parasit ini. Cryptosporidium telah diambil dari sampel air sungai dan disahkan kehadirannya menggunakan pengesanan immunopendaflour terhadap parasit ini. Sampel air yang diambil mengandungi Cryptosporidium yang kemudiannya diteruskan untuk pengekstrakan genomik DNA sebelum peringkat PCR. Komponen kimia PCR yang terdiri daripada MgCI₂, deoksinukleotida trifosfat (dNTPs), polimerase, air yang bebas DNase, primer umum dan larutan penimbal PCR telah dikaji dalam isipadu dan kepekatan yang berbeza. Setiap komponen kimia PCR dioptimakan secara berbeza dalam menghasilkan isipadu akhir 20 uL bagi setiap tindak balas. Nilai julat komponen kimia PCR terdiri daripada MgCI₂ (0.1 µM-0.5 µM), dNTPs (50-250 mM), air bebas DNase (5-10 µL), polimerase (0.2-0.5 U) dan primer primer umum (2-20 µM). Keputusan menunjukkan bahawa 0.2 µM MgCI₂, 100 mM dNTPs, air bebas DNase yang kurang daripada 10 µL, 0.5 U polimerase dan 10 mM primer umum adalah kombinasi terbaik untuk mendapatkan dapatan yang lebih baik bagi pengenalpastian molekul Cryptosporidium 18S rRNA. Kesimpulannya, kepekatan atau isipadu yang tepat dan sesuai bagi setiap komponen kimia PCR adalah penting untuk pengenalpastian molekul gen Cryptosporidium 18S rRNA. Bagi kajian masa depan, kajian ke atas kecerunan parameter suhu untuk menjalani PCR boleh dimasukkan untuk mengkaji sifat semulajadi kimia pada gen yang diamplifikasi untuk peringkat-peringkat penyahaslian, pelekatan atau penyambungan.

Kata kunci: kimia, Cryptosporidium, tindak balas rantai polimerase, gen 18S ribosomal RNA

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