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

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 MgCI 2 , 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 MgCI 2 (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 MgCI 2 , 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. AbstrakGen 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 MgCI 2 , 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 ISSN 1394 -2506 Mohd Aiman et al: CHEMICAL COMPONENTS OF POLYMERASE CHAIN REACTION IN 18S rRNA FOR DETECTION OF Cryptosporidium FROM RIVER WATER SAMPLES 402 berbeza dalam menghasilkan isipadu akhir 20 uL bagi setiap tindak balas. Nilai julat komponen kimia PCR terdiri daripada MgCI 2 (0.1 µM-0.5 µM), dNTPs (50-250 mM), ai...

Polymerase chain reaction (PCR) primer design to identify SNP rs7901695 transcription factor 7 like 2 (TCF7L2)

“…Proses identifikasi keberadaan SNP menggunakan PCR harus dilengkapi dengan primer DNA Polimerase mulai mensintesis DNA baru dari ujung primer. Nukleotida (dNTP atau deoksinukleotida trifosfat) -unit tunggal dari basa A, T, G, dan C, merupakan "blok bangunan" untuk untai DNA baru (Barudin et al, 2019).…”