grxB gene as a potential target for the development of a Cronobacter sakazakii detection method in infant formula milk using PCR
Muktiningsih Nurjayadi (a*), Dandy Akbar Juliansyah (a), Jefferson Lynford Declan (a), Gladys Indira Putri (a), Ismaya Krisdawati (a), Maharanianska Azzahra (a), Irvan Maulana (a), Adinda Myra Amalia Putri (a), Irma Ratna Kartika (a), Fera Kurniadewi (a), Dalia Sukmawati (b), Sri Rahayu (b), Vira Saamia (c), Dwi Anna Oktaviani Saputro (c), I Made Wiranatha (c), H. E. Enshay (d)(e)(f)

a) Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Gedung KH. Hasjim Asj^ari, 6th Floor, Jl. Rawamangun Muka, Jakarta Timur, 13220, Indonesia
*muktiningsih[at]unj.ac.id
b) Department of Biology, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Gedung KH. Hasjim Asj^ari, 9th Floor, Jl. Rawamangun Muka, Jakarta Timur, 13220, Indonesia
c) Center Forensic Laboratory of the Criminal Investigation, Police of the Republic of Indonesia, Ciapmbuan Babakan Madang, Bogor, 1681, Indonesia
d) Institute of Bioproduct Development, Universiti Teknologi Malaysia (UTM), Skudai, Johor Bahru, Malaysia.
e) School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia (UTM), Skudai, Johor Bahru, Malaysia.
f) City of Scientific Research and Technology Applications, New Burg Al Arab, Alexandria, Egypt.

Abstract

Cronobacter sakazakii is a pathogen that causes necrotizing enterocolitis, sepsis, and meningitis in infants or neonates with reported case-fatality rates varying from 40 to 80 percent. Therefore, it is necessary to develop fast and accurate detection of C. sakazakii. This study aims to get the primer pairs and optimal annealing temperature for the Polymerase Chain Reaction (PCR) method to detect C. sakazakii. The glutaredoxin B (grxB) gene was found to be suitable for the detection of Cronobacter sakazaki, and a primer pair based on the grxB gene sequence was designed and synthesized. The genomic DNA of Cronobacter sakazakii strain ATCC 29544 was isolated and then used as a template for PCR assay. Optimization of running PCR conditions was carried out by varying the annealing temperature at the temperature range between 53-62 degree Celsius and the template DNA concentration of C. sakazakii is 53 ng/micro Liter with a purity (A260/280) of 1,853. The results showed that the primer grxB gave optimal results at the annealing temperature range between 57-61 degree Celsius. In addition, the results also showed that the grxB primer pair could amplify the grxB C. sakazakii fragment to produce an amplicon with a size of 151 base pairs (bp) and only single band was formed. Furthermore, the results of the primer design and the optimum temperature obtained will be used for the development of a sensitive and specific rapid detection method of C. sakazakii in food samples using real time PCR.

Keywords: Cronobacter sakazakii, grxB Gene, Polymerase Chain Reaction

Topic: Chemistry