Current Issue

Volume 21, Number 1, Apr-Jun(Spring) 2019 , Serial Number: 81 Pages: 43-48

Detection of Mycoplasma Contamination of Cell Culture by A Loop-Mediated Isothermal Amplification Method


Zohre Soheily, M.Sc, 1, Mohammad Soleimani, Ph.D, 1, 2, *, Majidzadeh- Ardebili Keivan, M.D., M.PH., Ph.D., 3, 4,
Department of Microbiology, Qom Branch, Islamic Azad University, Qom, Iran
Department of Microbiology, Faculty of Medicine, AJA University of Medical Sciences, Tehran, Iran
Tasnim Biotechnology Research Center (TBRC), Faculty of Medicine, AJA University of Medical Sciences, Tehran, Iran
Motamed Cancer Institute, ACECR, Tehran, Iran
*Corresponding Address: P.O.Box: 14185611 Department of Microbiology Faculty of Medicine AJA University of Medical Sciences Tehran Iran Email:soleimanidor@yahoo.com

Abstract

Objective

Mycoplasmas are major contaminants of cell culture and affect in vitro biological and diagnostic tests. Mycoplasma detection is conducted using culture and molecular methods. These methods vary in terms of accuracy, reliably and sensitivity. Loop-mediated isothermal amplification (LAMP) is used to amplify target DNA in a highly specific and rapid manner. This study aimed to develop a LAMP method for rapid detection of Mycoplasma in culture samples.

Materials and Methods

In this descriptive laboratory study, for LAMP detection of Mycoplasma contaminations in cell culture, we used primers specifically designed for targeting the 16S rRNA conserved gene of Mycoplasma spp. For a positive control structure, 16S rRNA amplified based on PCR, was cloned in a plasmid vector and sequenced. The assay specificity was evaluated using Mycoplasma genomic DNA and a panel containing genomes of gram-positive and gram-negative organisms.

Results

In this study, the method developed for detection of Mycoplasma contamination of cell cultures was a rapid, sensitive and cost-effective LAMP approach. The results demonstrated that this method benefits from high specificity (100%) for amplification of Mycoplasma strains and high speed (multiplication within 60 minutes), while it does not require expensive laboratory equipment compared to those needed for polymerase chain reaction (PCR)-based detection.

Conclusion

Our study is the first report about application of LAMP assay based on 16S rRNA gene for detection of Mycoplasma strains; this technique could be considered a useful tool for rapid detection of contamination of cell culture.