Presence of β-Lactamase Gene TEM-1 DNA Sequence in Commercial Taq DNA Polymerase

Abstract

The development and spread of expanded-spectrum β-lactamases (ESBLs) that cause resistance to β-lactam antibiotics has contributed to great concern worldwide. Most ESBLs are derived from TEM and SHV β-lactamases by point mutations within the blaTEM and blaSHV genes, giving rise to extended-spectrum drug resistance (2, 3). The standard method for determining the specific ESBL gene for the more than 90 TEM-type and the more than 25 SHV-type ESBLs is PCR followed by nucleotide sequencing (2). We routinely applied the standard method to confirm that ESBL genes were present in clinical strains of Enterobacteriaceae (6). To avoid cross-contamination, we used separate rooms for sample preparation, PCR assembly, and agarose gel analysis. Recently, we noticed that the negative (water) controls used in PCR amplification for blaTEM genes produced positive results; this did not occur when the blaSHV gene was targeted. The PCR product was of the predicted size, and nucleotide sequencing revealed that it was the blaTEM-1 gene. After systematic analysis of pipette tips, microcentrifuge tubes, and reagents for PCR, we found that the Taq DNA polymerase was the source of contamination. As shown in Fig. ​Fig.1,1, Taq DNA polymerase from manufacturer A produced a strong signal, but that from manufacturer B did not. FIG. 1. PCR products amplified from negative (water) controls with primers targeting the blaTEM gene. The product was run on a 1% agarose gel with 0.5× Tris-acetate-EDTA buffer. All reactions were performed in duplicate. Lane M, DNA size marker; lanes ... PCR is widely used to detect specific DNA sequences for purposes of microbial identification, clinical diagnosis, and basic research. Because the method is extremely sensitive, a small amount of contaminating DNA can be a serious problem. Taq DNA polymerase is often expressed as a recombinant protein in Escherichia coli. For studies involving gene cloning and protein expression in E. coli, the blaTEM-1 gene has been the most commonly used selective marker for expression vectors that are generally present in multiple copies (9). It is likely that during Taq DNA polymerase purification, the DNA containing the blaTEM-1 gene was not completely removed. This failure may not be a rare occurrence, but the contamination would be detected only if primers specific for blaTEM gene were used. Several reports have documented the presence of exogenous DNA in commercial Taq DNA polymerases (1, 4, 5, 7, 8, 10). Sources of the contaminating DNA have ranged from bacteria (1, 4) and phage-like DNA (7) to both prokaryotes and eukaryotes (10); in other studies, it was determined that the contaminating DNA was not from E. coli or Thermus aquaticus (5, 8). In all of these previous reports, PCR amplification was performed with universal primers for the highly-conserved 16S rRNA gene, whereas in the present study, amplification was done with primers targeting the blaTEM gene. Nevertheless, investigators, especially those who work on TEM-type ESBLs, should be aware of the possibility that Taq DNA polymerase is contaminated with the blaTEM-1 gene.