The implementation of a universal 16S rRNA PCR can be hindered by problems with contamination of reagents which may be derived from a bacterial source, such as Taq DNA polymerase and uracil- N-glycosylase (UNG). This problem is exaggerated by the use of a highly conserved multiple-copy amplification target. PCR is capable of 10 6- to 10 7-fold amplification of a single copy of template DNA ( 29), making minor contamination of the PCR mixture with exogenous DNA a problem. In addition, there is sufficient variation within the 16S rRNA gene to provide species-specific discrimination of some of the major causative agents of meningitis and septicemia, namely, Neisseria meningitidis, Escherichia coli, Haemophilus influenzae, Streptococcus pneumoniae, and Listeria monocytogenes ( 24). A large amount of 16S rRNA sequence data is available, and these data indicate the highly conserved nature of the gene across the eubacterial kingdom. A gene target that is present in multiple copies increases the possibility of detection of small numbers of pathogens over an assay that detects a single copy gene target. Many bacterial species contain up to seven copies of the gene ( 3). The 16S rRNA gene is present in multiple copies in the genomes of all known human bacterial pathogens that belong to the eubacterial kingdom. Numerous workers ( 8, 16, 21, 24, 33) have used the 16S rRNA gene as a target for nonculture detection, and it has been the most widely used target for universal PCR amplification of DNAs from a broad range of organisms ( 17). This would confirm the necessity for antibiotic treatment and would influence patient management. Universal PCR can be used as a tool for the rapid detection of bacteria in normally sterile clinical samples and, as such, would be useful in differentiating bacterial from viral infections. This approach allows nonculture confirmation of meningitis and septicemia, which leads to improved disease surveillance and which provides guidance on appropriate antibiotic usage and patient management. The advent of molecular techniques, notably, PCR, makes it possible to identify the presence of bacterial DNA in culture-negative samples from patients with suspected infection ( 1, 4). Additionally, accurate disease surveillance is essential when polysaccharide-protein conjugate vaccines for meningococcal serogroup C ( 5, 26) and pneumococcal disease ( 23) are used and soon to be introduced into the national immunization schedules. Recently, PCR assays for the improved nonculture diagnosis of meningococcal and other bacterial diseases have been developed as a result of the growing discrepancy between the number of clinically diagnosed cases of meningococcal infection about which the Office of National Statistics is notified and culture-confirmed cases identified by the Public Health Laboratory Service Meningococcal Reference Unit ( 14). Without the development of ultrapure Taq DNA polymerase, ultrapure reagents, and plasticware guaranteed to be free of DNA, the implementation of a PCR for detection of eubacterial 16S rRNA with the TaqMan system will continue to be problematical. Restriction endonuclease treatment singly and together did not reduce the level of contaminating DNA. With UV irradiation there was a 4-log reduction in PCR sensitivity, with 8-methoxypsoralen activity facilitated by UV there was between a 5- and a 7-log reduction, and with DNase alone and in combination with restriction digestion there was a 1.66-log reduction. Certain treatments were more effective than others in eliminating the contaminating DNA however, to achieve this there was a decrease in sensitivity. In an attempt to overcome this problem, several methodologies were applied. This was compounded by the use of a small amplicon of approximately 100 bases, as is necessary with TaqMan chemistry. Contamination of reagents with bacterial DNA was a major problem exacerbated by the highly sensitive nature of the real-time PCR chemistry. During the development of this PCR, problems were noted with the use of this gene as an amplification target. A set of universal oligonucleotide primers specific for the conserved regions of the eubacterial 16S rRNA gene was designed for use with the real-time PCR Applied Biosystems 7700 (TaqMan) system.
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