Broad range DNA probes for detecting and amplifying eubacterial nucleic acids

Abstract

In this report we describe and characterize two oligomer probes that are broadly homologous to conserved eubacterial 16S ribosomal RNA (rRNA) sequences not present in human 18 rRNA or human mitochondrial 12S rRNA. One or both of the probes can detect all of 23 phylogenetically diverse eubacterial nucleic acids against which they were tested by dot blot hybridization. A sensitivity of about 1 bacterium per 10 eukaryotic cells was achieved. By using these oligomer sequences or their complements as primers in the polymerase chain reaction (PCR), the equivalent of 1 pg of E. coli DNA was detected in the presence of a large excess of eukaryotic DNA. Information useful for partial phylogenetic classification of detected organisms may be obtained by direct sequence analysis of the amplified DNA and comparison with known sequences or catalogs. Such broadly homologous probes offer advantages over more narrowly specific probes for detecting organisms whose identity is unknown. They could thus be employed for recognizing infection by organisms that cannot be cultured as may occur, for example, in tissue culture or in plant or animal diseases of unknown cause, provided the probes fail to hybridize with host nucleic acids.

Diagnosis of bacterial endophthalmitis by broad-range quantitative PCR

“…Chen et al 19 developed this PCR detection method for the eubacterial genome based on the conserved regions of the 16S rRNA sequence (16S rDNA) of Escherichia coli . As the universal primers chosen from 16S rDNA have a large amount of sequence information and highly conserved regions of the gene, primers can be synthesised for a wide variety of bacteria.…”

An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications

“…Forensic (for reviews [1], [2]), ancient DNA (for review [3]), environmental and conservation genetic studies (for review [4]) as well as analysis of DNA in processed food [5] deal with poorly preserved biological material in which DNA is often highly degraded, thus calling for highly sensitive amplification. Moreover, pathogens in clinical specimens can be detected and identified via PCR assays, (e.g., [6]). When only few initial target molecules are amplified via highly optimized and sensitive PCR procedures, contaminating DNA becomes a major problem since even low copy contamination will be amplified leading to false-positive results.…”

Enhanced Toxicity of Bacillus thuringiensis japonensis Strain Buibui Toxin to Oriental Beetle and Northern Masked Chafer (Coleoptera: Scarabaeidae) Larvae With Bacillus sp. NFD2

“…(Busti et al 2002); MDG74 F and MRW01 R (Greisen et al 1994); MP11P F and MP13P R (Chen et al 1989); MPLK1 F and MPLK2 R (Klaschik et al 2002); MfDl F, MrDl R, MrP2 R, MfD2 F, MrPl R, MfD3 F and MfD4 F (Weisburg et al 1991); and 16S rRNA F and 16S rRNA R (Integrated DNA Technologies, Inc., Coralville, IA). Primers were synthesized and obtained from Integrated DNA Technologies, Inc. PCR was conducted by adding 1 l of each primer (100 M), and 3 l of DNA isolate (1 ϫ 10 8 templates) into 45 l of Platinum PCR SuperMix 1.1ϫ (anti-TaqDNA polymerase antibody, Mg 2ϩ , dNTPs, and recombinant TaqDNA polymerase at concentrations sufÞcient to allow ampliÞcation during PCR) obtained from Invitrogen in a Þnal volume of 50 l. PCR vials were placed in a PTC-100 programmable thermal controller (MJ Research, Inc., Watertown, MA).…”