Detection of bacterial DNA in cerebrospinal fluid by an assay for simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and streptococci using a seminested PCR strategy

Rådström, Peter; Bäckman, Anders; Qian, Ny; Kragsbjerg, Peter; Påhlson, Carl; Olcén, Per

doi:10.1128/jcm.32.11.2738-2744.1994

Cited by 159 publications

(59 citation statements)

References 28 publications

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“…It has been reported that PCR amplification of numerous targets, such as the 16S rRNA gene, the 16S-23S rRNA interspacer region, the IS1106 region and the PorA gene, have been used to detect organisms in CSF and blood specimens from patients with meningitis (Radström et al, 1994;Hall et al 1995;Kotilainen et al 1998;Glustein et al 1999;Rantakokko-Jalava et al 2000;Seward and Towner, 2000). Molecular-based diagnostic approaches are now more frequently used alongside conventional microbiological techniques in the diagnostic clinical microbiology laboratory.…”

Section: Introductionmentioning

confidence: 99%

Employment of broad-range 16S rRNA PCR to detect aetiological agents of infection from clinical specimens in patients with acute meningitis - rapid separation of 16S rRNA PCR amplicons without the need for cloning

Millar

Moore

et al. 2003

J Appl Microbiol

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Aims: The aim of this study was to develop a polyacrylamide gel electrophoresis (PAGE) method for the rapid separation of 16S rRNA PCR amplicons from aetiological agents of acute meningitis. Methods and Results: Blood samples from 40 patients with suspected acute meningococcal meningitis were examined for the presence of causal agents, including Neisseria meningitidis employing two methods: (i) broad-range 16S rRNA PCR in conjunction with PAGE and automated sequencing and (ii) species-specific PCR employing ABI TaqMan technology for N. meningitidis. Analysis of clinical specimens employing 16S rRNA PCR yielded 33/40 (82AE5%) positive for the presence of bacterial DNA. Species-specific PCR yielded 30/40 (75%) clinical specimens positive for N. meningitidis. Prior to separation by PAGE, only 6/33 (18AE2%) amplicons were able to be identified by sequence analysis, the remaining amplicons (n ¼ 27) did not yield an identification due to the presence of mixed 16S rRNA PCR amplicons. Following separation, amplicons were re-amplified and sequenced, yielding 24/27 (88AE9%) positive for N. meningitidis and three specimens positive for Acinetobacter sp., Staphylococcus aureus and Streptococcus pneumoniae. One specimen was positive for both N. meningitidis and Streptococcus spp. and another specimen was positive for N. meningitidis and Pseudomonas sp., by broad-range PCR. Seven clinical specimens were negative for N. meningitidis and other eubacteria using both detection techniques. Conclusions: Clinical specimens including blood and cerebrospinal fluid from patients with suspected acute bacterial meningitis, may become contaminated with commensal skin flora, resulting in difficulties in downstream sequencing of pathogen plus contaminant DNA. This study allows for the rapid separation of amplified pathogen from contaminant DNA. Significance and Impact of Study: This study demonstrated the usefulness of the rapid separation of multiple 16S rRNA PCR amplicons using a combination of PAGE and automated sequencing, without the need of cloning. Adoption of this technique is therefore proposed when trying to rapidly identify pathogens in clinical specimens employing broad-range 16S rRNA PCR.

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Section: Introductionmentioning

confidence: 99%

Employment of broad-range 16S rRNA PCR to detect aetiological agents of infection from clinical specimens in patients with acute meningitis - rapid separation of 16S rRNA PCR amplicons without the need for cloning

Millar

Moore

et al. 2003

J Appl Microbiol

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“…The original method was modified as described by Zambardi et al [13] and makes use of the primers described by Frosh et al [14]. Subsequently, the method has been upgraded to detect simultaneously N. meningitidis, Streptococcus pneumoniae and Haemophilus influenzae [2,15]. In 2003, the National Reference Laboratory participated in an inter-laboratory comparison of PCR-based identification of N. meningitidis, which achieved a mean sensitivity and specificity of 90% and 93%, respectively [16].…”

Section: Discussionmentioning

confidence: 99%

Dynamics of PCR-based diagnosis in patients with invasive meningococcal disease

Bronská

Kalmusová

Džupová

et al. 2006

Clinical Microbiology and Infection

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Invasive meningococcal disease continues to be a life-threatening condition and rapid diagnosis is important for the administration of appropriate treatment. This study focused on the use of PCR for the diagnosis of meningococcal aetiology and the dynamics of PCR-based diagnosis over time in various biological samples. Sixty cerebrospinal fluid (CSF) and 144 serum samples collected during the first week of hospitalisation from 37 patients with laboratory-confirmed invasive meningococcal disease were investigated. Overall, 91.9% of CSF samples and 45.9% of serum samples were PCR-positive, while culture of CSF and blood was positive for only 35% and 39% samples, respectively. Positive PCR results were obtained until day 7 with CSF and until day 5 with serum. It is therefore recommended that samples for molecular diagnosis should be collected early in the course of suspected invasive meningococcal disease.

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“…The use of a universal PCR that amplifies conserved regions in various bacteria for DNA sequencing or probe preparation has been described (1,7,10,15). In 1989, Bottger (1) first demonstrated that a portion of the 16S rRNA gene from L. pneumophila, E. coli, or M. tuberculosis can be amplified by using one set of universal PCR primers and then sequenced to identify these bacteria.…”

Section: Discussionmentioning

confidence: 99%

“…Many different oligonucleotide probes were used to identify bacteria, including various probes specific for grampositive or gram-negative bacteria and 13 different speciesspecific probes. Radstrom et al (10) described the use of a seminested PCR method with genus-or species-specific primers to detect and identify H. influenzae, N. meningitidis, S. pneumoniae, S. agalactiae, and 24 different species of bacteria. All of these studies used multiple sets of PCR primers to detect or identify bacteria.…”

Section: Discussionmentioning

confidence: 99%

“…The main goal of this study is to develop an alternative method for detection and identification of bacteria in body fluids, such as blood and CSF. Since the 16S rRNA genes of almost all common bacterial pathogens found in body fluids have been sequenced, it is possible to design PCR primers capable of amplifying all eubacteria based on the conservative nature of the 16S rRNA genes (3,10). We have designed one set of such primers and found that this universal PCR can amplify all bacteria that we have examined to date.…”

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confidence: 99%

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Use of PCR with Universal Primers and Restriction Endonuclease Digestions for Detection and Identification of Common Bacterial Pathogens in Cerebrospinal Fluid

Lu¹,

Perng²,

Lee³

et al. 2000

J. Clin. Microbiol.

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We have designed a universal PCR capable of amplifying a portion of the 16S rRNA gene of eubacteria, including Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecium, Enterococcus faecalis, Mycobacterium tuberculosis, Legionella pneumophila, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Enterobacter cloacae, Pseudomonas aeruginosa, Acinetobacter baumannii, Proteus mirabilis, Haemophilus influenzae, and Neisseria meningitidis.The sizes of the amplified products from various bacteria were the same (996 bp), but the restriction patterns of most PCR products generated by HaeIII digestion were different. PCR products from S. aureus and S. epidermidis could not be digested by HaeIII but yielded different patterns when they were digested with MnlI. PCR products from S. pneumoniae, E. faecium, and E. faecalis yielded the same HaeIII digestion pattern but could be differentiated by AluI digestion . PCR products from E. coli, K. pneumoniae, S. marcescens, and E. cloacae also had the same HaeIII digestion pattern but had different patterns when digested with DdeI or BstBI. This universal PCR could detect as few as 10 E. coli or 250 S. aureus organisms. Compared with culture, the sensitivity of this universal PCR for detection and identification of bacteria directly from 150 cerebrospinal fluids was 92.3%. These results suggest that this universal PCR coupled with restriction enzyme analysis can be used to detect and identify bacterial pathogens in clinical specimens.

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Detection of bacterial DNA in cerebrospinal fluid by an assay for simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and streptococci using a seminested PCR strategy

Cited by 159 publications

References 28 publications

Employment of broad-range 16S rRNA PCR to detect aetiological agents of infection from clinical specimens in patients with acute meningitis - rapid separation of 16S rRNA PCR amplicons without the need for cloning

Employment of broad-range 16S rRNA PCR to detect aetiological agents of infection from clinical specimens in patients with acute meningitis - rapid separation of 16S rRNA PCR amplicons without the need for cloning

Dynamics of PCR-based diagnosis in patients with invasive meningococcal disease

Use of PCR with Universal Primers and Restriction Endonuclease Digestions for Detection and Identification of Common Bacterial Pathogens in Cerebrospinal Fluid

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