Detection of nucleic acid sequences from bacterial species with molecular genetic methods

Petershofen, Eduard K.; Fislage, Rainer; Faber, R.; Schmidt, Heidrun; Roth, W. Kurt; Seifried, Erhard

doi:10.1016/s0955-3886(00)00051-5

Cited by 21 publications

(14 citation statements)

References 20 publications

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“…With respect to commercially available Taq polymerase depleted of bacterial DNA, as already suggested by Rand et al [5], again in our tests this approach did not show any influence on the total sensitivity. Additionally variations in bacterial DNA contamination found in different batches of Taq polymerase have been reported by Petershofen et al [15]. Therefore, our discouraging findings may be due to the fact that only one batch of one specific distributor has been tested.…”

Section: Disclosurementioning

confidence: 69%

Obstacles of Multiplex Real-Time PCR for Bacterial 16S rDNA: Primer Specifity and DNA Decontamination ofTaqPolymerase

Philipp¹,

Huemer

Irschick³

et al. 2010

Transfus Med Hemother

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Background: The detection of a broad range of bacteria by PCR is applied for the screening of blood and blood products with special attention to platelet concentrates. For practical use it is desirable that detection systems include Gram-positive, Gram-negative and non-Gram-stainable bacteria. It is quite challenging to achieve high sensitivity along with a clear negative control with PCR reagents, because especially Taq polymerase is contaminated with traces of bacterial DNA. Methods: Bacterial DNA decontamination of Taq polymerase was attempted by two different methods using the restriction enzyme Sau 3A1 and microfiltration. Additionally a commercially available Taq polymerase depleted of bacterial DNA was included. A published real-time PCR specific for Gram-negative bacteria was adapted for Gram-positive bacteria, including certain Staphylococcus species and Mycobacteria, and was used to charge the three Taq polymer-ases depleted of bacterial DNA contamination Results: Despite published reports about successful DNA decontamination, all three approaches performed poorly in experiments done in this study. Sensitivity ranged at approximately 50–100 colony forming units (CFU) per PCR reaction for Escherichia coli and Staphylococcus epidermidis, corresponding to 1,250–2,500 CFU/ml sample material. Conclusion: It seems unsatisfying to accept detection limits that high for diagnostic bacterial PCR even if highly multiplexed. Reliable methods for DNA decontamination of Taq polymerase are needed and would present one important step towards bacterial DNA detection with high sensitivity.

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Section: Disclosurementioning

confidence: 69%

Obstacles of Multiplex Real-Time PCR for Bacterial 16S rDNA: Primer Specifity and DNA Decontamination ofTaqPolymerase

Philipp¹,

Huemer

Irschick³

et al. 2010

Transfus Med Hemother

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“…Previous studies have used 16S ribosomal DNA as a target for nucleic acid amplification technology (NAT) screening (6,7,29,33,35). Analyses of the sequences of 23S ribosomal DNAs revealed more variation between species, so they might be useful for identification or for specific detection with specific fluorescent probes.…”

Section: Discussionmentioning

confidence: 99%

“…Only automated bacterial blood culturing systems meet many of the requirements of an ideal test because they detect a wide range of organisms at concentrations of only 1 to 10 CFU per ml (5). Recently, molecular genetic techniques based on bacterial genomic detection were developed, with the 16S rRNA gene as a target (7,11,29,33). In real-time PCR, a sensitivity of about 30 CFU per ml was demonstrated (33).…”

mentioning

confidence: 99%

Two Novel Real-Time Reverse Transcriptase PCR Assays for Rapid Detection of Bacterial Contamination in Platelet Concentrates

2004

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The incidence of platelet bacterial contamination is approximately 1 per 2,000 units and has been acknowledged as the most frequent infectious risk from transfusion. In preliminary studies, the sterility of platelet concentrates (PCs) was tested with an automated bacterial blood culturing system and molecular genetic assays. Two real-time reverse transcriptase PCR (RT-PCR) assays performed in a LightCycler instrument were developed and compared regarding specificity and sensitivity by the use of different templates to detect the majority of the clinically important bacterial species in platelets. Primers and probes specific for the conserved regions of the eubacterial 23S rRNA gene or the groEL gene (encoding the 60-kDa heat shock protein Hsp60) were designed. During the development of the 23S rRNA RT-PCR, problems caused by the contamination of reagents with bacterial DNA were noted. Treatment with 8-methoxypsoralen and UV irradiation reduced the level of contaminating DNA. The sensitivity of the assays was greatly influenced by the enzyme system which was used. With rTth DNA polymerase in a one-enzyme system, we detected 500 CFU of Escherichia coli or Staphylococcus epidermidis/ml. With a two-enzyme system consisting of Moloney murine leukemia virus RT and Taq DNA polymerase, we detected 16 CFU/ml. With groEL mRNA as the target of RT-PCR under optimized conditions, we detected 125 CFU of E. coli/ml, and no problems with false-positive results caused by reagent contamination or a cross-reaction with human nucleic acids were found. Furthermore, the use of mRNA as an indicator of viability was demonstrated. Here we report the application of novel real-time RT-PCR assays for the detection of bacterial contamination of PCs that are appropriate for transfusion services.

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“…Contamination can enter the broad-range diagnostic protocol with the addition of each reagent (6) during the DNA extraction and amplification procedures. Specifically, we have noted four problem areas for bacterial DNA contamination, as follows: (i) lytic enzymes used for extraction of DNA from yeasts and bacteria, (ii) oligonucleotide primers, (iii) Taq polymerase (11), and (iv) water. Most manufacturers of consumable reagents do not guarantee their products to be DNA-free, although most would give an assurance that they are sterile (i.e., contain no viable organisms) and even DNase-free (for most molecular reagents).…”

Section: Evaluation Of Contamination Hazards Through Risk Assessment mentioning

confidence: 99%

Risk Assessment Models and Contamination Management: Implications for Broad-Range Ribosomal DNA PCR as a Diagnostic Tool in Medical Bacteriology

2002

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Detection of nucleic acid sequences from bacterial species with molecular genetic methods

Cited by 21 publications

References 20 publications

Obstacles of Multiplex Real-Time PCR for Bacterial 16S rDNA: Primer Specifity and DNA Decontamination ofTaqPolymerase

Obstacles of Multiplex Real-Time PCR for Bacterial 16S rDNA: Primer Specifity and DNA Decontamination ofTaqPolymerase

Two Novel Real-Time Reverse Transcriptase PCR Assays for Rapid Detection of Bacterial Contamination in Platelet Concentrates

Risk Assessment Models and Contamination Management: Implications for Broad-Range Ribosomal DNA PCR as a Diagnostic Tool in Medical Bacteriology

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