Internal Control DNA for PCR Assays Introduced into Lambda Phage Particles Exhibits Nuclease Resistance
| Autor: | Jörg Berg, Markus Stöcher |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Nuclease
biology Biochemistry (medical) Clinical Biochemistry Editorials Nucleic acid methods DNA Lambda phage biology.organism_classification Bacteriophage lambda Polymerase Chain Reaction Molecular biology law.invention Bacteriophage chemistry.chemical_compound chemistry law Nucleic acid biology.protein Deoxyribonuclease I Humans Multiplex Cloning Molecular Polymerase chain reaction |
| Zdroj: | Clinical Chemistry |
| ISSN: | 1530-8561 0009-9147 |
| DOI: | 10.1373/clinchem.2004.035519 |
| Popis: | Real-time PCR assays are widely used for the detection and quantification of pathogen-derived nucleic acids in clinical samples (1)(2)(3). Because clinical specimens contain PCR-inhibitory moieties that are not always reliably removed during nucleic acid extraction, real-time PCR assays are furnished with internal amplification controls (IACs) to identify those samples in which the PCR is inhibited (4)(5)(6)(7)(8)(9). Thus, false-negative test results or falsely diminished quantification results can be avoided. For pathogen-specific real-time PCR assays, the IAC is usually added to clinical samples, recovered in the process of nucleic acid extraction, and amplified in either a multiplex or competitive fashion with detection usually by discrimination of hybridization probes (4)(5)(6)(7)(8)(9). Plasmid-derived DNA is commonly used as IAC. In this instance, the procedure does not provide entire control of PCR assays because the lysis of pathogens during the nucleic acid purification procedure is not monitored. Plasmid-derived IACs consist of bare, unprotected DNA; therefore, the IAC may become degraded within the clinical specimen before nucleic acid extraction or during storage in the working stock, which could lead to unreliable amplification of IAC DNA, necessitating repetition of testing. In this report, we use a traditional lambda phage cloning procedure to generate phage particles containing target-specific IAC DNA, i.e., phage IAC. As IAC DNA we used the previously described multiple IAC that was generated for a panel of virus-specific real-time PCR assays with competitive IACs (10). We show that the obtained phage IAC contains one IAC DNA fragment, is resistant to DNase I digestion, and exhibits improved storage and handling properties as well as reliable amplification in the respective competitive real-time PCR assays. The IAC DNA was excised from the respective plasmid (pCR-II-TOPO) … |
| Databáze: | OpenAIRE |
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