Internal Control DNA for PCR Assays Introduced into Lambda Phage Particles Exhibits Nuclease Resistance

Stöcher, Markus; Berg, Jörg

doi:10.1373/clinchem.2004.035519

Cited by 24 publications

(26 citation statements)

References 11 publications

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“…418 When stored in SM buffer, the protected DNA was stable for at least a few months, but stability for more than 5 days in plasma at room temperature could not be achieved, which may be a result of greater susceptibility of lambda to plasma proteases compared to MS2. 418, 419 For ssDNA, encapsulation in fd phage was demonstrated with DNA taken from parvovirus B19. 420 The resultant controls were resistant to nuclease degradation and performed similarly to the native virus in PCR assays, with very similar growth curves observed.…”

Section: Applications Of Virus-based Particlesmentioning

confidence: 99%

Design of virus-based nanomaterials for medicine, biotechnology, and energy

2016

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Virus-based nanomaterials are versatile materials that naturally self-assemble and have relevance for a broad range of applications including medicine, biotechnology, and energy. This review provides an overview of recent developments in “chemical virology.” Viruses, as materials, provide unique nanoscale scaffolds that have relevance in chemical biology and nanotechnology, with diverse areas of applications. Some fundamental advantages of viruses, compared to synthetically programmed materials, include the highly precise spatial arrangement of their subunits into a diverse array of shapes and sizes and many available avenues for easy and reproducible modification. Here, we will first survey the broad distribution of viruses and various methods for producing virus-based nanoparticles, as well as engineering principles used to impart new functionalities. We will then examine the broad range of applications and implications of virus-based materials, focusing on the medical, biotechnology, and energy sectors. We anticipate that this field will continue to evolve and grow, with exciting new possibilities stemming from advancements in the rational design of virus-based nanomaterials.

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Section: Applications Of Virus-based Particlesmentioning

confidence: 99%

Design of virus-based nanomaterials for medicine, biotechnology, and energy

2016

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“…Plasmid-derived DNA may be degraded before extraction because it is unprotected and subjected to nucleases. 1 To protect the plasmid DNA, further packaging is required in a bacteriophage, which adds more time and expense to the control. Ribosomal 16 or 18S RNA is a convenient control for reverse transcription-polymerase chain reaction (PCR).…”

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confidence: 99%

Implementation of a T4 Extraction Control for Molecular Assays of Cerebrospinal Fluid and Stool Specimens

Gerriets

Greiner

Gebhart

2008

The Journal of Molecular Diagnostics

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The use of appropriate extraction and amplification controls for acellular specimens is not standardized in the clinical laboratory community. Extraction controls and checks for inhibitors of amplification in cellular specimens are most often accomplished by amplification of an internal human genomic target. This approach is not feasible for acellular specimens, which may contain little or no amplifiable genomic material. Other specimen types , such as stool, frequently contain amplification inhibitors. Failure to test for these inhibitors can result in the reporting of false-negative results. The goal of this study was to evaluate the use of a T4 bacteriophage as an extraction and amplification control for acellular specimens. The T4 bacteriophage assay was evaluated for use as a control in 290 specimens , including cerebrospinal fluid , serum , and filtered stool. The use of appropriate extraction and amplification controls for acellular specimens is not standardized in the clinical laboratory community. Inhibitors present in some patient specimens decrease the efficiency of amplification and, if not controlled, can result in the reporting of false-negative results. False-negatives may also arise from undetected extraction failures. Accrediting agencies expect diagnostic laboratories to control for these variables. To meet quality assurance standards recommended by accrediting agencies, a method to detect the presence of inhibitors, while simultaneously providing a control for extraction in acellular specimens, is needed. Extraction controls and amplification inhibitor checks for cellular specimens are most often accomplished by amplification of an internal human genomic target. This approach is not feasible for acellular specimens, which may contain little or no amplifiable genomic material. In our experience, genomic DNA targets are successfully amplified in only approximately 40% of cerebrospinal fluid (CSF) specimens. Other acellular specimen types, such as stool, frequently contain amplification inhibitors.Several forms of internal controls have been evaluated, including plasmid DNA, ribosomal RNA, armored RNA, and Lambda phage. Plasmid-derived DNA may be degraded before extraction because it is unprotected and subjected to nucleases. 1 To protect the plasmid DNA, further packaging is required in a bacteriophage, which adds more time and expense to the control. Ribosomal 16 or 18S RNA is a convenient control for reverse transcription-polymerase chain reaction (PCR). Armored RNA is a nucleic acid packaged in bacteriophage coat proteins that protect the RNA from ribonucleases. The RNA is released from its protective coat during the extraction procedure and can be used as a control for reverse transcription-PCR. 2 The disadvantage of this technology is that it is proprietary and expensive, and the reverse transcription process adds unnecessary time and expense to DNA-targeted assays. Additionally, inhibitors of reverse transcription and of PCR cannot be distinguished. Specially engineered Lambda phage DNA fr...

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“…One thousand lambda phage particles acting as a control for cell lysis, nucleic acid extraction and qPCR amplification 19 were added to sputum containing a range of MTB DNA from 0 to 50,000 genomic copies/ml. The average Cq of the cotJC assay was 27.4 ± 0.3 (range 26.9–28.2) across the range of MTB concentrations used indicating consistent performance.…”

Section: Resultsmentioning

confidence: 99%

XtracTB Assay, a Mycobacterium tuberculosis molecular screening test with sensitivity approaching culture

Reed

Basu

Butzler

et al. 2017

Sci Rep

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Nucleic acid amplification tests are increasingly used to diagnose tuberculosis (TB) due to their speed and sensitivity compared to sputum smear microscopy. However, these tests fail to equal culture’s sensitivity with sputum smear microscopy negative specimens and therefore cannot be used to rule out TB disease. For molecular tests to match culture’s sensitivity, they must detect ≤10 genomic copies of Mycobacterium tuberculosis (MTB) DNA, the limit of detection of culture, process ≥1 ml of sputum ensuring sufficient number of MTB are in the reaction, and efficiently remove sputum associated inhibitors from this large sample. Here we report the preliminary characterization of XtracTB Assay, a MTB testing protocol designed for inclusion in either an integrated point-of-care platform or a high throughput automated central laboratory system. The test combines DNA sequence specific sample prep to reduce the co-extraction of qPCR inhibitors with the amplification of two MTB specific loci (IS6110 and senX3-regX3) to increase test sensitivity and minimize the likelihood of false negatives. The analytical sensitivity of the XtracTB Assay was 5 genomic copies/ml of sputum rivaling that of culture. Furthermore, 142 valid test results yield clinical sensitivity of 94.9% (95% CI: 90.1–99.9) and specificity of 100% (95% CI: 90.0–100.0).

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Internal Control DNA for PCR Assays Introduced into Lambda Phage Particles Exhibits Nuclease Resistance

Cited by 24 publications

References 11 publications

Design of virus-based nanomaterials for medicine, biotechnology, and energy

Design of virus-based nanomaterials for medicine, biotechnology, and energy

Implementation of a T4 Extraction Control for Molecular Assays of Cerebrospinal Fluid and Stool Specimens

XtracTB Assay, a Mycobacterium tuberculosis molecular screening test with sensitivity approaching culture

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