Evaluation of the AmpliSensor PCR and the SHARP signal detection system for the early prediction of symptomatic CMV infection in solid transplant recipients

Rautenberg, Peter; Lübbert, Christoph; Weers, Wiebke; Boetel, Eric; Schweichler, Jörg; Zhou, Lin; Costard‐Jäckle, Angelika; H, Kraemer-Hansen; Harder, Timm C.

doi:10.1016/s1386-6532(99)00013-x

Cited by 8 publications

(4 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Blood samples are extensively used for PCR-based diagnosis of microbial infection, genetic disease, forensic analysis, and blood banking. [1][2][3][4] Prenatal genetic diagnosis using maternal plasma or serum has been developed recently. 5,6 However, when these techniques are applied to blood samples, it must be considered that PCR inhibitors in the specimens may lead to possible false-negative or reduced sensitivity despite advanced DNA purification methods.…”

mentioning

confidence: 99%

Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq

Zhang

Kermekchiev

Barnes

2010

The Journal of Molecular Diagnostics

124

View full text Add to dashboard Cite

PCR-based clinical and forensic tests often have low sensitivity or even false-negative results caused by potent PCR inhibitors found in blood and soil. It is widely accepted that purification of target DNA before PCR is necessary for successful amplification. In an attempt to overcome PCR inhibition , enhance PCR amplification , and simplify the PCR protocol, we demonstrate improved PCR-enhancing cocktails containing nonionic detergent , L-carnitine , D-(؉)-trehalose , and heparin. These cocktails , in combination with two inhibitor-resistant Taq mutants , OmniTaq and Omni Klentaq , enabled efficient amplification of exogenous , endogenous , and high-GC content DNA targets directly from crude samples containing human plasma , serum , and whole blood without DNA purification. In the presence of these enhancer cocktails , the mutant enzymes were able to tolerate at least 25% plasma , serum , or whole blood and as high as 80% GC content templates in PCR reactions. These enhancer cocktails also improved the performance of the novel Taq mutants in real-time PCR amplification using crude samples , both in SYBR Green fluorescence detection and TaqMan assays. The novel enhancer mixes also facilitated DNA amplification from crude samples with various commercial

show abstract

mentioning

confidence: 99%

Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq

Zhang

Kermekchiev

Barnes

2010

The Journal of Molecular Diagnostics

124

View full text Add to dashboard Cite

show abstract

“…Blood samples are extensively used for the PCR-based diagnosis of microbial infections and genetic diseases, as well as for forensic analysis and blood banking (14,35,40,42,50). However, when applying nucleic acid amplification techniques to blood samples, the amplification capacity can be dramatically reduced or blocked by the presence of PCR-inhibitory substances.…”

mentioning

confidence: 99%

Purification and Characterization of PCR-Inhibitory Components in Blood Cells

2001

View full text Add to dashboard Cite

In a recent study, immunoglobulin G in human plasma was identified as a major inhibitor of diagnostic PCR (W. Abu Al-Soud, L. J. Jönsson, and P. Rådström. J. Clin. Microbiol. 38:345-350, 2000). In this study, two major PCR inhibitors in human blood cells were purified using size exclusion and anion-exchange chromatographic procedures. Based on N-terminal amino acid sequencing and electrophoretic analysis of the purified polypeptides, hemoglobin and lactoferrin were identified as PCR-inhibitor components in erythrocytes and leukocytes, respectively. When different concentrations of hemoglobin or lactoferrin were added to PCR mixtures of 25 microl containing 10 different thermostable DNA polymerases and 1 ng of Listeria monocytogenes DNA as template DNA, AmpliTaq Gold, Pwo, and Ultma were inhibited in the presence of < or = 1.3 microg of hemoglobin and < or = 25 ng of lactoferrin, while rTth and Tli were found to resist inhibition of at least 100 microg of hemoglobin. In addition, the quantitative effects of seven low-molecular-mass inhibitors, present in blood samples or degradation products of hemoglobin, on real-time DNA synthesis of rTth using the LightCycler Instrument were investigated. A reaction system based on a single-stranded poly(dA) template with an oligo(dT) primer annealed to the 3' end was used. It was found that the addition of 0.25 to 0.1 mg of bile per ml, 2.5 mM CaCl2, 0.25 mM EDTA, 5 microM FeCl3, and 0.01 IU of heparin per ml reduced the fluorescence to approximately 76, 70, 46, 17, and 51%, respectively. Finally, the effects of nine amplification facilitators were studied in the presence of hemoglobin and lactoferrin. Bovine serum albumin (BSA) was the most efficient amplification facilitator, so that the addition of 0.4% (wt/vol) BSA allowed AmpliTaq Gold to amplify DNA in the presence of 20 instead of 1 microg of hemoglobin and 500 instead of 5 ng of lactoferrin. Including 0.02% (wt/vol) gp32, a single-stranded-DNA binding protein, in the reaction mixture of AmpliTaq Gold was also found to reduce the inhibitory effects of hemoglobin and lactoferrin.

show abstract

“…A major problem with PCR-based diagnostic tests of blood samples is the possibility of seeing false-negative reactions caused by PCR inhibitors present in the blood. Since, PCR-based diagnosis of microbial infection, genetic disease, forensic analysis, and blood banking [ [3] , [4] , [5] ] and prenatal genetic diagnosis are extensively used using direct blood/plasma/serum as the template [ 6 , 7 ], having a DNA polymerase that is resistant to PCR inhibitors appeared an attractive option for researchers worldwide. AmpliTaq Gold [ 8 ], KlenTaq [ 2 ], novel Taq mutants [ 9 ], Phusion Blood Direct PCR Kit, Phire Hot Start DNA polymerase, and Phire Hot Start DNA polymerase and KAPA Blood PCR kit [ 10 ] are some Taq mutants reported.…”

Section: Introductionmentioning

confidence: 99%

Novel properties of recombinant Sso7d-Taq DNA polymerase purified using aqueous two-phase extraction: Utilities of the enzyme in viral diagnosis

Sundarrajan¹,

Parambath²,

Suresh³

et al. 2018

Biotechnology Reports

View full text Add to dashboard Cite

HighlightsSso7d-Taq fusion protein purified using a single step of aqueous Two-Phase Extraction (ATPE) is >95% pure and is active.The S-Taq protein has higher thermostability and detergent tolerance over regular Taq polymerase and can be used for PCR's from direct whole blood.The PCR efficiency rate of S-Taq is higher than Taq polymerase and can be used to detect DNA viruses in a clinical setting efficiently.S-Taq can tolerate higher concentrations of magnesium ions and can be used for in-situ PCR’s.S-Taq can be used to carry out PCR’s of bacterial recombinants directly from the overnight culture since it is resistant to inhibition to Luria Bertani broth. This unique property of S-Taq will enable researchers to screen recombinants without the need to isolate the plasmid DNA of recombinants. This would be a huge cost savings for companies engaged in molecular biology research involving PCR’s.

show abstract

Evaluation of the AmpliSensor PCR and the SHARP signal detection system for the early prediction of symptomatic CMV infection in solid transplant recipients

Cited by 8 publications

References 38 publications

Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq

Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq

Purification and Characterization of PCR-Inhibitory Components in Blood Cells

Novel properties of recombinant Sso7d-Taq DNA polymerase purified using aqueous two-phase extraction: Utilities of the enzyme in viral diagnosis

Contact Info

Product

Resources

About