Spermine-mediated improvement of cycling probe reaction

Modrušan, Zora; Bekkaoui, Faouzi; Duck, P.

doi:10.1006/mcpr.1998.0156

Cited by 13 publications

(15 citation statements)

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“…Fusarium species because of its extremely high sensitivity and specificity. We used a combination of a chimera probe, composed of RNA and DNA, and an RNase (Bekkaoui et al, 1996;Modrusan et al, 1998), which can recognize even an SNP (Bekkaoui et al, 1996;Suzuki et al, 2011). A CPT-based real-time PCR for detecting Fusarium species has been previously reported (Itahashi et al, 2010).…”

Section: Discussionmentioning

confidence: 99%

Development of cycling probe-based real-time PCR system to detect Fusarium species and Fusarium solani species complex (FSSC)

Muraosa

Schreiber

Trabasso

et al. 2014

International Journal of Medical Microbiology

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In the present study, we developed a new real-time PCR system based on the cycling probe technology (CPT), which is composed of two single tube real-time PCR assays: the Fusarium genus-specific assay and the Fusarium solani species complex (FSSC)-specific assay with primers targeting the 28s ribosomal RNA gene. The Fusarium genus-specific assay was shown to be highly specific, detecting all reference Fusarium strains with no cross-reaction with other reference fungal strains, such as Aspergillus spp. and human DNA. The FSSC-specific assay also reacted very specifically with FSSC, except for a cross-reaction with Fusarium lunatum. To validate the real-time PCR system, we tested 87 clinical isolates of Fusarium spp. Identification results from the real-time PCR system were found to be 100% concordant with those from DNA sequencing of EF-1α gene. The sensitivity testing also demonstrated high sensitivity, enabling detection of one copy of standard DNA with good reproducibility. Furthermore, both assays were shown to be extremely sensitive even when fungal cells were mixed with human cells, detecting 3 germinated conidia spiked in 3mL of human blood. To apply our new real-time PCR system to the molecular diagnosis of fusariosis, we evaluated its efficacy using a mouse model of invasive F. solani infection. Plasma and whole blood samples of infected mice were tested using the real-time PCR system. The sensitivity of the real-time PCR system was found to be 100% (n=4) in plasma samples. In contrast, no amplification signal was detected in whole blood samples. This system could provide a rapid and precise diagnostic tool for early diagnosis, which is necessary for appropriate treatment and improvement of prognosis of disseminated fusariosis.

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Section: Discussionmentioning

confidence: 99%

Development of cycling probe-based real-time PCR system to detect Fusarium species and Fusarium solani species complex (FSSC)

Muraosa

Schreiber

Trabasso

et al. 2014

International Journal of Medical Microbiology

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“…It has been shown by Modrusan et al (12) that the presence of genomic DNA in CPT resulted in high background and was inhibitory. Addition of spermidine and EGTA into the CPT reaction greatly improved the assay (12).…”

Section: Detection Limit Of the Cpt Assaymentioning

confidence: 99%

“…Usually, the chimeric probe is labeled with radioisotope [ γ -32 P], and the cleaved probe percentage is evaluated by electrophoresis on a polyacrylamide gel (1,7,9,12,13). This method is time consuming, limits the number of samples that can be simultaneously analyzed and is not easily automated.…”

Section: Introductionmentioning

confidence: 99%

Colorimetric Detection of the Tuberculosis Complex Using Cycling Probe Technology and Hybridization in Microplates

et al. 2000

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Cycling probe technology (CPT) is a simple signal amplification method for the detection of specific target DNA sequences. CPT uses a chimeric DNA-RNA-DNA probe that is cut by RNase H when bound to its complementary target sequence. In this study, a hybridization assay was developed to detect biotinylated CPT products that result from the amplification of a Mycobacterium tuberculosis complex sequence. The chimeric probe was specifically designed to avoid the formation of secondary structures. The chosen capture probe was perfectly complementary to and was the same size as OL2, one of the two CPT products. The assay was based on the observation that a long sequence, such as the initial probe, was destabilized when bound to a small capture probe as a result of steric hindrance. The capture probe preferentially bound OL2 rather than the long initial probe. We added a prehybridization step with a helper DNA to enhance this discrimination between the two sequences. Colorimetric detection was performed using a peroxidase-streptavidin conjugate. After optimization, the non-isotopic hybridization assay allowed the detection of around 10 amol of target DNA. Besides being faster and easier to perform, this detection method was compared to electrophoresis separation and gave similar results.

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“…A target nucleic acid sequence can be amplified exponentially in vitro under isothermal conditions by using three enzymatic activities essential to retroviral replication: reverse transcriptase, RNase H (RNA cleavage) and a DNA-dependent RNA polymerase ( 8 ). Cycling probe technology represents a simple method for the detection of DNA target sequences, utilizing a chimeric DNA–RNA–DNA probe which is cleaved by RNase H when hybridized with its complementary target ( 9 ).…”

Section: Introductionmentioning

confidence: 99%

A novel endonuclease IV post-PCR genotyping system

Kutyavin¹,

Milesi²,

Belousov³

et al. 2006

Nucleic Acids Research

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Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA. The three component substrate is characterized by single-stranded DNA target, an oligonucleotide probe, separated from a helper oligonucleotide by a one base gap. The oligonucleotide probe contains a non-fluorescent quencher at the 5′ end and fluorophore attached to the 3′ end through a special rigid linker. Fluorescence of the oligonucleotide probe is efficiently quenched by the interaction of terminal dye and quencher when not hybridized. Upon hybridization of the oligonucleotide probe and helper probe to their complementary target, the phosphodiester linkage between the rigid linker and the 3′ end of the probe is efficiently cleaved, generating a fluorescent signal. In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated. High sensitivity and specificity are illustrated using single nucleotide polymorphism detection.

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Spermine-mediated improvement of cycling probe reaction

Cited by 13 publications

References 0 publications

Development of cycling probe-based real-time PCR system to detect Fusarium species and Fusarium solani species complex (FSSC)

Development of cycling probe-based real-time PCR system to detect Fusarium species and Fusarium solani species complex (FSSC)

Colorimetric Detection of the Tuberculosis Complex Using Cycling Probe Technology and Hybridization in Microplates

A novel endonuclease IV post-PCR genotyping system

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