Molecular Beacons as Diagnostic Tools: Technology and Applications

Abravaya, Klara; Huff, Jeffrey; Marshall, R L; Merchant, Barbara; Mullen, Carolyn; Schneider, George J.; Robinson, John

doi:10.1515/cclm.2003.070

Cited by 52 publications

(23 citation statements)

References 27 publications

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“…The start-up cost of setting up the real-time PCR may be prohibitive, and routine laboratories may need to refer specimens to reference laboratories. A multiplex qualitative real-time PCR assay that detects C trachomatis and N gonorrhoeae and contains an internal control has been developed (30).…”

Section: Amplification Methods Pcrmentioning

confidence: 99%

The Laboratory Diagnosis of Neisseria gonorrhoeae

Ng¹,

Martín²

2005

Canadian Journal of Infectious Diseases and Medical Microbiolog

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The present article describes the laboratory diagnosis of Neisseria gonorrhoeae by culturing of the organism from different types of clinical specimens followed by confirmatory tests. The success of culture methods requires good quality collection and transport of clinical specimens. The present guide describes the media requirements and cultural conditions for N gonorrhoeae growth and the characteristics for a presumptive identification of N gonorrhoeae. Confirmatory tests include biochemical tests, chromogenic enzyme substrate tests, immunoassays and nucleic acid methods. Nucleic acid detection methods include either amplification-based methods or nonamplification tests, and are increasingly used in clinical laboratories where a viable culture is not possible to obtain. Nucleic acid methods can also be used to detect the presence of low numbers in a specimen. Nucleic acid detection methods need confirmation with another amplification method or gene target. Controls must be included to ensure true positive and negative results, and to rule out nucleic acid contamination. Monitoring of antimicrobial susceptibilities of N gonorrhoeae is important to investigate treatment failure and to evaluate the efficacy of currently recommended therapies. Many methods for the characterization of N gonorrhoeae require cultures. The useful typing methods for determining strain relatedness include auxotyping, serotyping, plasmid profile analysis, DNA sequencing of the porB gene and pulsed-field gel electrophoresis. Quality assurance programs for diagnostic testing and antimicrobial susceptibility testing is reviewed.

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Section: Amplification Methods Pcrmentioning

confidence: 99%

The Laboratory Diagnosis of Neisseria gonorrhoeae

Ng¹,

Martín²

2005

Canadian Journal of Infectious Diseases and Medical Microbiolog

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“…Thus, HBV is similar to many retroviruses found in animals and pararetroviruses in plants [16,17] . After cloning and sequencing the HBV genome [18] , several related viruses were discovered in woodchucks (Marmota monax) [19] , ground squirrels (Spermophilus beecheyi) [20] and pekin duck (Anas domesticus) [21] .…”

Section: Taxonomymentioning

confidence: 98%

Hepatitis B virus taxonomy and hepatitis B virus genotypes

Schaefer¹

2007

WJG

313

235

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Hepatitis B virus (HBV) is a member of the hepadnavirus family. Hepadnaviruses can be found in both mammals (orthohepadnaviruses) and birds (avihepadnaviruses). The genetic variability of HBV is very high. There are eight genotypes of HBV and three clades of HBV isolates from apes that appear to be additional genotypes of HBV. Most genotypes are now divided into subgenotypes with distinct virological and epidemiological properties. In addition, recombination among HBV genotypes increases the variability of HBV. This review summarises current knowledge of the epidemiology of genetic variability in hepadnaviruses and, due to rapid progress in the field, updates several recent reviews on HBV genotypes and subgenotypes.

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“…Detection of target can be achieved using synthetic oligonucleotide probes or with the addition of a doublestranded DNA (dsDNA) 3 binding dye. Further specificity may be added by including a postamplification melting analysis step to confirm target when using a dsDNA binding dye or certain types of probes (3)(4)(5)(6)(7)(8). One method of real-time PCR detection and confirmation uses an unlabeled probe in combination with asymmetric amplification.…”

mentioning

confidence: 99%

Unlabeled Probes for the Detection and Typing of Herpes Simplex Virus

et al. 2007

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Background: Unlabeled probe detection with a doublestranded DNA (dsDNA) binding dye is one method to detect and confirm target amplification after PCR. Unlabeled probes and amplicon melting have been used to detect small deletions and single-nucleotide polymorphisms in assays where template is in abundance. Unlabeled probes have not been applied to low-level target detection, however. Methods: Herpes simplex virus (HSV) was chosen as a model to compare the unlabeled probe method to an in-house reference assay using dual-labeled, minor groove binding probes. A saturating dsDNA dye (LCGreen ® Plus) was used for real-time PCR. HSV-1, HSV-2, and an internal control were differentiated by PCR amplicon and unlabeled probe melting analysis after PCR. Results: The unlabeled probe technique displayed 98% concordance with the reference assay for the detection of HSV from a variety of archived clinical samples (n ‫؍‬ 182). HSV typing using unlabeled probes was 99% concordant (n ‫؍‬ 104) to sequenced clinical samples and allowed for the detection of sequence polymorphisms in the amplicon and under the probe. Conclusions: Unlabeled probes and amplicon melting can be used to detect and genotype as few as 10 copies of target per reaction, restricted only by stochastic limitations. The use of unlabeled probes provides an attractive alternative to conventional fluorescence-labeled, probe-based assays for genotyping and detection of HSV and might be useful for other low-copy targets where typing is informative.

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Molecular Beacons as Diagnostic Tools: Technology and Applications

Cited by 52 publications

References 27 publications

The Laboratory Diagnosis of Neisseria gonorrhoeae

The Laboratory Diagnosis of Neisseria gonorrhoeae

Hepatitis B virus taxonomy and hepatitis B virus genotypes

Unlabeled Probes for the Detection and Typing of Herpes Simplex Virus

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