A simple colorimetric assay for detection of amplified Mycobacterium leprae DNA

Vliet, G. M. E. Van Der; Wit, M. Y. L. De; Klatser, P. R.

doi:10.1006/mcpr.1993.1008

Cited by 9 publications

(5 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Quantified DNA standard A 530-bp fragment of the proline-rich antigen gene of M. leprae, which included the target sequence of our real-time PCR, was amplified by PCR using primers S 13 (CTCCACCTGGA-CCGGCGAT, nucleotide positin 175,442-175,424) and S 62 (GACTAGCCTGCCAAGTCG, position 174,912-174,929) [23] and cloned in E. coli using the pCR 2.1 cloning kit (Invitrogen, Karlsruhe, Germany). Plasmid concentration was determined photometrically.…”

Section: Selection Of Oligonucleotides and Optimization Of Pcrmentioning

confidence: 99%

Detection and quantification of Mycobacterium leprae in tissue samples by real-time PCR

Kramme

Bretzel

Panning

et al. 2003

Med Microbiol Immunol

View full text Add to dashboard Cite

Real-time PCR technology has improved molecular diagnostics of many pathogens, but no such test is available for Mycobacterium leprae. In this report we describe the establishment and the pre-clinical evaluation of such an assay. The test achieved a theoretical analytical sensitivity limit of 194 M. leprae cells per skin biopsy specimen and facilitated quantification of mycobacteria in tissue over a range of 54-54,000,000 cells per sample. In punch skin biopsies from 39 untreated Ugandan patients with newly diagnosed leprosy, the clinical diagnosis could be confirmed in 88.9% of multibacillary and 33.3% of paucibacillary (microscopically negative) patients. Real-time detection thus did not increase the clinical sensitivity of PCR as compared to conventional protocols, in spite of its evidently high analytical sensitivity. On the other hand, as still no culture system exists for M. leprae, the assay appears to be a robust tool for detection of the bacterium in selected clinical situations, as well as for quantitation in experimental settings.

show abstract

Section: Selection Of Oligonucleotides and Optimization Of Pcrmentioning

confidence: 99%

Detection and quantification of Mycobacterium leprae in tissue samples by real-time PCR

Kramme

Bretzel

Panning

et al. 2003

Med Microbiol Immunol

View full text Add to dashboard Cite

show abstract

“…r The diagnosis is most accurate when at least two other fragments are used in the Manual Supplementllll|ll FIGURE 1 Multiplex amplification of 9 exons in the human dystrophin gene. r The normal pattern of amplification, indicating the presence of these exons in the patient, is shown in lane C. Lane A is missing bands corresponding to exons 8, 12, 17, and 19, suggesting that the patient has a deletion in the region of exons [8][9][10][11][12][13][14][15][16][17][18][19]. The presence of some amplified bands serves to delineate the extent of the deletion and to indicate that the reaction has not failed.…”

Section: Indication Of Template Quantitymentioning

confidence: 99%

Multiplex PCR: advantages, development, and applications.

Edwards¹,

Gibbs²

1994

Genome Research

340

206

View full text Add to dashboard Cite

“…Briefly, different amounts of biotinylated PCR products were tested for adequate capture. Also several hybridisation-and wash buffers [Suzuki et al, 1993;King and Ball, 1993;Ossewaarde et al, 19941 were compared as well as different hybridisation temperatures [Tada et al, 1992;Van der Vliet et al, 1993;Ossewaarde et al, 19941. In addition, several probe concentrations [Suzuki et al, 1993;Ossewaarde et al, 19941 were tested and, finally, different substrate incubation times ranging from 1-4 hr to overnight were evaluated.…”

Section: Enzyme Immunoassaymentioning

confidence: 99%

A non-radioactive PCR enzyme-immunoassay enables a rapid identification of HPV 16 and 18 in cervical scrapes after GP5+/6+ PCR

et al. 1996

View full text Add to dashboard Cite

In previous studies, general primer mediated PCR (GP5+/6+ PCR) was applied successfully to detect a broad spectrum of human papillomaviruses (HPV) in cervical scrapes. In order to facilitate PCR based HPV detection and typing, a colourimetric microtitre plate based hybridisation assay was developed. The method utilised one biotinylated primer (bio-GP6+) in the GP-PCR. Biotinylated PCR products were captured on streptavidin coated microtitre plates, denaturated and hybridised to digoxigenin (DIG) labelled HPV specific internal oligo probes. The DIG labelled hybrids were detected using an enzyme immunoassay (EIA). Since HPV 16 and 18 are the most common HPV types found in cervical carcinomas, this approach was initiated for these two types. Cross-hybridisation reactions were not detected when the specificity of this PCR-EIA for HPV 16 and 18 was tested on a panel of 20 different HPV genotypes. The sensitivity of the assay was found to be between 10 and 100 HPV 16 and 18 viral genomes in a background of 100 ng cellular DNA. This was similar to the detection limit of Southern blot analysis of PCR products with radioactively labelled oligonucleotides. A group of cytomorphologically normal (n = 89) and abnormal (n = 96) cervical scrapes were composed of HPV 16 and HPV 18 positive and HPV negative scrapes. All HPV 16 and 18 positive smears were detected by PCR-EIA. These results indicate that PCR-EIA has the potential for a rapid and sensitive HPV DNA test for day-to-day routine examination of cervical scrapes.

show abstract

A simple colorimetric assay for detection of amplified Mycobacterium leprae DNA

Cited by 9 publications

References 0 publications

Detection and quantification of Mycobacterium leprae in tissue samples by real-time PCR

Detection and quantification of Mycobacterium leprae in tissue samples by real-time PCR

Multiplex PCR: advantages, development, and applications.

A non-radioactive PCR enzyme-immunoassay enables a rapid identification of HPV 16 and 18 in cervical scrapes after GP5+/6+ PCR

Contact Info

Product

Resources

About