Overcoming Polymerase Chain Reaction Inhibition in Old Animal Tissue Samples Using Ethidium Bromide

1995 ◽  
Vol 225 (1) ◽  
pp. 169-172 ◽  
Author(s):  
L.M. Hall ◽  
E. Slee ◽  
D.S. Jones
2007 ◽  
Vol 41 (3) ◽  
pp. 288-294 ◽  
Author(s):  
Tanusree Sen ◽  
Sirsendu Jana ◽  
Sen Sreetama ◽  
Uttara Chatterjee ◽  
Sasanka Chakrabarti

2009 ◽  
Vol 21 (5) ◽  
pp. 701-706 ◽  
Author(s):  
Ho To ◽  
Tomohiro Koyama ◽  
Shinya Nagai ◽  
Kotaro Tuchiya ◽  
Tetsuo Nunoya

Quantitative real-time polymerase chain reaction (qPCR) assays were developed and validated in combination with enrichment culture for the detection and discrimination of Erysipelothrix rhusiopathiae and other Erysipelothrix species from tissue samples. The targets for SYBR green qPCR assays were the 16S ribosomal RNA gene for Erysipelothrix species and a gene involved in capsular formation for E. rhusiopathiae. The specificity of the assays was assessed with Erysipelothrix species and other related bacterial species. The limit of detection was found to be 5 colony-forming units per reaction. Amplification of DNA extracted from spleen and joint samples spiked with increasing quantities of Erysipelothrix cells was shown to be equally sensitive to DNA extracted from a pure bacterial culture. The assays were evaluated with 88 tissue samples from 3 experimentally infected pigs and 50 mice and with 36 tissue samples from 3 naturally infected pigs and 11 noninfected pigs. Results were compared with those of direct qPCR and conventional culture. The qPCR after enrichment increased the diagnostic sensitivity over that of culture and qPCR, thereby significantly reducing the total time taken for the detection of E. rhusiopathiae and other Erysipelothrix species. Therefore, this technique could be used for practical applications.


2003 ◽  
Vol 127 (2) ◽  
pp. 213-217 ◽  
Author(s):  
Jennifer L. Hunt ◽  
Patricia Swalsky ◽  
E. Sasatomi ◽  
Laura Niehouse ◽  
Anke Bakker ◽  
...  

Abstract Context.—A recurring problem in surgical pathology practice is specimen mix-up and floater contamination. While many cases can be resolved histologically, a significant number remain unclear and may have serious clinical and medicolegal implications. Objectives.—To design a microdissection and genotyping assay to identify contaminating floater tissues in paraffin-embedded tissues that is optimized for small samples, and to use the assay to resolve a series of clinical cases with floater tissues. Materials and Methods.—Twenty-one cases of possible tissue floater contamination in paraffin-embedded tissue blocks were included. Using 4 unstained, 4-μm-thick histologic sections, multiple sites were microdissected under direct visualization either by hand or by laser capture microdissection. Nonneoplastic and neoplastic tissues were sampled. Polymerase chain reaction was performed for a panel of 10 polymorphic microsatellite markers at 1p34, 3p26, 5q21, 9p21, 10q23, and 17p13. Allele size and content were analyzed semiquantitatively by fluorescent capillary electrophoresis, and the genotypes for the tissues in the paraffin-embedded tissue blocks were compared for identity. Results.—Tissue identification was successful in all cases, despite small tissue sample size and fixation effects. Comparative analysis of neoplastic tissue floaters and the presumptive source tumor was performed when possible to control for possible allelic loss or microsatellite instability. Conclusions.—Microdissection and genotyping are effective and reliable means to objectively resolve problems of possible floater contamination. Even minute tissue samples provide sufficient DNA template for polymerase chain reaction microsatellite analysis. Because of the potential clinical implications of floaters, we recommend that all suspected floaters that would change a diagnosis from benign to malignant be subjected to genotyping assay to confirm the identity of the floater tissue.


Sign in / Sign up

Export Citation Format

Share Document