Extraction of microsporidial DNA from modified trichrome-stained clinical slides and subsequent species identification using PCR sequencing

Parasitology ◽  
2008 ◽  
Vol 135 (6) ◽  
pp. 701-703 ◽  
Author(s):  
K. S. CHAN ◽  
T. H. KOH
Keyword(s):  
Polymerase Chain Reaction ◽  
Species Identification ◽  
Environmental Samples ◽  
Species Level ◽  
Molecular Techniques ◽  
Chain Reaction ◽  
Clinical Specimens ◽  
Polymerase Chain ◽  
Pcr Conditions ◽  
Hiv Negative

SUMMARYMolecular techniques involving polymerase chain reaction (PCR) and sequencing provide a relatively simple and objective means of identifying microsporidia to species level. We modified previously described methods of DNA extraction and PCR conditions for identification of microsporidia from museum slides, clinical specimens and environmental samples and successfully identifiedVittaforma corneaein 11 out of 13 cases of microsporidial infection from used trichrome-stained slides of corneal scrapings from HIV-negative patients with keratoconjunctivitis.

Limitations of Molecular Biological Techniques for Assessing the Virological Safety of Foods†

1999 ◽  
Vol 62 (6) ◽  
pp. 691-697 ◽  
Author(s):  
GARY P. RICHARDS
Keyword(s):  
Polymerase Chain Reaction ◽  
Environmental Samples ◽  
Hepatitis A ◽  
Enteric Viruses ◽  
Molecular Techniques ◽  
Technical Expertise ◽  
Chain Reaction ◽  
Assay Sensitivity ◽  
Testing Laboratories ◽  
Polymerase Chain

Enteric viruses, including hepatitis A, Norwalk, and Snow Mountain viruses, Hawaii agent, and rotaviruses have been associated with outbreaks of foodborne illness. Classical culturing procedures are available for poliovirus; however, hepatitis A, Norwalk, and many of the other viruses and agents cannot be propagated in cell culture, therefore, molecular biological tools have emerged as a possible means to detect enteric viruses in foods and environmental samples. There are limitations however in the application of polymerase chain reaction and reverse transcription polymerase chain reaction that restrict their usefulness for measuring the virological safety of foods. The most serious limitation is that molecular techniques fail to discriminate between viable and inactivated viruses even though inactivated viruses pose no threat to the consumer and may be present at levels substantially higher than the virulent forms. Other disadvantages include a lack of assay sensitivity and specificity, high assay costs, and a level of technical expertise not available in most food-testing laboratories. Overall, scientific advances in the development of molecular biological tools have outpaced the demonstration of their validity in assessing the virological safety of foods.

Polymerase chain reaction amplification and typing of rotavirus in environmental samples

1995 ◽  
Vol 31 (5-6) ◽  
pp. 371-374 ◽  
Author(s):  
R. Gajardo ◽  
R. M. Pintó ◽  
A. Bosch
Keyword(s):  
Polymerase Chain Reaction ◽  
Environmental Samples ◽  
Polymerase Chain Reaction Amplification ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Group A ◽  
Reaction Amplification ◽  
Stool Specimens ◽  
Polymerase Chain

A reverse transcription polymerase chain reaction (RT-PCR) assay is described that has been developed for the detection and serotyping of group A rotavirus in stool specimens and concentrated and non-concentrated sewage specimens.

Diagnostic Accuracy of a Direct Panfungal Polymerase Chain Reaction Assay Performed on Stained Cytology Slides

2021 ◽  
pp. 030098582199156
Author(s):  
Alexandra N. Myers ◽  
Unity Jeffery ◽  
Zachary G. Seyler ◽  
Sara D. Lawhon ◽  
Aline Rodrigues Hoffmann
Keyword(s):  
Polymerase Chain Reaction ◽  
Diagnostic Accuracy ◽  
Molecular Techniques ◽  
Fungal Culture ◽  
Chain Reaction ◽  
Identification Of Fungi ◽  
Fungal Dna ◽  
Polymerase Chain ◽  
Panfungal Pcr ◽  
Pcr Targeting

Molecular techniques are increasingly being applied to stained cytology slides for the diagnosis of neoplastic and infectious diseases. Such techniques for the identification of fungi from stained cytology slides have not yet been evaluated. This study aimed to assess the diagnostic accuracy of direct (without nucleic acid isolation) panfungal polymerase chain reaction (PCR) followed by sequencing for identification of fungi and oomycetes on stained cytology slides from dogs, cats, horses, and other species. Thirty-six cases were identified with cytologically identifiable fungi/oomycetes and concurrent identification via fungal culture or immunoassay. Twenty-nine controls were identified with no cytologically or histologically visible organisms and a concurrent negative fungal culture. Direct PCR targeting the internal transcribed spacer region followed by sequencing was performed on one cytology slide from each case and control, and the sensitivity and specificity of the assay were calculated. The sensitivity of the panfungal PCR assay performed on stained cytology slides was 67% overall, 73% excluding cases with oomycetes, and 86% when considering only slides with abundant fungi. The specificity was 62%, which was attributed to amplification of fungal DNA from control slides with no visible fungus and negative culture results. Direct panfungal PCR is capable of providing genus- or species-level identification of fungi from stained cytology slides. Given the potential of panfungal PCR to amplify contaminant fungal DNA, this assay should be performed on slides with visible fungi and interpreted in conjunction with morphologic assessment by a clinical pathologist.

scholarly journals Prevalence and diversity of Arcobacter spp. in retail chicken meat in Turkey

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Celenk Molva ◽  
Halil Ibrahim Atabay
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Chicken Meat ◽  
Species Level ◽  
Waterborne Pathogens ◽  
Chain Reaction ◽  
Retail Markets ◽  
Specific Polymerase Chain Reaction ◽  
Polymerase Chain ◽  
Retail Chicken Meat

Arcobacters are food and waterborne pathogens associated with human and animal infections. The objective of the present study was to investigate the prevalence and diversity of <em>Arcobacter</em> spp. in commercially sold chicken meat in İzmir region of Turkey. For this purpose, 100 samples including legs (n=40), 17 chicken quarters (n=17), drumstickers (n=16), breasts (n=11), wings (n=10), and carcasses (n=6) were collected from different retail markets. A total of 65 isolates were confirmed as <em>Arcobacter</em> spp. from 55 samples by genus-specific polymerase chain reaction (PCR). The prevalence of <em>Arcobacter</em> spp. was 32.5, 81.3, 64.7, 72.7, 83.3, and 50% for legs, drumstickers, chicken quarters, breasts, carcasses and wings, respectively. Based on the multiplex-PCR, most of the isolates were identified as <em>A. butzleri</em> (n=45, 80%), followed by <em>A. cryaerophilus</em> (n=2, 3.6%), <em>A. skirrowii</em> (n=1, 1.8%) and 17 isolates (30.9%) could not be identified at the species level.

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