Use of a nested PCR-enzyme immunoassay with an internal control to detect Chlamydophila psittaciin turkeysReportar como inadecuado

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BMC Infectious Diseases

, 5:76

First Online: 26 September 2005Received: 03 June 2005Accepted: 26 September 2005


BackgroundLaboratory diagnosis of Chlamydophila psittaci, an important turkey respiratory pathogen, is difficult. To facilitate the diagnosis, a nested PCR-enzyme immunoassay PCR-EIA was developed to detect the Cp. psittaci outer membrane protein A ompA gene in pharyngeal swabs.

MethodsThe fluorescein-biotin labelled PCR products were immobilized on streptavidin-coated microtiter plates and detected with anti-fluorescein peroxidase conjugate and a colorimetric substrate. An internal inhibition control was included to rule out the presence of inhibitors of DNA amplification. The diagnostic value of the ompA nested PCR-EIA in comparison to cell culture and a 16S-rRNA based nested PCR was assessed in pharyngeal turkey swabs from 10 different farms experiencing respiratory disease.

ResultsThe sensitivity of the nested PCR-EIA was established at 0.1 infection forming units IFU. Specificity was 100%. The ompA nested PCR-EIA was more sensitive than the 16S-rRNA based nested PCR and isolation, revealing 105 out of 200 52.5% positives against 13 and 74 for the latter two tests, respectively. Twenty-nine 23.8% out of 122 ompA PCR-EIA negatives showed the presence of inhibitors of DNA amplification, although 27 of them became positive after diluting 1-10 the specimens in PCR buffer or after phenol-chloroform extraction and subsequent ethanol precipitation.

ConclusionThe present study stresses the need for an internal control to confirm PCR true-negatives and demonstrates the high prevalence of chlamydiosis in Belgian turkeys and its potential zoonotic risk. The ompA nested PCR-EIA described here is a rapid, highly sensitive and specific diagnostic assay and will help to facilitate the diagnosis of Cp. psittaci infections in both poultry and man.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2334-5-76 contains supplementary material, which is available to authorized users.

Marnix Van Loock, Kristel Verminnen contributed equally to this work.

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Autor: Marnix Van Loock - Kristel Verminnen - Trudy O Messmer - Guido Volckaert - Bruno M Goddeeris - Daisy Vanrompay


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