Sawyer J, Mealing D, Dalley D, Davé D, Lesellier S, Palmer S, Bowen-Davies J, Crawshaw T R, Chambers M A
Technology Transfer Unit, Biotechnology Department, Veterinary Laboratories Agency Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom.
J Clin Microbiol. 2007 Aug;45(8):2398-403. doi: 10.1128/JCM.00292-07. Epub 2007 May 30.
A real-time PCR assay for the measurement of gamma interferon (IFN-gamma) mRNA in European badger (Meles meles) blood cultures was developed. The levels of IFN-gamma mRNA in blood cultures stimulated with either bovine or avian tuberculin or specific mycobacterial antigens were compared with those in a nonstimulated control blood culture as the basis for determining the tuberculosis (TB) status of live badgers. The assay was validated by testing 247 animals for which there were matching data from postmortem examination and culture of tissues. Relative changes in the levels of IFN-gamma mRNA in response to bovine tuberculin and specific antigens were found to be greater among badgers with tissues positive for TB on culture. The test was at its most accurate (87% of test results were correct) by using blood cultures containing bovine tuberculin as the antigen and when the response to avian tuberculin was taken into account by subtracting the avian tuberculin response from the bovine tuberculin response. At a specificity of 90.7%, the test was 70.6% sensitive. At the same specificity, the current serological enzyme-linked immunosorbent assay for TB in badgers was only 53% sensitive. This work demonstrates that measurement of IFN-gamma mRNA by real-time PCR is a valid method for the detection of TB in live badgers and may provide an alternative to the current serological methods of diagnosis, the Brock test. The testing procedure can be completed within 5 h of receipt of the blood culture samples. In addition, the use of a molecular biology-based test offers the potential to fully automate the testing procedure through the use of robotics.
开发了一种用于测量欧洲獾（Meles meles）血液培养物中γ干扰素（IFN-γ）mRNA的实时PCR检测方法。将用牛结核菌素或禽结核菌素或特异性分枝杆菌抗原刺激的血液培养物中IFN-γmRNA的水平与未刺激的对照血液培养物中的水平进行比较，以此作为确定活体獾结核病（TB）状态的依据。通过对247只动物进行检测验证了该检测方法，这些动物有来自死后组织检查和培养的匹配数据。发现在培养时组织中TB呈阳性的獾中，对牛结核菌素和特异性抗原反应时IFN-γmRNA水平的相对变化更大。当使用含有牛结核菌素作为抗原的血液培养物，并且通过从牛结核菌素反应中减去禽结核菌素反应来考虑对禽结核菌素的反应时，该检测最为准确（87%的检测结果正确）。在特异性为90.7%时，该检测的敏感性为70.6%。在相同特异性下，目前用于獾结核病检测的血清学酶联免疫吸附试验的敏感性仅为53%。这项工作表明，通过实时PCR测量IFN-γmRNA是检测活体獾结核病的有效方法，并且可能为目前的血清学诊断方法——布罗克试验提供替代方法。检测程序可以在收到血液培养样本后的5小时内完成。此外，使用基于分子生物学的检测方法有可能通过使用机器人技术使检测程序完全自动化。
