The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis.
The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis.
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DOI：
10.1186/s12879-016-1696-7
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发表时间：
2016-08-03
影响因子：
3.7
通讯作者：
Huggett JF
中科院分区：
文献类型：
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作者：
Devonshire AS;O'Sullivan DM;Honeyborne I;Jones G;Karczmarczyk M;Pavšič J;Gutteridge A;Milavec M;Mendoza P;Schimmel H;Van Heuverswyn F;Gorton R;Cirillo DM;Borroni E;Harris K;Barnard M;Heydenrych A;Ndusilo N;Wallis CL;Pillay K;Barry T;Reddington K;Richter E;Mozioğlu E;Akyürek S;Yalçınkaya B;Akgoz M;Žel J;Foy CA;McHugh TD;Huggett JF
基于实时荧光定量PCR （qPCR）的方法，如Xpert MTB/RIF，正越来越多地用于结核病的诊断。虽然定性方法足以用于诊断，但对结核病患者的治疗性监测需要目前使用涂片显微镜进行的定量方法。定量分子测量用于治疗监测的潜在用途已经进行了研究，但结果是可变的和不确定的。缺乏适当的参考方法和参考资料是理解这种分歧来源的障碍。数字PCR （dPCR）提供了一种精确的方法来定量参考物质中的特定DNA序列，可用于评估用于结核病治疗监测的定量分子方法。为了评估一种开发质量保证材料的新方法，我们使用dPCR来量化一系列原型参考物质中的特定DNA序列，并评估不同实验室和仪器之间的准确性。这些材料随后也被用于评估qPCR和Xpert MTB/RIF在8个临床检测实验室的定量性能。发现dPCR提供的结果与其他测试方法非常一致，并且即使在使用不同仪器时，在实验室之间无需校准也具有高度可重复性。当临床实验室使用qPCR和Xpert MTB/RIF对标准物质进行分析时，所有实验室都能正确地根据浓度对标准物质进行排序，但测量的量级存在显着差异。结核病是一种病原体的定量可以导致更好的患者管理的疾病，qPCR方法提供了快速进行这种分析的潜力。然而，我们的研究结果表明，当使用精确表征的材料来评估qPCR方法时，测量结果差异太大，无法确定结核分枝杆菌的分子定量是否能提供临床有用的读数。本研究中描述的方法提供了一种方法，通过该方法可以独立于临床变异性评估定量分子方法的技术性能，以提高测量结果的准确性。这将有助于最终提高这些方法用于改善结核病患者管理的可能性。本文的在线版本（doi:10.1186/s12879-016-1696-7）包含补充材料，可供授权用户使用。
Real-time PCR (qPCR) based methods, such as the Xpert MTB/RIF, are increasingly being used to diagnose tuberculosis (TB). While qualitative methods are adequate for diagnosis, the therapeutic monitoring of TB patients requires quantitative methods currently performed using smear microscopy. The potential use of quantitative molecular measurements for therapeutic monitoring has been investigated but findings have been variable and inconclusive. The lack of an adequate reference method and reference materials is a barrier to understanding the source of such disagreement. Digital PCR (dPCR) offers the potential for an accurate method for quantification of specific DNA sequences in reference materials which can be used to evaluate quantitative molecular methods for TB treatment monitoring. To assess a novel approach for the development of quality assurance materials we used dPCR to quantify specific DNA sequences in a range of prototype reference materials and evaluated accuracy between different laboratories and instruments. The materials were then also used to evaluate the quantitative performance of qPCR and Xpert MTB/RIF in eight clinical testing laboratories. dPCR was found to provide results in good agreement with the other methods tested and to be highly reproducible between laboratories without calibration even when using different instruments. When the reference materials were analysed with qPCR and Xpert MTB/RIF by clinical laboratories, all laboratories were able to correctly rank the reference materials according to concentration, however there was a marked difference in the measured magnitude. TB is a disease where the quantification of the pathogen could lead to better patient management and qPCR methods offer the potential to rapidly perform such analysis. However, our findings suggest that when precisely characterised materials are used to evaluate qPCR methods, the measurement result variation is too high to determine whether molecular quantification of Mycobacterium tuberculosis would provide a clinically useful readout. The methods described in this study provide a means by which the technical performance of quantitative molecular methods can be evaluated independently of clinical variability to improve accuracy of measurement results. These will assist in ultimately increasing the likelihood that such approaches could be used to improve patient management of TB. The online version of this article (doi:10.1186/s12879-016-1696-7) contains supplementary material, which is available to authorized users.