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首页> 外文期刊>Journal of Applied Life Sciences International >Optimization and Evaluation of Triplex Real-time PCR Assay for Detection of Genes Encoding Staphylococcal Virulence and Methicillin Resistance Using Two Different Multi-channel Emission Instruments
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Optimization and Evaluation of Triplex Real-time PCR Assay for Detection of Genes Encoding Staphylococcal Virulence and Methicillin Resistance Using Two Different Multi-channel Emission Instruments

机译：使用两种不同的多通道发射仪器检测三重实时荧光定量PCR检测葡萄球菌毒力和甲氧西林抗性基因的优化和评估

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摘要

Aim of Study: To optimize a triplex real-time PCR assay developed elsewhere and to evaluate the performance characteristics of two different multi-channel real-time PCR systems on the newly optimized assay. Methodology: A triplex real-time PCR assay developed for three key genes encoding virulence and antibiotic resistance in Staphylococcus aureus , namely, lukSF-PV , mecA , and spa was optimized and evaluated using two different real-time PCR instruments (7500SDS and LightCycler 480). Bacterial strains (N=230), including staphylococcal and non-staphylococcal isolates, were used for the study. Results: Following optimization, cycling and data analysis completed within one hour compared with the former three hours. Assay specificity became 100% on both instruments. The negative predictive value (NPV) and the positive predictive value (PPV) rose to 100%. Conclusion: The optimized triplex real-time PCR assay is highly reproducible across the two systems without loss of speed, sensitivity and specificity. The results also suggested that user-familiarization with each machine operational system would allow assay performance across various real-time PCR platforms currently being used.
机译：研究目的：优化在其他地方开发的三重实时PCR分析，并在新优化的分析上评估两个不同的多通道实时PCR系统的性能特征。方法：针对三种编码金黄色葡萄球菌的毒力和抗生素抗性的关键基因开发的三重实时PCR分析方法，使用两种不同的实时PCR仪器（7500SDS和LightCycler 480）进行了优化和评估，该三个关键基因分别是lukSF-PV，mecA和spa ）。细菌菌株（N = 230），包括葡萄球菌和非葡萄球菌分离株，用于研究。结果：经过优化，循环和数据分析在一小时内完成，而前三个小时完成了。两种仪器的测定特异性均达到100％。阴性预测值（NPV）和阳性预测值（PPV）升至100％。结论：优化的三重实时荧光定量PCR检测方法在两个系统中都具有很高的重现性，而不会降低速度，灵敏度和特异性。结果还表明，用户熟悉每个机器操作系统将允许跨当前使用的各种实时PCR平台进行化验。

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