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首页> 外文期刊>Journal of Medical Virology >Improved quantitative PCR protocols for adenovirus and CMV with an internal inhibition control system and automated nucleic acid isolation
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Improved quantitative PCR protocols for adenovirus and CMV with an internal inhibition control system and automated nucleic acid isolation

机译：带有内部抑制控制系统和自动核酸分离的改进的腺病毒和CMV定量PCR方案

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With the establishment of routine virus load (DNAemia) screening for Human adenovirus (HAdV) and Cytomegalovirus (CMV) in post-transplant care quality standards for quantitative PCR-assays are increasing. Established real-time PCR assays were improved with a fully automated DNA-extraction and with a competitive internal control DNA packaged into a lambda phage which serves as an extraction and amplification control in each sample. HAdV and CMV DNA were detected and quantified simultaneously in various types of diagnostic samples like blood, feces or respiratory tract materials. Inhibition was observed in 0.33-0.66% of over 14,000 diagnostic samples, an infrequent but nevertheless not negligible event, which is observed mainly in stool samples. CMV viral load in broncho-alveolar lavage fluid (BALF) ranged between positive but below the quantitation limit of 1,000 copies/ml up to 1.8×10 7 copies/ml with a median of 6.0×10 3 copies/ml. Forty-one (4.7%) BALF samples had a viral load above 5.0×10 5 copies/ml, which was proposed as a threshold for the diagnosis of pneumonia. HAdV viral loads ranged between positive but below the quantitation limit of 1,000 copies/ml to a very high concentration of 1.3×10 11 copies/ml in stool and BALF samples. A HAdV-DNAemia of 10 4 copies/ml was found only in patients with stool viral load of above 10 5 copies/ml. These data support the hypothesis that quantitation in diagnostic materials other than blood may give valuable diagnostic information and that further evaluation of this approach is reasonable.
机译：随着常规病毒载量（DNAemia）的建立，在移植后护理中对人类腺病毒（HAdV）和巨细胞病毒（CMV）的筛查，用于定量PCR分析的质量标准正在提高。已建立的实时PCR检测方法通过全自动DNA提取和竞争性内部对照DNA进行了改进，该DNA被包装到λ噬菌体中，可作为每个样品的提取和扩增对照。在各种类型的诊断样本（例如血液，粪便或呼吸道材料）中同时检测和定量了HAdV和CMV DNA。在超过14,000个诊断样品中，有0.33-0.66％的样品被抑制，这种情况很少见，但还是可以忽略不计，主要是在粪便样品中观察到。支气管肺泡灌洗液（BALF）中的CMV病毒载量介于阳性之间，但低于1,000拷贝/ ml的定量极限，直至1.8×10 7拷贝/ ml，中位数为6.0×10 3拷贝/ ml。 41例（4.7％）BALF样品的病毒载量高于5.0×10 5拷贝/ ml，被提议作为诊断肺炎的阈值。在粪便和BALF样品中，HAdV病毒载量介于阳性之间，但低于1,000拷贝/ ml的定量极限至1.3×10 11拷贝/ ml的极高浓度。仅在粪便病毒载量高于10 5拷贝/ ml的患者中发现HAdV-DNAemia> 10 4拷贝/ ml。这些数据支持以下假设：定量分析血液以外的其他诊断材料可能会提供有价值的诊断信息，并且对该方法进行进一步评估是合理的。

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