Improved quantitative PCR protocols for adenovirus and CMV with an internal inhibition control system and automated nucleic acid isolation

被引:28
作者
Henke-Gendo, C. [1 ]
Ganzenmueller, T. [2 ]
Kluba, J. [2 ]
Harste, G. [2 ]
Raggub, L. [2 ]
Heim, A. [2 ]
机构
[1] Hannover Med Sch, Inst Med Microbiol & Hosp Epidemiol, D-30625 Hannover, Germany
[2] Hannover Med Sch, Inst Virol, D-30625 Hannover, Germany
关键词
viral load; quantitation; TaqMan; stool; broncho-alveolar lavage fluid; REAL-TIME PCR; LUNG-TRANSPLANT RECIPIENTS; EPSTEIN-BARR-VIRUS; CYTOMEGALOVIRUS DNA; DIAGNOSTIC PCR; LOAD; ASSAYS; AMPLIFICATION; SAMPLES; PLASMA;
D O I
10.1002/jmv.23285
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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 0.330.66% 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? x 10(7) copies/ml with a median of 6.0 x 10(3) copies/ml. Forty-one (4.7%) BALF samples had a viral load above 5.0 x 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 x 10(11) copies/ml in stool and BALF samples. A HAdV-DNAemia of >104 copies/ml was found only in patients with stool viral load of above 105 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. J. Med. Virol. 84:890896, 2012. (C) 2012 Wiley Periodicals, Inc.
引用
收藏
页码:890 / 896
页数:7
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