Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-06-06
1999-03-02
Myers, Carla J.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 435 9121, 435 915, 435 9151, 536 2431, 536 2433, 935 8, 935 78, C12Q 168, C07H 2104, C12P 1934
Patent
active
058769374
DESCRIPTION:
BRIEF SUMMARY
DESCRIPTION
The subject invention lies in the field of nucleic acid and is directed in particular at methods wherein nucleic acid is isolated from cells, in particular eukaryotic cells. As any person skilled in the art will acknowledge methods using nucleic acid are frequently time consuming and uncertain in outcome due to the instability of nucleic acid material. In particular this is the case for RNA which is extremely sensitive to degradation. In particular in tests which are directed at determining whether a specific nucleic acid sequence is present in a sample it is important to be able to determine whether the sequence to be detected was in fact absent in the original sample or has been degraded during the method steps the sample has been subjected to, thereby leading to a false negative result. In particular in clinical diagnoses on the nucleic acid level a quality control of the nucleic acid that is extracted from such a sample is an important issue. For clinical samples containing cells, a cellular messenger RNA can be used as control to test the extracted material. Unsuccessful amplification of such message is indicative of lack of integrity of the messenger RNAs in that extract.
A number of control marker nucleic acid sequences are known in the state of the art. However, all these known markers exhibit the disadvantage that they are not markers that can be used universally for a large variety of types of cells. In addition, such markers are frequently expressed to such a high degree in the cells that their absence obviously is indicative of degradation but that their presence can still occur even though degradation has taken place of nucleic acid in the sample. Such degradation can however occur to an insufficient degree to ensure that all the marker nucleic acid has also been degraded. This latter aspect is naturally vital in the case of clinical diagnosis as this could lead to a false negative report of the presence of, for example, an infection or a tumor.
The importance of RNA detection in samples is increasing due to a number of discoveries where the presence of DNA in infections need not necessarily be clinically relevant. Infections considered to be latent may be detected with DNA analysis. For example, in the Journal of Medical Virology 42:164-169 (1994) by Velzing et al., the presence of cytomegalovirus (CMV) immediate early antigen (IEA) DNA and mRNA in peripheral blood leukocytes detected by PCR was investigated and related to the appearance of CMV pp65 antigen, CMV serology, and clinical status. In this test, keratin type I mRNA and the ssu rRNA gene served as internal controls. Two of seven seronegative samples were CMV EA positive. No relation was found between serology and the presence of CMV IEA DNA as determined in 37 samples. Five of 32 samples that could be analyzed were positive for CMV IEA mRNA of which four were also positive in the pp65 antigen detection technique. A clear relation was found between the presence of CMV IEA mRNA and CMV pp65 antigen in leukocytes and in the clinical findings as well. Velzing et al. concluded that detection of CMV mRNA may have a role in diagnosis of an active clinically relevant CMV infection. We have tested the same panel of patient samples and discovered different results, which is indicative that the presence of CMV IEA mRNA and CMV pp65 antigenicity are not related.
As human cytomegalovirus (CMV) infections are a major cause of morbidity and mortality in patients with decreased cellular immunity, such as recipients of organism transplants and patients with AIDS, the diagnosis of CMV infection often poses great challenges. Isolation of the virus is time consuming and may be unrelated to disease, since shedding of the virus frequently occurs. Also in particular the immune response is often delayed or even completely absent in immunocompromised patients so that reliance on a specific IgM response, commonly used for diagnosis of a primary CMV infection could therefore be difficult. In order to detect active viral replication, i.e the prese
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Akzo Nobel N.V.
Gormley Mary E.
Myers Carla J.
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