Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-07-21
2002-12-03
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06489110
ABSTRACT:
The present invention is related to the detection of bacteria, such as Mycobacteria, in human or animal body fluids such as blood, sputum and urine. The present invention provides a method for assessing the viability of bacteria such as Mycobacterium tuberculosis without the need for propagation of the bacteria.
For example, tuberculosis (TB) caused by Mycobacterium tuberculosis is a major public health problem in many countries world-wide with particular significance in developing countries. Tuberculosis control programmes are faced with an increased burden of cases, a shift towards diagnostically more difficult categories of patients such as extrapulmonary and smear-negative cases, and the emergence of multidrug-resistant strains of
M. tuberculosis.
Improved diagnosis would be a valuable contribution in the struggle to solve this global public health emergency.
The method of the present invention is concerned with the amplification of specific nucleic acid sequences.
Nucleic acid amplification reactions promise to reduce the time for diagnosis from weeks to hours, while surpassing the sensitivity and specificity of the classical methods. Besides their potential value in diagnosis, amplification reactions offer the possibility of a rapid identification and drug-susceptibility determination. Amplification of DNA target molecules to a detectable level by the polymerase chain reaction (PCR) is the best analyzed system for detecting Mycobacteria.
The “Polymerase Chain Reaction” (PCR) is described in European patent applications EP 200362 and EP 201148. PCR is a cyclic process which has double stranded DNA as target. Each cycle in the PCR process starts with the separation of a double stranded DNA target in its two complementary strands. To each strand a primer will anneal and DNA polymerases present will extend the primers along the DNA strand to which it annealed thus forming two new DNA duplexes. When the reaction mixture is heated the strands of the DNA duplexes will be separated again and a new PCR cycle can start. Thus, the PCR process produces multiple DNA copies of a DNA target. Amplification using PCR, can also be based on an RNA template. The actual PCR needs to be preceded by a reverse transcription step to copy the RNA into DNA (RT-PCR). However, if RT-PCR is used for the detection of transcripts differentiation of mRNA- and DNA-derived PCR products is necessary. DNAse treatment prior to RT-PCR can be employed (Bitsch, A. et al., J Infect. Dis 167, 740-743., 1993; Meyer, T. et al., Mol. Cell Probes. 8, 261-271., 1994), but sometimes fails to remove contaminating DNA sufficiently [Bitsch, A. et al., 1993].
More recently a different class of nucleic acid amplification methods namely the “transcription based amplification techniques” was developed. The techniques involve the transcription of multiple RNA copies from a template comprising a promoter recognized by an RNA polymerase. Said copies are used as input for further amplification. Such methods have been described by Gingeras et al. in WO88/10315 and Burg et al. in WO89/1050. Isothermal transcription based amplification techniques have been described by Davey et al. in EP 323822 (relating to the NASBA method), by Gingeras et al. in EP 373960 and by Kacian et al. in EP 408295 (the TMA method). Transcription based amplification reactions may also be performed with thermostable enzymes. Such a thermostable method is described in EP 682121 filed in the name of Toyo Boseki KK.
The isothermal transcription based nucleic acid amplification techniques have been utilized to detect mycobacteria, such as the NASBA method [Vliet, G. M. E. van der, Schukkink, R. A. F., Gemen, B. van, Schepers, P. and Klatser, P. R. (1993) Nucleic acid sequence-based amplification (NASBA) for the identification of mycobacteria. J. Gen. Microbiol. 139, 2423-2429.] and another transcription-mediated RNA amplification test (TMA)[Jonas, V., Alden, M. J., Curry, J. I., Kamisango, K., Knott, C. A., Lankford, R., Wolfe, J. and Moore, D. F. (1993) Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by amplification of rRNA. J. Clin. Microbiol. 31, 241] both targeted at 16S ribosomal RNA.
Amplification reactions targeted at the 16S rRNA or the gene encoding it are usually directed to a conserved region which comprises species-specific variable sequences [Vliet, G. M. E. van der, Schukkink, R. A. F., Gemen, B. van, Schepers, P. and Klatser, P. R. (1993) Nucleic acid sequence-based amplification (NASBA) for the identification of mycobacteria. J. Gen. Microbiol. 139, 2423-2429., Jonas, V., Alden, M. J., Curry, J. I., Kamisango, K., Knott, C. A., Lankford, R., Wolfe, J. and Moore, D. F. (1993) Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by amplification of rRNA. J. Clin. Microbiol. 31, 241]. They have the advantage that a single amplification reaction can identify the mycobacterial species. An additional advantage of the transcription-mediated RNA amplification assays targeted at 16S rRNA, is the high number of target molecules per cell−±2000; sensitivity is thereby favoured.
Since RNA, especially mRNA, has a generally much shorter half-life time than DNA, its detection may be useful for the assessment of the viability of mycobacteria [Moore, D. F., Curry, J. I., Knott, C. A. and Jonas, V. (1996) Amplification of rRNA for assessment of treatment response of pulmonary tuberculosis patients during antimicrobial therapy. J. Clin. Microbiol. 34, 1745-1749., Vliet, G. M. E. van der, Schepers, P., Schukkink, R. A. F., Gemen, B. van and Klatser, P. R. (1994) Assessment of mycobacterial viability by RNA amplification. Antimicrob. Agents Chemother. 38, 1959-1965.], which is relevant to the problems of resistance against drugs and contagiousness of the patient.
The present invention is based on the detection of mRNA encoding the elongation factor EF-Tu.
The elongation factor EF-Tu is essential in (myco)bacterial translation. Elongation factors play an ancillary role in the elongation step of translation and are thus an indicator of the cell's metabolic activity. For every translation EF-Tu is required. The amount of EF-Tu protein can be as high as 50% of their total protein content in active proliferating cells.
EF-Tu encoding gene sequences (DNA) have been used as a marker to detect the presence of bacterial cells.
In EP133288 a method is disclosed for the detection of bacterial DNA with a probe comprising a base sequence of at least a portion of one of the strands of a tuf or fus gene. Southern blot hybridization of the digested mycoplasmal DNAs with the elongation factor (EF-Tu) gene tuf of
E.coli
was used as a basis to detect polymorphism in mycoplasma strains.[Yogev et al. FEMS Microbiol.Lett., 50(2-3), 145-9, 1988].
A PCR based assays for the detection of Mycoplasma tuberculosis, and Mycoplasma fermentans using the gene encoding elongation factor Tu (tuf) as the target sequence had also been described [Berg et al.,Mol.Cell.Probes, 10(1), 7-14, 1996 and Luneberg et al., J.Clin.Microbiol., 31(5), 1088-94, 1993].
The present invention, however, is concerned with the detection of EF-Tu mRNA as a marker for bacterial viability.
The present invention thus provides a method for the assessment of bacterial viability whereby mRNA coding for the elongation factor EF-Tu is used as a target in a nucleic acid amplification reaction and the presence and/or amount of said mRNA is determined.
The presumably short-lived mRNA coding for the EF-Tu is most likely highly abundant in the (myco)bacterial cell and a decrease therein will indicate a decline in metabolic activity. Furthermore, because of the EF-Tu's essential role, it is plausible to assume that it is present in all mycobacterial species, allowing the development of a general amplification system with species-specific primers and/or probes, analogous to the 16S rRNA NASBA design [Vliet, G. M. E. van der, Schukkink, R. A. F., Gemen, B. van,
Klatser Paul R.
Oudshoorn Pieter
bioMérieux, B.V.
Fredman Jeffrey
Myers Bigel & Sibley & Sajovec
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