Mircroorganism quantitation and detection method and kit...

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage

Reexamination Certificate

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C435S006120, C435S091200

Reexamination Certificate

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06277560

ABSTRACT:

The present invention relates to the quantitation and detection of microorganisms. In particular, it relates to the quantitative determination and/or detection of microorganisms having a DNA or RNA genome.
The invention applies to all known microorganisms such as viruses, bacteria, bacilli, protozoa, hematozoa which have a DNA or RNA genome and affect humans, animals or plants, as well as to microorganisms such as those existing in humans, animals and plants in a pathological or normal state; and it also applies to the quantitation and detection of these same microorganisms in products originating from animals—including humans—and plants, as well as of those capable of existing in water.
Further on, the method of the invention will be described for the quantitation of infection with RNA-containing human immuno-deficiency virus (HIV) and infection with the human DNA-containing hepatitis B virus (HBV).
BACKGROUND OF THE INVENTION
At present, the study of the epidemiology, treatment and prophylaxis of human, animal and plant diseases is a priority objective of human and veterinary medicine and of agronomy or the plant hygiene industry. Identification and quantification of microorganisms in media such as tissues, biological fluids and in-vitro preparations are steps that are indispensable for the proper conduct of such projects. Taking into account the size of most microorganisms, particularly viruses, and their concentration in biological media (generally of the order of 10
1
to 10
12
microorganisms per mL), the only current technological tools that permit both molecular identification and quantitative evaluation are those which make use of genome fragment amplification. The method of genomic amplification most frequently used at the present time is called polymerase chain reaction or PCR. Thus, it is possible, by means of an enzyme, polymerase, to amplify a DNA-containing genome fragment. Likewise, it is possible to carry out a reverse transcription of RNA into DNA, and an amplification with the aid of the reverse transcriptase+polymerase couple or by an enzyme performing these two operations. Once amplified, this genome fragment or “amplificate” is specifically identified by various methods (radioactive or colored probe, fragment size).
Methods of quantitation of HIV by PCR are described, e.g., by M. Holodniy et al.,
J. Infect. Dis.
163, 862-866 (1991); S. Aoki-Sei et al.,
J. AIDS Res. Hum. Retrovirus
8, 1263-1270 (1992); P. Bagnarelli et al.,
J. Virol.
66, 7328-7335 (1992); M. Piatak Jr. et al.,
Science
259, 1749-1755 (1993); S. M. Bruisten et al.,
AIDS
7 (Suppl. 2), S15-S20 (1993).
Methods of quantitation of hepatitis C virus RNA have been described, e.g., by U. Kumar et al.,
J. Virol. Methods
47(1-2), 95-102 (1994); A. Mazin et al.,
J. Clin. Microbiol.
32(8), 1939-44 (1994); A. Ravaggi et al.,
J. Clin Microbiol.
33(2), 265-9 (1995); K. K. Young et al.,
J. Clin Microbiol.
33(3), 654-7 (1995).
Methods of quantitation of hepatitis B virus DNA have been described, e.g., by P. Lehtovaara et al.,
PCR Methods Appl.
3(3), 169-75 (1993); J. Wu et al.,
J. Virol. Methods
49(3), 331-41 (1994); H. L. Zaaijer et al.,
J. Clin Microbiol.
32(9), 2088-91 (1994); S. Kaneko et al., J. Clin. Microbiol. 27(9), 1930-33 (1989).
However, the relationship between amplified product and initial DNA or RNA is not really constant from one experiment to another; hence, the necessity of being concerned with the problem of measuring a standard whose concentrations, moreover, are known.
Until now, the PCR amplification capacities could be evaluated by means of cells wherein the genome to be determined is present in a constant number of copies only, or by means of plasmids each having a single gene fragment. In this way, it could be established that, thanks to PCR, even a single gene fragment can be specifically identified by PCR. It has also been established that a proportionality ratio exists between DNA concentrations of a plasmid that are subjected to amplification and the DNA concentrations measured after amplification. However, this proportionality ratio has been found to change from one experiment to another, because PCR amplification is a biological process, resulting from the action of polymerase, which is not controlled in such complete fashion as to make it purely physical technique.
This has led researchers and engineers of biotechnological companies to introduce standardization into every viral quantitation experiment. Until now, the standard has consisted in a series of concentrations (generally three to five) of a standard plasmid whose DNA fragment to be amplified has the same primers, and, depending on the technologies, is either of a partly different sequence or of identical sequence. The amplification ratio of the DNA of equal concentrations of two plasmids of identical primers but of different lengths and thus of different sequences (1) may be different from one case to another and (2) is not necessarily identical to that of the DNA originating from the identical native virus concentrations within the same experiment.
The problem is even more complex if it is sought to quantitate or identify RNA viruses, such as, e.g., that of AIDS (HIV). For RNA viruses, the PCR amplification step must, in effect, be combined with a reverse transcription step (abbreviated RT) which is frequently carried out either with the aid of another enzyme, reverse transcriptase, or with an enzyme possessing both actions (reverse transcription and DNA amplification). In both cases, the enzymes currently available on the market make it possible to obtain reverse transcription ratios that can vary from one experiment to another from 10:1 (that is to say, 10 copies of RNA give one copy of DNA) to 100:1 (100 copies of RNA give 1 copy of DNA). Until now, the resulting problem, namely the noncomparability of results obtained in successive experiments, has been solved by simultaneous incubation of a concentration range of a standard RNA fragment (standard transcript) using the same primers, but whose amplified part is either identical if it is used as an external standard, or different in sequence or length if it is used in co-amplification, the transcript then being incubated in the same tube as the RNA of the target microorganism to be determined. Unfortunately, it seems that the concentrations of two transcripts can be different, and be different even from those originating from RNA obtained from the identical virus concentrations.
In conclusion:
The known reverse transcription and/or amplification techniques do not furnish identical results from one experiment to another for the same sample;
It is imperative to use a standard, which may be either internal or external;
An external standard composed either of a plasmid for a DNA microorganism or a transcript RNA for an RNA microorganism permits only a relative quantitation, because the amplification ratio is not necessarily identical to that of the DNA or RNA of the complete microorganism;
Moreover, a co-amplified internal standard involves different lengths or sequences of the plasmid or the transcript, and has the same drawbacks as an external standard using a plasmid or a transcript.
In the present state of the art, amplification of the DNA of a plasmid is not truly proportional to that undergone by viral DNA. As for the standard currently used for RNA viruses, it is a transcript which has the same drawbacks as a plasmid, to which is added the possibility of free RNA degradation.
Hence, there has been a need for microorganism quantitation and detection tests yielding absolute values of the number of target microorganisms.
SUMMARY OF THE INVENTION
It has now been found that it is possible to quantitate or detect in an absolute fashion any DNA or RNA microorganism, provided that:
(1) one knows or is otherwise able to determine a standard concentration of the microorganism or the concentration of the DNA or RNA contained in this microorganism, called standard microorganism; and
(2) one compares the quantity of the product of reverse transcr

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