MXPA98003500A - Best nucleic acid experiments - Google Patents

Abstract

The present invention relates to the detection of specific nucleic acid sequences, either by a process of amplification of specific nucleic acid sequences or not. More particularly, the invention provides improved compositions and methods to reduce the risk of contamination in the handling of reagents, internal amplification controls and the use of automated devices for the automated detection of one or more amplified nucleic acid sequences.

Description

EXPERIMENTS OF IMPROVED NUCLEIC ACID Field of the Invention The present invention relates to the detection of specific nucleic acid sequences in a target test sample.

In particular, the present invention relates to the automated detection of specific nucleic acid sequences, which are unamplified or amplified nucleic acid sequences (amplicons).

In addition, the present invention relates to the use of automated amplification, methods and compositions for monitoring successful amplification, improved methods to reduce the possibility of contamination and the use of unified reaction regulators and individual dose aliquots of the reaction components for the amplification.

Finally, the present invention also relates to the unique construction and methods for the conventional or automated detection of one or more of a different nucleic acid sequence in a single experiment.

Background of the Invention The development of techniques for the manipulation of nucleic acids, the amplification of said nucleic acids when necessary, and the subsequent detection of specific sequences of nucleic acids or amplicons, has generated extreme sensitivity and specific nucleic acid sequence experiments for the diagnosis of diseases and / or identification of pathogenic organisms in a test sample.

Amplification of Nucleic Acids When necessary, the enzymatic amplification of nucleic acid sequences will increase the ability to detect such nucleic acid sequences. Generally, the currently known amplification schemes can be broadly grouped into two classes based on the enzymatic amplification reactions being handled by the continuous cyclization of the temperature between the denaturation temperature, the first tempering temperature and the amplicon synthesis temperature. (product of the enzymatic amplification of the nucleic acid), or if the temperature remains constant through the process of enzymatic amplification (isothermal amplification). The typical cyclic nucleic acid amplification technology (thermocyclic) and the polymerase chain reaction (PCR) and the ligase chain reaction (LCR). Specific protocols for such reactions are mentioned, for example, in Short Protocols in Molecular Biology. second edition, A Compendium of Methods from Current Protocols in Molecular Biology. (Editorials Ausubel et al., John Wiley &Sons, New York, 1992) Chapter 15. Reactions that are isothermal include: transcription-mediated amplification (TMA), amplification based on nucleic acid sequence (NASBA) and amplification by filament displacement (SDA).

U.S. Patent documents describing nucleic acid amplification include: 4,638,195; 4,683,202; 5,130,238; 4,876,187; 5,030,557; 5,399,491; 5,409,818; 5,485,184; 5,409,818; 5,554,517; 5,437,990 and 5,554,516 (each of which is included herein by reference) It is well known that methods such as those described in those patents allow the amplification and detection of nucleic acids without requiring cloning, and are responsible for the most experiments for nucleic acid sequences. However, it is equally well recognized that together with the detection sensitivity possible with the amplification of the nucleic acid, the ease of contamination by minimal amounts of undesired exogenous nucleic acid sequences is extremely great. The contamination by nucleic acids of unwanted exogenous DNA or RNA is equally likely. The utility of the amplification reactions will be improved by methods for controlling the introduction of undesired exogenous nucleic acids and other contaminants.

Prior to the discovery of thermostable enzymes, methods using thermocycling were extremely difficult due to the requirement of the addition of fresh enzyme after each step of denaturation, because initially the high temperatures required for denaturation, also inactivated the polymerases. Once the thermostable enzymes were discovered, the cyclic amplification of the nucleic acids became an even more simplified procedure where the addition of the enzyme was the only necessary at the beginning of the reaction. In this way, it was not necessary to open the tubes and it was not necessary to add a new enzyme during the reaction, which allowed an improvement in the efficiency and accuracy, as well as reducing the risk of contamination, as well as the cost of the enzymes. An example of a thermostable enzyme is the polymerase isolated from the organism of Thermophilus aquaticus.

In general, isothermal amplification may require the combined activity of multiple enzymatic activities for which optimal thermostable variables have not been described. The initial step of an amplification reaction will usually require the denaturation of the target nucleic acid, for example, in the TMA reaction, the initial denaturation step is usually > 65 ° C, but can typically be > 95 ° C, and it is used when it is required to eliminate the secondary structure of the target nucleic acid.

The reaction mixture is then cooled to a lower temperature, which allows the first quenching and is the optimum reaction temperature for the combined activities of the amplification enzymes. For example, in TMA the enzymes are generally a T7 RNA polymerase and a reverse transcriptase (which includes endogenous RNase activity). The temperature of the reaction is kept constant through the subsequent isothermal amplification cycle.

Due to the lack of appropriate thermostable enzymes, some isothermal amplifications will generally require the addition of enzymes to the reaction mixture after denaturation at high temperature, and cooling to a lower temperature. This requirement is inconvenient, and requires the opening of the reaction tube of the amplification, which introduces a greater opportunity for contamination.

Thus, it would be more useful if said reactions could be more easily carried out with a reduced risk of contamination by methods that would allow integrated denaturation and amplification without the need to manually transfer the enzymes.

Amplifier Regulator and Single Reaction Reaction Aliquot Typical reaction protocols require the use of several different regulators. designed to optimize the activity of the particular enzyme that is used in certain steps in the reaction, or by optimal resuspension of the components of the reaction. For example, while a typical PCR amplifier lOx PCR will contain 500 mM KCl and 100 mM Tris HCl, pH 8.4, the concentration of MgCl2 will depend on the target nucleic acid sequence and the first interest situation. The reversible transcription regulator (5x) typically contains 400 mM Tris-Cl, pH 8.2; 400 mM KCl and 300 mM MgCl2, whereby the reversible transcriptase regulator of the Maloney Murine Leukemia virus (5X) typically contains 250 mM Tris-Cl, pH 8.3; 375 mM KCl; 50 mM DTT (Dithiothreitol) and 15 mM MgCl2.

While such reaction regulators can be prepared in volume of chemicals in stock, most commercially available amplification products provide packaged reagents in volume and specific regulators for use with the amplification protocol. For example, commercially available manual amplification experiments for the detection of significant clinical pathogens (for example, Chlamydia and Mycobacterium tuberculosis Gen-Probé Inc. detection experiments) require several manual manipulations to perform the experiment, including dilution of the sample test in a sample dilution regulator (SDB), the combination of the diluted sample with the reagents of the amplification reaction, said oligonucleotides and oligonucleotide-specific promoters / primers that have been reconstituted in an amplification reconstitution regulator (ARB ), and finally, the addition to this mixture of the reaction of reconstituted enzymes in an enzyme dilution regulator (EDB).

The preparation and use of multiple regulators requires multiple manual additions to the reaction mixture, which introduces a greater possibility of contamination. It would be more useful to have a simple unified controller that could be used in all phases of an amplification protocol. In particular, with the commercially available TMA experiments described above, the requirement of three regulators greatly complicates the automation of said protocol.

The packaging by volume of the enzyme or other components of the reaction by the manufacturers, may require the reconstitution of the components in large quantities, and the use of quantities in the presence of multiple reagents, may be wasted when at least the maximum number of reactions are gone. to carry out, since some of these compounds can be stable for only a short time. This reconstitution process also requires multiple manipulations by the user of the reagents in existence, and making aliquots of individual reaction quantities of reagents from stock, creates a greater opportunity for contamination.

Methods and compositions for the preparation of bulky amounts of conserved proteins are known, see for example: U.S. Patents 5,098,893; 4,762,857; 4,457,916; 4,891,319; 5,026,566 and the International Patent Publications WO 89/06542; WO 93/00806; WO 95/33488; WO 89/00012, all of which are incorporated herein by reference in their entirety. However, the use of preserved and pre-aliquoted reagent compounds in simple reaction quantities / doses is very useful and economical. The simple aliquots of the enzyme reagents prevent the multiple use of volume reagents, reduce waste and reduce the possibility of contamination to a greater degree. In addition, said simple reaction aliquots are more appropriate for the automation of the reaction process.

The requirements of many regulator changes and the multiple additions of reagents complicate the automation of these reactions. A single dose unit of the reaction regulator mixture and a unified combination regulator will simplify the automation of the process and reduce the opportunity for contamination.

Automation of Nucleic Acid Detection with or without Amplification. Experiments to test nucleic acid, and a combination of amplification / test experiments can be rapid, sensitive, highly specific and usually require precise handling to minimize contamination with nonspecific nucleic acids, and are thus the first candidates for automation. As with conventional nucleic acid detection protocols, it is generally required to use an oligonucleotide sequence detection test which is bound by some means to a compound that generates a signal that can be detected. A possible test detection system is described in U.S. Patent No. 4,581,333, incorporated herein by reference in its entirety.

In addition, the automation of a nucleic acid detection system targets an amplified or unamplified nucleic acid, or a combined automated amplification detection system will be generally adaptable for use as oligonucleotides that capture nucleic acids that are bound to some form of nucleic acid. solid support system. Examples of such linkages and methods for attaching nucleic acids to solid supports are found in U.S. Patent Nos. 5,489,653 and 5,510,084, both of which are incorporated herein by reference.

The automation of amplification, detection and a combination of amplification and detection is desirable to reduce the interaction requirements of multiple users with the experiment. Apparatus and methods for optically analyzing test materials are described, for example, in U.S. Patent No. 5,122,284 (incorporated herein by reference). It is believed that automation is more economical, efficient, reproducible and accurate for the processing of clinical experiments. Thus, with the superior sensitivity and specificity of nucleic acid detection experiments, the use of amplification of nucleic acid sequences and automation in one or more phases of an experimental protocol can improve the utility of the experimental protocol and its usefulness in a clinical situation.

Advantage of Internal Control Sequences. The amplification of the nucleic acid is highly sensitive to the reaction conditions, and since it can not amplify and / or detect any specific nucleic acid sequence in a sample, it may be due to the error in the amplification process, as much as it should be to the absence of desired target sequence. The amplification reactions are notoriously sensitive to the reaction conditions and have generally required to include control reactions with known target nucleic acids and first compounds in separate reaction vessels treated at the same time. However, the internal control sequences added in the test reaction mixture would truly control for the success of the amplification process in the reaction mixture being tested and would be more useful. U.S. Patent No. 5,457,027 (incorporated herein by reference) shows certain internal control sequences which are useful as a standard internal oligonucleotide in the isothermal amplification reactions for Mycobacterium tuberculosis.

However, it would be extremely useful to have a general method of generating internal control sequences, which would be useful as internal controls of the various amplification procedures, which are specifically designed so that they are not affected by the nucleic acid sequences present in the organism. target, the host organism or the nucleic acids present in the normal flora or in the environment. Generally, such internal control sequences should not be substantially similar to any nucleic acid sequence present in a clinical situation, including humans, pathogenic organisms, intestinal flora organisms or environmental organisms which could interfere with the amplification and detection of the internal control sequences.

Detection of more than one Nucleic Acid Sequence in a Single Experiment In general, the reaction of a simple experiment for the reaction of nucleic acid sequences is limited to the detection of a single objective nucleic acid sequence. This simple goal limitation increases the costs and time required to perform clinical diagnostic experiments and verification of control reactions. Detecting more than one nucleic acid sequence in a sample using a simple experiment would greatly increase the efficiency of the sample analysis and would be of great economic benefit in reducing costs, for example, helping to reduce the need for multiple clinical experiments.

The detection of multiple analysis in a single experiment has been applied to the detection of antibody analysis as in, for example, International Patent Publication No. WO 89/00290 and WO 93/21346, which are incorporated in their entirety to the present as a reference.

In addition to the cost reduction and time required, the detection of more than one nucleic acid target sequence in a single experiment would be the reduction of erroneous results. In particular multiple detection, it would greatly increase the utility and benefit using internal control sequences and allow rapid validation of negative results.

Summary of the Invention. The present invention comprises methods for automated isothermal amplification and detection of a specific nucleic acid in a test sample to be tested comprising: a) combining a test sample to be tested with a regulator, a mixture of free nucleotides, first specific oligonucleotides and optionally the thermostable nucleic acid polymerase enzyme, in a first reaction vessel and placing the reaction vessel in an automated apparatus such that: b) the automated apparatus heats the vessel of the first reaction to a temperature and for a sufficient time to denature, if necessary, the nucleic acid in the sample to be tested; . c) the automated apparatus cools the vessel of the first reaction at a temperature that the first oligonucleotides can specifically tune the target nucleic acid; d) the automated apparatus transfers the reaction mixture from the vessel of the first reaction to a second reaction vessel and puts the reaction mixture in contact with the thermolabile nucleic acid amplification enzyme; e) the automated apparatus maintains the vessel temperature of the second reaction which allows primer-mediated amplification of the nucleic acid; f) the automated apparatus contacts the amplified nucleic acid in the vessel of the second reaction with a capture nucleic acid specific for the nucleic acid to be tested, so that they form a test complex that captures the specifically bound nucleic acid; g) the automated apparatus optionally washes the specifically captured amplified nucleic acid so that the non-specifically bound nucleic acid is washed out of the test complex that captures the nucleic acid that binds specifically; h) the automated apparatus contacts the test complex that captures the nucleic acid that specifically binds to a test-identified nucleic acid by the amplified nucleic acid, such that a complex is formed between the specifically amplified nucleic acid and the test identified nucleic acid; i) the automated apparatus cleans the test complex that captures the nucleic acid that binds specifically, so that the identified test nucleic acid not specifically bound, is washed out of the specifically bound complex; j) the automated apparatus contacts the specifically linked complex with a solution where a detection reaction between the test identified nucleic acid is made between the solution and the identification linked to the nucleic acid so that a detectable signal is generated from the sample in proportion to the amount of amplified nucleic acid specifically bound in the sample; where steps h, i and j can occur sequentially or simultaneously; k) the automated device detects the signal and optionally displays a value for the signal, or optionally registers a value for the signal.

As used herein, the term "test sample" includes samples taken from living patients, from non-living patients, from surfaces, gas, vacuum or liquid, from tissues, body fluids, body surface samples or cavities and any similar source . The term regulator as used herein encompasses suitable regulator formulations which can support the effective activity of an identification, for example, an enzyme placed in said regulator when treated at a temperature appropriate for the activity and given the appropriate enzyme substrate and It is tempered as necessary. The term "first specific oligonucleotide nucleic acid" means an oligonucleotide having a nucleic acid sequence which is substantially complementary to, and will hybridize / specifically tune to, a target nucleic acid of interest and may optionally contain a promoter sequence recognized by RNA. polymerase The term "reaction vessel" means a container in which a chemical reaction can be carried out and preferably capable of withstanding any temperature from -80 ° C to 100 ° C.

The present invention further provides for the method described above, wherein the regulator of the reaction in a unified regulator and as such is suitable for the denaturation of nucleic acids and the tuning of nucleic acids, and is further capable of sustaining the enzymatic activity of the polymerization of nucleic acid and the amplification enzyme. Further encompassed by the invention is the method wherein the nucleic acid amplification enzyme is administered in the second reaction chamber as a single dose of experiment amount in a lyophilized pill and the reaction chamber is sealed before the amplification step.

The invention shows an apparatus for the automated detection of more than one target nucleic acid sequence or amplicons comprising a solid phase receptor (apparatus such as SPR pipette) covered with at least two distinct zones of a capture nucleic acid oligonucleotide.

The invention shows a method for the automated detection of more than one target nucleic acid sequence comprising contacting a solid phase receptor (apparatus such as SPR pipette) covered with at least two different zones of a nucleic acid oligonucleotide of capture in a single or multiple zone to be tested and detect a signal (s) from the specifically linked test. In one example of the invention, the SPR is covered with two distinct zones of capture nucleic acid oligonucleotides which are made specific by different target nucleic acid sequences. In another example of the invention, the SPR is covered with at least one capture test for a target nucleic acid sequence and a capture test for an amplification control nucleic acid sequence, which when detected confirms that the amplification take place.

The present invention also comprises a randomly generated internal amplification positive control nucleic acid including the nucleic acid sequence of R1C1 and a second internally amplified positive control nucleic acid having the nucleic acid sequence of R1C2.

The present invention further comprises a method for generating an internal amplification positive control nucleic acid consisting of: generating random nucleic acid sequences of at least 10 nucleotides in length, testing said random nucleic acid sequence and selecting by specific functionality, combining in collaboration a number of said nucleic acid sequences functionally selected and testing the combined nucleic acid sequence and optionally selecting against the formation of intro-filament nucleic acid dimer or the formation of hair tip structures.

BRIEF DESCRIPTION OF THE DRAWINGS The presently preferred specimens of the invention will be described together with the illustrations in the appendix, where as reference numbers they refer to similar elements in various views and in which: Figure 1 is a graph illustrating the stability of the temperature of the single dose reagent pill .; Figure 2 illustrates the general TMA protocol; Figure 3A is a schematic representation of a reaction vessel in a disposable double chamber and the heating steps associated therewith to effect a TMA reaction according to a possible exemplary of the invention; Figure 3B is a schematic representation of an alternative form of the invention in which two separate reaction chambers combine to form a double chamber reaction vessel.

Figure 3C is a schematic representation of two alternative specimens of a double chamber reaction vessel that is put on a test strip to be processed with a solid phase receiver and optical equipment according to the preferred reaction specimen; Figure 4 is a schematic representation of an alternative example of a double chamber reaction vessel formed of two separate chambers that are combined in a way that allows a liquid sample in one chamber to be transferred to the other chamber, with the vessel combined double chamber placed on a test strip as illustrated in Figure 3C; Figure 5 is a perspective view of a single amplification process station for the test strips having the double chamber reaction vessels according to a currently preferred form of the invention; Figure 6 is a perspective view of one of the amplification modules of the modules of Figure 4, as viewed from behind the module; Figure 7 is a perspective view of the module of Figure 5; Figure 8 is another perspective view of the module of Figure 7; Figure 9 is a detailed perspective view of a part of the test strip holder and the Peltier heating systems at 95 ° C of the module of Figures 6 - 8; Figure 10 is an isolated perspective view of the support of the test strip of Figure 9 showing two test strips installed in the support of the test strips; Figure 11 is a detailed perspective view of the support of the test strip or tray of Figure 7; Figure 12 is a block diagram of the electronic parts of the amplification process station of Figure 7; Figure 13 is a diagram of the vacuum subsystem for the amplification process station of Figure 6; Y Figure 14 is a graph of the thermal cycle of the station of Figure 6; Figure 15 illustrates a scheme of the operation of multiple VIDAS detection.

Figure 16 illustrates the production of SPR with two different capture zones; Figure 17 illustrates the configuration of the VIDAS apparatus strip for multiple detection; Figure 18 illustrates and graphs the results of the multiple VIDAS protocol verification by detecting only the target Neisseria gonorrhoeae NG; Figure 19A is a graph showing the results when the 1 x 1012 targets of Chlamydia trachomatis (CT) are mixed with 0.1 x 109, 1 x 1010, 1 x 10n, or 1 x 1012, the NG targets, and they detect with the VIDAS instruments using the multiple protocol and covered with SPRs with CT capture tests at the bottom of the SPR zone, and the NG capture tests in the upper area of SPR.

Figure 19B illustrates the results when the 1 x 1012 CT targets are mixed with 0.1 x 109, 1 x 1010, 1 x 10a, or 1 x 1012, the NG targets, and detected with the VIDAS instruments using the protocol multiple and covered with SPRs with CT capture tests at the bottom of the SPR zone, and the NG capture tests in the upper area of SPR.

Figure 20A is a diagram showing the detection of the M.tb nucleic acid by the VIDAS apparatus after amplification; Figure 20B is a diagram showing the detection of the M.tb nucleic acid by the VIDAS apparatus.

Figure 21 is a diagram showing the detection of the M.tb nucleic acid by the VIDAS apparatus after amplification; Figure 22 is a graph showing the detection of the M.tb nucleic acid by the VIDAS apparatus after amplification using a binary / double chamber protocol.

, Figure 23 illustrates the results generated by the method described showing a collection of strands of nucleic acid sequences and tests for specific functional parameters.

Figure 24 shows the nucleic acid sequence of Internal Random Control 1 (RIC1) with the possible first / oligonucleotide test for the amplification and detection of the control sequence.

Figure 25 shows an analysis of the possible secondary structural compounds of the sequence RIC1.

Figure 26 shows the nucleotide acid sequence of Internal Random Control 2 (RIC2) with the possible first / oligonucleotide test for amplification and detection of the control sequence.

Figure 27 shows an analysis of the possible secondary structural compounds of the RIC2 sequence.

Figure 28 illustrates the results of RIC1 DNA detection, where ran21 is the capture test and ran33 is an enzyme-linked detector test and shows that amplification and detection occurs under standard test conditions.

Figure 29 shows that the RIC1 RNA amplified by TMA and the detected signal activated chemically in a VIDAS instrument (bioMérieux Vitek, Inc.) using the enzyme-linked detection system, has a sensitivity limit of approximately 1000 RIC1 RNA molecules. (without the optimization of conditions).

Figure 30 shows the nucleic acid sequence for internal control oligonucleotides designed for assays to detect: Chlamydia trachomatis (CT) identified as CRIC-2; for Neisseria gonorrhoeae (NG) identified as SIRG; for Mycobacterium tuberculosis (MT) identified as MIRC; and internal control for HIV identified as HRIC.

Description of the Invention The following examples are provided to better illustrate certain copies of the present invention without intending to limit the scope of the invention.

Example 1 Single Dose Reagents and Unified Regulator The implementation of a TMA reaction (see U.S. Patent 5,437,990) online in a VIDAS apparatus or in line to a separate instrument (when detection occurs in a VEDAS instrument) requires the modification of the chemistry used to carry out the reaction manually. First, the reagents packaged in volume have been modified in single aliquot doses, and second, the reaction regulator compounds have been altered to form a comprehensibly simple multifunctional unified regulator solution.

Under the current manual technology, reagents are prepared as freeze-dried "cakes" of multiple test quantities. The amplification and enzyme reagents of this form must be reconstituted in volume and formed in aliquots for individual experiments.

Thus, the automated form of TMA in the VIDAS system is improved in the previous manual method by using single-dose pills of lyophilized reaction components that can be resuspended in a single unified regulator which will support sample dilution, denaturation of nucleic acids, the tuning of nucleic acids and the desired enzymatic activity.

A) Unified Regulator and Single Dose Reagents To test the feasibility of single dose amplification reagents, the standard TMA amplification for Chlamydia and the enzymatic reagents (Gen - Probé Inc.), the reagents in volume are reconstituted in 0.75 ml. of water. 12.5 μl of the amplification reconstituted with water or the enzyme reagent (ie, a single-dose aliquot) are aliquoted into microcentrifuge tubes. These tubes are placed in a vacuum centrifuge with low heating to remove the water. The final result of this procedure is a microcentrifuge tube containing a small brown cake of enzymes or amplification reagents at the bottom of the tube.

The combined unified regulator used in this example, consists of a combination of Sample Dilution Regulator (SDB) from commonly available Gen-Probé Inc., Reconstitution Amplification Regulator (ARB) and Enzyme Dilution Regulator (EDB) in a ratio of 2: 1: 1. For each dry amplification reagent microfuge tube, lOOμl of the combined Unified Regulator and positive control nucleic acid (+) is added and overlaid with lOOμl of silicone oil. The tube was then heated to 95 ° C for 10 minutes and then cooled to 42 ° C for 5 minutes. The total volume of 200 μl is then transferred to a tube containing the dry enzyme reagent. This is then slowly mixed to resuspend the enzyme reagent and the solution is heated for one hour at 42 ° C.

The control reactions are prepared using Gen - Probe control reagents, which are reconstituted in the 1.5 ml normal of ARB or EDB according to the instructions given in the Gen - Probé kit. In each control reaction, 25 μl of the reconstituted amplification reagent is combined with 50 μl of SDB with the positive control nucleic acid (+). The mixture is also heated to 95 ° C for 10 minutes and then cooled to 42 ° C for 5 minutes. To this is added 25 μl of the reconstituted enzymatic reagent and incubated at 42 ° C for one hour. The negative control does not have nucleic acid.

The (Unified) reactions of the Unified Test Regulator and the standard control reactions (control) are then subjected to the protocol of the Hybridization Protection Experiment (HPA) standard of Gen-Probé Inc. Gradually, add 100 μl of a specific nucleic acid test of Chlamydia trachomatis to each tube and allow to hybridize for 15 minutes at 60 ° C. Then, 300 μl of the Selection Reagent is added to each tube and allowed to occur for 10 minutes. Minutes the differential hydrolysis of the hybrid and unhybridized test. Then, the tubes are read in a Leader 50 luminometer from Gen-Probe and the resulting information is recorded as Relative Light Units (RLU) detected from the label, as shown in table 1 below. The information is reported as RLU, reaction TMA HPA C. Trachomatis.

Table 1; Unified single-dose aliquot of amplification and enzyme reagents The information in Table 1 demonstrates that comparable results are obtained when single dose aliquots of dry amplification and enzymatic reagents are used. In addition, the information shows that the results are comparable using three separate regulators (ARB, EDB and SDB) and a unified combined regulator (SDB, ARB and EDB combined in a ratio of 2: 1: 1) to resuspend the reagents and perform The reactions.

B, Single Dose Reagent Pelletizing To simplify the manufacture of single dose aliquots, methods are used that will allow the pelletization of these reagents in single dose aliquots. Pills (or pellets) can be made little by little by aliquoting an aqueous solution of the chosen reagent (which has been combined with an appropriate excipient, such as D (+) Trehalose (a-D-Glucopyranosyl-d-D-glucopyranoside , obtained from Pfanstiehl Laboratories, Inc., Waukegan, IL) in a cryogenic fluid and then sublimation is used to remove the water from the pill Once the reagent / trehalose mixture is made by aliquots (drops) in a cryogenic liquid A frozen spherical pill is formed, these pills are then placed in a lyophilizer where the frozen water molecules are sublimated during the vacuum cycle.The result of this procedure are small, stable pills with no reagents on the sides that can be distributed in appropriate packaging: Single dose aliquots of reagents containing RT, T7 and sugar are subjected to a wide range of temperatures to examine the stability of the pill. After undergoing a temperature test for 10 minutes, the pills are then used for CT amplification. The results are expressed in the graph of Figure 1. The results show that single-dose reagent pills remain stable even after exposure to high temperatures for 10 minutes.

The extraordinary stability of the dried enzymes in trehalose has been previously reported (Colaco, et al., 1992, Bio / Technology, 10, 1007) which has renewed interest in long-term protein research, has become a topic of interest (Franks, 1994, Bio / Technology, 12, 253). The resulting pills of the amplification reagents and the enzymatic reagents were tested by using TMA / HPA reactions of C. trachomatis.

The prepared amplification pills are placed in a tube to which 75 μl of a mixture of ARB and SDB (mixed in a ratio of 1: 2) are added with positive control nucleic acid. This sample was then heated at 95 ° C for 10 minutes and then cooled to 42 ° C for 5 minutes. To this is added 25 μl of enzymatic reagent, which has been reconstituted using a standard procedure of Gen-Probé Inc. This mixture is allowed to incubate for one hour at 42 ° C. Then the reactions are analyzed by the HPA procedure, as described above. The results of this test are reported as RLU in Table 2 and are identified as enzymatic (+) pills. Control reactions are prepared using standard reagents from Gen-Probé Inc. following the standard procedure. The information reported as RLU, standard TMA / HPA reaction of C. trachomatis.

TABLE 2 Aliquots of Single Dose of Pelleted Amplification and Enzymatic Reagents.

The information in table 2 shows that there is no significant difference when using standard reagents from Gen - Probé Inc. or dry pills, prepared from the single dose amplification reagent or the enzyme reagent pill. Thus, the aliquots of single doses of the reagents are suitable for use with a simple unified regulator to be applied to an automation using a VIDAS system.

Example 2 Automated Isothermal Amplification Using Thermollabile Enzymes. To automate the reaction of the isothermal amplification experiment for use with clinical experiment apparatus, such as a VIDAS instrument (bioMérieux Vitek, Inc.), a new dual-chamber reaction vessel has been designed to implement the use of the unified regulator and the single reaction aliquot reagent pills described above in the isothermal amplification experiment of the test samples which can be used additionally in combination with a self-sufficient process station.

A) Double Reaction Chambers The use of two chambers will facilitate keeping the heat-stable amplification / sample reagent (containing the first oligonucleotides and nucleotides) of the heat-labile enzymatic components (ie, the reversible RNA transcriptase, RNA polymerase RNase H).

Figure 3A is a schematic representation of a disposable double chamber reaction vessel 10 and the heating steps associated therewith for effecting a TMA reaction according to a possible exemplary of the invention. Chamber A contains the mixture of the amplification, namely, nucleotides, first oligonucleotides, MgCl 2 and other salts and regulator compounds. Chamber B contains the amplification enzyme that catalyzes the amplification reaction, for example T7 and / or RT. After addition of the targets (or patient sample) within Chamber A, heat is applied to denature the nucleic acid targets of the DNA and / or remove the RNA secondary structure. The temperature of Chamber A is then allowed to cool to allow the first tempering. Subsequently, the solution of chamber A is brought into contact with chamber B. Chambers A and B now in liquid communication with each other, are kept at an optimum temperature for the amplification reaction, for example, 42 ° C. chamber A of chamber B and applying heat for denaturation only to chamber A, the heat-labile enzymes in chamber B are protected from inactivation during the denaturation step.

Figure 3B is a schematic representation of an alternative form of the invention in which two separate reaction chambers 12 and 14, combine to form a double-chamber reaction vessel 10. As the example of Figure 3A, the chamber A is pre-loaded during a manufacturing step with an amplification mixture, namely, nucleotides, first oligonucleotides, MgCl2 and other salts and regulator compounds. Chamber B is pre-loaded during manufacture with the amplification enzyme that catalyzes the amplification reaction, for example T7 and / or RT. The liquid sample is then introduced into chamber A. The targets are heated for denaturation at 95 ° C in chamber A. After cooling chamber A at 42 ° C, the solution in chamber A is placed in contact with the enzymes in chamber B to activate the isothermal amplification reaction.

If the reaction vessel is designed as such, after having put the contents of cameras A and B in contact, the amplification chamber does not allow any exchange of materials with the environment, it is noted that a closed amplification system that minimizes the risk of contaminating the amplification reaction with heterogeneous targets or products of amplification of previous reactions.

Figure 3C is a schematic representation of two alternative double-chamber vessels 10 and 10 'which are placed on a test strip 19 when carrying out the process with a solid-phase receiver and optical equipment according to the preferred sample of the reaction. In the example of Figure 3, a unidirectional flow system is provided. The sample is first introduced into chamber A to heat to denaturation temperature. Chamber A contains the mixture of dry amplification reagent 16.

After cooling, the liquid is transferred to chamber B which contains the dry enzyme 18 in the form of a pill. Chamber B is maintained at 42 ° C. after the sample is introduced into chamber B. The amplification reaction takes place in chamber B at the optimum reaction temperature (42 ° C). After the reaction is completed, the test strip 19 is processed in a machine such as the VEDAS instrument available from bioMérieux Vitek, Inc., the assignee of the present invention. People with skill in the art are familiar with the V DAS instrument.

The heating and cooling steps of the chamber A can be carried out prior to the insertion of the double chamber disposable reaction vessel 10 or 10 'into the test strip 19, or, alternatively, appropriate heating elements can be placed adjacent to the end of the left hand 24 of the test strip 19 to provide adequate temperature control of the reaction chamber A. The remaining amplification process station of Figures 4 - 14, which is described below, incorporates appropriate heating elements and control systems to provide adequate temperature control for the control vessel 10.

Figure 4 is a schematic representation of an alternative example of a double chamber reaction vessel 10"formed of two separate interlocked vessels 10A and 10B that are combined in a manner that allows a sample of liquid in a chamber to flow towards the chamber. another, with the combined double chamber vessel 10"placed on the test strip 19 as described above in Figure 3C. The liquid sample is introduced into chamber A, which contains the mixture of dry amplification reagent 16. The vessel A is then heated to 95 ° C. and then cooled to 42 ° C. The two vessels A and B are combined by means of a conventional closure that fits between the complementary closure surfaces on the projection of tube 26 in chamber B and recessive conduit 28 in chamber A. Mixing the sample solution of chamber A with the enzyme of the chamber B occurs because the two chambers are in a liquid communication with each other, as indicated by arrow 30. Then the sample can be amplified in the disposable double-chamber combined reaction vessel 10", off-line or within of the line to close the combined disposable cup 10"inside a modified VIDAS strip. The VIDAS instrument could effect the detection of the amplification reaction in a known way.

B. Amplification Station Figure 5 is a perspective view of a single residence amplification process system 200 for the test strips 19 having the double chamber vessels according to the current preferred form of the invention. The system 200 consists of two identical amplification stations 202 and 204, a power source, a power source module 206, a control circuit module 208, a vacuum tank 210 and connectors 212 for the power source module. 206. Tank 210 has hoses 320 and 324 to provide vacuum to amplification stations 202 and 204 and ultimately to a variety of vacuum tests (one per strip) in the manner described above to facilitate fluid transfer from the first camera to the second camera. The vacuum subsystem is described below along with Figure 14.

The amplification stations 202 and 204 each have a tray for receiving at least one of the associated strips and temperature control, the valve activation and vacuum subsystems for heating the reaction orifices of the strip at the appropriate temperatures. , transferring liquids from the first chamber in the reaction holes of the double chamber to the second chamber, and activating a chamber as a thimble valve or preferably a pellet valve to open the liquid channel and let the liquid flow between two cameras.

The stations 202 and 204 are designed as amplification stations that remain alone for the performance of the amplification reaction in an automated manner after the patient or clinic sample has been added to the first chamber of the reaction vessel of the double camera described above. The processing of the strips after the reaction is completed with a SPR, takes place in a separate machine, such as a VIDAS instrument. Specifically, after the strips have been placed in stations 202 and 204 and the reaction is carried out in the stations, the strips are removed from stations 202 and 204 and placed in a VIDAS instrument for the subsequent process and analysis in the known form.

The entire system 200 is under the control of a microprocessor by an interface board of the amplification system (not shown in Figure 5). The control system is shown in the form of a block diagram in Figure 12 and will be described later.

Referring now to Figure 6, one of the amplification stations 202 is shown in a perspective view. The other amplification station is of identical design and construction. Figure 7 is a perspective view of the front of the module of Figure 6.

Regarding these figures, the station includes a vacuum test slide motor 222 and a vacuum test slide camshaft 246 which operates to slide a vacuum test set 244 (shown in FIG. 7) so that the Thimble valves rise and fall in relation to the sliding of the vacuum tests 246 to open the thimble valves and apply the vacuum so that the liquid is released from the first chamber of the reaction vessel 10 to the second chamber. Vacuum tests 244 in reciprocity within the annular recess provided in the slip tests 246. Obviously, it is necessary to observe the proper registration of the tip structure and the vacuum test 244 with the corresponding structure in the test strip as it is installed in the trays you need to observe.

The station includes side walls 228 and 230 that provide a frame for station 202. Control board 229 is mounted between side walls 228 and 230. The electronic module for station 202 is installed in control tray 229 of the tray .

A set of thermal protection covers of the trays 220 are part of a thermal subsystem and is provided for wrapping a tray 240 (Figure 7) that receives one or more of the test strips. The protective covers 220 help maintain the temperature of the tray 240 at the appropriate temperatures. The thermal subsystem also includes a Peltier 242 heating vessel at 42 ° C, a part of which is placed adjacent to the second chamber in the reaction chamber of the double chamber in the test strip to maintain that chamber at the temperature appropriate for the enzymatic amplification reaction. A heating vessel 250 at 95 ° C is provided for the front of the tray 240 to maintain the first chamber of the reaction orifice in the test strip at the denaturation temperature.

Figure 8 is another perspective view of the module of Figure 7, showing a heating container 250 at 95 ° C and a set of fins 252. It should be noted that the heating container 250 at 95 ° C is placed at front and slightly down the tray 240. The heating vessel 242 at 42 ° C is placed behind the heating vessel 250.

Figure 9 is a detailed perspective view of a portion of the tray 240 that holds the test strips (not shown) as shown from above. The tray 240 includes a front part having a base 254, a plurality of parallel rough structures arising in discontinuous fashion 256 with recessive slots 258 for receiving the test strips. The base of the front 254 of the tray 240 is in contact with the heating vessel 250 at 95 ° C. The side walls of the parallel raised ridges 256 at positions 256A and 256B are placed as close as possible to the first and second positions. reaction vessel chambers 10 of Figure 3 A to reduce the thermal resistance. The base of the rear part of the tray 240 is in contact with the Peltier heating container at 42 ° C, as seen in Figure 8. The part 256B of the raised ridges for the rear part of the tray is physically isolated from the part 256A for the front of the tray and the part 256B is in contact with the heating vessel at 42 ° C to maintain the second chamber of the reaction vessel in the test strip at the appropriate temperature.

Continuing to refer to Figure 9, the vacuum tests 244 include a rubber gasket 260. When the vacuum tests 244 are decreased by the vacuum test motor 222 (Figure 6), the gaskets 260 are placed} on the upper surface of the test strip that surrounds the vacuum port in the double chamber reaction vessel to tighten the seal and allow the vacuum to be released into the second chamber.

Figure 10 is an isolated perspective view of the test strip holder of the tray 240 of Figure 9, showing two test strips installed in the tray 240. The tray 240 has a plurality of lines or slots 241 that receive up to six test strips 19 for a simultaneous process. Figure 10 shows the heating containers 242 and 250 for maintaining the respective parts of the tray 240 and the ridges 256 at the appropriate temperature.

Figure 11 is a detailed perspective view of the test strip holder 240 as seen from above. The Peltier heating container at 95 ° C below the front 254 has been removed to better illustrate the rear heating container 242 under the rear of the tray 240.

Figure 12 is a block diagram of the control and electronic system of the amplification process system of Figure 5. The control system is divided into two boards 310 and 311, section A 310 at the top of the diagram which is for the amplification module or station 202 and the other board 311 (section B) which is for the other module 204. The two boards 310 and 311 are identical and only the high section 310 will be contemplated.

The two boards 310 and 311 are connected to an interface board of the amplification station 300.

The interface board 300 communicates with a personal computer 304 via a high-speed information transport 302. The personal computer 304 is a conventional IBM compatible computer with a hard disk drive, video monitor, etc. In a preferred exemplary, stations 202 and 204 are under control by interface board 300.

The board 310 for station 202 controls the front tray 240 which is maintained at a temperature of 95 ° C. by two modules of Peltier heating vessels, a pair of fans and temperature sensors incorporated within the front part 254 of the tray 240. The rear part of the tray is maintained at a temperature of 42 ° C. by two Peltier modules and a temperature sensor. The movement of the vacuum tests 244 is controlled by the test engine 222. The position of the sensors is provided to give input signals to the tray controller board as the position of the vacuum tests 244. The controller board of the Tray 310 includes a set of handlers 312 for the active and passive compounds of the system which receives information from the temperature and position sensors and emission commands for the active compounds, ie motors, fans, Peltier modules, etc. The handlers are responsible for control from the amplification interface board 300. The interface board also issues the commands to the vacuum pump for the vacuum subsystems, as shown.

Figure 13 is a diagram of the vacuum subsystem 320 for the stations of the amplification process 202 and 204 of Figure 5. The subsystem includes a one liter plastic vacuum tank 210 which is connected by an input line 322 to a vacuum pump 323 to generate a vacuum in tank 210. A vacuum supply line 324 is provided to vacuum a pair of solenoid valves to tighten 224 (see Figure 6) by means of supply lines 324A and 324B provide vacuum to a manifold 226 that distributes the vacuum to the vacuum tests 244. Note that the pointed tips 245 of the vacuum tests 244 for cutting the film or membrane 64 covering the strip 19. The vacuum system 320 also includes a transducer of differential pressure 321 to monitor the presence of vacuum in tank 210. Transducer 321 provides pressure signals to interface board 300 of Figure 12.

Figure 14 is a graph representative of the thermal cyclic profile of the station of Figure 5. As indicated on line 400, after an initial upward ramp 402 in which the temperature lasts less than one minute, a first temperature is reached TI (for example, a denaturation temperature) which is maintained for a predetermined period of time, such as 5 to 10 minutes, at which time a reaction occurs in the first chamber of the reaction vessel. Then, a downward movement of temperature occurs as indicated at 404, and the temperature of the reaction solution in the first chamber of the reaction vessel 10 is cooled to the temperature T2. After a certain amount of time after cooling to temperature T2, a liquid transfer occurs in which the solution in the first chamber is transported to the second chamber. The temperature T2 is maintained for an adequate amount of time for the reaction of interest, such as one hour. At time 406, the temperature rapidly increases to a temperature T3 or 65 ° C. at the top of the amplification reaction.

For a TMA reaction, it is important that the time increment, from time 406 to time 408, is short, that is, less than 2 minutes and preferably less than one minute. Preferably, all increases and decreases in temperature occur in less than one minute.

Other examples of the reaction vessels and components of the amplification station are also conceived, and such examples of said alternative examples are included in the present invention.

Example 3 Automated VIDAS Test for Amplified and Non-Amplified Detection? E Mycobacterium tuberculosis (M.tb) Using the VIDAS instrument (bioMérieux Vitek, Inc.) modified at 42 ° C, we have developed a fast and simple online amplification and detection experiment for clinical laboratory for the detection of M.tb in test samples, which can be completed In a short time. The entire experiment is designed to take place on a simple test strip, minimizing the potential for contamination to targets or amplicons. The experiment based on the amplification is capable of the detection of M.tb where the sample contains only 5 cells similar to the sensitivity achieved by the commercial kit of Gen-Probé.

. The amplification-based experiment uses unique rRNA sequence target for Transcription Mediated Amplification (TMA), followed by hybridization and fluorescent detection linked by enzymes from the nucleic acid test (amplicon) in the instrument LIVES The amplification / detection experiment can detect approximately 1 Fg of M.tb rRNA or less than one MTB organism per test, and is specific for all elements of the M.tb complex. Specific tests for the detection of M.tb can be found in C. Mabilat, 1994, J. Clin. Microbiol. 32, 2707.

Standard smears for the acid fast bacillus are not always reliable as a diagnostic tool, and even when a mycobacterium other than MTB can be positive. Currently, standard methods for TB diagnosis require growing the slow-growing bacteria, and it can take six weeks or more. During this time, the patient is usually isolated. The initial result is that this automated test matches or exceeds the clinical sensitivity of the culture method and offers a highly sensitive method to detect M.tb rapidly (in less than three hours) in infected samples, thus aiding diagnosis, fast isolation and treatment.

A) Sample Preparation A volume of 450 μl of specimen is added to 50 μl of specimen regulator dilution in a lysate tube containing crystal beads, sonicated for 15 minutes at room temperature to lyse organisms, inactivated by Heat for 15 minutes at 95 ° C. When required, the isothermal amplification is performed by a commercially available manual experiment set (Gen - Probé Inc.), following game instructions using standard game reagents. However, similar experiments can be carried out using the modified compounds as described in the previous examples.

B. Detection To operate the automated detection experiment, the detection system requires the hybridization of the target nucleic acid or amplicon to a specific capture nucleic acid bound to a solid support, (in the VIDAS system it is called "a solid phase receptor" which is an apparatus such as SPR pipette) and an identified detection test nucleic acid (eg, where the identification can be alkaline phosphatase, a chemiluminescent signal compound, or other reagent that allows specific detection of a binding test) .

In an automated system such as the VIDAS, after several washing steps to remove the unbound test, the SPR transfers the target test hybrid to an enzymatic substrate, whereby the detectable signal is activated from the link test and detected by the instrument of the experiment. In one specimen, the detection test is conjugated with alkaline phosphatase and once placed with the methyl umbelliferyl phosphate substrate (MUMP), the substrate is converted to 4-methyl umbelliferone (4 MU) by alkaline phosphatase. The 4-MU produces a fluorescence that is measured and recorded by the standards of a VEDAS instrument as relative fluorescence units (RFU). When the target nucleic acid is not present, no detection test is attached and no substrate is converted, so no fluorescence is detected.

C. Analytical Sensitivity Controls. The controls are usually prepared in a specimen dilution regulator array with positive controls containing 5 fg of M.tb rRNA of about 1 Mie cell. The sensitivity of the automated test experiment can be determined by testing the dilutions of the M. tb cells used. Cell lysates can generally be prepared with a spiral of 1 μl of cells (the assumption being that there are approximately lxlO9 unit forming colonies (CFU) per a full 1 μl spiral, based on previous trituration and CFU experiments). Dilutions of the M.tb lysates can be tested with the automated test experiment.

Figure 20A is a graph showing the detection of amplicons M.tb according to the Gen-Pro Set. Figure 20B is a graph showing the detection of ampoules M.tb of the same reactions as in Figure 20A by the LIVES instrument.

Figure 21 is a graph showing the amplification and detection of the M.tb nucleic acids in the modified VIDAS apparatus. The enzyme is used in liquid form and the amplification is carried out in line with the VIDAS experiment instrument.

Figure 22 is a graph showing the amplification and detection of M.tb nucleic acids in the modified VIDAS apparatus using the disposable binary / double chamber reaction vessel. The denaturation step is carried out offline of the VIDAS instrument, amplification and amplicon detection is carried out in line with the VIDAS instrument.

Example 4 Automated VIDAS Test for Chlamydia Amputed Detection Using the VIDAS instrument (bioMérieux Vitek Inc.), we have developed a simple, highly specific, fully automated experiment for the rapid detection of Chlamydia trachomatis (CT) from simple samples. The test uses unique sequences of isothermal TMA targets of the rRNA followed by hybridization and fluorescent detection linked to the enzyme. The automated test specifically detects all clinically important Chlamydia trachomatis (CT) serovars from urogenital specimens in less than two hours. We obtained an analytical sensitivity of 0.5 fg rRNA or the equivalent of approximately one tenth (1/10) of an elementary body of Chlamydia trachomatis (CT). An agreement between the automated test and the amplified CT test of Gen - Probé for two hundred and seven (207) endocervical clinical samples and urine showed a complete agreement.

Chlamydia trachomatis (CT) infection is the leading cause of sexually transmitted diseases in the United States and Europe. It is currently estimated that four million new CT infections occur each year in the United States.

Chlamydia trachomatis (CT) is a small obligate intracellular parasite that causes infections in men and women, adults and newborns. The greatest challenge for the control of CT infection is that up to 75% of infected women and 50% of infected men are asymptomatic. This results in a large reservoir of unrecognized infected individuals that can transmit the CT infection. The rapid and simple detection of the CT infection would help in a greater degree to assist in the identification of infected individuals.

A, Preparation of Samples and Patient Specimens. The coded samples (n = 207) are obtained from patients with symptoms that coincide with the CT infection. The cervical samples are collected with the Gen - Probé sample collection kit containing the Gen - Probé transport medium; Urine samples are collected in standard urine collection devices. All samples are stored at 4 ° C.

The cervical samples are centrifuged at 425xg for 5 minutes to put all the liquid at the bottom of the tube. The samples are then treated with 40 μl of the Gen-Probe Specimen Preparation Reagent and incubated at 60 ° C. for 10 minutes. 20 μl of the treated sample is pipetted into 400 μl of sample dilution regulator (SDB).

Two Mi. of each urine sample is set at 37 ° C for 10 minutes and microfuge at 12,000xg. for 5 minutes. The supernatant is removed and 300 μl of the sample dilution regulator is added to each specimen. The 15 CT serovars are used for included samples, the specimens are quantified and 20 μl of specimens containing 4x102 IFU / ml are added. (inclusion forms the units per my.) of each serovar is added to 400 μl of SDB. A panel of exclusive urogenital microorganisms is obtained and quantified and 20 μl of 2 x 109 / ml are pipetted. microorganisms in 400 μl of SDB. The positive control containing 0.5 fg rRNA or the equivalent of 0.1 CT of elementary body is diluted in SDB.

B Sample Amplification v VIDAS Detection Samples are amplified using the TMA protocol and the rRNA targets are hybridized to conjugated oligomers for AMVE copolymers and an alkaline phosphatase-conjugated oligomer. See, for example, U.S. Patent No. 5,489,653 and ,510,084. As described above, the solid phase receptor (apparatus similar to a SPR pipette) takes the linked hybrids through successive washing steps and finally into the 4-MUP substrate. The alkaline phosphatase converts the substrate to fluorescent 4-MU which is detected by the VIDAS experiment machine and recorded as relative fluorescence units.

Table 2 below illustrates CT detection by the automated VIDAS experiment after amplification as RFV (RFV = RFU - background of RFU) against rRNA CT concentrations. Dilutions of C. trachomatis purify the rRNA from 0 to 200 molecules are amplified (n = 3) and detected in the automated VIDAS test experiment. The limit of detection is 20 molecules of purified rRNA.

TABLE 2B Detection of CT by VIDAS C- Analytical Specificity v Results The amplifications and detections are carried out in the presence of each of the following ATCC organisms with detections reported as RFV in Table 3 below: TABLE 3 Exclusivity Panel for CT Anaesthetic specificity for information on Chlamydia serovars reported as RFV is shown in Table 4 below: TABLE 4 Inclusivity Panel for CT Table 5 below illustrates the results of specimen tests of clinical cervical samples for CT comparing the results of the AMP-CT experiment of the Gen-Probe manual and the automated VIDAS test experiment.

TABLE 5 CT Detection of the Amplified Clinical Cervical Specimen AMP-CT Experiment of the Gen-Probé Manual Outside the VEDAS line + Automated Test + 35 0 Experiment 0 85 Table 6 below illustrates the results of the clinical urine specimen testing by comparing the results of the manual AMP-CT experiment and the automated VIDAS test experiment.

TABLE 6 CT Detection in Amplified Clinical Urine Specimen AMP-CT Experiment of Gen-Probé Manual Out of the line of VEDAS + Automated Test + 25 0 Experiment - 0 62 In this way, there was a perfect agreement in the results of the experiment between the automated test experiment using the VIDAS experiment and the Gen-Probé AMP-CT experiment manual.

Example 5 Detection of Multiple Nucleic Acid (multiple sequence). The value of diagnostic tests based on nucleic acid tests can be substantially increased through the detection of multiple different nucleic acid molecules and the use of internal positive controls. An automated method has been invented for use with the VIDAS instrument (bioMérieux Vitek Inc.) which can discretely detect at least two different nucleic acid sequences in an experiment reaction and the multiple protocol is determined. In this manner, a nucleic acid amplification method or a processed test sample can be protected for more than one nucleic acid target amplified in the same experiment. This method relies on the separation of discrete nucleic acid tests which can specifically capture different sequences (amplicons) of target nucleic acid in the SPR pipette apparatus of the V DAS instrument. The SPR is a tip similar to a disposable pipette that allows the movement of the equids that also acts as a solid support for affinity capture. Multiple capture by SPR is demonstrated using specific capture tests for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG).

Figure 15 illustrates a scheme of the multiple VIDAS detection operation. The SPR tips are covered in two distinct zones with oligonucleotide nucleic acid sequences that are used to specifically capture complementary nucleic acid sequences (ampucons) with their corresponding reporter nucleic acids or specific detector identified with alkaline phosphatase (AKP). After washing to remove the unlinked report tests, the AKPs located for the SPR background are detected with the fluorescent substrate 4 - MUP. The AKP is made from the bottom of the SPR with NaOH or other reagents that promote the denaturation of nucleic acid hybrids or inactive alkaline phosphate activity. The enzymatic reaction orifices are emptied, washed and refilled with fresh 4-MUP. To confirm that the AKP is removed from the SPR mooring, the new substrate is exposed to the bottom of the SPR and the residual fluorescence is measured. Finally, the AKP reporter test that joins the upper part of the SPR is detected by immersion of the SPR in 4 -MUP and that represents the presence of the second amplicons.

Figure 16 illustrates the production of SPR with two different capture zones. The SPR is first inserted with the tip into a silicone inlet, which is supported on the hanger. The differential pressure is used to uniformly release a test solution in about 1 ug / ml of specific capture, conjugated to the AMVE copolymer, within all the SPRs at the same time. The amount of fluid attracted in each SPR, and thus the size of the zone, is controlled by regulating the amount of pressure in the system. The conjugate binding of the SPR surface is achieved by the passive absorption of several hours at room temperature. After washing and drying, the SPR is covered with a small adhesive disc and inserted into new hangers with a tip orientation downwards. The lower part of the SPR is then similarly covered with a second capture test conjugate. The SPR is stable when stored dry at 4 ° C.

Figure 17 illustrates a preferred specimen of the strip configuration of the apparatus LIVES for multiple detection. The strip can be pre-assembled with 200 μl of an AKP test mixture (1 x 10 12 molecules of each test) in a hybridization regulator in an XI hole, 600 μl of washing buffer in holes X3, X4, X5, 600 μl of strip reagent for holes X6 and X7 and 400 μl of AKP substrate in X8 and sealed with foil. The foil-sealed optical cuvette (XA) containing 300 μl of 4-MUP is closed inside the strip and the strips are inserted into the VIDAS instrument at 37 ° C. The multiple VIDAS protocol is then executed using SPRs covered with two Catch tests in different areas.

The multiple protocol of VIDAS can involve several steps. For example, the routing test protocol contains thirteen (13) basic steps as described below: 1. Transfer 230 μl of XO target to AKP tests on XI. 2. Hybridize and capture all the SPR. 3. Wash SPR (316 μl) twice with PBS / Tween (X3, X4), 4. 4 - MUP at the bottom of SPR (86.9 μl) in XA for 5.3 minutes and then read the signal, 5. 4 - MUP at bottom of SPR (86.9 μl) in XA for 14.8 minutes and then read the signal (optional), 6. Transfer the substrate used from XA to X2 (5 x 67.1 μl), 7. The AKP strip from the bottom of SPR (112.6 μl) ) with NaOH (X7), 8. Wash XA with fresh NaOH (3 x 112.65 μl, X6 to XA to X6), 9. Wash XA with PBS / Tween (3 x 112.65 μl, X6 to XA to X6), 10. Transfer 4 - MUP from X8 to XA (6 x 48 μl), 11. 4 - MUP to the bottom of SPR (86.9 μl) in XA for 10.7 minutes and then read the signal, 12. 4 - MUP to the upper part of SPR (294 μl) in XA for 5.5 minutes and then read the signal, 13. 4 - MUP to the upper part of SPR (294 μl) in XA for 15 minutes and then read the signal (optional).

All steps hybridization, substrate, washing and strips, involve multiple pipetting cycles of the respective solution within the SPR maintaining the solution for a defined period of time and pipetting the solution outside the SPR. The sustained times for hybridization, substrate and washing or striping are 3.0, 0.5 and 0.17 minutes, respectively. The fluorescence signal detected by the device. The total experimentation time for the research protocol is 1.75 hours but it can be reduced to around 75 minutes.

Figure 18 illustrates and graphs the verification results of the VEDAS multiple protocol executed as described above, except where SPR is homogeneously covered with only a simple capture test for Neisseria gonorrhoeae (NG). The number of oligonucleotide targets of NG in the test sample varies from 0.1 x 1010 or 1 x 1011 molecules in the test sample. The information shown are averages of the replicated samples. The graph as illustrated is divided into two parts: the left and right halves show the results of two fluorescent measurements from the lower and upper regions of the SPR, respectively. The measurements taken from the lower area after making the strips in the lower area of the binding nucleic acid and the exposure for approximately 11 minutes in the fresh 4-MUP substrate is approximately 46 RFU for all tested samples and is equivalent to the previous measured fluorescence. This measurement is shown by the time point 0 in the center of the graph. In this way, the graph illustrates two sequential sets of fluorescence measurements of a single SPR, the first set of measurements is taken from the lower half of the SPR (left half of the graph) and a second set of measurements taken from the upper part of the SPR (to the right of the graph). This experiment validates that the multiple protocol and the SPR procedure covered in zones produce essentially identical results, as indicated by the fluorescence intensities in the left and right parts of the graph, from the upper and lower portions of the SPR Figure 19 illustrates the multiple detection of the CT and NG oligonucleotide targets in different access amounts. Figure 19A is a graph showing the results when 1 x 1012 of the CT lenses are mixed with 0.1 x 109, 1 x 1010, 1 x 1011, 1 x 1012, the NG targets and those detected with the instruments LIVES using the multiple protocol and the SPR covered with the CT capture tests in the low area of the SPR and the NG capture tests in the upper area of the SPR. The information is plotted as above, where the graph illustrates two sequential sets of fluorescence measurements of a single SPR, the first set of measurements is taken from the lower half of the SPR (left half of the graph), and a second set of measurements taken from the upper part of the SPR (to the right of the graph) with verifications of the SPR strips in the center of the graph. Importantly, this experiment shows that the two SPR zones act independently in the multiple protocol, since the high fluorescence signals from one zone do not interfere with the signals produced from the second zone, regardless of whether these latter signals are high (1 x 1012) or low (1 x 109 barely detectable) or negative.

Table 7 below summarizes the information obtained by multiple VIDAS detection of CT and NG in a sample at various levels of targets, reported in RFUs.

TABLE 7 Detection of CT and NG targets in the sample.

: The information is reported in RFUs, after ~ 5 minutes of exposure of 4 - MUP to link the AKP B test: Columns are information for that number of NG targets in the sample. c: Horses are the information for that number of the CT objectives of the samples. D: The first value reported is RFU detected from the part of the CT experiment. E: The second value reported is RFU detected from the part of the NG experiment.

In this way, the multiple protocol of VIDAS is clearly operative and allows the rapid and discrete detection of more than one different nucleic acid in a sample. This protocol, and the SPR cover can be manipulated in many formats to present areas covered by different surface areas with different sizes of spaces between two or more of the detection zones. The SPR can be covered with nucleic acids which are designed to capture different regions of the same nucleic acid sequence to detect, for example, expression of truncated genes, different alleles or alternately spliced genes. The SPR can be coated to capture ampUcons of the internal control molecules of the nucleic acid, which can be used to successfully detect and confirm the amplification reactions of the nucleic acid. In this way, the multiple V DAS protocol is a flexible method for the detection of more than one nucleic acid sequence in the same sample, in a single experiment.

EXAMPLE 6 Internal Control Sequence and Its Method The construction of internal control sequences composed of sequences of blocks constructed functionally chosen by randomly generated nucleic acid sequences to be used as internal positive controls of amplification reaction, ideally requires that the sequences of control are specifically designed to be used by the various nucleic acid amplification protocols including, but not limited to PCR, LCR, TMA, NASBA and SDA. The internal control nucleic acid sequence, in combination with the appropriate sequence specific for first oligonucleotides or first promoters, will generate a positive amplification signal if the amplification reaction is successfully completed.

Ideally, the internal control nucleic acid is useful with respect to the nucleic acid sequences present in the normal flora or in an environment. Generally, the internal control sequences should not be substantially similar to any nucleic acid sequence present in a clinical situation, including humans, pathogenic organisms, organisms of the normal flora or organisms of the environment that could interfere with the amplification and detection of the internal control sequences.

The internal control sequences of the present invention are composed of blocks of sequences chosen from a list of randomly generated nucleic acid sequences. The functional blocks are segments that provide a special property necessary to allow the amplification, capture and detection of the amplification product. For example, in a TMA reaction, the internal control sequences are most useful when the functional blocks reach certain functional requirements of the amplification protocol, such as: a) a first binding site in the filaments; b) a capture site; c) a detector test link site; d) a T7 promoter that contains the first binding site in the sensitive filament. Each of these functional elements has its own particular repressions, such as length, content in% GC, Tm, the lack of homology for known sequences and the absence of secondary structural features (ie, free of the formation of dimers or hair tip structures) which can be used to select the appropriate sequence. In this way, blocks of randomly generated functional sequences can be tested for the desired functional properties before being used in the construction of internal control sequences.

To construct an internal control sequence having the desired properties comprising a specific number of functional blocks and satisfying the desired repressions within each block, a random sequence generator is used to generate number strings; Each number is limited to the range of 0.000 to 4.000. The length of the strings is flexible and is chosen based on desired lengths of the functional blocks.

Each number in the string (ie, ni, n2, n3, n4 ... nx where x is the length of the string) is then assigned to a corresponding nucleotide as follows: guanosine (G) if 0 < n < l; adenosine (A) yes 1 < n < 2; Thymidine (T) yes 2 < n < 3; and cytosine (C) if 3 < n < 4. A large collection of such strings is produced and tested by those who achieve the sequence and structural requirements of each functional block. Figure 23 illustrates the results generated by the described method showing a collection of strings of nucleic acid sequences and tests for specific functional parameters. The internal control sequence can include DNA, RNA, or modified UONucleotides or any combination of nucleic acids, so that the illustrated sequences using DNA nomenclature can be easily adapted as desired to the appropriate nucleic acid.

The Internal Potential Control (IC) sequences are then constructed by assembling the functional blocks (randomly selected) in the proper order. Finally, assembled internal control sequences are also examined to ensure that the structural constraints of global sequences are maintained. For example, in TMA reaction, the internal control sequence should not have significant base-pair potential between the first two binding sites or the 3 'dimer structures stably. Those internal control sequences that pass through these test layers are then physically produced using superimposed oligonucleotides and tested for their performance in the actual amplification / detection experiments.

Although any functional block may have some homology to the sequences present in a clinical situation (a perfect combination of a block of 21 nucleotides at a random frequency 4e21 of 1 in sequences or approximately 4xl012 is expected, the generated sequences are compared with the base of GenBank data) it is not likely that all functional blocks have a substantial homology. Because the internal control nucleic acid sequences are constructed from a group of functional blocks put together, the possibility that a natural nucleic acid sequence has an identical strand of nucleic acid sequence block in the same group organization It is remote.

Two specific internal control sequences have been constructed using the method described above. The Internal Random Control 1 (RIC1) is shown in Figure 24 with the possible first / oligonucleotide tests for ampufication and detection of the control sequence. Figure 25 shows an analysis of the possible secondary structure of the RIC1 molecule. RIC1 is constructed using the randomly generated strings ran 16, ranl9, ran21 and ran33. The functional blocks require the first link to be reached at row 16 and row 19, while the capture site is satisfied by row 21 and the test link site of the detector is reached by row33. The option of a capture test sequence designation or detection test can be exchanged, provided that the appropriate binding molecule is coupled to the appropriate test, where a reporter test oligonucleotide is attached to a means to generate a detectable signal , and the oligonucleotide of the capture test is attached to means for adhering the capture test to an appropriate support. The tests and oligos are described on the understanding that in the case of a two-stranded DNA, the complementary strand may be the target or it may be converted as is appropriate for use as the detection strand. Thus in the right circumstance, one skilled in the art will be able to modify the sequences as presented to generate tests and alternative primers that are appropriate for use in a manner equivalent to that described herein.

The Random Internal Control 2 (RIC2) is shown in Figure 26 with possible first / oligonucleotide tests for amplification and detection of the control sequence. Figure 27 shows an analysis of the possible components of the secondary structure of the RIC2 sequence. Similarly, RIC1 and RIC2 are constructed using randomly generated cords ran27, ran32, ran39 and ran51. Thus, it is illustrated that it is also possible that the functional blocks require a first link, capture test link, a detector test link that can be reached by the alternative random sequences generated by the method described above.

Figure 28 illustrates the results of RIC1 DNA detection where ran21 is the capture test and ran33 is an enzyme-linked detector test and shows that detection occurs under standard test conditions with the expected fluorescence intensities. Figure 29 shows that RNA from RIC1, amplified by TMA and detected by a VIDAS instrument (bioMérieux Vítel, Inc.) using the enzyme linkage detection system, has a sensitivity limit of approximately 1000 RNA molecules of RIC1 ( without the optimization of conditions). Similar analysis of the RIC2 sequences was performed and found to be similar to RIC1. It is significant that the amplification and detection systems of the internal control work effectively under the conditions optimized for the selected objective.

As an alternative approach for Multiple detection using internal controls (IC), the SPR can be covered homogeneously with a mixture of different capture nucleic acid sequences in a single area completely SPR. For example, two capture sequences can be combined in one zone, one specific for an objective test sequence and one specific for an internal control sequence. The target ampUcons, if present, and the internal control amplicons hybridize simultaneously to the SPR. In the presence of the identified test nucleic acid sequences specific for the target test nucleic acid sequence. After washing, a first signal reading is made so that the presence or absence of identification in the SPR is determined to confirm the presence of the test target. A second hybridization (sequential hybridization) is then made to the SPR using a specific detection identification test for internal control. The SPR is washed to remove the excess detection test without binding and the second identification is measured to indicate the presence or absence of internal control. If the first signal is negative, a positive signal from the second IC reading confirms the functionality of the amplification / detection system. If the first signal is positive, this is sufficient to confirm the functionality of the amplification / detection system and the second signal is not material (positive result). If the first and second identification are the same, an additive signal will result from the first positive reading and the second positive reading from IC. If the first signal is negative and the second IC signal is also negative, then the amplification / detection function failed, which could be due to, for example, sample interference or mechanical failure. In this case, the results of the test are reported invalid (false negative) and it is recommended to retest if the labels are different then no step of hybridization or sequential detection would be necessary.

There is a great interest in the use of internal controls, the fundamental national being that "... if the sample does not support the amplification of the internal control, it is not likely to support the amplification of the internal control of the target nucleic acid sequence. . " (Document NCCLS MM3-A, Molecular Diagnostic Methods for Infectious Diseases, Approved Guide, page 55, March 1995).

Using a sequential hybridization approach, with multiple detection tests, it has been possible to design protocols that allow for the discrete detection of the first reading signal (ie, the pure CT signal) and a second reading of the additional "mixed" signal ( that is, additional to CT and IC signals, see Table 7 A below). This protocol will not need to make strips. For example, Table 7A shows when the different mixtures of synthetic CT and IC targets are first captured with homogenously covered SPRs and hybridized first with the CT detector test. After the first reading, hybridization is performed with an IC detector test, followed by a second reading (with the same substrate).

This type of protocol can also be used for a combined internal GC / CT control experiment, if a test approach is allowed (no discrimination between GC and / or CT positives during the first reading). The specific signals of GC and CT have to be solved when carrying out specific CT and GC experiments in positive test samples (from 5% to 10% of cases, depending on the prevalence) the SPR would be covered homogeneously with 3 tests of capture (internal control of CT / GC). As an alternative, the IC could share a capture test with either CT or GC.

TABLE 7A Detection of Homogeneously Covered Multilevel SPR In this way, the internal control sequences described above are useful for the application with the VEDAS apparatus with the SPR covered and the use of the multiple system to give a combined experiment of nucleic acid detection and monitoring control for a successful reaction.

EXAMPLE 7 Internal Control Sequence The refinement of the internal control sequences generated in a random manner will allow the optimization of said internal control sequences for specific test systems. Following the methods described above, the internal control nucleic acid sequences have been designed and engineered for use in various amplification and detection systems that include an internal control for a Chlamydia trachomatis (CT) assay identified as CRIC-2; for Neisseria gonorrhoeae (NG) identified as SIRG; for Mycobacterium tuberculosis (MT) identified as MIRC. An internal control was generated for HIV assays identified as HRIC, where both the sequence of capture probes and the sequence of reporter probes were derived from the random sequence. The sequence of the internal control, and the corresponding target sequence are illustrated in Figure 30. In each of these internal control sequences, the Random Sequence Probe # 1082 can be used as the reporter probe, when properly conjugated with a reporter molecule as described above. In internal HIV control, a Random Sequence Oligonucleotide Capture Sequence # 1081 was designed for use in the capture control sequence, for improved quantification of termination between the target ampUcons and the IC amplicons by a capture test common.

LIST OF SEQUENCES (1) General Information: (i) Applicant: Luigi, Catanzrariti Kluttz, Biyan W Vera-García, Marcela Burg, J.L. Moe, James G. McKinley, Geoff A. (ii) Title of the Invention: ACID EXPERIMENTS IMPROVED NUCLEICO (iii) Number of Sequences: 18 (iv) Correspondence Address: (A) Recipient: McDonnell Boehnen Hulbert &; BerghofF (B) Street: 300 S. Wacker drive, office 3200 (C) City: Chicago (D) State: Illinois (E) Country: United States of America (F) Zip Code: 60606 (v) Way to read on computer : (A) Media Type: Flexible Disk (B) Computer: PC compatible with D3M (C) Operating System: PC-DOS / MS-DOS (D) Computing Program: Patentln Version # 1.0, Version # 1.30 (vi) Current Application Data: (A) Application Number: (B) Filing date: (C) Classification: (vii) Previous Application Information: (A) Application number: US 08/850, 171 (B) Date of submission: May 2, 1997 (C) Reference number / certificate: 97, 195 (ix) Telecommunications information: (A) Telephone number: 312-913-0001 (B) Fax number: 312-913-0002 (2) Information for sequence identification number: 1: (i) Sequence characteristics: (A) Length: 87 basic pairs (B) Type: nucleic acid (C) Chains: both (D) Topology: unknown (ii) ) Type of molecule: other nucleic acid (A) Description: / desc = "random internal control 1" (ix) Feature: (A) Name / Password: misc feature (B) Position: 4..24 (D) Other Information: / note = "RAN21 AMVE-probe, amino link at 5 'end" (ix) Feature (A) Name / Password: misc feature (B) Position: 25..45 (D) Other information: / note = "RAN33 AKP - I tried, amino link at 'end "(xi) Sequence description: Sequence identification number: 1: GGGAGCGAAT GTTAGGGCAC ACTCATGGGT GAGCAAGTCT TTCTGTAAGG GCTGATGTCA GGCGTATTGA CAAGCATGAC GACCAGA 87 (2) Sequence description: sequence identification number: 2: (i) Sequence characteristics: (A) Length: 48 basic pairs (B) Type: nucleic acid (C) Chains: single (D) Topology: linear (ii) Type of molecule: other nucleic acid (A) Description: / desc = "random internal control 1 detection oUgo '(xi) Sequence description: Sequence identification number: 2: CAATACGCCT GACATCAGCC CTTACAGAAA GACTTGCTCA CCCATGAG 48 (2) Sequence description: sequence identification number: 3: (i) Sequence characteristics: (A) Length: 56 basic pairs (B) Type: nucleic acid (C) Chains: single (D) Topology: linear (ii) Type of molecule: other nucleic acid (A) Description: / desc = "RIC1 top oligo" (ix) Characteristic: (A) Name / Password: misc feature (B) Position: 3..22 (D) Other information: / note = "T3 Promoter" (xi) Sequence description: Sequence identification number. 3 : GCAATTAACC CTCACTAAAG GGAGCGAATG TTAGGGCACA TCATGGGTGA GCAGTC 56 (2) Sequence description: sequence identification number: 4: (i) Sequence characteristics: (A) Length: 64 basic pairs (B) Type: nucleic acid (C) Chains: single (D) Topology: linear (ü) Type of molecule: other nucleic acid (A) Description: / desc = "RIC1 lower oUgo" (xi) Sequence description: Sequence identification number: 4: TCTGGTCGTC ATGCTTGTCA ATACGCCTGA CATCAGCCCT TACAGAAAGA CTTGCTCACC CATG 64 (2) Sequence description: sequence identification number: 5: (i) Sequence characteristics: (A) Length: 48 basic pairs (B) Type: nucleic acid (C) Chains: single (D) Topology: linear (ii) Type of molecule: other nucleic acid (A) Description: / desc = "promoter T7 / RAN19 primer" (ix) Feature: (A) Name / Password: misc feature (B) Position: 28..48 (D) Other information: / note = "RAN 19 primer" (xi) Sequence description: Sequence identification number: 5: AATTTAATAC GACTCACTAT AGGGAGATCT GGTCGTCATG CTTGTCAA 48 (2) Sequence description: sequence identification number: 6: (i) Sequence characteristics: (A) Length: 96 basic pairs (B) Type: nucleic acid (C) Chains: both (D) Topology: unknown (ii) Type of molecule: other nucleic acid (A) Description: / desc = "Random internal control 2" (ix) Feature: (A) Name / Password: misc feature (B) Position: 1.96 (D) Other information: / note = "RIC2 objective" (ix) Characteristic: (A) Name / Password: misc feature (B) Position: 1..26 (D) Other information: / note = "RAN51 TMA primer" (ix) Feature: (A) Name / Password: misc feature (B) Position: 28..48 (D) Other information: / note = "RAN27 AMVE-probe, amino link at end 5" (ix) Feature: (A) Name / Key: misc feature (B) Position: 49..69 (D) Other information: / note = "RAN32 AKP probe, amino link at the 5 'end" (xi) Sequence description: Sequence identification number: 6 : CAGTAGAGGT AGGGGCTGCT AGGAGTATAA CAGAAGCCAG TGTACGGAAC GACTCAGCAC GGCGAATACT TTGCTACCAG ACCTAGAGGA GTGCGT 96 (2) Sequence description: sequence identification number: 7: (i) Sequence characteristics: (A) Length: 47 basic pairs (B) Type: nucleic acid (C) Chains: single (D) Topology: linear (ii) Type of molecule: other nucleic acid (A) Description: / desc = "RIC2 oligo detection)" (xi) Sequence description: Sequence identification number: 7: AAGTATTCGC CGTGCTGAGT CGTTCCGTAC ACTGGCTTCT GTTATAC 47 (2) Sequence description: sequence identification number: 8: (i) Sequence characteristics: (A) Length: 67 basic pairs (B) Type: nucleic acid (C) Chains: single (D) Topology: linear (ii) Type of molecule: other nucleic acid (A) Description: / desc = "RIC2 oligo superior" (ix) Characteristic: (A) Name / Password: misc feature (B) Position: 3..228 (D) Other information: / note = "T3 promoter" (xi) Sequence description: Sequence identification number: 8: GCAATTAACCA CTCACTAAAG GGCAGTAGAG GTAGGGGCTG CTAGGAGTAT AACAGAAGCC AGTGTAC 67 (2) Sequence description: sequence identification number: 9: (i) Sequence characteristics: (A) Length: 66 basic pairs (B) Type: nucleic acid (C) Chains: single (D) Topology: linear (ii) Type of molecule: other nucleic acid (A) Description: / desc = "RIC 2 lower oligo" (xi) Sequence description: Sequence identification number: 9: ACGCACTCCT CTAGGTCTGG TAGCAAAGTA TTCGCCGTGC TGAGTCGTTC CGTACACTGG CTTCTG 66 (2) Sequence description: sequence identification number: 10: (i) Sequence characteristics: (A) Length: 52 basic pairs (B) Type: nucleic acid (C) Chains: single (D) Topology: üneal (ii) Type of molecule: other nucleic acid (A) Description: / desc = "promoter T7 / RAN39 primer" (ix) Characteristic: (A) Name / Password: misc feature (B) Position: 28..52 (D ) Other information: / note = "RAN39 first" (xi) Sequence description: Sequence identification number: 10: AATTTAATAC GACTCACTAT AGGGAGAACG CACTCCTCTA GGTCTGGTAG CA 52 (2) Sequence description: sequence identification number: 11: (i) Sequence characteristics: (A) Length: 111 basic pairs (B) Type: nucleic acid (C) Chains: both (D) Topology: unknown (ii) Type of molecule: other nucleic acid (A) Description: / desc = "CT internal control objective" (xi) Sequence description: Sequence identification number: 11: CGGAGUAAGU UAAGCACGCG GACGAUUGGA GAGUCCGUA GAGCGAUGAG AACGGUUAGA AGGCAAAUCC GCUAACAUAA GAUCAGGUCG CGAUCAAGGG GAAUCUUCGG G 111 (2) Sequence description: sequence identification number: 12: (i) Sequence characteristics: (A) Length: 111 basic pairs (B) Type: nucleic acid (C) Chains: BOTH (D) Topology: unknown (ii) Type of molecule: other nucleic acid (A) Description: / desc = "Internal CT control" (ix) Characteristic: (A) Name / Password: misc feature (B) Position: 35..54 (D) Other information: / note = "Random sequence probe 2 (reporter)" (xi) Sequence description: Sequence identification number: 12: CGGAGUAAGA UAAGCACGCG GACGAUUGGA AGAAUGGGUG AGCAAGUCUU UCUGGUUAGU AGGCAAAUCC GCUAACAUAA , GAUCAGGUCG CGAUCAAGGG GAAUCUUCGG G 111 (2) Sequence description: sequence identification number: 13: (i) Sequence characteristics: (A) Length: 110 basic pairs (B) Type: nucleic acid (C) Chains: both (D) Topology: unknown (ii) Type of molecule: other nucleic acid (A) Description: / desc = "target of internal control NG" (xi) Sequence description: Sequence identification number: 13: GGCGAGUGGC GAACGGGUGA GUAACAUAUC GGAACGUACC GGGUAGCGGG GGAUAACUGA UCGAAAGAUC AGCUAAUACC GCAUACGUCU UGAGAGGGAA AGCAGGGGAC 110 (2) Sequence description: sequence identification number: 14: (i) Sequence characteristics: (A) Length: 110 basic pairs (B) Type: nucleic acid (C) Chains: both (D) Topology: unknown (ii) Type of molecule: other nucleic acid (A) Description: / desc = "internal control NG" (ix) Characteristic: (A) Name / Password: miscjfeature (B) Position: 29..49 (D) Other information : / note = "Random sequence probe Reporter" "(xi) Sequence description: Sequence identification number: 14: GGCGAGUGGC GAACGGGUGA GUAACAUAAU GGGUGAGCAA GUCUUUCUGG GGAUAACUGA UCGAAAGAUC AGCUAAUACC GCAUACGUCU UGAGAGGGAA AGCAGGGGAC 110 (2) Sequence description: sequence identification number: 15: (i) Sequence characteristics: (A) Length: 125 base pairs (B) Type: nucleic acid (C) Chains: double (D) Topology: Linear unknown (ii) Type of molecule: other nucleic acid (A) Description: / desc = "internal control objective MT" (xi) Sequence description: Sequence identification number: 15: GGGAUAAGCC UGGGAAACUG GGUCUAAUAC CGGAUAGGAC CACGGGAUGC AUGUCUUGUG GUGGAAAGCG CUUUAGCGGU GUGGGAUGAC CCCGCGGCCU AUCAGCUUGU UGGUGGGGUG ACGGC (2) Sequence description: identification number sequence: 16: (i) Sequence characteristics: (A) Length: 96 basic pairs (B) Type: nucleic acid (C) Chains: double (D) Topology: unknown (ii) Type of molecule: other nucleic acid (A) Description: / desc = "internal control MT" (ix) Characteristic: (A) Name / Password: misc feature (B) Position: 54..74 (D) Other information: / note = "Random sequence probe (reporter) "(xi) Sequence description: Sequence identification number: 16: GGGAUAAGCC UGGGAAACUG GGUCUAAUAC CGGAUAGGAC CACGGGAUGC AUGAUGGGUG AGCAAGUCUU UCUGAGCTTG TTGGTGGGGT GACGGC 96 (2) Sequence description: sequence identification number: 17: (i) Sequence characteristics: (A) Length: 109 basic pairs (B) Type: nucleic acid (C) Chains: double (D) Topology: unknown (ii) Type of molecule: other nucleic acid (A) Description, / desc = "internal HIV target control" (xi) Sequence description: Sequence identification number: 17: ACAGCAUACA AAUGGCAGUA UUCAUCCACA AUUUUAAAAG AAAAGGGGGG AUUGGGGGGU ACAGUGCAGG GGAAAGAAUA GUAGACAUAA UAGCAACAGA CAUACAAAC 109 (2) Sequence description: sequence identification number: 18: (i) Sequence characteristics: (A) Length: 90 basic pairs (B) Type: nucleic acid (C) Chains: double (D) Topology: unknown (ü) Type of molecule: other nucleic acid (A) Description: / desc = "HIV internal control" (ix) Characteristic: (A) Name / Password: misc feature (B) Position: 20..40 (D) Other information: / note = "Random sequence probe 1082 (Reporter) "(ix) Feature: (A) Name / CLave: misc_feature (B) Position: 41..61 (C) Other information: / note =" Random sequence probe 1081 (Capture) "(xi) Sequence description: Sequence identification number: 18: ACAGCAUACA AAUGGCAGUA UGGGUGAGCA AGUCUUUCUG UAAGGGCUGA UGUCAGGCGU AGUAGACAUA AUAGCAACAG ACAUACAAAC 90

Claims (29)

1. CLAIMS: 1. A unified regulator suitable for the denaturation of double-stranded nucleic acids, and for the softening of nucleic acids, which is also capable of sustaining the enzymatic activity of the nucleic acid polymerization enzyme, said regulator comprises dilution regulator of sample, regulator of reconstitution of the amplification and regulator of dilution of enzymes.
2. 2. A unified regulator as in Claim 1, wherein the components of said regulator are encapsulated.
3. 3. A unified regulator as in claim 1, wherein the components of said regulator are lyophilized.
4. 4. A method for generating an internal nucleic acid of universal positive amplification control, said method comprises: generating random nucleic acid sequences of at least a length of 10 nucleotides, testing said random nucleic acid sequences and selecting them for their specific functionality, combining in group over a said functionally selected nucleic acid sequence, and testing the combined nucleic acid sequence and optionally selecting it against the formation of internal filament nucleic acid dimers, or the formation of hair tip structures.
5. 5. An internal control nucleic acid sequence of universal positive amplification, said nucleic acid sequence comprises a nucleic acid sequence that is generated randomly.
6. 6. An internal universal positive amplification control nucleic acid sequence as in Claim 5, further comprising a nucleic acid sequence selected from the group comprising a specific first control nucleic acid sequence or capture test nucleic acid sequence. specific.
7. 7. An internal nucleic acid sequence of universal positive amplification control as in Claim 5 having the nucleic acid sequence chosen from the group consisting of RIC1, RIC2, CRIC-2, SICGR, MRIC, and HRIC.
8. 8. A nucleic acid specific for an internal nucleic acid of universal positive amplification control of Claim 7 having the nucleic acid sequence chosen from the group consisting of RAN33, RAN19, RAN16, RAN51, RAN27, RAN32, RAN39, test # 1081 and test # 1082.
9. 9. A method for detecting the presence or absence of a single-stranded or double-stranded nucleic acid in a sample, by isothermal amplification of said first nucleic acid in a double-chamber reaction vessel, wherein said reaction vessel with The double chamber comprises two reaction chambers, a first and a second, which can be placed in fluid communication with each other, by means of which said fluid communication can be interrupted as a control, said method comprises: a) combining said first reaction chamber: a sample, said sample potentially containing said first nucleic acid, reaction regulator, a mixture of Ubrs nucleotides, a first and second primer of specific oligonucleotides and placing said reaction vessel in an automated apparatus such as that; b) the automated apparatus heats the first reaction chamber at a sufficient temperature, and for a time sufficient to make any double-stranded nucleic acid in the sample by testing sufficient single-stranded nucleic acid so that a hybridization product can be formed , said hybridization product comprises said nucleic acid and at least one of said first and second first UONucleotide; c) the automated apparatus then cools the first chamber to a temperature sufficient to form a hybridization product, if said first nucleic acid is present; d) the automated apparatus then transfers the reaction mixture via said controllable communication, so that the reaction mixture is contacted with the nucleic acid polymerization enzyme; e) the automated apparatus maintains the temperature of the second reaction chamber at a temperature sufficient to allow amplification mediated by the first specific oligonucleotide of said first nucleic acid, if present; f) the automated apparatus then contacts any amplicon and said first nucleic acid in the second reaction chamber with a capture nucleic acid specific for said nucleic acid so that a capture probe hybridization complex specifically linked to the nucleic acid can be formed; g) the automated apparatus optionally washes the complex hybridization mixture so that the nonspecifically linked nucleic acid is washed from the capture probe complex with specifically linked nucleic acid; h) the automated apparatus contacts the capture probe complex with nucleic acid specifically linked to a labeled nucleic acid probe specific for said first nucleic acid so that a probe-labeled capture probe-specifically linked nucleic acid probe complex can be formed; i) the automated apparatus optionally washes the labeled probe-capture probe-specifically linked nucleic acid complex so that the labeled and linked probe nucleic acid is not specifically washed from the probe-labeled capture probe-specifically linked nucleic acid probe complex; j) and the automated apparatus detects the presence or absence of said general signal and optionally displays a value for the signal, and optionally registers a value for the signal, wherein the automated device contacts the labeled probe-probe complex. capture- nucleic acid specifically bound to a solution wherein a detectable signal of the sample is proportional to the amount of said first nucleic acid in the sample; wherein each of the steps h, i, e j can be carried out sequentially or concurrently.
10. 10. A method as in Claim 9 wherein the nucleic acid ampuficating enzyme is placed in said second reaction chamber as a single test dose quantity in a lyophilized pill, and said reaction chamber is sealed before being used.
11. 11. A method as in claim 9 wherein the nucleic acid amplification enzyme is a thermostable enzyme.
12. 12. A method as in Claim 9 wherein the nucleic acid amplification enzyme is placed in the first reaction chamber.
13. 13. A method as in Claim 9 which also incorporates internal control molecules.
14. 14. A method as in Claim 9 further comprising target nucleic acid sequence of amplification and detection.
15. 15. A method as in Claim 9 which further includes detection of first control sequences.
16. 16. A method for the automated detection specific to one or more viral nucleic acids or microorganisms in a sample, said method comprises: a) Using at least one microorganism in said sample, if present, to release target nucleic acid; b) amplifying said nucleic acid to form amplicons; c) contacting said sample with a solid phase receptacle covered with a capture nucleic acid, wherein said capture nucleic acid can form a hybridization complex with said amplicons; d) allowing said hybridization complex to be formed; e) contacting said hybridization complex with a detection nucleic acid, wherein said detection nucleic acid can form a specific hybridization detection complex with said target nucleic acid, and is conjugated with a means to generate a detectable signal from from the group consisting of enzyme, chromophore, chemiluminescent compound, radioisotope and fluorophore; f) allowing said detection complex to be formed, and generating said detectable signal; g) detecting said detectable signal if said amplicon is present in said sample; h) wherein, optionally, between each step, said hybridization complex can be washed to remove excess bound nucleic acid non-specifically.
17. 17. A method as in claim 16, wherein said microorganism or virus is selected from the group consisting of Mycrobacterium tuberculosis (m.tb), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Human Immunodeficiency Virus (HIV).
18. 18. An apparatus for the automated detection of a first target nucleic acid and a second target nucleic acid, said apparatus comprises a solid phase receptacle, wherein said receptacle is covered with a first capture nucleic acid that can form a specific hybridization complex with said first nucleic acid and a second capture nucleic acid which can form a specific hybridization complex with said second nucleic acid.
19. 19. An apparatus as in Claim 18 wherein said first capture nucleic acid and said second capture nucleic acid are covered in a solid phase receptacle in two distinct zones.
20. 20. A device as in Claim 18 wherein said first nucleic acid and said second nucleic acid are covered in said solid phase receptacle in a single zone.
21. 21. An apparatus as in Claim 18, modified for the detection of more than two nucleic acids, further comprising additional capture nucleic acids which can each form a specific hybridization complex with additional target nucleic acid other than said first and second nucleic acids in said sample.
22. 22. A method for the automated detection of the presence or absence of a first target nucleic acid and a second target nucleic acid in a sample, said method comprising: a) contacting said sample with a solid phase receptacle, wherein said phase receptacle solid is covered with a first capture nucleic acid, which can form a hybridization complex with said first nucleic acid, and a second capture nucleic acid which can form a specific hybridization complex with said second nucleic acid; b) allowing said specific hybridization complex to be formed if said nucleic acid is present; c) contacting said solid phase receptacle hybridization complex with a first detection nucleic acid, wherein said detecting nucleic acid can form a specific hybridization detection complex with said first nucleic acid, and is conjugated with a means for generating a detectable signal from the group consisting of enzyme, chromophore, chemiluminescent compound, radioisotope and fluorophore; d) allowing said detection complex to be formed, and then generating said detectable signal; e) detecting said detectable signal if said first nucleic acid is in said sample; f) contacting said hybridization complex in a single-phase receptacle with a second detection nucleic acid, wherein said detection nucleic acid can form a specific hybridization detection complex with said second nucleic acid, and is conjugated with a means for generate a detectable signal from the group consisting of enzyme, chromophore, chemiluminescent compound, radioisotope and fluorophore; g) allowing the specific detection complex to be formed, and then generating said detectable signal; h) detecting said signal if said second nucleic acid is in said sample; i) and wherein optionally, between the steps, said hybridization complex can be washed to remove excess non-specific binding nucleic acid; j) wherein the absence of a detectable signal correlates with the absence of said nucleic acid in said sample.
23. 23. A method as in Claim 22 that also incorporates internal control molecules.
24. 24. A method as in Claim 22 which further includes ampUfication and detection of a target nucleic acid sequence.
25. 25. A method as in Claim 22 which further includes detection of first control sequences.
26. 26. A method as in Claim 22, wherein one or more of said steps c and f are carried out concisely, said steps d and g are carried out concurrently and the steps e and h are carried out concurrently.
27. 27. A method as in Claim 22, wherein said means for generating a detectable signal is the alkaline phosphate enzyme, and the generation of said detectable signal comprises contacting said media with a substrate of phosphate umbelliferyl methyl.
28. 28. A method as in Claim 22, modified for the detection of more than two nucleic acids, wherein said solid phase receptacle is further covered with additional specific capture nucleic acid, wherein said specific capture nucleic acid can form a hybridization complex. specific with additional target nucleic acid other than said first and second nucleic acids in said sample, and wherein said method contacts the additional hybridization capture complex with additional specific detection nucleic acid, wherein said additional detection nucleic acid can form a specific hybridization detection complex with said additional nucleic acid, and conjugates with a means for detecting a detectable signal chosen from the group consisting of enzymes, chromophore, chemiluminescent compound, radioisotope and fluorophore.
29. 29. A method as in Claim 28 wherein said first nucleic acid is specific for Chlamydia trachomatis (CT), and said second nucleic acid is specific for Neisseria gonorrhoeae (NG). EXTRACT OF THE INVENTION The present invention relates to the detection of specific nucleic acid sequences, whether by a process of amplification of specific nucleic acid sequences or not. More particularly, the invention provides improcompositions and methods to reduce the risk of contamination in the handling of reagents, internal amplification controls and the use of automated devices for the automated detection of one or more amplified nucleic acid sequences.