PT103767A - NUCLEIC PEPTIDE ACID PROBE (NAP), CARTON AND METHOD FOR SPECIFIC DETECTION OF HELICOBACTER PYLORI AND THEIR APPLICATIONS. - Google Patents

Abstract

The present invention relates to the design of a nucleic acid peptide (PNA) probe for the specific detection of Helicobacter pylori. THIS PROBLEM IS APPLIED TO A PROCESS BASED ON MOLECULAR BIOLOGY TECHNIQUES, ESPECIALLY IN FLUORESCENT IN SITU HYBRIDIZATION (FISH) APPLICABLE IN THE DIAGNOSIS AND QUANTIFICATION OF H. PYLORI IN DIFFERENT TYPES OF SAMPLES, INCLUDING BIOPLASMS, BLOOD, AIR, FOOD, WATER AND OTHER ENVIRONMENTAL SAMPLES. DUE TO THE PHYSICAL-CHEMICAL CHARACTERISTICS INHERENT IN ITS STRUCTURE, THESE PROBES ALLOW A FASTER AND SENSITIVE ANALYSIS THAN USING A DNA PROBE. OTHER OF THE ASPECTS OF THE PRESENT INVENTION IS RELATED TO THE DEVELOPMENT OF A BRIEF, BASED ON THE PNA PROBE AND THE REFERRED PROCESS, WHICH HAVE AS OBJECTIVE TO DETECT, IDENTIFY AND / OR QUANTIFY H. PYLORI IN BIOLOGICAL, CLINICAL AND / OR ENVIRONMENTAL SAMPLES .

Description

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DESCRIPTION " NUCLEIC PEPTIDE ACID PROBE (PNA), CASE AND METHOD FOR SPECIFIC DETECTION OF HELICOBACTER PYLORI AND THEIR APPLICATIONS "

Field of the Invention

This invention relates to a method of detecting clinically relevant microorganisms, having developed a nucleic acid peptide (PNA) probe for specific detection of Helicobacter pylori. Another aspect of the present invention relates to the application of said probe and said method of detection to a kit for the detection, identification and / or quantification of H. pylori in biological samples thus having application in the pharmaceutical industry.

BACKGROUND OF THE INVENTION Helicobacter pylori is an important pathogenic microorganism that causes chronic gastritis and is associated with the development of peptic ulcers, atrophic gastritis and gastric cancer. H. pylori infection can be urease diagnosed either by invasive techniques or by non-invasive techniques. The invasive ones require the accomplishment of a superior endoscopy, and are usually accompanied by the collection of gastric biopsies. Noninvasive tests are the most common because they are acceptably clinically effective, cheaper and more convenient for the patient. The most commonly used tests / kits are those which detect a substantially specific H. pylori enzyme, α (US2003068656 and EP0920531). 2

However, in addition to not being totally specific or sensitive, these tests provide no further information on either the localization of the bacterium in the stomach or on the histopathological lesion underlying the presence of the bacterium. Thus, there are situations where it is convenient to perform invasive techniques to perform a more complete diagnosis.

The biopsies obtained by upper endoscopy can be analyzed in order to obtain information on the presence of H. pylori either by culture methods or by molecular methods.

More recently, molecular methods, including RAPD (" random amplification of polymorphic DNA "), PCR (" Polymerase Chain Reaction " - polymerase chain reaction) (JP9065899 and W09109049) and fluorescent (FISH) for the identification of different bacteria have been imposed on the most time-consuming methods of cultivation. FISH is probably the most common method for the detection and localization of a microorganism or groups of microorganisms in a sample. The technique detects nucleic acid sequences through a probe attached to a fluorochrome that specifically hybridizes with the complement of its sequence within intact cells.

Until very recently, FISH methods were based on the use of conventional DNA oligonucleotides of about 20 bases. More recently, nucleic acid peptide (PNA) probes for bacterial detection have been developed and optimized. 3

PNA molecules are mimics of DNA, in which the negatively charged sugar-phosphate structure is replaced by an achiral and electrically neutral formed by repeated N- (2-aminoethyl) glycine units. The PNA is able to hybridize to complementary nucleic acid sequences, following Watson-Crick's rules (US5539082). When compared to the traditional DNA probes, and because of its electrically uncharged structure, PNA has superior hybridization characteristics, exhibiting faster and stronger binding with the target sequence and a lack of electrostatic repulsion. As such, the optimal length of the PNA probe is around 15 bases.

It has been shown that this technique is rapid, sensitive and very specific for microbial detection and may be an important alternative or complement to the culture media since they produce faster results and provide additional information on the location of the bacteria in gastric samples. Since adaptation of non-invasive methods is impossible for the detection of bacteria in other environments, this method is also more comprehensive in its application. Various probes have been designed and disclosed through patent documents for microorganisms of clinical interest such as Mycoplasma, Acholeplasma, or Ureaplasma (US2006252081; WO2006122049); Staphylococcus species other than Staphylococcus aureus (W02005054516; EP1709198); Enterococcus faecalis and / or other Enterococcus species (W02005018423; US2005153307); Pseudomonas (US2004259132) Escherichia coli, Klebsiella pneumoniae, 4

Klebsiella oxytoca, Streptococcus agalactiae, fungus, and Acinetobacter (W02006130872). For H. pylori only a PNA probe was disclosed which, although very specific, was not very sensitive (Table 1), possibly due to the wide genetic variety observed for H. pylori.

Table 1 - Specificity and theoretical sensitivity of the existing PNA probes for the detection of H. pylori.

The specificity was calculated as NHp / (TN) x 100, where NHp represents the number of strains of H. pylori and TN o. total number of bacterial strains detected by the probe. b Sensitivity was calculated as NHp / (TNHp) x100, where NHp represents the number of H. pylori strains detected by the probe and TNHp the total number of H. pylori strains in the databases. The process of the present invention, with the application of the PNA probe, does not involve the use of reagents or enzymes for the formation of pores in the cell membranes prior to the hybridization step and consequently the PNA probe can be applied directly immediately following the preparation of the sample on a slide.

However, some of the more common compounds used in hybridizations are still required, and as such, the probe is usually included in kits that facilitate the FISH process by the users. Cases using PNA probes for the electrophoretic separation of DNA samples are already disclosed (US2005053944, W09712995, EP1477572).

Summary of the Invention The present invention relates to a nucleic acid peptide (PNA) probe for the specific detection of Helicobacter pylori. PNA probes have physicochemical characteristics inherent to their structure, which when applied to a method based on FISH technology, allow a faster and more sensitive analysis than using a DNA probe. Another aspect of the present invention relates to the development of a kit, based on the application of this probe to the above methodology, which allows the detection of H. pylori in biological samples in a simple and rapid manner.

General Description of the Invention

PNA probes have physicochemical characteristics inherent in their structure that allow a faster and more sensitive analysis than using a DNA probe, particularly when applied to FISH. As in FISH DNA, the functional part of the probe is the nucleobase sequence designed so that it can hybridize to an existing sequence in the rRNA of the target cells. Successful hybridization allows then, for example by fluorescence microscopy, to gauge the presence / absence and concentration of the microorganism in question. To achieve this objective, the probe will have to take into account the specific requirements for each assay, particularly at the length and sequence level, so that a stable complex with the target is formed under suitable hybridization conditions. The addition of the signal emitting fluorescence molecule, such as fluorescein or the Alexa fluorochromes family, is generally achieved by the use of a chemical compound called 8-amino-3,6-dioxaoctanate which bridges the two The hybridization process consists of three steps: fixation, hybridization and washing.

In this case, for ten advisable, the use of paraformaldehyde and ethanol each minute to fix the sample is but not required. between 30 and 120 sequence in a 5'-solution

During the hybridization, which usually takes minutes, the probe of this invention with GAGACTAAGCCCTCC-3 'is mixed called the hybridization solution.

A b c d e c e c e b c e c e c e c e c e c e c e c e c e c e c e c e c e c e c e c e c e c e c c e c c e c c e c e c c e c c e c c e c c e c c e c c e c c e c c e c c e c c e n (ie ionic strength), temperature, detergent concentration, concentration of the probe. The optimum process for obtaining a particular probe / target sequence complex is often found by the well-known technique of setting several of the factors mentioned above and then determining the The same factors can be modeled for the hybridization of PNA with DNA, with the difference that this hybridization is relatively independent of the ionic strength of the solution. For this probe, with a solution of 10% (wt. / vol) dextran sulfate, 10 mM NaCl, 30% (vol / vol) formamide, 0.1% (wt / vol) sodium pyrophosphate, 0.2% (w / v) polyvinylpyr rolledone, 0.2% (wt / vol) Ficol, 5 mM di-sodium EDTA, 0.1% (vol / vol) Triton X-100, 50 mM Tris-7 HGl (pH 7.5) and 200 nM of the PNA probe is obtained hybridization with success at 59 ° C.

Finally, during washing, the not fully hybridized probe is withdrawn from the sample to ensure the specificity of the process. In general, the closer the other sequences present in the target sequence sample, the more important this step is. For this case, immersion of the sample in a solution of 5 mM Tris Base, 15 mM NaCl and 1% (vol / vol) Triton X (pH 10) at the hybridization temperature for 30 minutes is one of the suitable ones to guarantee the desired specificity.

After this process, the sample can then be observed under a fluorescence microscope with suitable filters for the detection of the fluorochrome attached to the probe. For samples where the target sequence is not present, the microscope does not pick up any signal emission. The process is applicable to the diagnosis and quantification of H. pylori in various types of samples, including biopsies, blood, air, food, water and other environmental samples.

In addition, PNA probes can also be used in real-time PCR, a process in which the amount of product is continuously evaluated during the amplification process, thus allowing the quantification of genetic material in the initial sample.

In its simplest form, the PCR product is detected by linking dyes with the double strand of DNA. Since they are not specific, these dyes recognize any product formed in this process, which requires the DNA of interest to be purified prior to the amplification step. To solve this problem, DNA probes that recognize specific sequences have been developed, and as for FISH, also here the PNA molecules demonstrated additional advantages.

DETAILED DESCRIPTION OF THE INVENTION 1. Sample Preparation

Samples may come from biopsies, blood, air, food and water. In the case of biopsies, the samples are cut into strips of 3 to 5 Dm and placed on slides. In the event that E. pylori is in suspension, such as in water, air and blood samples, the samples are filtered by a black polycarbonate membrane or equivalent. The membranes are then placed on slides. For food samples, it is necessary to withdraw H. pylori from the food matrix using ultrasonication or a mechanical blasting method of flat blades in the sample, which is soaked in water or sterile buffer solution. From the moment H. pylori is in suspension, the technique applied is similar to that of water, air, and blood. Alternatively, samples where H. pylori is in suspension may be directly hybridized.

2. Design of PNA probes

For the identification of oligonucleotides that could be used as probes, 16S rRNA sequences from various databases were aligned. Since there were several 15-base sequences that were capable of detecting the majority of H. pylori strains, additional criteria were used for selection of the final sequence. These included the absence or moderate existence of complementary sequences in the probe sequence and also the detection of the fewest number of microorganisms possible. According to these criteria, the 5'-GAGACTAAGCCCTCC-3 'sequence was selected.

3. Hybridization of the PNA 3.1. Fixation

In order to prevent the loss of 16S rRNA during the hybridization process, the sample is exposed to a solution of 4% (wt / vol) paraformaldehyde and 50% (vol / vol) ethanol for ten minutes each. 3.2. Hybridization

During this step, a hybridization solution containing the probe is brought into contact with the sample. During this time, the probe penetrates the cell membranes and binds to 16S rRNA complementary sequences. Hybridization usually takes place between 30 and 120 minutes at 59øC and may be carried out for either H. pylori in suspension or adhered. In the first case, a hybridization solution is added in concentrated form so that the concentrations of the compounds in the final solution are as follows. In the second, the volume of the hybridization solution needed to completely cover the sample is applied directly thereto. During the hybridization period, it is essential that the hybridization solution does not evaporate. To this end, a coverslip is added above the hybridization solution and the environment in the incubation is kept moist through the insertion of moistened paper around the blade.

Hybridization solution 10

A solution of 10% (w / v) dextran sulfate, 10 mM NaCl, 30% (vol / vol) formamide, 0.1% (wt / vol) sodium pyrophosphate, 0.2% (wt / vol) polyvinylpyrrolidone, 0.2% (w / v) Ficol, 5 mM di-sodium EDTA, 0.1% (vol / vol) Triton X-100, 50 mM Tris-HCl (pH 7.5) and 200 nM of the PNA probe. 3.3. Cleaning

During this step, the unhybridized probe is removed from the sample by immersing it in a solution of 5 mM Tris Base, 15 mM NaCl and 1% (vol / vol) Triton X (pH 10) at the hybridization temperature for 30 minutes . 3.4. Obtaining results

The results are obtained by observation under a fluorescence microscope with filters suitable for detection of the probe-linked fluorochrome, with no signal being detected in situations where the target sequence is not present.

Lisbon, June 21, 2007

Claims (11)

A nucleic peptide acid probe for detecting Helicobacter pylori, characterized in that it is up to 86% identical to the sequence 5'-GAGACTAAGCCCTCC-3 'and has a length between 8 and 18 nucleobases. Use of the PNA probe according to the preceding claim, characterized in that it is applied in a methodology for the detection, identification and / or quantification of Helicobacter pylori in biological and / or environmental biological samples. A method of detecting, identifying and quantifying Helicobacter pylori in a sample, characterized in that use is made of a PNA probe according to the previous claims, and includes the following steps: PNA probe contact with the sample Hybridization of the probe of PNA with the target sequence of the microorganisms present in the sample Detection of the hybridization as being indicative of the presence, identity and / or amount of microorganisms in the sample. A method according to claim 3, characterized in that the sample in question is derived, preferably but not exclusively, from biopsies, blood, air, food and water. A method according to claim 3, characterized in that the step of hybridizing the PNA probe to the target sequence is carried out at 59 ° C for one and one half hours and washing is carried out at the same temperature for half an hour. A method according to claim 5, characterized in that the hybridization is fluorescent and performed in situ. A method according to claim 3, characterized in that the presence, identity and / or amount of Helicobacter pylori in the sample is detected through a conjugate, a radioisotope, an enzyme or a luminescent or fluorescent compound. Use of the method according to claims 3 to 7, characterized in that it is intended for the detection, identification and / or quantification of Helicobacter pylori in biological, clinical and / or environmental samples. quantification, described in the method of A kit for the detection, identification and of Helicobacter pylori in samples characterized by containing the probe claim 1 and based on claims 3 to 7. Use of the kit according to claim 9, characterized in that it is applied in a real-time PCR assay. Use of the kit according to claim 10, characterized in that it is applied to the detection, identification and quantification of Helicobacter pylori in clinical samples such as biopsies and / or environmental. Lisbon, June 21, 2007