PT106672A - PROOFS OF NUCLEIC PEPTIDE ACID, CASE AND METHOD FOR DETECTING GENUS OF GENDER LACTOBACILLUS SPP. AND / OR GARDNERELLA SPP. AND THEIR APPLICATIONS - Google Patents

Abstract

The present invention is directed to the design of two nucleoside peptides (PNAs) for detecting Lactobacillus bacteria and / or Gardnerella spp. These probes are applied to a process based on techniques of molecular biology, especially fluoride-in-situ hybridization (FISH) FOR THE DIAGNOSIS OF BACTERIAL VAGINOSIS OR THE DETECTION AND QUANTIFICATION OF THESE BACTERIAL GENES IN DIFFERENT TYPES OF SAMPLES, INCLUDING BLOOD, FOODS, BIOPSIES, FERTILIZERS, WATER AND OTHER CLINICAL, ENVIRONMENTAL SAMPLES OR FROM THE AGRICULTURAL OR FOOD INDUSTRY.

Description

NUCLEIC PEPTIDE ACID PROBES, CASE AND METHOD FOR DETECTING SPECIES OF THE GENUS LACTOBACILLUS SPP. AND / OR GARDNERELLA SPP. AND THEIR APPLICATIONS "

Field of the Invention

This invention is part of a process for the detection of clinically relevant microorganisms for the evaluation of the infectious process known as bacterial vaginosis, and two peptide nucleic acid probes (PNA) have been developed to detect Lactobacillus spp. and / or Gardnerella spp. in several types of samples.

Another aspect of the present invention relates to the application of such probes and said method of detection to a kit for the detection and quantification of Lactobacillus spp. and / or Gardnerella spp. in biological samples and, therefore, clinical application for the diagnosis of bacterial vaginosis.

Background of the Invention

Lactobacillus spp. and Gardnerella spp. constitute instrinsic bacteria of the vaginal mucosa. In normal cases, bacteria of the genus Lactobacillus spp. are found in high numbers whereas bacteria of the genus Gardnerella spp. or they are absent. Although planktonic bacteria of the genus Gardnerella spp. are present in the vaginal microflora of healthy women, these may eventually form biofilms thus occurring cases of bacterial vaginosis. The biofilm of Gardnerella spp. induces a change in phenotype expression and, consequently, a greater virulence. Simultaneously, the number of bacteria Lactobacillus spp. decreases dramatically in cases of bacterial vaginosis, favoring the microbial growth of pathogenic anaerobes in the vaginal mucosa. In this way, it is necessary to use the two nucleic acid peptide probes to evaluate the vaginal microflora. The diagnosis of bacterial vaginosis infection may be based on time-consuming methods which imply a clinical examination and a laboratory examination which usually involves performing a microbial culture of the patient's vaginal exudate. Clinical diagnosis is the most frequently used, although it is error-prone and extremely susceptible to physician interpretation. This examination consists of the identification of at least three positive results in four symptoms / signs characteristic of this vaginal infection, namely, pH level of vaginal discharge equal to or greater than 4.5, the presence of epithelial cells covered with gram negative bacteria, appearance typical discharge or vaginal discharge, and the release of a characteristic odor of nitrogenous products by the addition of a 10% potassium hydroxide solution. In turn, the microbial culture laboratory test consists of inoculating the patient's vaginal exudate in a medium enriched with 5-10% of blood, such as Human Blood Medium (HBA) or Brucella Blood Medium (BBA), for 48-72 hours at 37 ° C in an atmosphere of 5% carbon dioxide. Then, bacterial growth is usually quantified by the four growth zones in the Petri dish and observation is performed under the microscope after Gram staining. During the microscopic observation of Gram 2 staining, the criteria of Nugent et al., 1991, are applied, thus classifying the sample observed in one of the three possible diagnoses: normal vaginal microflora; intermediate vaginal microflora; and bacterial vaginosis. However, the lack of sensitivity / specificity of clinical examination and slowness in the analysis of microbial culture imply a complex procedure for the correct clinical diagnosis of bacterial vaginosis in patients.

Other laboratory assays employ methods based on PCR (" Polymerase Chain Reaction " - polymerase chain reaction) for the identification of bacterial strains in bacterial vaginosis. But this technique requires some care at the level of DNA contamination that, if not followed, can translate into a false positive or even a false negative. In addition, due to the presence of Gardnerella spp. in clinical samples of healthy women, although in a low number, thus prevents the application of this methodology as a clinical diagnostic procedure.

Recently, nucleic acid peptide (PNA) probes for bacterial detection have been developed and optimized. 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.

Although the PNA molecule lacks pentoses, specific hybridization occurs between the PNA sequences and the complementary nucleic acid sequences, by hydrogen bonds. This hybridization obeys Watson-Crick's rules. The electrically neutral nature of the PNA is responsible for the more robust hybridization characteristics of this molecule with respect to the high stability in the PNA / target sequence bindings (rRNA or double stranded DNA) when compared to the DNA probes traditionally used . Thus, because of their high affinity, the PNA probes have relatively short nucleotide sequences, preferably between 8 to 17 nucleotides. A DNA probe is usually at least 18 nucleotides, owing to its poor stability and lower melting temperature (Tm), also requiring additional processes for attachment and permeabilization with enzymes or other agents. PNA molecules also have a higher resistance to proteases and nucleases than the natural DNA molecule.

When coupled to a fluorochrome, the PNA probes can be detected by microscopy or flow cytometry by fluorescence in situ hybridization (FISH) technique. This technique produces faster results in clinical samples when compared to traditional culture methods, and their efficacy, speed, sensitivity and specificity have been proven. Its application is also very comprehensive and can be used in several areas of microbiology, including the detection of pathogenic microorganisms in human, food and environmental samples.

Some examples of probes already designed and published / patented for some microorganisms of clinical interest, such as probes developed for the detection of the genus Salmonella, Bacillus anthracis, Staphylococcus coagulase negative species, Papillomavirus 4 human, and the genus Candida, among others, demonstrate the increasing interest that this technique provides.

For bacterial vaginosis a FISH methodology has been published with DNA probes for detection in vaginal exudates of various strains involved in the infection (Fredicks et al., 2005). However, the probes used corresponded to larger DNA oligonucleotides and with different target sequences, although both probes had their target oligonucleotides in ribosomal RNA (16S). In this study, the probe (Eub-338-Cy5 5'-GCTGCCTCCCGTAGGAGT-Cy5-3 ') was used to identify the bacteria Lactobacillus spp. was non-specific and presented nineteen nucleotides, detecting the order Bacillales. In turn, the probe for Gardnerella vaginalis (G.vag-198-Cy3 5'-CCACTAAACACTTTCCCAACAAGA-Cy3-3 ') had twenty-five nucleotides. Both probes operated at a hybridization temperature (Tm) of 45 ° C, possibly leading to false positives. Finally, the fluorochromes used in the work corresponded to cyanine number 5 (Cy5) and number 3 (Cy3), which are lower fluorescence than the fluorochromes Alexa Fluor (Alexa Fluor 488 and 594) used in the probes presented here. Thus, this methodology revealed specificity and sensitivity lower than the method proposed here. Bacterial vaginosis is a common vaginal infection in women of reproductive age and may initially occur asymptomatically. The etiology of this infection remains unknown, however, bacterial vaginosis is associated with a change in vaginal microflora, characterized by the increasing substitution of the normal flora of Lactobacillus species by certain anaerobic bacteria such as Gardnerella vaginalis. In developed countries, the incidence rate of bacterial vaginosis (bacterial infection) is approximately double that of genital candidiasis (fungal infection) in symptomatic women and even more common than trichomoniasis (protozoal infection). Thus, a method of detection that allows the rapid visualization of the microorganisms responsible for a vaginal infection and the amount of bacteria present in the vaginal exudate, more properly the relationship between Lactobacillus spp. and Gardnerella spp. The present invention describes a reliable and feasible method of systematically identifying bacteria present in normal vaginal samples and in pathogenic cases of bacterial vaginosis. Therefore, it is a significant contribution to the rapid selection of appropriate treatment, sparing discomfort, risks, prolongation of treatments and costs to the patient.

On the other hand, hygiene and quality control in the food and agricultural industry plays an increasingly prominent role in our day. Foodstuffs, such as milk, yogurt and cheese, require an efficient and rapid method of detection and / or quantification for certain micro-organisms, eg Lactobacillus spp. . In fact, the direct application of the PNA probes to Lactobacillus spp. in food products, in particular milk, makes the present invention quite practical and dynamic in the food and agricultural industry. In this manner, the present invention facilitates the control process by avoiding time-consuming and costly treatments in the preparation of food products for their hygiene and quality analysis.

Summary of the Invention The present invention relates to nucleic acid peptide (PNA) probes or set of PNA probes for detecting and / or quantifying bacteria present in normal vaginal samples and involved in pathogenic cases of bacterial vaginosis. The probes recognize the 16S rRNA of the genus of each type of bacterial species in question or the genomic sequences corresponding to the rRNA mentioned. PNA probes have physicochemical characteristics inherent in their structure which, when applied to a method based on FISH technology, allow a faster, more robust and specific analysis than using a DNA probe.

One of the problems solved by the present invention is the detection and visualization of the ratio / ratio between the existing species of Lactobacillus spp. and Gardnerella spp. in vaginal samples. In addition to providing a reliable and expeditious method for the determination of the presence of Lactobacillus spp. and / or Gardnerella spp. in a biological sample, also allows the quantification of these species, thus allowing the classification of vaginal microflora (normal microflora, intermediate microflora or bacterial vaginosis) and initiate convenient treatment quickly and safely.

Another aspect of the present invention relates to the development of a kit based on the application of these probes to the fluorescence in situ hybridization (FISH) technique, which allows the detection of Lactobacillus spp. and / or Gardnerella spp. in biological samples, in a simple and rapid manner. The two probes may or may not be used simultaneously. The present invention describes a set of PNA probes which detect, i.e. identify and / or quantify the presence of Lactobacillus spp. and / or Gardnerella spp. These PNA probes comprise one of at least 80% nucleotide sequences identical to SEQ ID Nos. 1 to 6.

In a preferred embodiment the PNA probes described in the present invention detect the target sequence in the rRNA, the rDNA or in the complementary sequences of the Lactobacillus spp. and / or Gardnerella spp ..

In an even more preferred embodiment the present invention encompasses the following sequences: at least one of the nucleotide sequences at least 80% identical to SEQ ID No. 1 to 3 which detects Lactobacillus spp. ; at least one of the nucleotide sequences, at least 80% identical to SEQ ID No. 4 to 6 detecting Gardnerella spp.

In an even more preferred embodiment, the sequences are linked to at least one type of detectable moiety. The type of detectable moiety to be used may be selected from one of the following groups: a conjugate, a branched detection system, a chromophore, a fluorophore, a radioisotope, an enzyme, a hapten or a luminescent compound, among others .

In an even more preferred embodiment the fluorophore group may be at least one of the following: Alexa fluorophores, cyanines, 5- (and -6) Carboxy-2 ', 7'-dichlorofluorescein, 5-ROX (5-carboxy-X trihydammonium salt), among others. Still further object of the present invention is a kit for detecting Lactobacillus spp. and / or Gardnerella spp. in biological samples, which comprises at least one of the probes previously described.

In a more preferred embodiment the kit may further comprise at least one of the following solutions: a fixative solution, a hybridization solution and a wash solution.

In still more preferred embodiment the fixing solution may comprise paraformaldehyde and ethanol, namely 2-8% (w / v) paraformaldehyde and 25-75% (vol / vol) ethanol, and / or the hybridization solution may comprise formamide . It is also an object of the present invention to provide a method of detecting Lactobacillus spp. and / or Gardnerella spp. in biological samples, which uses at least one of the aforementioned PNA probes and comprising the following steps: contacting the PNA probe with biological samples; the hybridization of the PNA probe with the target sequence of the microorganisms present in the biological samples; The detection of hybridization as indicative of said detection and quantification in the biological samples.

However, said biological samples may be derived from blood, vaginal exudate, food, water, biopsies, among others.

In an even more preferred embodiment the detection of the hybridization may be carried out by fluorescence. Further object of the present invention is the use of the PNA probes described above, the use of the kits described above and the methodology to be applied in a method of detecting Lactobacillus spp. and / or Gardnerella spp. in biological samples.

The present invention encompasses PNA probes, reagents, methods and kit intended for the detection or quantification of strains of Lactobacillus spp. and / or Gardnerella spp. The higher specificity of PNA probes (relative to DNA probes) allows better discrimination of nucleotide sequences related to one or two nucleotides of difference. In the present invention this aspect assumes particular relevance, since the difference between the genera belonging to the same family of the genus Lactobacillus spp. since they share a certain level of genomic similarity, in particular 16S RNA.

The PNA probes described in the present invention are capable of detecting rRNA, rRNA (rDNA) corresponding genomic sequences, or sequences complementary thereto, allowing specific detection of the target genus.

The probes of this invention are used for in situ hybridization analysis of Lactobacillus spp. and / or

Gardnerella spp. optionally present in the sample, preferably by the fluorescent in situ hybridization technique.

The nucleotide sequences of the PNA probes described in this invention are selected from a group formed with at least 80% identical to the following sequences: 5'-ACA TGG AGT TCC ACT-3 '(SEQ ID No. 1 Lactobacillus spp.), 5 AGC ATG-3 '(SEQ ID No. 2 Lactobacillus spp.), 5' -TTC TCA GTT CGG ACT-3 '(SEQ ID No. 3 Lactobacillus spp.), 5'-CAG CAT TAC CAC (SEQ ID No.5 Gardnerella spp.), 5'-GCT CCA GAA TAG CTC-3 '(SEQ ID No.4 Gardnerella spp.), 5'-ATG CTC CAG AAT AGC-3' ID No.6 Gardnerella spp.), The development of new PNA-FISH probes is now performed empirically. In this case, we started by testing the ideal number of bases for the operation of each of the probes. A high number of bases causes the probes to have a high affinity for the target and thus function at too high temperatures, whereas too small a number implies that the binding free energy is not sufficient for hybridization to occur. For this case, the best results were for the range of 12-18 base pairs. In addition, the best sequences for the detection of each of the above-mentioned bacterial genera have been selected. It was found that when the probes contained a higher specificity with sequences of fifteen nucleotides, presenting Gibbs energy values (AG °) at the theoretical target values for a more robust hybridization. Thus, the PNA probe intended for the detection and / or quantification of Gardnerella spp. (SEQ ID No. 4) showed in silico a specificity and sensitivity of 100%. However, the PNA probe intended for the detection and / or quantification of Lactobacillus spp. (SEQ ID No. 1) shows a specificity of 78.86% and a sensitivity of 92.76%, which may hybridize to some species of other bacterial genera but which demonstrate a rather distinct morphology of the species Lactobacillus spp. and therefore easily distinguishable by microscopy.

For each case the optimal conditions of the FISH process also had to be studied since the success of the hybridizations of the PNA-FISH probes is dependent on hybridization conditions as well as fixation / permeabilization and washing. Initially, the conditions of the previously published invention (WO2008155742-A2; PT103767-A1), which is intended for the detection of Helicobacter pylori, were considered.

Thus, the present laboratory investigation began by analyzing parameters such as temperature, the concentration of formamide, as well as the time of hybridization and washing. Since the conditions for the operation of the previous probe might not be ideal for the new set of probes proposed here. Since it is necessary to optimize the parameters simultaneously without having an idea of which one (or even one of the others) is negatively affecting the PNA-FISH method, this process is quite complicated and time-consuming. Moreover, in some studies published by other authors it is possible to verify that many probes tested do not prove to be efficient and as such are useless for the PNA-FISH process. In this case, the most favorable time and temperature, formamide concentration conditions were identified as being 90 minutes of hybridization and 30 minutes of washing at 60 ° C and 30% formamide. It is to be noted that due to the relationship between temperature and formamide, it is expected that at intermediate concentrations of formamide the hybridization will also operate at a temperature range of 55-65 ° C.

Subsequently, the other parameters were studied as fixation / permeabilization with different percentages of paraformaldehyde (1-20%) and ethanol (10-90%). A higher efficiency was obtained with 4% paraformaldehyde and 50% ethanol.

A successful hybridization then allows, for example, by fluorescence microscopy, flow cytometry or real-time PCR, to gauge the presence / absence, concentration and characterization of each of the bacterial genera under study in the analyzed samples.

For this it is also important to consider the fraction of the PNA probe that allows the detection / identification of the existence of a stable complex formed by the probe and the target. Such a detectable moiety of the probe is selected from one of the following groups: a conjugate, a branched detection system, a chromophore, a fluorophore, radioisotope, an enzyme, a hapten or a luminescent compound. 13

Different probes may be coupled to the same detectable group (e.g., a fluorophore) in order to detect bacterial species of the genus Lactobacillus spp. , while a further optional probe with another different detectable molecule, in order to identify, for example, bacterial species of the genus Gardnerella spp. The method described in the present invention comprises contacting a sample with one or more PNA probes described above. According to the method, the microorganisms in a sample are detected, identified or quantified, relative to their bacterial genus, correlating the hybridization, performed under the appropriate hybridization conditions, of the PNA sequence with the target sequence. Consequently, the analysis is based on a single test with a final opinion. In contrast, current routine methods for the analysis of microorganisms are based on multiple characteristics involving multiple tests, as previously described. Still further object of the present invention is a kit suitable for carrying out the assay to determine, i.e. detect, identify or quantify bacterial species belonging to the genus Lactobacillus spp. and / or Gardnerella spp. The kit of the invention includes one or more PNA probes and other reagents or compounds selected for carrying out the in situ hybridization assays.

In an even more preferred embodiment, that of a kit suitable for carrying out the assay to detect, identify or quantify bacterial species belonging to genus 14

Lactobacillus spp. and / or Gardnerella spp. it also contains a fixation, hybridization and wash solution.

Preferably, this method is intended to replace the current laboratory methods associated with the clinical examination performed by the patient. Thus, from obtaining a sample of the patient and the identification / quantification of bacterial species belonging to the genus Lactobacillus spp. and / or Gardnerella spp., the clinician may properly classify the patient's vaginal microflora and, accordingly, recommend appropriate treatment. Thus, the implementation of this method of laboratory diagnosis is essential for a greater efficiency of the contemporary clinical diagnosis.

PNA probes can be applied directly to the slide prepared sample, since the application of these probes does not involve the use of reagents or enzymes for the permeabilization of the cell membranes prior to hybridization. However, it needs some of the compounds most commonly used in hybridizations. Thus, the probes are usually included in kits allowing for easier handling by the users.

Brief description of the figures

For ease of understanding of the present application, there are attached figures which represent preferred embodiments which, however, are not intended to limit the technique disclosed herein.

Figure 1 - Detection of the 16S rRNA gene of species

Lactobacillus spp. (a) and Gardnerella spp. (b), respectively, in samples of vaginal exudates (SOI to S10).

Legend - λ, lambda DNA marker; - negative PCR control; +, positive internal PCR control; SOI to S10, clinical samples of vaginal exudates from 1 to 10.

DETAILED DESCRIPTION OF THE INVENTION I - Definitions a) As used herein, the term " nucleotide " includes natural and unnatural molecules commonly known to those using nucleic acid related technology, to thereby generate polymers that specifically bind to nucleic acids. b) When the term " nucleotide sequence " is used, it is the same as referring to a segment of a polymer containing subunits, in this case nucleotides. c) The term " target sequence " means a nucleotide sequence of the bacterial species belonging to the genus Lactobacillus spp. or Gardnerella spp. which is intended to be detected in the assay, wherein the nucleotide portion of one of the probes is designed to hybridize. d) The term " PNA probe " means a polymer of PNA subunits which has a nucleotide sequence and is specific for hybridizing to a target sequence of the microorganism of interest. 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. E) When the term " detectable moiety " is used, it refers to molecules which can be attached to the probe, thereby to render the probe detectable by an instrument or method. f) The term " sample " refers to any biological sample that may contain the target micro-organism or sequence for detection. Preferably the biological samples are in the liquid form (for example: water, food, blood, vaginal exudate, urine, etc.) or as a tissue sample (eg biopsy specimens, in particular vaginal biopsies). II - Description

Design of PNA probes:

The PNA probes of this invention have as oligonucleotide target sequences belonging to the 16S rRNA zones of the bacterial species of the genera Lactobacillus spp. and / or Gardnerella spp. Thus, 16S rRNA sequences from various databases, such as Ribosomal Database Project II (RDPII, version 10.0; http://rdp.cme.msu.edu/, Cole et al. 2009), were aligned for each of the bacterial genera under study. The probe Lac663 (SEQ ID No. 1) is intended for identification / quantification of species of the genus Lactobacillus spp. and its target sequence is located at position 663 to 677 of the 16S rRNA sequence of the strain Lactobacillus spp. strain MDL2

Genebank: HM753265.1). In turn, the Gardl62 probe (SEQ ID No. 4) was designed for the identification / quantification of species of the genus Gardnerella spp. and its target sequence is located at position 162 to 176 of the rRNA 16S17 sequence of strain Gardnerella vaginalis 409-05 (RDPII Number: S001872672).

Preferably, the PNA probes of this invention comprise 15 nucleotide sequences. In addition to the probes mentioned above, other probes for the detection / quantification of Lactobacillus spp. (SEQ ID No. 2 and 3) and Gardnerella spp. (SEQ ID No. 5 and 6) have also been developed within the scope of this invention.

Table 1 - PNA probes of this invention. SEQ ID No. Organisms Target Nucleotide sequence 1 Lactobacillus spp. 5'-ACA TGG AGT TCC ACT-3 '2 Lactobacillus spp. 5'-AGG CTC GAA AGC ATG-3 '3 Lactobacillus spp. 5'-TTC TCA GTT CGG ACT-3 '4 Gardnerella spp. 5'-CAG CAT TAC CAC CCG-3 '5 Gardnerella spp. 5'-ATG CTC CAG AAT AGC-3 '6 Gardnerella spp. 5'-GCT CCA GAA TAG CTC-3 '

Alternatively, this invention also contemplates variations in the nucleotide sequences of the probes. Such variations may include deletions, insertions among others. In general, a nucleotide sequence homology of 80% or greater is sufficient for the proper functioning of the method.

PNA probe detectable fraction: Not limited to the following examples, the detectable fraction of the PNA probe may include different types of molecules, such as dextran conjugates, chromophores, fluorophores, 18 radioisotopes, enzymes, hapten, chemiluminescent compound among others.

As an example, the class of fluorophores are preferred for use (but not limited to): Alexa fluorophores, Alexa Fluor series, cyanines, 5- (and -6) Carboxy-2 ', 7'-dichlorofluorescein, 5- ROX (5-carboxy-X-rhodamine, triethylammonium salt). METHOD The present invention provides a method for the identification / quantification of species of the genus

Lactobacillus spp. and / or Gardnerella spp. The PNA probes used comprise at least one of the nucleotide sequences at least 80% identical to SEQ ID Nos. 1 to 6. PNA probes described herein with the target sequence of the bacterium under suitable hybridization conditions or suitable in situ hybridization conditions (as addressed in EXAMPLE 1). Preferably, fluorescent in situ hybridization (FISH or PNA-FISH) or real-time PCR are the assay formats for the analysis of species of the genus Lactobacillus spp. and / or Gardnerella spp. The method can thus be divided into: sample preparation, cell attachment, hybridization, washing and visualization of the results (see example 1). The method can be performed on adhered or suspended cells.

Hybridization Conditions

There are several factors that impose or control the stringency of hybridization of the PNA probe to target sequences. These include the percentage of formamide used (or other denaturing chemical reagent), the salt concentration and hence the ionic strength, the hybridization temperature, the detergent concentration, pH, among others.

In order to detect optimal hybridization conditions it may be necessary to fix the different factors and vary each factor alone until a desired degree of discrimination is found.

The closer a target sequence to another target is to the sample, the greater the degree of accuracy in defining the different factors influencing hybridization. In this invention non-target sequences may have only one nucleotide apart from the target sequences (since the genus Lactobacillus spp. Is found in a family with several bacterial genera related to each other and sharing a high genomic similarity), a high level of discrimination in order to avoid non-specific hybridizations of the PNA probe to non-target sequences. Probes of this invention which hybridize to target sequences relative to species from two distinct bacterial genera can be used together since both bacterial genera are found in vaginal exudates. However, each probe type (for Gardnerella spp. Or Lactobacillus spp.) May optionally be used alone or in combination with optimized hybridization conditions without making non-specific binding in the target sequences of the remaining probes.

Preparation of samples:

The samples to be analyzed may come from vaginal exudates, blood, urine, among others. In the case of vaginal exudates, swabs with vaginal samples 20 are vortexed in 1 ml of saline (0.9% NaCl) or phosphate buffered saline (PBS) and centrifuged at 10,000 rpm for 5 minutes at room temperature. The supernatant is then discarded and the decanted cells resuspended in 1 ml of saline (0.9% NaCl) or in phosphate buffer (PBS) again. 20 to 50 Dl of the above suspension is collected and placed on immunofluorescence slides. In case the bacteria under study are found in more complex samples, such as in urine, milk and blood samples, the samples are centrifuged at 10,000 rpm for 5 min at room temperature, the supernatant is discarded and the decanted cells are resuspended in 1 ml of saline (0.9% NaCl) or in phosphate buffer (PBS). Eventually, the samples may still be diluted 1 to 10 again in saline or PBS in order to eliminate potential autofluorescent compounds present in the initial samples. Alternatively, the suspensions may further be filtered by a polycarbonate membrane or equivalent. Finally, 20 to 50 Dl of the suspension is collected and placed on immunofluorescence slides. The membranes are then placed on slides. It is further important to note that the saline solution and the PBS buffer must be pre-sterilized by autoclaving at 121øC for 15-20 minutes.

The present invention contemplates a kit which allows performing the assay which analyzes the presence and quantity of species of the genus Lactobacillus spp. and / or Gardnerella spp. . 21

The PNA probes to be used in the kit, its characteristics, the method involved were previously referred to herein. The kit in this invention comprises at least two probes, wherein the first comprises one of the nucleotide sequences at least 80% identical to SEQ ID Nos. 1 to 3 (recognize Lactobacillus spp.); while the second comprises comprising one of the nucleotide sequences at least 80% identical to SEQ ID NOs. 4 to 6 (recognize species Gardnerella spp.). It is important to note that in this invention also comprises the other reagents or compositions which are selected to carry out the assay.

The PNA probes, their characteristics, methods and kit of this invention are suitable for the analysis of nucleic acids present, or not, internally in the organisms of interest. Thus, this invention can be used for both analysis of organisms or analysis of extracted nucleic acids or derivatives of the organisms of interest, so that the source of the target sequences is not a limitation in this invention.

The following examples illustrate different situations and various steps of application of the invention, are preferred embodiments of the present invention, without intending to be limiting in any of them: EXAMPLE 1: Detection of species of the genera Lactobacillus spp. and / or Gardnerella spp ..

Table 2 - Sequences of the PNA probes used. 22

Target Organs Nucleotide sequence of the probe Lactobacillus spp. Lac663 Alexa 488-OO-ACA TGG AGT TCC ACT Gardnerella spp. Gardl62 Alexa 594-OO-CAG CAT TAC CAC CCG

Legend - 00 = 8-Amino-3,6-dioxaoctanate (double bond that binds the two molecules, the fluorophore and the nucleotide sequence of the probe).

Alexa Fluor 488nm / 594nm - fluorophore (detectable moiety). Bacterial strains:

Different bacterial genera were obtained from clinical isolates, whose identity was verified by the sequencing of 16S rRNA, and several reference collections (see table 3), such as American Type Culture Collection, Manassas (ATCC), Spanish Type Culture Collection ( CECT), Deutsche Sammlung von Mikroorganismen (DSM), Culture Collection University of Goteborg (CCUG), among others.

All collection bacterial species were incubated 20-24 h in fresh growth medium plates prior to laboratory experiments under ideal growth and atmospheric temperature conditions (aerobic, anaerobic and microfilia) for each particular case. The species Lactobacillus spp. were grown on Man plates,

Rogosa and Sharpe agar (MRS), whereas the species of Atopobium spp. and Gardnerella spp. were cultured on Brucella Blood Agar (BBA). The remaining bacterial species were inoculated into Brain Heart Infusion agar (BHI). 23

For each bacterial strain, one drop of culture was placed on immunofluorescence slides with wells of 8 mm in diameter. The slide was then placed about 10 minutes in the oven at 60 ° C.

Fixation:

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

Hybridization:

During this step, a drop of hybridization solution was placed in contact with the sample containing 10% (w / v) dextran sulfate, 10 mM NaCl, 30% (vol / vol) formamide, 0.1% (w / v) (wt / vol) polyvinylpyrrolidone, 0.2% (wt / vol) Ficol, 5 mM di-sodium EDTA, 0.1% (vol / vol) Triton X-100, 50 mM Tris-HCl (pH 7.5) and 200 nM of each of the PNA probes. The sample was covered with a coverslip to ensure uniform spreading of the probe. The slides were then placed in the oven for 90 minutes. During this time, the probes were able to penetrate cell membranes and bind to 16S rRNA complementary sequences. During the hybridization period, it is essential that the hybridization solution does not evaporate. To this end, the presence of the coverslip and wet paper around the blade were required. 24

Cleaning:

After the hybridization time the slides were removed and the slides were immersed in preheated wash solution containing 5 mM Tris Base, 15 mM NaCl and 0.1% (vol / vol) Triton X (pH 10), placing in the oven at the temperature of hybridization for 30 minutes.

Results:

The results were obtained by observation under a fluorescence microscope with filters suitable for the detection of the fluorochromes attached to the probes (i.e., which contemplate the wavelengths emitted by the probe coupled fluorochromes), and no signal was detected in situations in which the target sequence is not present. The filter 488 allows fluorescein uptake promoted by the Alexa fluorochrome 488, which is a signal for the hybridization of Lacto663 probe (SEQ ID No. 1) to the target sequence characteristic of the genus Lactobacillus spp. . In turn, the filter 594 allows to capture the fluorescence promoted by the fluorochrome Alexa 594, which is a demonstration of the hybridization of probe Gardl62 (SEQ ID No. 4) with the characteristic target sequence of the genus Gardnerella spp ..

Table 3 - Fluorescence microscopy results with filters suitable for the detection of the fluorochromes attached to the probes. Bacterial Species Strain Genus Lac663 Probe efficiency Gardl62 Probe efficiency L. pentosus CECT4023 Lactobacillus spp. ++++ 'L. casei CECT5275 Lactobacillus spp. ++++ 25 L. rhamnosus CECT288 Lactobacillus spp. ++++ - L. coryniformis sub. torquens CECT4129 Lactobacillus spp. L. paracasei CECT227 Lactobacillus spp. ++++ - L. acidophilus ATCC4356 Lactobacillus spp. ++++ - L. agilis CCUG 31450 Lactobacillus spp. ++++ - L. anlmalis ATCC350 4 6 Lactobacillus spp. +++ - L. bifermentans ATCC354 0 9 Lactobacillus spp. +++ - L. brevis ATCC14869 Lactobacillus spp. ++++ - L. buchneri ATCC4005 Lactobacillus spp. +++ - L. fermentum ATCC11739 Lactobacillus spp. +++ - L. crispatus ATCC33820 Lactobacillus spp. ++++ - L. curvatus sub. curvatus ATCC25601 Lactobacillus spp. ++++ L. delbrueckll sub. delbrueckii ATCC9649 Lactobacillus spp. +++ L. delbrueckii sub. lactis ATCC12315 Lactobacillus spp. +++ L. farciminis DSM20182 Lactobacillus spp. ++++ - L. fructivorans ATCC8288 Lactobacillus spp. +++ - L. gallinarum CCUG31412 Lactobacillus spp. ++++ - L. gasseri ATCC9857 Lactobacillus spp. ++++ - L. graminis DSM20719 Lactobacillus spp. ++ - L. hamsteri ATCC43851T Lactobacillus spp. +++ - L. helveticus ATCC15009 Lactobacillus spp. ++++ - L. hilgardii NCFB962 Lactobacillus spp. +++ " 26 L. intestinalis ATCC4 9335 Lactobacillus spp. +++ - L. johnsonii ATCC11506 Lactobacillus spp. ++++ - L. murinus ATCC35020 Lactobacillus spp. ++++ - L. parabuchneri ATCC12936 Lactobacillus spp. ++++ - L. paracasei sub. paracasei CCUG27320 Lactobacillus spp. +++ L. plantarum NCIMB8 8 2 7 Lactobacillus spp. +++ - L. reuteri NCFB2656 Lactobacillus spp. +++ - L. rhamnosus ATCC7469 Lactobacillus spp. ++++ - L. ruminis ATCC2 7 7 81 Lactobacillus spp. ++++ - L. sakei sub. carnosus CCUG8045 Lactobacillus spp. ++ L. salivarius DEVRIESE94 / 438 Lactobacillus spp. +++ - L. plantarum NCCB4 60 43 Lactobacillus spp. +++ - Lactococcus lactis 53 Lactococcus spp. - / ++ - Streptococcus s thermophilus Streptococcus spp. Streptococcus s thermophilus B Streptococcus spp. +++ Leuconostoc mesenteroide s Leuconostoc spp. - / + Bacillus subtilis DSM 7-10 Bacillus spp. Enterococcus faecium CECT410 Enterococcus spp. Enterococcus faecalis CECT184 Enterococcus spp. - - Gardnerella vaginalis 51 - Gardnerella spp. - ++++ Gardnerella vaginalis 101 Gardnerella spp. ++++ 27

Gardnerella vaginalis AMD Gardnerella spp. Gardnerella vaginalis ATCC Gardnerella spp. - ++ + + Atopobium vaginae CCUG 38953T Atopobium spp. - - Atopobium vaginae CCUG 42099 Atopobium spp. - - Atopobium vaginae CCUG 44116 Atopobium spp. - - Atopobium vaginae - Atopobium spp. - - Bacillus cereus - Bacillus spp. - - Enterobacter aerogenes CECT 684 Enterobacter spp. - - Salmonella enterica Salmonella spp. - - Escherichia coli 0157-.H7 NCTC 12900 Escherichia spp. - - Staphylococcus aureus CECT 976 Staphylococcus spp. - - Staphylococcus aureus CECT 86 Staphylococcus spp. - - Shlgella ATCC 12022 Shlgella spp. - - Listeria monocytogene s Listeria spp. Klebsiella pneumoniae sub. Ozaenae ATCC 11296 Klebsiella spp. Salmonella Typhl - Salmonella spp. - - Listeria seeligeri CECT 917 Listeria spp. " " Escherichia coli CECT 434 Escherichia spp. - - Listeria monocytogene s CECT 5873 Listeria spp.

Legend - The efficiency of the PNA probes was tested in triplicate for each of the strains. This hybridization efficiency was assessed qualitatively as follows: (-) hybridization absent; (+) weak hybridization; (++) moderate hybridization; (+++) efficient hybridization; (++++) excellent hybridization. The table shows the average value obtained 28

Lactobacillus spp. and Gardnerella spp. from vaginal exudates. Since the aim of the present invention is the rapid identification / quantification of Lactobacillus spp. and Gardnerella spp. in clinical samples, among others.

Results:

The results were obtained by fluorescence microscopy observation with filters suitable for the detection of the fluorochromes attached to the probes.

Table 4 - Results of the mixed samples under fluorescence microscopy with filters suitable for the detection of the fluorochromes attached to the probes.

Species present in mixed samples Strain code in mixed samples Multiplex with PNA probes Lac663 Probe efficiency Gardl62 Probe efficiency L. pentosus; G. vaginalis 51 CECT4023; - ++++ ++++ L. casei; G. vaginalis 101 CECT5275; - ++++ ++++ L. rhamnosus; G. vaginalis AMD CECT288; - ++++ ++++ L. crispatus; G. vaginalis ATCC ATCC 33820; - ++++ ++++ L. delbrueckií sub. delbrueckii; Atopobium vaginae ATCC9649; CCUG 38953T +++ L. acidophilus; A. vaginae ATCC4356; CCUG 42099 ++++ L. gasseri; A. vaginae ATCC9857; CCUG 44116 ++++ L. paracasei sub. paracasei; L. lactis 53 CCUG27320; - +++ - / + L. rhamnosus; E. faecium ATCC7469; CECT410 ++++ - L. reuteri; E. coli 0157-.H7 NCFB2656; NCTC 12900 +++ - S. aureus; G. vaginalis 51 CECT 976; - _ ++++ Shigella; G. vaginalis 101 ATCC 12022; - _ ++++ L. seeligeri; G. vaginalis AMD CECT 917; In the triplicate experiments for each of the bacterial species analyzed. EXAMPLE 2: Detection of species of the genus Lactobacillus spp. and / or Gardnerella spp. by Multiplex FISH.

This example is intended to illustrate the possibility of detecting Lactobacillus spp. and Gardnerella spp. from a single FISH assay through the simultaneous use of two PNA probes in mixed samples, i.e. with two or more bacterial strains. Since the aim of the present invention is the rapid identification / quantification of species

Lactobacillus spp. and / or Gardnerella spp. in complex samples.

For this purpose, a considerable number of mixed samples were made from the bacterial strains used in EXAMPLE 1 (see table 4). For the efficient accomplishment of the FISH multiplex protocol, the same parameters discussed above were re-tested to ascertain the maximum efficiency of the intended assay. Indeed, the efficiency remained the same under the same conditions set forth in the protocol of EXAMPLE 1. Therefore, the hybridization protocol used for the multiplex was the same as in EXAMPLE 1.

The probes used in this analysis of bacterial genera in mixed samples were the same probes previously reported in Table 2 of EXAMPLE 1. However, in this assay, the aliquot of use consisted of the elaboration of a single aliquot with the two PNA probes, more specifically , the Lacto663 probe (SEQ ID No. 1) and the Gardl62 probe (SEQ ID No. 4). Finally, some changes may be made to optimize the visualization of the probes together in the same assay, namely, the exposure time for each filter in order to maximize the fluorescence of the hybridized probe. In addition, the mixed samples were observed by the probe with the lower excitation energy fluorochrome, in this case the Gardl62 probe, avoiding the involuntary excitation of the other PNA probe of the multiplex assay, namely the Lac663 probe.

This example is intended to illustrate the possibility of directly detecting Lactobacillus spp. and Gardnerella spp. from vaginal exudates. Since the aim of the present invention is the rapid identification / quantification of Lactobacillus spp. and Gardnerella spp. in clinical samples, among others.

In addition, a considerable number of vaginal exudates (vaginal samples of ten control volunteers, SOI through S10) were acquired. Before performing the FISH protocol, the swabs with the vaginal exudates were pre-vortexed in 1 ml of saline (0.9% NaCl) or in phosphate buffered saline (PBS) and centrifuged at 10,000 rpm for 5 minutes at room temperature. The supernatant was then discarded and the decanted cells resuspended again in 1 ml of saline (0.9% NaCl) or in phosphate buffer (PBS). Finally, the remainder of the procedure was similar to that described above in the bacterial collection strains.

This experimental increase possibility of detecting is intended to illustrate directly the species 30

Lactobacillus spp. and Gardnerella spp. from vaginal exudates. Since the aim of the present invention is the rapid identification / quantification of Lactobacillus spp. and Gardnerella spp. in clinical samples, among others.

Results:

The results were obtained by fluorescence microscopy observation with filters suitable for the detection of the fluorochromes attached to the probes.

Table 4 - Results of the mixed samples under fluorescence microscopy with filters suitable for the detection of the fluorochromes attached to the probes.

Species present in mixed samples Strain code in mixed samples Multiplex with PNA probes Lac663 Probe efficiency Gardl62 Probe efficiency L. pentosus; G. vaginalis 51 CECT4023; - ++++ ++++ L. casei; G. vaginalis 101 CECT5275; - ++++ ++++ L. rhamnosus; G. vaginalis AMD CECT288; - ++++ ++++ L. crispatus; G. vaginalis ATCC ATCC 33820; - ++++ ++++ L. delbrueckii sub. delbrueckii; Atopobium vaginae ATCC9649; CCUG 38953T +++ L. acidophilus; A. vaginae ATCC4356; CCUG 42099 ++++ L. gasseri; A. vaginae ATCC9857; CCUG 44116 ++++ 31 L. paracasei sub. paracasei; L. lactis 53 CCUG27320; - +++ - / + L. rhamnosus; E. faecium ATCC7469; CECT410 +++ + - L. reuteri; E. coli 0157: H7 NCFB2656; NCTC 12900 +++ - S. aureus; G. vaginalis 51 CECT 976; - - +++ + Shigella; G. vaginalis 101 ATCC 12022; - E. aerogenes; G. vaginalis ATCC CECT 684; - ++++ L. pentosus; G. vaginalis ATCC; E. faecalis CECT4023; - r CECT184 ++++ L. casei; G. vaginalis AMD; A. vaginae CECT5275; - r CCUG 38953T ++++ L. rhamnosus; G. vaginalis 101; A. vaginae CECT288; - r CCUG 42099 ++++ ++++ L. críspatus; G. vaginalis 51; A. vaginae ATCC 33820; "R CCUG 44116 ++++ ++++ L. casei; L. mesenteroides; A. vaginae CECT5275; f CCUG 38953T ++ + +

Legend - This hybridization efficiency was assessed qualitatively as follows: (-) missing hybridization; (+) weak hybridization; (++) moderate hybridization; (+++) efficient hybridization; (++++) excellent hybridization. The table shows the average value obtained in the triplicate experiments for each of the bacterial species analyzed.

Table 5 - Results of vaginal exudate samples under fluorescence microscopy with suitable filters for the detection of the fluorochromes attached to the probes. 32

Sample No. Lac663 Probe Code Gardl62 Probe vaginal sample efficiency efficiency 1 o fu} k_ > v > 444 444 · - > S02 4444 - 3 SOS 444 444 4 S04 44 444 R yj S 0 5 4444 - 6 soe 44 44 7 S07 + 4 44 O O S 0 3 444 444 9 S 0 9 44 44 10 S10 44 -

Legend - This hybridization efficiency was assessed qualitatively as follows: (-) missing hybridization; (+) weak hybridization; (++) moderate hybridization; (+++) efficient hybridization; (++++) excellent hybridization. The table shows the average value obtained in the triplicate experiments for each of the bacterial species analyzed.

In the case of vaginal exudate samples, the results of the FISH assays (Table 5) were confirmed by PCR (see Figure 1). More specifically, the presence of Lactobacillus spp. was confirmed by the band of * 62 bp in figure 1 (a), while the presence of Gardnerella spp. was verified by the * 111 bp band in figure 1 (b). All samples were pre-treated by heat shock, which consisted of 5 minutes at 100 ° C followed by 5 minutes on ice, prior to performing the 30 cycle cycle at 50 ° C (melting temperature, tm).

The PCR results were in agreement with the results obtained previously by FISH (table 5). 33

LIST OF SEQUENCES

< 110 > MINHO'S UNIVERSITY

&lt; 120 &gt; &quot; NUCLEIC PEPTIDE ACID PROBES, CASE AND METHOD FOR DETECTING GENUS SPECIES OF LACTOBACILLUS SPP. AND / OR GARDNERELLA SPP. AND THEIR RESPECTIVES &lt; 210 &gt; SEQ ID NO 1 APPLICATIONS &quot; 5'-ACA TGG AGT TCC ACT-3 '(SEQ ID No. 1), and have a length between 8 to 18 nucleotides &lt; 210 &gt; SEQ ID NO 2 5'-AGG CTC GAA AGC ATG-3 '(SEQ ID No. 2), and have a length between 8 to <210> SEQ ID NO: 3 18 nucleotides 5'-TTC TCA GTT CGG ACT-3 '(SEQ ID No. 3), and have a length between 8 to &lt; 210 &gt; SEQ ID NO 4 18 nucleotides 5'-CAG CAT TAC CAC CCG-3 '(SEQ ID No. 4), and have a length between 8 at &lt; 210 &gt; SEQ ID NO 5 18 nucleotides 5'-ATG CTC CAG AAT AGC-3 '(SEQ ID No. 5), and having a length between 8 to 18 nucleotides &lt; 210 &gt; SEQ ID NO 6 5'-GCT CCA GAA TAG CTC-3 '(SEQ ID NO.) And have a length between 8 to 18

No. 6), nucleotides 35

References

Nugent R., Krohn M. and Hillier S. Reliability of Diagnosing Bacterial Vaginosis Is Improved by a Standardized Method of Gram Stain Interpretation. Journal of Clinical Microbiology, 1991, 29 (2): 297-301.

Fredricks, D., Fiedler, T. and Marrazzo, J. Molecular Identification of Bacteria Associated with Bacterial Vaginosis. N Engl J Med 2005,353: 1899-911.

Cole, J. R., Q. Wang, E. Cardenas, J. Fish, B. Chai, R. J. Farris, A. S. Kulam-Syed-Mohideen, D. M. McGarrell, T. Marsh, G. M. Garrity, and J. M. Tiedje. The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res. 2009, 37 (Database issue): D141-D145; doi: 10.1093 / nar / gkn879.

Lisbon, March 17, 2014 36

Claims (15)

A PNA probe characterized by detecting / quantifying the presence of species of the genus Lactobacillus spp. and / or Gardnerella spp., which comprises at least one of the nucleotide sequences at least 80% identical to SEQ ID NOs. 1 to 6. A PNA probe according to claim 1, characterized in that it detects the target sequence in the rRNA, the rDNA or in the complementary sequences of the rRNA of species of the genus Lactobacillus spp. and / or Gardnerella spp .. A PNA probe according to any one of claims 1 to 2, characterized in that it is attached to at least one type of detectable moiety. A PNA probe according to claim 3, characterized in that the detectable moiety of the probe is selected from one of the following groups: a conjugate, a branched detection system, a chromophore, a fluorophore, a radioisotope, an enzyme , a hapten or a luminescent compound. PNA probe according to claim 4, characterized in that the fluoro group is at least one of the following: Alexa fluorophores, Alexa Fluor series, cyanines, 5- (and -6) Carboxy-2 ', 7' dichlorofluorescein, 5-ROX (5-carboxy-X-rhodamine, triethylammonium salt). 6. Kit for the detection of species of the genus Lactobacillus spp. and / or Gardnerella spp. in biological samples. A PNA probe characterized by detecting / quantifying the presence of species of the genus Lactobacillus spp. and / or Gardnerella spp., which comprises at least one of at least 80% nucleotide sequences structurally similar to SEQ ID NOs. 1 to 6. A PNA probe according to claim 1, characterized in that it detects the target sequence in the rRNA, in the rDNA or in the complementary sequences of the rRNA of species of the genus Lactobacillus spp. and / or Gardnerella spp. . A PNA probe according to any one of claims 1 to 2, characterized in that it is attached to at least one type of detectable moiety. A PNA probe according to claim 3, characterized in that the detectable moiety of the probe is selected from one of the following groups: a conjugate, a branched detection system, a chromophore, a fluorophore, a radioisotope, an enzyme , a hapten or a luminescent compound. PNA probe according to claim 4, characterized in that the fluoro group is at least one of the following: Alexa fluorophores, Alexa Fluor series, cyanines, 5- (and -6) Carboxy-2 ', 7' dichlorofluorescein, 5-ROX (5-carboxy-X-rhodamine, triethylammonium salt). Characterized in that it comprises at least one of the probes described in any one of claims 1 to 5. A kit according to claim 6, characterized in that it further comprises at least one of the following solutions: a fixing solution, a hybridization solution and a washing solution. A kit according to claim 7, characterized in that the fixing solution comprises paraformaldehyde and ethanol, in particular 2-8% (w / v) of paraformaldehyde and 25-75% (vol / vol) of ethanol. A kit according to claim 8, characterized in that the hybridization solution comprises formamide. A method of detecting species of the genus Lactobacillus spp. and / or Gardnerella spp. in biological samples, characterized by using at least one of the PNA probes described in any one of the preceding claims and comprising the following steps: a. contacting the PNA probe with said samples; B. hybridization of the PNA probe to the target sequence of the microorganisms present in said samples; W. detection of the hybridization as indicative of said detection and quantification in said samples. A method according to claim 10, wherein said biological sample is derived from vaginal exudates, blood, food, water or biopsies. 2 A method according to claim 11, characterized in that the hybridization is by fluorescence. Use of the PNA probes as described in any one of claims 1 to 5, characterized in that species of the genus Lactobacillus spp. and / or Gardnerella spp. in biological samples. Use of the kit described in any one of claims 6 to 9, characterized in that a species of the genus Lactobacillus spp. and / or Gardnerella spp. in biological samples. Use of the method described in any one of claims 10 to 12, characterized in that it is applied in the detection of species of the genus Lactobacillus spp. and / or Gardnerella spp. in biological samples. Lisbon, March 17, 2014 3