PT106672B - 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

DESCRIPTION

NUCLEIC PEPTIDE ACID PROBES, CASE AND METHOD FOR DETECTION OF LACTOBACILLUS SPP SPECIES. AND / OR GARDNERELLA SPP. AND THEIR APPLICATIONS

Field of the Invention

This invention is part of a method of detecting clinically relevant microorganisms for the evaluation of the infectious process called bacterial vaginosis, and two nucleic acid peptide (PNA) probes have been developed to detect Lactobacillus spp. and / or Gardnerella spp. on various types of samples.

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

Background of the Invention

Lactobacillus spp. and Gardnerella spp. constitute instrumental bacteria of the vaginal mucosa. In normal cases, bacteria of the genus Lactobacillus spp. are in large numbers while bacteria of the genus Gardnerella spp. exist in small numbers or are even absent. Although bacteria in planktonic form of the genus Gardnerella spp. If they are present in the vaginal microflora of healthy women, they may eventually form biofilms and bacterial vaginosis occurs. Gardnerella spp. Biofilm. induces a change in phenotype expression and, consequently, 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. Thus, the need to use the two nucleic acid probe probes for vaginal microflora evaluation is highlighted.

Diagnosis of bacterial vaginosis infection can be based on time-consuming methods that involve a clinical examination and a laboratory examination that typically involves performing a microbial culture of the patient's vaginal exudate. Clinical diagnosis is most often used, although it is prone to errors and extremely susceptible to physician interpretation. This test consists of identifying at least three positive results in four characteristic symptoms / signs of this vaginal infection, namely, vaginal discharge pH level of 4.5 or greater, the presence of epithelial cells covered with Gram negative bacteria, appearance discharge or discharge from vaginal examination and the release of a characteristic odor of nitrogenous products by the addition of a 10% potassium hydroxide solution. In turn, laboratory examination for microbial culture consists of inoculating the patient's vaginal exudate in a medium enriched with

-10% blood, such as Human Blood Medium (HBA) or

Brucella Blood Medium (BBA) for 48-72h at 37 ° C in a 5% carbon dioxide atmosphere. Next, bacterial growth is usually quantified by the four growth zones in the petri dish and observed under the microscope after Gram staining. During microscopic observation of Gram stain the criteria of Nugent et al.

1991, thus classifying the observed sample into one of three possible diagnoses:

normal vaginal microflora;

intermediate vaginal microflora; and bacterial vaginosis. However, lack of sensitivity / specificity of clinical examination and lengthy analysis of microbial culture imply a complex procedure for the correct clinical diagnosis of bacterial vaginosis in patients.

Other laboratory assays employ PCR (Polymerase Chain Reaction) based methods for the identification of bacterial strains in bacterial vaginosis. However, this technique requires some care in 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. However, in a small number of healthy women clinical samples, this method is therefore prevented from being applied as a clinical diagnostic procedure.

Recently, nucleic acid peptide (PNA) probes have been developed and optimized for bacterial detection. PNA molecules are mimics of DNA, in which the negatively charged sugar-phosphate structure is replaced by an electrically neutral, achiral formed by repeated N- (2-aminoethyl) glycine units.

Although the PNA molecule lacks pentoses, specific hybridization occurs between PNA sequences and complementary nucleic acid sequences by hydrogen bonds. This hybridization follows the WatsonCrick 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 PNA / target sequence (rRNA or double stranded DNA) binding when compared to traditionally used DNA probes. Thus, because of their high affinity, PNA probes have relatively short nucleotide sequences, preferably between 8 and 17 nucleotides. A DNA probe typically has at least 18 nucleotides, due to its poor stability and lower melting temperature (Tm), also requiring additional fixation and permeabilization procedures with enzymes or other agents. PNA molecules also exhibit greater resistance to proteases and nucleases than the natural DNA molecule.

When coupled to a fluorochrome, PNA probes can be detected by microscopy or flow cytometry using the in situ fluorescent hybridization (FISH) technique. This technique yields faster results in clinical specimens when compared to traditional cultivation methods and has been proven to be effective, rapid, sensitive and specific. Its application is also very broad and can be used in many 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 genus Salmonella, Bacillus anthracis, coagulase negative Staphylococcus species, human Papillomavirus, and genus Candida, among others, demonstrate the growing interest that this technique provides.

For bacterial vaginosis, a DNA probe FISH methodology has already been published for the detection in vaginal exudates of various strains involved in the infection (Fredicks et al., 2005). However, the probes used corresponded to larger DNA oligonucleotides 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 ') used to identify Lactobacillus spp. it was nonspecific and had nineteen nucleotides, detecting the order Bacillales. The Gardnerella vaginalis probe (G.vag-198-Cy3 5'-CCACTAAACACTTCCCAACAAGA-Cy3-3 '), in turn, had twenty-five nucleotides. Both probes operated at a hybridization temperature (Tm) of 45 ° C and could lead to false positives. Finally, the fluorochromes used in the study corresponded to cyanine number 5 (Cy5) and number 3 (Cy3), which are

fluorescence lower than fluorochromes Alexa Flúor (Alexa Fluor 488 and 594) used in the probes herein presented. Thus, this methodology revealed specificity and sensitivity lower than the method here

proposed.

Bacterial vaginosis is a common vaginal infection in women of reproductive age and may initially occur asymptomatically. The etiology of this infection is still unknown, but bacterial vaginosis is associated with a change in vaginal microflora and is characterized by the growing replacement of the normal flora of Lactobacillus species with certain anaerobic bacteria such as Gardnerella vaginalis. In developed countries, the incidence rate of bacterial vaginosis (bacterial infection) is approximately double the genital candidiasis (fungal infection) in symptomatic women and even more common than trichomoniase (protozoal infection). Thus, a detection method that allows a quick visualization of the microorganisms responsible for a vaginal infection and the amount of bacteria present in the vaginal exudate, the relationship between Lactobacillus spp. and Gardnerella spp ..

The present invention describes a reliable and feasible method of systematically applying identification of bacteria present in normal vaginal samples and in pathogenic cases of bacterial vaginosis. Therefore, it is a significant contributor to the rapid selection of appropriate treatment, saving you the hassle, risk, prolonged treatment and patient costs.

On the other hand, hygiene and quality control in the food and agricultural industry is playing an increasingly prominent role today.

Food products such as milk, yoghurt and cheese require an effective and rapid detection and / or quantitation method for certain microorganisms, eg species

Lactobacillus spp.

In fact, the direct application of PNA probes to Lactobacillus spp.

In food products, in particular milk, the present invention makes it very practical and dynamic in the food and agricultural industry. In this manner, the present invention facilitates the control process by avoiding time consuming and expensive 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 PNA probe sets, 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 concerned or the genomic sequences corresponding to the mentioned rRNA. PNA probes have inherent physicochemical characteristics that, when applied to a method based on FISH technology, allow for 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 relationship / ratio between the existing species of Lactobacillus spp. and Gardnerella spp. in vaginal samples. In addition to providing a reliable and expeditious method for determining the presence of Lactobacillus spp. and / or Gardnerella spp. In a biological sample, it is also possible to quantify these species, thus allowing the classification of vaginal microflora (normal microflora, intermediate microflora or bacterial vaginosis) and to 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 fluorescent in situ hybridization (FISH) technique, which allows detection of Lactobacillus spp. and / or Gardnerella spp. in biological samples, simply and quickly. Both probes may or may not be used at the same time.

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 the nucleotide sequences at least 80% 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 Lactobacillus spp rRNA, rDNA or complementary RNA sequences. 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 NOs. 1 to 3 which detect Lactobacillus spp. ;

at least one of the nucleotide sequences, at least 80% identical to SEQ ID NOs. 4 to 6 which detect Gardnerella spp.

In an even more preferred embodiment, the sequences are linked to at least one type of detectable fraction. 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 series fluorophores, cyanines, 5- (and -6) Carboxy-2 ', 7' dichlorofluorescein, 5-ROX (5-carboxy-X- rhodamine, triethylammonium salt), among others.

It is further object of the present invention a detection kit of Lactobacillus spp. and / or Gardnerella spp. in biological samples, which comprises at least one of the previously described probes.

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

In an even more preferred embodiment the fixation solution may comprise paraformaldehyde and ethanol, namely 2-8% (w / vol) paraformaldehyde and 25-75% (vol / vol) ethanol and / or the hybridization solution may comprise formamide. .

It is also the object of the present invention to describe a method for detecting Lactobacillus spp. and / or Gardnerella spp. in biological samples using at least one of the above-mentioned PNA probes and comprising the following steps:

contacting the PNA probe with biological samples;

hybridization of the PNA probe to the target sequence of microorganisms present in biological samples;

detection of hybridization as indicative of said detection and quantitation in biological samples.

However, such biological samples may come from blood, vaginal exudate, food, water, biopsies, among others.

In an even more preferred embodiment the detection of hybridization may be done by fluorescence.

It is a further object of the present invention to use the above described PNA probes, the use of the previously described kits and the methodology to be applied in a Lactobacillus spp detection methodology. and / or Gardnerella spp.

in biological samples.

General Description of the Invention

The present invention encompasses probes of

PNA, reagents, methods and kit for detection or quantification of Lactobacillus spp.

and / or Gardnerella spp .. The highest specificity of the PNA probes

DNA) allows for better discrimination of nucleotide sequences related to one or two nucleotides of difference. In the present invention this aspect is of particular relevance since the difference between genders belonging to the same family as

Lactobacillus spp.

since they share a certain level of genomic similarity, in particular

16S RNA.

The probes of

PNAs described in the present invention are capable of detecting rRNAs, rRNA-corresponding genomic sequences (rDNAs), or sequences complementary thereto, permitting 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 fluorescent in situ hybridization technique.

The nucleotide sequences of the PNA probes described in this invention are selected from a group formed of at least 80% identical to the following sequences:

5'-ACA TGG AGT CBT ACT-3 ' (SEQ ID At the . 1 Lactobacillus spp ), 5'-AGG CTC GAA AGC ATG-3 ' (SEQ ID At the . 2 Lactobacillus spp ), 5'-TTC TCA GTT CGG ACT-3 ' (SEQ ID No. 3 Lactobacillus spp

),

5'-CAG CAT TAC CAC CCG-3 ' (SEQ ID No. 4 Gardnerella spp.), 5'-ATG CTC CAG AAT AGC-3 ' (SEQ ID No.5 Gardnerella spp.), 5'-GCT CCA GAA Tag CTC-3 ' (SEQ ID NO.6 Gardnerella spp.),

The development of new PNA-FISH probes is currently being done empirically. In this case, we started by testing the ideal number of bases for the operation of each probe. Too many bases make the probes have a high affinity for the target and thus operate at too high temperatures, while too few implies that the binding free energy is not sufficient for hybridization to occur. For this case, the best results were for the range 12-18 bp. In addition, the best sequences for detecting each of the aforementioned bacterial genera had to be chosen. It was found that when the probes contained greater specificity with fifteen nucleotide sequences, they had Gibbs energy values (AG °) at the ideal theoretical values for more robust hybridization. Thus, the PNA probe for detection and / or quantification of Gardnerella spp. (SEQ ID No. 4) showed in silico a specificity and sensitivity of 100%. However, the PNA probe for detection and / or quantification of Lactobacillus spp. (SEQ ID No. 1) shows 78.86% specificity and 92.76% sensitivity and may hybridize to some species of other bacterial genera but which show a very distinct morphology from Lactobacillus spp. and therefore easily distinguishable by microscopy.

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

Thus, the present laboratory investigation began by analyzing parameters such as temperature, formamide concentration, as well as hybridization and washing time. Since the conditions for the operation of the previous probe could not be ideal for the new set of probes proposed here. Since it is necessary to optimize the parameters simultaneously without having an idea which one (or even one of them) is negatively affecting the PNA-FISH method, this process is quite complicated and time consuming. Moreover, in some works published by other authors it is possible to verify that many probes tested do not turn out to be efficient and therefore useless for the PNA-FISH process. In this case, the most favorable time and temperature conditions, formamide concentration were identified to be 90 minutes hybridization and 30 minutes wash at 60 ° C and 30% formamide. Note that due to the relationship between temperature and formamide, it is expected that at intermediate concentrations of formamide hybridization will also function 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%). Higher efficiency was achieved with 4% paraformaldehyde and 50% ethanol.

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

For this purpose it is also important to consider the fraction of the PNA probe that allows detection / identification of the existence of a stable complex formed by the probe and the target. Such a detectable fraction 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.

Different probes may be coupled to the same detectable group (eg a fluorophore) to detect bacterial species of the genus Lactobacillus spp. , while another optional probe with another different detectable molecule, 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, microorganisms in a sample are detected, identified or quantified for their bacterial genus by correlating the hybridization, performed under the appropriate hybridization conditions, of the PNA sequence to the target sequence. Consequently, the analysis is based on a single trial with a definitive opinion. In contrast, current routine methods for microorganism analysis are based on multiple characteristics involving multiple tests as described above.

It is further object of the present invention 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 performing in situ hybridization assays.

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

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

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

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

Brief Description of the Figures

For an easier understanding of this application,

if attached figures, at which are, represent achievements Preferred whereas, however, do not intend to limit the technique disclosed here • Figure 1 Detection of gene 16S rRNA from species Lactobacillus spp. (The) and Gardnerella spp. (B),

respectively, in vaginal exudate samples (S01 to S10).

Caption - λ, lambda DNA marker; -, negative PCR control; +, PCR positive internal control; S01 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 used the term nucleotide sequence is the same as referring to a segment of a polymer that contains subunits, in this case the nucleotides.

c) The term target sequence means a nucleotide sequence of bacterial species belonging to the genus Lactobacillus spp. or Gardnerella spp. which is intended to be detected in the assay, where the nucleotide portion of one of the probes is designed to hybridize.

d) The term PNA probe means a PNA subunit polymer that 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 backbone is replaced by an electrically neutral, achiral formed of repeated N- (2-aminoethyl) glycine units.

e) When the term detectable fraction is used, it refers to molecules that can be attached to the probe to thereby make the probe detectable by an instrument or method.

f) The term sample refers to any biological sample that may contain the target microorganism or sequence for detection. Preferably the biological samples are in liquid form (e.g., water, food, blood, vaginal exudate, urine, etc ...) or as a tissue sample (e.g., biopsy specimens, namely vaginal biopsies).

II - Description

PNA Probe Design:

PNA probes of this invention target oligonucleotides belonging to 16S rRNA zones of 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 bacterial genus under study. The Lac663 probe (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 Lactobacillus spp. strain MDL2 (Genebank Number: 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 Gardnerella vaginalis 409-05 strain rRN16 sequence (RDPII number: S001872672).

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

Table 1 - PNA probes of this invention.

SEQ ID No. Target Organisms 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 ' 5th Gardnerella spp. 5'-ATG CTC CAG AAT AGC-3 ' 6th Gardnerella spp. 5'-GCT CCA GAA TAG CTC-3 '

Alternatively, this invention also contemplates variations in probe nucleotide sequences. Such changes may include deletions, insertions, among others. Generally speaking, a nucleotide sequence homology of 80% or above, is sufficient for the proper functioning of the method.

Fraction detectable PNA probe:

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, radioisotopes, enzymes, hapten, chemiluminescent compound among others.

As an example, among the class of fluorophores are preferable for use (but not limited to): fluorophores from Alexa series, 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 discussed in EXAMPLE 1). Preferably, fluorescent in situ hybridization (FISH or PNA-FISH) or real-time PCR are assay formats for the analysis of Lactobacillus spp. and / or Gardnerella spp ..

The method can thus be divided into: sample preparation, cell fixation, hybridization, washing and visualization of results (see example 1). The method may be performed on adhered or suspended cells.

Hybridization Conditions

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

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 is to another non-target sequence in the sample, the more accurate the definition of the different factors influencing hybridization will have to be. In this invention non-target sequences may have only one nucleotide difference from the target sequences (since the genus Lactobacillus spp. Is in a family with several related bacterial genera and sharing a high genomic similarity), requiring a high degree of genomic similarity. level of discrimination to avoid non-specific hybridization of the PNA probe to non-target sequences. Probes of this invention that hybridize to target sequences relative to species of two distinct bacterial genera can be used together, as both bacterial genera are in vaginal exudates. Optionally, however, each probe type (for Gardnerella spp. Or Lactobacillus spp.) Can be used uniquely or uniquely, along with optimized hybridization conditions, without performing nonspecific binding on the target sequences of the remaining probes.

Sample Preparation:

The samples to be analyzed may come from vaginal exudates, blood, urine, among others. For vaginal exudates, swabs with vaginal samples are vortexed in 1 ml saline (0.9% NaCl) or phosphate buffer (Phosphate Buffer 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 saline (0.9% NaCl) or phosphate buffer (PBS). 20 to 50 Dl of the above suspension is collected and placed on immunofluorescence slides. If the bacteria under study are in more complex samples, such as urine, milk and blood samples, the samples are centrifuged at 10,000 rpm for 5 min at room temperature, discarding the supernatant and resuspending the decanted cells. in 1 ml saline (0.9% NaCl) or phosphate buffer (PBS). Eventually, samples may further dilute 1 to 10 again in saline or PBS to eliminate potential autofluorescent compounds present in the initial samples. Alternatively, the suspensions may further be filtered through 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 also important to note that saline and PBS buffer should be pre-sterilized by autoclaving at 121 ° C for 15 to 20 minutes.

Case:

The present invention contemplates a kit for carrying out the assay that analyzes the presence and quantity of species of the genus Lactobacillus spp. and / or Gardnerella spp. .

The PNA probes to be used in the kit, their characteristics, the method involved were previously referred to herein.

The kit in this invention comprises at least two probes, the first of which comprises one of the nucleotide sequences at least 80% identical to SEQ ID Nos. 1 to 3 (recognize Lactobacillus spp. Species); while the second comprises one nucleotide sequence at least 80% identical to SEQ ID Nos. 4 to 6 (recognize Gardnerella spp. Species). Importantly, in this invention it also comprises the other reagents or compositions that are selected to perform the assay.

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 organism analysis or analysis of nucleic acids extracted or derived from 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 application steps of the invention, are preferred embodiments of the present invention, not intended to be limiting in any of them:

EXAMPLE 1: Detection of Lactobacillus spp. and / or Gardnerella spp ..

Table 2 - Sequences of the PNA probes used.

Target Organisms Probe nucleotide sequence Lactobacillus spp. Lac663 Alexa 488-OO-ACA TGG AGT TCC ACT Gardnerella spp. Gardl62 Alexa 594-OO-CAG CAT TAC CAC CCG

Caption - 00 = 8-amino-3,6-dioxaoctanate (double bond which bonds the two molecules, the fluorophore and the nucleotide sequence of the probe).

Alexa Fluor 488nm / 594nm - fluorophore (detectable fraction).

Bacterial Strains:

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

All bacterial species in the collection were incubated 20-24h in fresh growth medium plates prior to laboratory experiments under ideal growth temperature and atmospheric conditions (aerobiosis, anaerobiosis and microfilia) for each particular case. The species Lactobacillus spp. were grown in plates of Man, Rogosa and Sharpe agar (MRS), while Atopobium spp. and Gardnerella spp. grown on Brucella Blood Agar (BBA). The remaining bacterial species were inoculated into Brain Heart Infusion Agar (BHI).

For each bacterial strain, one drop of the culture was placed on 8 mm diameter immunofluorescence slides.

The slide was then placed about 10 minutes in the oven at 60 ° C.

Fixation:

In order to prevent loss of 16S RNA 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% (wt / vol) dextran sulfate, 10 mM NaCl, 30% (vol / vol) formamide, 0.1% (wt / vol) sodium pyrophosphate, 0.2% (wt / vol) polyvinylpyrrolidone, 0.2% (wt / vol) Ficol, 5 mM disodium 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. Then the slides were placed in the oven for 90 minutes. During this time, probes could penetrate cell membranes and bind to complementary 16S rRNA sequences. During the hybridization period, it is essential that the hybridization solution does not evaporate. For this, the presence of the coverslip and wetted paper around the slide was required.

Washing:

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

Results:

The results were obtained by observing the fluorescence microscope with filters suitable for the detection of probe-bound fluorochromes (ie, wavelengths at which the probe-coupled fluorochromes are emitted), and no signal was detected in situations where the target sequence is not present.

The filter 488 allows to capture fluorescence promoted by the fluorochrome Alexa 488, which reveals the hybridization of Lacto663 probe (SEQ ID No. 1) with the characteristic target sequence of the genus Lactobacillus spp. . Filter 594, in turn, captures the fluorescence promoted by the fluorochrome Alexa 594, which reveals Gardl62 probe hybridization (SEQ ID No. 4) with the characteristic target sequence of the genus Gardnerella spp.

Table 3 - Fluorescence microscope results with filters suitable for detecting fluorochromes attached to probes.

Bacterial Species Strain Genus Lac663 Probe efficiency Gardl62 Probe efficiency L. pentosus CECT4023 Lactobacillus spp. ++++ L. married CECT5275 Lactobacillus spp. ++++

L. rhamnosus CECT288 Lactobacillus spp. ++++ L. coryníformís sub. torquens CECT4129 Lactobacillus spp. L. paracasei CECT227 Lactobacillus spp. ++++ L. acidophilus ATCC4356 Lactobacillus spp. ++++ L. agilis CCUG 31450 Lactobacillus spp. ++++ L. anmalis ATCC35046 Lactobacillus spp. +++ L. bifermentans ATCC35409 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. delbrueckii sub. delbrueckii ATCC9649 Lactobacillus spp. +++ L. delbrueckii sub. lactis ATCC12315 Lactobacillus spp. +++ L. farcimínís DSM20182 Lactobacillus spp. ++++ L. fructivorans ATCC8288 Lactobacillus spp. +++ - L. gallinarum CCUG31412 Lactobacillus spp. ++++ L. gasseri ATCC9857 Lactobacillus spp. ++++ - L. gramínís DSM20719 Lactobacillus spp. ++ - L. hamsteri ATCC43851T Lactobacillus spp. +++ - L. helveticus ATCC15009 Lactobacillus spp. ++++ - L. hílgardíí NCFB962 Lactobacillus spp. +++

L. Intestlnalls ATCC49335 Lactobacillus spp. +++ L. johnsoníí ATCC11506 Lactobacillus spp. ++++ L. murinus ATCC35020 Lactobacillus spp. ++++ L. parabuchnerí ATCC12936 Lactobacillus spp. ++++ L. paracasei sub. paracasei CCUG27320 Lactobacillus spp. +++ L. plantarum NCIMB8827 Lactobacillus spp. +++ L. reuteri NCFB2656 Lactobacillus spp. +++ L. rhamnosus ATCC7469 Lactobacillus spp. ++++ L. ruminis ATCC27781 Lactobacillus spp. ++++ L. sakei sub. carnosus CCUG8045 Lactobacillus spp. ++ L. sallvarius DEVRIESE94 / 438 Lactobacillus spp. +++ L. plantarum NCCB46043 Lactobacillus spp. +++ Lactococcus lactis 53 Lactococcus spp. - / ++ Streptococcu s thermophílus A Streptococcus spp. Streptococcu s thermophilus B Streptococcus spp. +++ Leuconostoc mesenteroí s Leuconostoc spp. - / + Bacillus subtilis DSM 7-10 Bacillus spp. Enterococcus faecíum CECT410 Enterococcus spp. Enterococcus faecalis CECT184 En terococcus spp. - - Gardnerella vaglnalls 51 - Gardnerella spp. - ++++ Gardnerella vaginalls 101 Gardnerella spp. ++++

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. - Staphylococc us aureus CECT 976 Staphylococcus spp. - Staphylococc us aureus CECT 86 Staphylococcus spp. - Shigella ATCC 12022 Shigella spp. - - Listeria monocytogene s Listeria spp. Klebsiella pneumoniae sub. Ozaenae ATCC 11296 Klebsiella spp. Salmonella Typhi Salmonella spp. - Listeria seeligeri CECT 917 Listeria spp. - Escherichia coli CECT 434 Escherichia spp. - Listeria monocytogene s CECT 5873 Listeria spp.

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

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

Results:

Results were obtained by fluorescence microscope observation with filters suitable for detection of fluorochromes attached to the probes.

Table 4 - Results of the mixed samples under the fluorescence microscope with filters suitable for the detection of probe-bound fluorochromes.

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. críspatus; G. vaginalis ATCC ATCC33820; - ++++ ++++ L. delbrueckii sub. delbrueckií; 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 triplicate experiments for each of the bacterial species analyzed.

EXAMPLE 2: Detection of 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 by simultaneously using two PNA probes on mixed samples, ie with two or more bacterial strains.

Since the purpose of the present invention is rapid identification / quantification of species

Lactobacillus spp. and / or Gardnerella spp.

in complex samples.

To this end, a considerable number of mixed samples were performed from the bacterial strains used in EXAMPLE 1 (see table 4). For the effective accomplishment of the FISH multiplex protocol, the same parameters as above were re-tested to obtain the maximum efficiency of the desired assay. In fact, the efficiency remained maximal under the same conditions as in the EXAMPLE 1 protocol. Therefore, the hybridization protocol used for the multiplex was the same as that of EXAMPLE 1.

The probes used in this analysis of bacterial genera in mixed samples were the same probes previously referred to in the table of

EXAMPLE

1. However, in this assay, the aliquot of use consisted of producing a single aliquot with the two PNA probes, more specifically the Lacto663 probe (SEQ ID probe).

Gardl62 (SEQ ID) some changes can be made to optimize the visualization of the probes together in the same assay, namely the exposure time for each filter to maximize the fluorescence of the hybridized probe. mixed samples by the fluorochrome probe with lower excitation energy, in this case the Gardl62 probe, avoiding involuntary excitation of the other multiplex assay PNA probe, namely the Lac663 probe.

This example is intended to illustrate the possibility of directly detecting Lactobacillus spp.

Gardnerella spp. from vaginal exudates.

Since the purpose of the present invention is rapid identification / quantification of species

Lactobacillus spp.

and Gardnerella spp. in clinical samples, among others.

In addition, a considerable number of vaginal exudates were acquired (vaginal samples from ten control volunteers, S01 to FISH experimental, swabs with vaginal exudates were previously vortexed into 1 ml saline (0.9% NaCl) or phosphate buffer (Phosphate minutes at room temperature. At 10,000 rpm for 5 then the supernatant was discarded and the decanted cells were resuspended in 1 min.

NaCl) or phosphate buffer the rest of the procedure was similar to that previously reported in the bacterial strains of collection.

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

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

Results:

Results were obtained by fluorescence microscope observation with filters suitable for detection of fluorochromes attached to the probes.

Table 4 - Results of the mixed samples under the fluorescence microscope with filters suitable for the detection of probe-bound fluorochromes.

Species present in mixed samples Strain code in mixed samples Multiplex with PNA Probes Lac663 Probe efficiency Gardl62 Probe efficiency L. pentosus; G. vagínalís 51 CECT4023; - ++++ ++++ L. casei; G. vagina 101 CECT5275; - L. rhamnosus; G. vaginal vagina AMD CECT288; - ^{++++ ++++} L. críspatus; G. vaginaalis ATCC ATCC33820; - ^{++++ ++++} L. delbrueckii sub. delbrueckii; Atopobum 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. reuterí; E. coli 0157: H7 NCFB2656; NCTC 12900 +++ - S. aureus; G. vagínalís 51 CECT 976; - ++++ Shigella; G. vaginálís 101 ATCC 12022; - ⁺⁺⁺⁺ E. aerogenes; G. vaginaalis ATCC CECT 684; - ⁺⁺⁺⁺ L. pentosus; G. vagina anal ATCC; E. faecalís CECT4023; ^- f CECT184 L. casei; G. vaginal vagina AMD; A. vaginae CECT5275; ^- f CCUG 38953T L. rhamnosus; G. vaginalis 101; A. vaginae CECT288; ^- f CCUG 42099 ++++ ++++ L. críspatus; G. vagínalís 51; A. vaginae ATCC33820; ^- f CCUG 44116 L. casei; L. mesenteroids; A. vaginae CECT5275; f CCUG 38953T

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

Table 5 - Results of vaginal exudate samples under the fluorescence microscope with filters suitable for detection of probe-bound fluorochromes.

No. of samples vaginal code of sample Lac663 Probe efficiency Gardl62 Probe efficiency 1 S 01 ++ + ++ r 2 The Ú di +++ + - 3 S 3 +++ 4 S04 + -Γ ++ r 5th S05 + - 6th S 0 6 ++ 7th S G 7 -i- + 8th S 0 8 +++ ++ and 9th S09 + -Γ -r + 1 0 S10 -

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

For 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 ~ 62 bp band in figure 1 (a), while the presence of Gardnerella spp. was proved by the band of ~ 111 bp in figure 1 (b). All samples underwent heat shock pretreatment, which consisted of 5 minutes at 100 ° C followed by 5 minutes on ice, before performing 30 cycles PCR at 50 ° C (melting temperature, tm).

The PCR results were consistent with the results previously obtained by FISH (Table 5).

LIST OF SEQUENCES <110> UNIVERSITY OF MIN <120> NUCLEIC PETTITE ACID PROBES, KIT AND METHOD TO DETECT SPECIES OF LACTOBACILLUS SPP. AND / OR GARDNERELLA SPP. AND APPLICATIONS <210> SEQ ID NO 1

5'-ACA TGG AGT TCC ACT-3 ' (SEQ ID No. 1), and have a length between 8 to 18 nucleotides <210> SEQ ID NO 2 5'-AGG CTC GAA AGC ATG-3 ' (SEQ ID No. 2), and have a length between 8 to 18 nucleotides <210> SEQ ID NO 3 5'-TTC TCA GTT CGG ACT-3 ' (SEQ ID No. 3), and have a length between 8 to 18 nucleotides <210> SEQ ID NO 4 5'-CAG CAT TAC CAC CCG-3 ' (SEQ ID No. 4), and have a length between 8 to 18 nucleotides <210> SEQ ID NO 5 5'-ATG CTC CAG AAT AGC-3 ' (SEQ ID No. 5), and have a length between 8 to 18 nucleotides

<210> SEQ ID NO 6

5'-GCT CCA GAA TAG CTC-3 '(SEQ ID NO: 6), and have a length of 8 to 18 nucleotides

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.

Claims (14)

1. PNA probe characterized by detecting / quantifying the presence of species of the genus Lactobacillus spp. and / or Gardnerella spp., The sequences of structurally semc which comprises at least one of 80% nucleotides slhante at SEQ at least ID We. 1 to 6. 2. PNA probe, of a deal with The claim 1, characterized in that it detects the target sequence in rRNA, rDNA or complementary RNA sequences of species of the genus Lactobacillus spp. and / or Gardnerella spp. . PNA probe according to any one of claims 1 to 2, characterized in that it is linked to at least one type of detectable fraction. PNA probe according to Claim 3, characterized in that the type of detectable fraction 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 luminescent compound. PNA probe according to Claim 4, characterized in that the fluorophore group is at least one of the following: Alexa series 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, further comprising at least one of the following solutions: a fixation solution, a hybridization solution and a wash solution. A kit according to claim 7, characterized in that the fixing solution comprises paraformaldehyde and ethanol, namely 2-8% (weight / vol) paraformaldehyde and 25-75% (vol / vol) ethanol. A kit according to claim 8, characterized in that the hybridization solution comprises formamide. 10. Method of detection of species of the genus Lactobacillus spp. and / or Gardnerella spp. in biological samples, characterized in that it uses at least one of the PNA probes described in any one of the preceding claims and comprises the following steps: The. 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 hybridization as indicative of said detection and quantitation in said samples. Method according to claim 10, characterized in that said biological sample comes from vaginal exudates, blood, food, water or biopsies. Method according to Claim 11, characterized in that the hybridization is by fluorescence. Use of PNA probes as described in any one of claims 1 to 5, characterized in that they are applied in a detection methodology to 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 it is applied for the detection of 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 for the detection of species of the genus Lactobacillus spp. and / or Gardnerella spp. in biological samples.