Primers, control probes, method and kit for diagnosis of urinary tract infections

Abstract

The present invention relates to primers and control probes for the detection of pathogenic bacteria responsible of urinary tract infections by LAMP-PCR.

Description

[0001] PRIMERS, CONTROL PROBES, METHOD AND KIT FOR DIAGNOSIS OF URINARY TRACT INFECTIONS

[0002] Technical Field

[0003] This application relates to the field of biotechnology and specifically primers and oligonucleotide probes for use in a relevant method and kit for the simultaneous and real-time amplification and quantification of nucleic acid molecules and the subsequent simultaneous and unambiguous identification of one or more pathogens (viruses and / or bacteria) such as Escherichia coli (spp), Proteus mirabilis (spp), Klebsiella pneumoniae (spp), Enterococcus faecalis (spp) and / or the presence of one or more mutations in said pathogens associated with antibiotic resistance comprising the so-called CTX-M group 1 and the so-called CTX-M group 9.

[0004] Background Art

[0005] Urinary Tract Infections (UTIs) are a wide range of infections that affect any part of the urinary tract, which comprises the kidneys, ureters, bladder and urethra. UTIs are one of the most common infections in the world, affecting millions of people each year causing increased costs to health care systems and contributing to the emergence of antibiotic-resistant bacterial strains (Agudelo, J. et al. (2018), Journal of the American Medical Association, 320(11), 1125-1134; Ferreira, E. M. et al. (2019), Clinics, 74(11), el438; Kumarasamy, N. et al. (2010), Antibiotic resistance: Global report on surveillance. World Health Organization).

[0006] UTIs can be caused by a variety of bacteria, among which the main and most common ones are Escherichia coli (spp), Proteus mirabilis (spp), Klebsiella pneumoniae (spp) and Enterococcus faecalis (spp) (Nat Rev Microbiol. 2015 May; 13(5): 269-284). Bacteria enter the urethra through the skin or rectum and infect the urinary tract. Infections can affect different parts of the urinary tract, but the most common type is bladder infection (cystitis). Kidney infection (pyelonephritis) is another type of UTIs. These are less common but more serious than bladder infections (https: / / www.cdc.gov / antibiotic- use / uti.html#:~:text=UTIs%20are%20common%20infections%20that,is%20ano ther%20type%20of%20UTI). The prevalence of bacteria producing CTX-M extended spectrum 0-lactamase (ESBL) has increased worldwide, and some of them have become a major cause of infections such as those of the bloodstream or of the urinary tract (UTI). In this regard, a loop-mediated isothermal amplification (LAMP) assay is known for the simple, rapid and sensitive detection of the two most common CTX-M groups, namely CTX-M groups 1 and 9 (Rivoarilala O.L. et al. (2018), PLoS One, 13(7): e0200421).

[0007] The overuse and often misuse of 0-lactam antibiotics are the most important factors promoting the selection and emergence of 0-lactam-resistant bacteria in both human and veterinary medicine. The first 0-lactamases, TEM-1, TEM-2 and SHV-1, were found in strains of Escherichia coli and Klebsiella pneumoniae and described in the early 1960s, about 15 years after the widespread use of penicillin. A few years later, these 0-lactamases spread worldwide and were found in several species of Enterobacteriaceae. TEM-1, TEM-2 and SHV-1 were named narrowspectrum 0-lactamases because of their ability to hydrolyze penicillin and the first generation of cephalosporins, such as cephalothin, cephaloridine or cefazolin. Twenty years later, new 0-lactam antibiotics, particularly extended spectrum cephalosporins, were developed to mitigate the emergence of these narrowspectrum 0-lactamase enzymes. However, selective pressure from overuse of broad spectrum cephalosporins has selected new 0-lactamase variants. These enzymes are known as extended spectrum 0-lactamases (ESBLs) because of their broader spectrum of activity. However, ESBL-producing bacteria remain sensitive to carbapenems, cefamycins (e.g., cefoxitin) and 0-lactamase inhibitors such as clavulanic acid. ESBLs are usually mediated by plasmids that facilitate their transfer among bacteria. Currently, more than 600 ESBLs have been described, most of which belonging to the CTX-M families (for cefotaximase) and to the mutants of 0-lactamases TEM-1 / 2 and SHV-1. The 0-lactamases of the TEM-, SHV-, and CTX-M-type belong to a fairly heterogeneous lineage of molecular class A 0-lactamases with active site in serine. The 0-lactamases of the CTX-M type can be further differentiated into at least six sub-lineages or groups, namely CTX-M- 1, CTX-M-2, CTX-M-8, CTX-M-9, CTX-M-25 and KLUC. ESBLs are among the most significant determinants of resistance that are spreading worldwide. Several studies have confirmed the spread of 0-lactamases of the CTX-M type in many countries, especially CTX-M-14 (CTX-M group 9) and CTX-M-15 (CTX-M group 1) enzymes. Diagnosis of resistance to ESBLs is recommended for the treatment of patients and necessary for surveillance.

[0008] Therefore, the public health system needs to rapidly track and monitor the presence of urinary tract infections (Ferreira, E. M. et al. (2019), Clinics, 74(11), el438).

[0009] UTIs can be diagnosed by means of urinalysis or ultrasound of the urinary tract. Traditional diagnostic methods for UTIs are based on the identification of bacteria in urine. These methods include:

[0010] Urine test in clinical chemistry. Urinalysis is a laboratory test used to detect the presence of bacteria, white blood cells and other signs of infection in urine.

[0011] Urine culture: Urine culture is a laboratory test used to grow bacteria in urine. This test allows identifying the type of bacteria causing the infection and determining its sensitivity to antibiotics.

[0012] Traditional diagnostic methods can be useful in diagnosing UTIs, but they may have some limitations. For example, urinalysis may be negative even if an infection is present, especially in the early stages of infection. Urine culture, on the other hand, can take several days to provide results, which can delay the start of treatment or, worse yet, if treatment is needed immediately, can lead to the use of broad-spectrum antibiotics on resistant or insensitive bacteria.

[0013] In recent years, several alternative diagnostic methods for UTIs have been developed. These methods are based on innovative technologies which allow detecting bacteria in urine more quickly and accurately than traditional methods. One such technology is the polymerase chain reaction (PCR). PCR is a laboratory method which allows selectively amplifying a DNA fragment. PCR can be used to detect the presence of bacteria in urine by amplifying a bacterium-specific DNA fragment. Another alternative diagnostic method for UTIs is rapid molecular diagnosis (RMD). RMD is a laboratory method which allows rapid identification of bacteria in urine using a combination of technologies, including PCR and mass spectrometry.

[0014] PCR and RMD are promising diagnostic methods for UTIs. These methods are faster and more accurate than traditional methods and can be used to diagnose UTIs even in the early phases of infection (Arora, A. et al. (2022), Indian Journal of Urology, 38(1), 1-11; Chowdhury, A. et al. (2022), International Journal of Pediatrics, 10(2), 223-232).

[0015] As known, the genetic structure of an organism is organized into triplets of nitrogenous bases (four types: Adenine, Guanine, Cytosine and Tyrosine or Uracyl), phosphate groups and sugars (two types: Deoxyribose and Ribose) arranged in filaments with direction (way) 5’ - 3’ (where, by biological convention, the end 5’ represents the head or beginning of the filament and the end 3 ’ represents the tail thereof), named nucleic acid, the nature of which can be DNA (if they contain Tyrosine and deoxyribose as sugar) wherein each molecule is formed of two strands with antiparallel pattern complementary to each other so as to distinguish them into parallel and antiparallel strands, or RNA (if they contain Uracyl and ribose as sugar), each molecule formed of single strands, with varying structure, length and content depending on the organism itself.

[0016] As part of diagnostic or research needs, nucleic acid molecules can be studied and specific genetic markers (or parts thereof) can be highlighted. In addition to sequencing, which allows total reading and recognition of the nucleic acid strands, the main detection of DNA strands, in the diagnostic context, is done by PCR (Green, M.R. et al. (2019), Cold Spring Harbor Protocols).

[0017] PCR is based on using the ability of the enzyme DNA polymerase to synthesize (copy / amplify) a new DNA strand which is complementary to the template strand. Since DNA polymerase can add a nucleotide only on a pre-existing group 3’, it needs a known sequence primer to which the first nucleotide can be added. This requirement makes it possible to delineate a specific region of the template sequence that the researcher wishes to amplify. Each amplification cycle will produce an identical copy of the complementary strand with respect to the strand to be amplified. At the end of x number of amplification cycles, the amount of copies obtained will be equal to 2x, by means of a procedure described by the term logarithmic exponential amplification.

[0018] At the end of each cycle of the PCR reaction, the specific sequence outlined at the ends by the primer sequences also named “amplicon” will be recognized by specific probes labeled with fluorescent molecules. The molecule will not fluoresce until the subsequent phase of elongation by polymerase releases the fluorophore. Binding to the labeled probe then will allow quantification of the newly amplified nucleic acid because the fluorescent signal generated will be, simultaneously and in real time, sensed by a dedicated instrument called a Real Time PCR thermocycler: the excitation and emission signal is distinctive and detected by an optical group in the instrument.

[0019] The increase in fluorescence (which will occur in a linear fashion until the elements needed for copying are completely depleted) will be measured by the increased presence of the specific nucleic acid sequence that is to be traced (Yoshimura, M. et al. (2005), Methods in molecular medicine).

[0020] An advanced methodology falling under the family of quantitative real-time reverse-transcription polymerase chain reactions (qRT-PCR) includes the allelic discrimination assay (Malkki, M. et al. (2012), Methods in Molecular Biology) wherein, in addition to the components used for classical qRT-PCR, oligonucleotide sequences are added that compete with each other for binding to a specific region of interest if it differs from the discriminated form by one or more mutations and / or deletions and / or base insertions.

[0021] Loop-mediated isothermal amplification polymerase chain reaction (LAMP- PCR) is a nucleic acid amplification technique that uses constant temperature amplification. LAMP-PCR is different from traditional PCR in that it does not require heating and cooling cycles. Instead, LAMP-PCR uses a series of four enzymes working together to amplify the target nucleic acid at a constant temperature of 65 °C. LAMP-PCR is a rapid and sensitive technique which can be used for a variety of applications, including diagnostics, research and bioengineering (Zhang, X. et al. (2012), Methods 58(3), 164-172. Wang, Y. et al. (2014), Analytical and Bioanalytical Chemistry, 406(12), 3519-3531. Li, L. et al. (2013), Analytical and Bioanalytical Chemistry, 405(11), 3493-3503.).

[0022] The L AMP-related benefits are:

[0023] Shorter reaction time: LAMP-PCR takes only 30-60 minutes, compared with 2-4 hours required for traditional PCR.

[0024] Increased sensitivity: LAMP-PCR can detect very low amounts of nucleic acid, down to 0.5 copies.

[0025] Lower complexity: LAMP-PCR requires fewer reagents and equipment than traditional PCR.

[0026] Along with the ability to detect the presence of the initial strains of major pathogens as etiologic causes of UTIs, it is therefore critical to detect the presence of any antibiotic resistance.

[0027] In light of what is known in the state of the art, there is a strong need to develop new effective diagnostic methods for simultaneous and unambiguous detection of one or more pathogens (viruses and / or bacteria) such as Escherichia coli (spp), Proteus mirabilis (spp), Klebsiella pneumoniae (spp), Enterococcus faecalis (spp) and / or the presence of one or more mutations in said pathogens associated with antibiotic resistance by including the so-called CTX-M group 1 and the so-called CTX-M group 9 and in particular that they are also capable of detecting the presence of strains characterized by antibiotic resistance, characterized by specific mutations and / or deletions and / or insertions which allow the detection thereof, since the currently available tests are not capable of detecting the presence thereof effectively and efficiently.

[0028] It is now clear that in patients with infection a slow diagnosis in urinary tract infections (UTIs) can have serious consequences for the patient. In particular, it can lead to: Risk of infection progression: If the infection is not treated early, it can spread to deeper parts of the urinary tract, such as the kidneys. This can lead to more serious complications, such as pyelonephritis.

[0029] Risk of developing recurrent infections: Slow diagnosis may increase the risk of developing recurrent urinary tract infections.

[0030] Risk of developing antibiotic resistance: If the infection is not treated properly, bacteria can become resistant to antibiotics.

[0031] This may make it more difficult to treat future infections (Agudelo, J. Et al. (2018), Journal of the American Medical Association, 320(11), 1125-1134. Ferreira, E. M. et al. (2019), Clinics, 74(11), el438. Gupta, K. et al. (2019).

[0032] In addition, slow diagnosis can result in:

[0033] Prolonged discomfort and pain: UTI symptoms, such as pain or burning during urination, can be very uncomfortable. Slow diagnosis can prolong these symptoms, causing discomfort and pain for the patient.

[0034] Loss of productivity: UTI symptoms can make it difficult to concentrate or perform daily activities. Slow diagnosis can lead to a loss of productivity for the patient.

[0035] Higher economic costs: UTI treatment can be expensive, especially if the infection progresses or becomes recurrent. Slow diagnosis can lead to higher economic costs for the patient.

[0036] The need for rapid, highly informative diagnostics with sensitivity levels close to 100% highlighted by the situation in the UTIs also finds application in additional scenarios given by diseases, often endemic, for which the current diagnosis brings with it the same limitations and problems described above (Agudelo, J. et al. (2018), Journal of the American Medical Association, 320(11), 1125-1134; Ferreira, E. M. et al. (2019). Clinics, 74(11), el438; Gupta, K. et al. (2019), UpToDate).

[0037] Description of the Invention

[0038] The inventors of the present invention have identified specific primers and oligonucleotide probes that allow for the identification and amplification of pathogens such as Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Enterococcus faecalis and / or the presence of one or more mutations in said pathogens associated with antibiotic resistance including the so-called CTX-M group 1 and the so-called CTX-M group 9.

[0039] Therefore, the present invention is an optimal and necessary solution to provide for rapid, safe, efficient and cost-effective diagnostic tools that addresses a real and immanent need in the monitoring and rapid diagnosis of the etiological agents of urinary tract infections which can help fix other critical health issues.

[0040] The invention is an alternative to assays currently in use for the detection and monitoring of urinary tract infections, including current PCR and LAMP PCR techniques.

[0041] The aforementioned objects are achieved by the sequences SEQ.ID 1 to 44.

[0042] A further object of the present invention is the use of sequences selected from the consistent group of SEQ.ID 45 to 62, and mixtures thereof as primers in nucleic acid amplification techniques, preferably in constant temperature loop-mediated polymerase chain amplification reaction techniques.

[0043] An additional object of the present invention is the use of the sequences selected from the consistent group of SEQ. ID 63 to 67.

[0044] A further object of the present invention is the use of the sequences selected from the consistent group of SEQ. ID 63 to 67 as control probes, to be used preferably and according to the technical choices as positive amplification standards in parallel reactions. The use of SEQ. ID 63 to 67 will allow calibrating the operation of the kit, whenever necessary, by means of nucleic acid identification and / or quantification techniques, more preferably constant temperature loop-mediated polymerase chain amplification reaction techniques.

[0045] Yet another object of the present invention is the use of the sequences selected from the consistent group of SEQ. ID 1 to 44 and 45 to 62 as primers in constant temperature loop-mediated polymerase chain amplification reaction techniques.

[0046] The present invention relates to a mixture comprising at least three sequences per type, preferably at least six, selected from the consistent group of SEQ. ID 1 to 62, and at least one control probe selected from the consistent group of SEQ. ID 63 to 67.

[0047] The present invention also relates to an in vitro method for simultaneous diagnosis of pathogenic etiological agents of urinary tract infections.

[0048] The method is carried out by constant temperature loop-mediated polymerase chain amplification reaction and does not require sample processing, or where any sample processing is carried out by filtration with a special syringe filter.

[0049] The procedure involves: a) Loading the sample into a pre-filled test tube containing the appropriate mixture of necessary probes, control probes, primers and reagents. b) Loading the test tube onto an instrument with thermocycler and analyzer functions. c) Initiation of the appropriate test protocol on the thermocycler (isothermal PCR). d) Reading the signal possibly generated by the presence of pathogens in the sample, within one hour after the start of the analytical protocol.

[0050] The present invention still relates to a kit for the simultaneous diagnosis of pathogens (bacteria) such as Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Enterococcus faecalis and / or the presence of one or more mutations in said pathogens associated with antibiotic resistance including the so-called CTX-M group 1 and the so-called CTX-M group 9 comprising: the sequence probe SEQ. ID. NO. 63, for the definition of the correct detection of the Escherichia coli (spp) target gene; the sequence probe SEQ. ID. NO. 64, for the definition of the correct detection of the Proteus mirabilis (spp) target gene; the sequence probe SEQ. ID. NO. 65, for the definition of the correct detection of the Klebsiella pneumoniae (spp) target gene; the sequence probe SEQ. ID. NO. 66, for the definition of the correct detection of the Enterococcus faecalis (spp) target gene; the sequence probe SEQ. ID. NO. 67, for the definition of the correct detection of one or more sequences of said pathogens associated with the antibiotic resistance including the so-called CTX-M group 1 and the so-called CTX-M group 9; a group of primers (at least three primers, preferably at least six) complementary to the parallel and antiparallel strands of a sequence Escherichia coli (spp), Proteus mirabilis (spp), Klebsiella pneumoniae (spp), Enterococcus faecalis (spp) selected from the consistent group of SEQ. ID. NO. 1 to 62; recombinant enzyme with polymerase functions; deoxyribonucleotide triphosphate; reaction buffer and suitable additives.

[0051] Brief Description of the Drawings

[0052] Additional characteristics will become clear from the detailed description that follows with reference to the proposed experimental data and attached figures.

[0053] Figure 1 shows the organization of the primers in the target sequence and the LOOP generation that mediates their amplification.

[0054] Figure 2 shows the PCR and LAMP techniques compared.

[0055] Embodiments of the Invention

[0056] With particular reference to these figures, a detailed description of the invention is given below.

[0057] Primers comprise: external primers (F3 and B3); internal primers (FIP and BIP); and loop primers (LF and LB).

[0058] Primers are designed according to the bacterial target of interest.

[0059] Primers are selected from the group consisting of sequences SEQ. ID. 1 to 62.

[0060] In particular, the sequences SEQ. ID. 1 to 44 contain degenerate sequences, N- type or others, aimed at identifying possible variants of the same sequence, without the need to update the kit composition from time to time.

[0061] In detail, degenerate sequences are mixtures of sequences of similar primers that incorporate and insist on actual or plausible variations at specific positions to account for the degeneracy in the genetic code. This approach is useful when the precise nucleotide sequence of the target DNA is unknown or when variable sequences might be present in the genetic code for various reasons. A useful solution that also allows for less sequence-specific recognition in such cases is to use a degenerate primer (with one or more degeneracies), which is a mixture of similar primers with different bases at the variable positions. Within a mixture of degenerate primers, only a limited number of primer molecules complete the template. Target regions are characterized by being greater than 50 bp (base pairs) and are selected from the consistent group of:

[0062] The controls for each region represent alternatives to each other. They may be used uniquely and exclusively based on the experimental needs.

[0063] The person experienced in the field will be able to appropriately choose the intercalants with which to detect the reaction from those known in the art and / or commercially available.

[0064] Optionally, the mixtures in addition to primers and probes and control probes contain suitable salts, enzymes and additives.

[0065] Salts, enzymes, reaction buffers and additives can be appropriately selected by the person skilled in the art from those known in the art and commercially available.

[0066] In one embodiment of the procedure, lOul of sample corresponding to amounts of DNA equal to about 10-50 ng are analyzed, and the reaction is carried out in a volume equal to 20 pl in which there are three primer pairs in the following concentrations: 0.2 pM external primers, 0.8 pM loop primers and 1.6 pM internal primers.

[0067] The reaction mixture also contains 2.0 pl buffer for 10 x Bst DNA polymerase enzyme [lx containing 20 mM Tris-HCl, 10 mM KC1, 10 mM (NHI)SO4, 2 mM MgSC , 0.1% Triton X-100], 1 pl of an 8-U / pl concentration of Bst DNA polymerase (New England Biolabs, Inc, MA), 2 pl 2 mM MgSCU, 5 pl betaine (Sigma-Aldrich, St. Louis, MO) and 5 pl of sample.

[0068] Test tubes or plates containing one target per well are loaded into the thermocycler instrument for the evaluation of the amplification and quantification in which reaction cycles occur.

[0069] The reaction conditions are: temperature of between 30°C and 95°C, preferably 60°C; time of between 0 and 60 min, preferably 60 min; number of cycles of between 1 and 50, preferably 1;

[0070] After each cycle, fluorescence is measured by software and normalized fluorescence (ARn) is calculated as a function of negative control. The result of amplification can also be detected through the use of intercalating dyes. These dyes include propidium iodide, ethidium bromide, methylene blue, acridine orange and Sybr green. Among the various dyes, Sybr green produced the best visual discrimination. For both dyes, 1 pl of the diluted dye was added to 19 pl of the reaction mixture to develop the color reaction.

[0071] In one preferred embodiment, the kit contains: at least three primers in the following concentrations:

[0072] 0.1-1 pM (0.2 pM) external primers (F3 and B3);

[0073] 0.2-2.2pM (0.8 pM) loop primers (LF and LB);

[0074] 0.8-3.8pM (1.6 pM) internal primers.

[0075] 2.5 pl of 10x Bst DNA polymerase reaction buffer [lx containing 20 mM Tris-HCl, 10 mM KC1; 10 mM (NH2)SO4, 2 mM MgSO4, 0.1% Triton X- 100];

[0076] 1 pl of an 8-U / pl concentration of Bst DNA polymerase (New England Biolabs, Inc, MA);

[0077] 2 mM MgSO4(2 pl), 5 pl betaine (Sigma-Aldrich, St. Louis, MO);

[0078] The embodiments for making the kits (of which, from the primer tables at least three alternatives, preferably at least six, will be selected from the .1 or .2 group) are listed below.

[0079] Mixture 1:

[0080] Primers:

[0081] Control probe:

[0082] Mixture 2: Primers:

[0083] Control probe:

[0084] Mixture 3:

[0085] Primers: Control probe:

[0086] Mixture 4:

[0087] Primers:

[0088] Control probe:

[0089] Mixture 5: Primers: Control probe:

[0090] Mixture 6:

[0091] Primers:

[0092] Control probe:

[0093] All mixtures contain suitable salts, enzymes and additives. Example 1

[0094] Samples are from urine samples, from patients who have signed an informed consent.

[0095] Samples are either processed directly or subjected to filtration with a “SuperSai” filter or similar.

[0096] For each reaction, 10 pl of sample (corresponding to about 10-50 ng of bacterial / viral genome) is used, and the final reaction carried out in a volume of 20 pl, the remaining 10 pl is made up as follows:

[0097] - 1.5pM of a primer solution with the following characteristics: 0.2 pM external primers, 0.8 pM loop primers and 1.6 pM internal primers.

[0098] - 2.0 pl of 10x Bst DNA polymerase reaction buffer [lx containing 20 mM Tris-HCl, 10 mM KC1, 10 mM (NH2)SO4, 2 mM MgSCU, 0.1% Triton X- 100],

[0099] - 1 pl of an 8-U / pl concentration of Bst DNA polymerase (New England Biolabs, Inc, MA), 2 mM MgSCti (2 pl),

[0100] - 5 pl betaine (Sigma-Aldrich, St. Louis, MO)

[0101] - hi-pure sterile water for molecular biology, up to volume completion.

[0102] Test tubes or plates containing one target per well are loaded into the thermocycler instrument for the evaluation of the amplification and quantification in which the reaction cycles occur. Reaction cycles then take place in the thermocycler instrument.

[0103] The software analyzes the fluorescence level after each cycle and calculates the normalized dye fluorescence (ARn) depending on the negative control.

[0104] The reaction consists of a polymerase amplification reaction using primers specific to the region that wants to be sampled by probes for the presence of the pathogen(s) (virus and / or bacteria) or for the presence of the specific mutations. Bacterial genomic regions are deposited on open source GISAID database. Total sequences are converted to FASTA sequences and analyzed using Serial Cloner 2.6 software (Serialbasics).

[0105] The probes that are used to increase the specificity of LAMP PCR are designed to consist of sense and antisense sequences from the region they belong to. The detection can be done with intercalants.

[0106] In that case, the oligonucleotide is intercalated with a fluorophore with emission, depending on the fluorophore, at a wavelength of between 513 nm and 707 nm for absorption of between 490 n and 683 nm. The fluorophore will emit the signal only at the time when it is intercalated.

[0107] The resulting primers were evaluated by melting temperature, stability, length, formation of homo- and hetero-dimers and formation of hairpain sequences. In silico analysis was carried out with the IDTdna Oligoanalyser tool (IDTdna) software. All sequences that passed this quality control were then analyzed with NCBI’s Primer-Blast software in order to exclude redundant sequences within other organisms or other regions of the same pathogen and to associate, with each primer or probe sequence, the uniqueness of binding on all genome deposition databases of all the already sequenced species. The sequences of the specific genes were studied according to the same procedures described above, isolated and conceived for the primer and probe design and evaluated from a qualitative and unique point of view according to the same methodology as described above. For antibiotic resistances, the same procedure was used starting from the regions on GISAID which were obtained and cloned.

[0108] It has in practice been ascertained that the described invention achieves the intended objects.

Claims

CLAIMS1) One or more of the sequences of SEQ. ID. NO. 1 to 44 and 45 to 62.2) Use of at least one of the sequences of SEQ. ID. 1 to 62 as primers in nucleic acid amplification techniques.3) Use of at least one of the sequences of SEQ. ID. 63 to 67 as control probes in nucleic acid amplification techniques.4) Use of at least three of the sequences of SEQ. ID. 1 to 62, at least one of the sequences of SEQ. ID. 63 to 67, mixtures thereof, in nucleic acid amplification techniques.5) Use according to claim 4, wherein fluorescent intercalants are present in the mixture where there are the sequences.6) Use according to any one of claims 2 to 5, wherein the nucleic acid amplification technique is constant temperature loop-mediated polymerase chain amplification reaction.7) Mixture comprising at least three primer sequences selected from the consistent group of SEQ. ID. NO. 1 to 62, and at least one control probe selected from the consistent group of SEQ. ID .NO. 63 to 67.8) Mixture according to claim 7, characterized by the fact that it comprises a fluorescent intercalant.9) Mixture according to claim 7 or 8, characterized by the fact that it comprises salts, enzymes and additives.10) In vitro method for the simultaneous diagnosis of pathogenic etiological agents of the urinary tract infections comprising the following phases: supply of a urine sample; mixing of said urine sample with at least three primer sequences selected from the consistent group of SEQ. ID. NO. from 1 to 62, and at least one probe selected from the consistent group of SEQ. ID. NO. from 63 to 67; amplification of the nucleic acid sequences coupled to the probes by means ofLAMP-PCR; quantification of the amplification product.11) Method according to claim 10, characterized by the fact that that it does notcomprise a phase of DNA extraction from said urine sample.12) Method according to claim 10, characterized by the fact that said phase of supply comprises a step of processing said urine sample by means of filtration.13) Method according to claim 10, characterized by the fact that said phase of mixing comprises a step of adding at least one fluorescent intercalant.14) Kit for the simultaneous in vitro diagnosis of pathogens, particularly Escherichia coli (spp), Proteus mirabilis (spp), Klebsiella pneumoniae (spp), Enterococcus faecalis (spp) and / or for the detection of one or more mutations in said pathogens associated with antibiotic resistance, particularly CTX-M group 1 and CTX-M group 9, comprising:- the probes of SEQ. ID. NO. 63 to 67, for the detection of at least one of: the Escherichia coli target gene (spp), the Proteus mirabilis target gene (spp), the Klebsiella pneumoniae target gene (spp), the Enterococcus faecalis target gene (spp), one or more mutations in said pathogens associated with the antibiotic resistance including the so-called CTX-M group 1 and the so-called CTX-M group 9; a group of primers complementary to the parallel and antiparallel strands of an Escherichia coli (spp), Proteus mirabilis (spp), Klebsiella pneumoniae (spp), Enterococcus faecalis (spp) sequence selected from the consistent group of SEQ. ID. NO. from 1 to 62; recombinant enzyme with polymerase functions; deoxyribonucleotide triphosphate; reaction buffer and suitable additives; fluorescent and non-fluorescent intercalants.

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