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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/16—Primer sets for multiplex assays

Definitions

• the present invention relates to the field of biotechnology, and in particular concerns a method and a kit of reagents used in a simple way for the identification of biological fluids in a sample of any kind, for example consisting of forensic findings, clinical samples, swabs from outpatient or hospital, findings at a crime scene and / or traces of interest to forensic science investigations of police and private investigators.
• the purpose of the present invention is therefore to provide a method and a kit for the identification of a range of biological fluids that can be used in a simple way and with the aid of instrumentation commonly present in small and medium molecular biology laboratories, disseminated in the territory.
• the method can however easily be adapted to other instruments and methods for the analysis of the DNA including the latest techniques for NGS (next generation sequencing). Said aim is achieved with a method and a kit whose main features are specified in the first claim and other features are specified in subsequent claims.
• the kit according to the present invention is based on molecular biology techniques customary in forensic laboratories and applicable also to traces of biological fluids of forensic interest, and it is based on the technique of polymerase chain reaction and hybridization techniques, but the present invention differs from what is currently available primarily because it relies on the amplification l and identification not of human genomic or non-mitochondrial (mtDNA) sequences, but of different genome sequences belonging to various species that dwell the human body regions of origin of biological fluids under consideration and that constitute the microflora (mfDNA).
• mtDNA human genomic or non-mitochondrial
• the vaginal mucosa for example, is characterized by a complex microbial population that in non-pathological conditions, is made up by elements belonging to the genus Lactobacillus, such as L. iners, L.
• vaginalis in cases of bacterial vaginosis (CS Bradshaw, SN Tabrizi, CK Fairley, AN Morton, and Rudland, SM Garland. The association of Atopobium vaginae and Gardnerella vaginalis with bacterial vaginosis and recurrence after oral metronidazole therapy. J Infect Dis. Sep 15 2006 , 194 (6) :828-36. Epub 2006 Aug 16). According Zozaya-Hinchliffe and colleagues (Zozaya-Hinchliffe M, Lillis R, Martin DH, MJ Ferris. Quantitative PCR assessments of bacterial species in women with and without bacterial vaginosis. J Clin Microbiol.
• the DNA is more resistant, but the techniques of analysis of the human genome currently used in forensic and biomedical laboratories, do not allow, to date, to distinguish the source of fluids, since the primary structure of the DNA of an individual is identical in different tissues.
• Frumkin (2010) based on the methylation of the human genome in different tissues
• Fleming and Harbison (2010) who have suggested alternative approaches, including those based use of differences in gene expression in cells of the vaginal mucosa or into cells of the microflora, having also proposed a multiple amplification of bacterial mRNA.
• characterization of the DNA of the microflora from specific sequences for one or more microbial species vaginal and at the same time also for one or more bacteria from other possible microflora, allowing to identify or exclude the vaginal origin of a fluid in a sample, and indicating the possible contamination with possible other biological fluids.
• a method of determining the origin of fluids or biological specimens and kits of reagents for their identification in a sample has been developed by using as an indicator of molecular components and in particular microbial genomic sequences of the microflora present in the fluids and which dwell in the respective mucosae of origin.
• a set of protocols and reagents properly defined and optimized provide a kit applicable for forensic purposes and other applications such as laboratory analysis and / or research studies in various sectors.
• the kit allows the identification of a biological fluid, or its traces on a matrix, starting from the identification of genomic sequences of bacterial DNA, and includes a component (component A) for the multiplex amplification and a second component (component B) for hybridization.
• the component B includes at least one probe specific for each bacterial species considered for the identification of biological fluid in a sample.
• kit Other elements that compose the kit are the usual reagents and instruments used for these types of analyses, as well as instructions for use.
• the kit according to the present invention by identifying characteristic properties of the microflora of the biological fluid from the genome allows, in fact, to make a diagnosis of compatibility and / or exclusion compared to other biological fluids.
• This arrangement makes it possible to define a compatibility between a finding and a biological fluid, even if the finding was not immediately picked up and / or has been exposed to adverse environmental conditions, since the resistance of the bacterial DNA is high enough, as it is known .
• the kit can be applied in conventional molecular biology procedures, already used for analysis of traces of human DNA by applying already available technologies and skills and providing added value to the investigation.
• kits according to the present invention compared to other methods based on polymerase chain reaction, consists in the fact that it does not require the use of restriction enzymes, of apparatus for electrophoresis or dying of the DNA, but it is based on the hybridization with a probe, detectable by a classic tool for real-time amplification, allowing to search for multiple microbial indicators simultaneously in a single test -qualitative and quantitative-allowing also an automation of the analysis.
• restriction enzymes of apparatus for electrophoresis or dying of the DNA
• kits according to the present invention consists in the fact that the amplification for one or more biological fluids can be realized in a single operation even with more indicators microbial parallel, adding to a mixture of standard reaction only the A and B components and DNA sample diluted in water.
• the kit according to the present invention comprises two components for each biological fluid.
• the first of these components includes the reagents for the multiple amplification, and the second (component B) reagents for hybridization.
• Component A consists of an amplification mixture that includes all the necessary reagents according to the known process for the polymerase chain reaction, commonly known as PCR and described inter alia in U.S. patents 4,683,195 and 4,683,202.
• reagents are: the reaction buffer that allows to create the conditions for the functionality of the enzyme (pH, salt concentration), the dNTP ie deoxynucleotide triphosphates consisting of nitrogenous bases (adenine, guanine, cytosine, thymidine) monomers required for the reaction polymerase; magnesium chloride (MgC12) necessary to ensure specificity and efficiency of the reaction (annealing of primers and functionality of the polymerase), and the mixture of the primers that oligonucleotides of specific sequence, required to initiate the amplification of DNA complementary to each other according to the well- known principle of the polymerase chain reaction.
• MgC12 magnesium chloride
• the mixture A includes the primers corresponding to different biological species, for example for the vaginal fluid sequences belonging to the genome of Lattobacillus gasseri and Lattobacillus crispatus or Lattobacillus iners; for saliva to Streptococcus salivarius and Streptococcus mutans ; for rectal mucus to Escherichia coli and Enterococcus faecalis.
• each mixture contains about 20 parts of a 100 micromolar solution of the primers corresponding to each species. These rates may of course vary within certain limits, which can also be of about 10%, without impairing the effectiveness of the mixture and its stability.
• the primers can be ordered from several companies including, for example, Sigma- Aldrich, MWG, PRIMM. Some 'primers sequences used from 5 -3, forward and reverse
• the primers corresponding to other characteristics of the different species microflora human and / or animal and / or belonging to specific environmental matrices and / or foodstuffs interest in a differential diagnosis, and also in a method of diagnosis by exclusion.
• a solution 100 micromolar of the following primers 5'-3 'corresponding to the species Bos taurus is added approximately 10 parts of a solution 100 micromolar of the following primers 5'-3 'corresponding to the species Bos taurus:
• Probe means an oligonucleotide of known sequence specific for the biological species sought, which can be done by synthesizing by outsourcing to appropriate companies or synthesized directly in the laboratory with the use of an instrument called
• oligonucleotide synthesizer The important feature of the probe is that the sequence of bases A, C, G, T (adenine, cytosine, guanine, thymine) is defined.
• the kit includes probes corresponding to different human biological fluids, for example, vaginal fluid, saliva, mucus, rectal, and is thus allowed the identification of at least two different human biological fluids.
• the probes have the function of linking the DNA segments that are amplified using the mixtures of component A. These segments will undergo a hybridization reaction allowing to identify the biological fluids sought.
• known methods and standard solutions can be used and for the detection any system can be used.
• at least two triads primer-probe for each biological fluid have been used simultaneously, corresponding respectively to at least two bacterial indicators.
• probes corresponding to other components of microflora useful in the further characterization of the same fluid or other identifying matrices can be added in order to obtain a reaction that can allow the kit according to the present invention to identify the same procedure with the same fluid with more probes and / or other possible biological contaminants of interest in a differential diagnosis of exclusion.
• the components A and B will be added to the necessary reagents for the amplification reaction, according to known protocols.
• individual reagents are acquirable by different companies (SIGMA, Fermentas, Promega), and are typically used under the following final concentrations: lOmM Tris-HCl pH 8.3 (20 0 C), 50 mM KC1, 1.5 MgCl mM, 0.2 mM dNTP (Also, it is also possible to improve the yield and stability, adding stabilizers and / or a reference dye, acquired by different companies, and to be dosed according to protocols already known).
• ready made solutions can be also used, which are obtainable from different companies (Sigma, Applied Biosystems).
• FIGURE I referring to Example 1, relates to the identification of vaginal fluid.
• the sample of DNA extracted from vaginal swab was positive for the presence of L. gasseri and L. crispatus, while it does not appear significant signal for the indicators of other biological fluids such as saliva and / or rectal mucus.
• FIGURE ⁇ referring to Example 3, relates to the calibration curve (E. faecalis, serial dilutions) ... EXAMPLE 1
• component A 20 parts of a solution 100 ⁇ of each primer were diluted in ultrapure water.
• oligonucleotides were synthesized (SIGMA-Aldrich), received lyophilized and resuspended in sterile ultrapure water to a final concentration of 100 ⁇
• component A for the identification of vaginal fluid was carried out in the following way: in a test tube of 2 ml 100 ⁇ of ultrapure sterile water, and respectively 100 ⁇ of the primer with sequence GCACTAACAGCCGAAGAAGG (SEQ ID 1) , ⁇ of the primer with sequence TTCGGATATCTCCGGATCAC (SEQ ID 2), 100 ⁇ of the primer with sequence AGATTGAAGAGCGACCGAGA (SEQ ID 3), ⁇ of the primer with sequence
• component A for the identification of saliva was carried out in the following way: in a test tube containing 2 ml 100 ⁇ f ultrapure sterile water, and respectively 100 ⁇ of the primer with sequence CGGTTCTCAGCAAGACATGA (SEQ ID 7), 100 ⁇ of the primer with sequence ATGGTACCCAATCCGCAATA (SEQ ID 8), 100 ⁇ of the primer with sequence GATGCCAAGGGTGAAGTTGT (SEQ ID 9), 100 ⁇ of the primer with sequence
• component A for the identification of rectal mucus was carried out in the following way: in a test tube containing 2 ml of ultrapure sterile water, and respectively 100 ⁇ of the primer with sequence CCCTTACGCTGAAGAGATGC (SEQ ED 11) 100 ⁇ of the primer with sequence GAGGTTAAAGCCGACAGCAG (SEQ ED 12), 100 ⁇ of the primer with sequence AGAAATTCCAAACGAACTTG (SEQ ED 13), 100 ⁇ of the primer with sequence
• CAGTGCTCTACCTCCATCATT (SEQ ED 14) were added.
• component B For the preparation of component B, 5 parts of a solution 100 ⁇ of each probe were diluted in ultrapure water.
• oligonucleotides labeled at 5 'with a fluorophore and the 3' end with a quencher were synthesized (SIGMA-Aldrich), received lyophilized and resuspended in ultrapure sterile water to a final 100 ⁇ concentration
• component B for the identification of vaginal fluid was carried out in the following way: in a test tube containing 2 ml, 450 ⁇ of ultrapure sterile water, and respectively 25 ⁇ were added of each of the following labeled probes of sequence:
• component B for the identification of saliva was carried out in the following way: in a test tube containing 2 ml , 450 ⁇ of ultrapure sterile water, and respectively 25 ⁇ were added of each of the following labeled probes of sequence:
• component B for the identification of mucus rectal ⁇ we proceeded as follows: in a test tube containing 2 ml , 450 ⁇ of ultrapure sterile water, and respectively 25 ⁇ were added of each of the following labeled probes of sequence:
• GenElute Bacterial Genomic DNA kit (Sigma- Aldrich NA2100) has been used with some modifications to the protocol. Briefly, the swab was washed in 500 ul of sterile PBS for 45 minutes under stirring at room temperature.
• the eluate was centrifuged and the pellet frozen at -20 0 C for 20 minutes. Then, Glass Beads (Sigma-Aldrich G1145) were added together with 200 ul Lysozime solution. With a sterile pestle we disintegrated accurately the pellet and then incubated at 37 ° C for 30 minutes. Then it was proceeded by adding RNase and Proteinase K, reaching the elution up to a final volume of 50 1.
• the amplification reactions were conducted on an ABI 7000 instrument from Applied Biosystems, in a volume of 25 ul total, programmed as follows: denaturation 95 ° C for 10 minutes, followed by 40 cycles at 95 0 C for 15 seconds and 60 0 C for one minute.
• Each of the DNA samples described above and of the positive and negative controls was analyzed in parallel for the search of three biological fluids: vaginal, salivary, rectal, using the respective components A and B prepared as described in Example 1.
• For each sample were added 10 1 of DNA in a test tube 0.2 ml thin wall in which they were aliquoted 1.25 ⁇ of component A, 1.25 ⁇ of component B and 12.5 ⁇ of amplification reaction 2X of the company Applied Biosystems.
• Each DNA sample was amplified in parallel in three tubes using respectively the components A and B for the vaginal fluid, for saliva and rectal mucus. The results observed ( Figure 1) made it possible to correctly assign each sample to the fluid of origin.
• probes and primers for Lactobacillus gasseri, and Enterococcus faecalis have been used.
• the DNA samples were extracted and processed as described in example 2, except that has been used a 1:1 mixture of component A and component B, from which 2.5 ⁇ in each tube were aliquoted.
• Chloroflexi spp. Clostridiales, Dialister spp., Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Finegoldia magna, Gardnerella vaginalis, Lactobacillus acidophilus phage kc005, kc007 phage Lactobacillus acidophilus, Lactobacillus acidophilus phage kc012, kc021 phage Lactobacillus acidophilus, Lactobacillus casei, coleohominis Lactobacillus, Lactobacillus coleohominis,
• Lactobacillus crispatus Lactobacillus delbrueckii phage kc023, kc031 Lactobacillus delbrueckii phage, phage kc039 Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus iners, Lactobacillus jensenii,
• Lactobacillus rhamnosus Lactobacillus salivarius, Lactobacillus spp , Lactobacillus vaginalis, Leptotrichia spp., Megasphaera spp., Olsenella spp., Pediococcus acidilactici, Peptoniphilus indolicus, Peptostreptococcus anaerobius, Porphyromonas asaccharolytica, Prevotella bivia, Shuttleworthia spp., Staphylococcus capitis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Streptobacillus spp. , Streptococcus agalactiae,
• Streptococcus anginosus Streptococcus mutans, Streptococcus salivarius, Tricomonas Faetus, Tricomonas vaginalis
• Granulicatella adiacens Granulicatella elegans, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus crispatus Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus ultunensis, Lactobacillus rhamnosus, Naegleria fowleri, Olsenella uli , Porphyromonas gingivalis, Porphyromonas micra, Porphyromonas endodontalis, Prevotella baroniae, Prevotella denticola, Prevotella spp., Pseudoramibacter spp., dentocariosa Rothia, Streptococcus gordonii, Streptococcus intermedius, Streptococcus oralis,
• Example of other microorganisms found in the intestinal microflora be used alone or in admixture among them:
• Acinetobacter baumannii Actinomyces meyeri, Actinomyces radingae, urogenitalis Actinomyces, Aerococcus christensenii, Aerococcus viridans, Agrobacterium radiobacter, Alloscardovia omnicolens, Anaerococcus tetradius, Anaerococcus vaginalis, Atopobium vaginae,
• Bacteriofagoides lambda, M13 Bacteriofagoides, Bacteroides uniformis, Balantidium coli,
• Staphylococcus epidermidis Staphylococcus haemolyticus, Staphylococcus hominis,
• Staphylococcus warneri streptococcal phage CI Streptococcus agalactiae, Streptococcus anginosus, Streptococcus bovis, Streptococcus intermedius, Streptococcus mitis group, Streptococcus parasanguinis, Streptococcus salivarius, Trichomonas hominis, Trichomonas vaginalis,
• Staphylococcus hominis Staphylococcus capitis, Staphylococcus epidermidis, Streptococcus epidermidis, Streptococcus hominis, Trichophyton, Xanthomonadaceae,
• the components A and B for each biological fluid may be mixed in a single solution (component AB).
• primers and probes can be increased in number within the limits of the equipment used and the number of fluorochromes available for the marking of the probes within a single tube.
• the amplification mixture can be prepared according to protocols known by the user, or purchased ready-made from companies such as Sigma or Applied Biosystems, having the user simply to add the DNA of the sample to be examined, and the mixture of primers and probes (component A and component B or component AB).
• the kit in its application in instruments for real-time PCR provided by different companies (Biorad, Applied Biosystems, WVR), as well as the equipment calibrated and suitably programmed for the amplification conditions, it will be necessary to employ the positive and negative controls tubes and appropriate criteria for the interpretation of results, but all this material and general procedures are already known in the general methodology and therefore need no special description. If appropriate, the completion of the kit can be carried out by the diagnostic laboratory itself, which can simply adapt the protocol to its instrument adapting to their needs the use of components A and B according to the present invention.

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