Patent Application
20200123598
Embodiments of the present disclosure relate to primers, oligonucleotides, chemical components and methods for a multiplex Polymerase Chain Reaction (PCR) analysis able to discriminate and genotype different strains of the same pathogen, different pathogens belonging to separated genus and genetic variations in a single reaction. In particular, embodiments of the present disclosure relate to primers, oligonucleotides, methods and conditions to detect and characterize different strains of pathogens responsible for sexually transmitted diseases, such as, but not limited to, high-risk human papilloma virus (HPV), through PCR using High Resolution Melting (HRM) technology in one multiplex assay.
Polymerase Chain Reaction (PCR) is a robust technique used for diagnostic applications worldwide. PCR consists of a primer extension reaction that amplifies specific nucleic acids in vitro. The reaction exploits a thermostable DNA polymerase and it is based on several cycles including different temperature steps, that allow DNA denaturation, primer annealing, and polymerase-mediated elongation of the target DNA.
PCR can be combined with fluorescent dyes able to intercalate with double stranded DNA, or fluorescently-labelled DNA probes; in this way, the signal derived from newly synthesized amplicons can be quantitatively acquired in real-time through fluorescence acquisition.
High Resolution Melting (HRM) is an additional post-PCR analysis step that further characterizes the amplicons by studying thermal denaturation of double-stranded DNA. This occurs through the analysis of amplicon disassociation (melting) behaviour in a ramp of temperatures usually ranging from 65° C. to 95° C., with a fluorescence acquisition rating of 0.1° C./sec or less. This method allows to discriminate sequence variations and features among different amplicon, and even single nucleotide polymorphisms (SNPs) can be observed.
HRM is used in diagnostics, for example in the context of genetic tests able to identify SNPs in polymorphic alleles and it has been proposed for a variety of applications including pathogen detection.
Nowadays HRM analysis requires highly pure extracted DNA: this restricts its application mostly to high-income settings where DNA extraction is automatized.
Moreover, to achieve high specificity, HRM is performed in PCR reactions requiring target-dedicated primers and probe sets, that are not usually a quite affordable reagent. Nevertheless, HRM has an enormous unexplored potential for useful applications also in low-income settings, such as the characterization of human pathogens.
It is well known that Human Papillomavirus (HPV) is the main cause of cervical cancer. This tumour is the second most frequent tumour in women with more than 500.000 cases worldwide1 and it is mostly determined by persistent infection of high risk HPV (HR-HPV) genotypes; approximately 20 cancer-associated HR-HPV have been described so far, being HPV16 and HPV18 the most frequent (70% of all invasive cervical cancer)2,3. Effective cervical cancer screening programs have reduced the incidence and the mortality of this tumor4; initially screening was performed with the Papanicolau test (Pap test), a cytological test able to identify precancerous cells after collection of cervical specimen by endocervical brush, and after plating and staining the specimen on a microscopic slide. This technique shows a very high positive predictive value but it has poor average sensibility5 and low negative predictive value.
Current studies indicate that HPV DNA testing is more efficient for the prevention of invasive cervical cancer than the Pap test screening alone, given the higher negative predictive value of the HPV DNA test6-8. In addition, given the variability of positive predictive values for cancer development among different HR-HPV genotypes, HPV genotyping is useful for the triage of HPV-positive patients, both in cervical cancer and in other HPV-related cancers (such as ano-genital and oropharyngeal tumours)9-13. It might as well discriminate the transient infections from the persistent ones, being the latter the true risk factor for cancer transformation. Finally, genotyping test might represent an important tool for vaccine monitoring and herd immunity in population study, to assess the variation of the prevalence of each genotype in vaccinated and not-vaccinated population14.
Nowadays the most diffused tests can be classified into two groups15:
All these tests require dedicated instruments and are not affordable enough for a widespread diffusion especially in developing countries.
PCR multiplexing is often a tricky analysis due to the use of several primer set that lead to fluorescence aspecific amplification signals (such as ones derived from primer dimers formation). This can be limited thanks to fluorescently-labelled probes (e.g. Taqman probes, Life Technologies), that ensures a higher specificity compared to intercalating dyes that does not make distinction between amplicons. However, fluorescently-labelled probes dramatically increase the cost of the assay, affect test robustness and more than one fluorescence channel is required; this means that the tests can be executed only by dedicated instruments with peculiar specificities.
There is therefore a need to improve molecular fingerprinting methods to detect DNA targets through polymerase chain reaction (PCR), which overcome at least one of the drawbacks in the art.
One aim of the present disclosure is to provide molecular fingerprinting methods to detect DNA targets or genotypes through PCR.
In particular, one aim of the present disclosure is to discriminate pathogens, in particular viral Human Papillomavirus genotypes through a specific HRM analysis in a single reaction.
One further aim of the present disclosure is to increase the amplification of specific PCR amplicons only, reducing aspecific signal emission, e.g. fluorescent, background.
One further aim of the present disclosure is to avoid primer dimers.
Still one further aim of the present disclosure is to provide primers, oligonucleotides, chemical components and methods for a multiplex PCR that overcome aspecific fluorescence signal problems as above discussed.
Yet one further aim of the present disclosure is to set each real-time PCR machine for a correct and precise melting analysis required to genotype the pathogen.
According to embodiments, a molecular fingerprinting method to detect and genotype DNA targets in a sample through Polymerase Chain Reaction (PCR) is provided. The method of the present disclosure is able to discriminate and genotype different strains of the same pathogen, different pathogens belonging to separated genus and genetic variations in a single reaction. In one embodiment, the method includes:
According to further embodiments, a diagnostic kit to detect and genotype DNA targets is provided, comprising a PCR reaction mixture that can be used to perform PCR amplification and a subsequent a HRM analysis on the PCR reaction mixture previously subjected to real-time PCR. In one embodiment, the PCR reaction mixture comprises two or more pairs of amplification primers for amplifying, in a multiplex approach, two or more target nucleic acids,
wherein said primers are designed in order to generate amplicons with a different melting temperature each other in order to discriminate, in the HRM analysis, each amplicon by observing the specific melting temperature of each amplicon.
According to still further embodiments, amplification primers are provided for performing a molecular fingerprinting method to detect and genotype DNA targets through PCR, wherein the method is able to discriminate and genotype different strains of the same pathogen, different pathogens belonging to separated genus and genetic variations in a single reaction. In one embodiment, the amplification primers are provided for amplifying in a multiplex approach two or more target nucleic acids in a PCR amplification, wherein said primers are designed in order to generate amplicons with a different melting temperature each other in order to discriminate, in a HRM analysis following the PCR amplification, each amplicon by observing the specific melting temperature of each amplicon.
According to yet further embodiments, an apparatus to perform a molecular fingerprinting method for detection and genotyping of DNA targets in a sample through PCR is provided. The method is able to discriminate and genotype different strains of the same pathogen, different pathogens belonging to separated genus and genetic variations in a single reaction. In one embodiment, the apparatus includes:
According to embodiments, combinable with all embodiments described herein, the DNA target that can be detected and genotyped is a pathogen DNA target.
Advantageously, the proposed technique exploits common DNA intercalating molecules or compounds, such as for instance intercalating dyes, that are much more affordable than fluorescently-labelled probes.
Moreover, according to the present disclosure, the post-PCR HRM analysis does not require a dedicated instrument but it can also be performed in any thermocycler with a HRM resolution of at least 0.1° C./sec or less. In this context primer design is crucial and the primers designed according to the present disclosure are found to be fully successful in ensuring the highest specificity for each single target, given the multiplex assay format.
In advantageous embodiments, the melting fingerprinting technology according to the present disclosure has been applied to successfully improve the detection of HPV DNA, the main cause of cervical cancer.
Advantageously, embodiments described herein may use specific buffer and condition, specific DNA primers and innovative hybrid primers.
Embodiments described herein allow to discriminate, for example, viral HPV genotypes through a specific HRM analysis.
Advantageously, embodiments described herein allow to detect up to 37 HPV types, including for instance 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73 (MM9), 81, 82 (MM4), 83 (MM7), 84 (MM8), IS39, CP6108. The viral genome amplification reaction can be coupled to the amplification of a DNA loading control target (human beta-globin gene for example).
Other pathogens that can be detect according to the present disclosure include for instance other pathogens than HPV responsible for sexually transmitted diseases, such as the bacterium Treponema pallidum subspecies pallidum, responsible for the syphilis infection, the bacterium Neisseria gonorrhoeae, responsible for the gonorrhoea infection, the bacterium Chlamydia Trachomatis, responsible for the chlamydia infection, and the HIV virus.
Embodiments described herein also provide a melting calibrator that is required to set each real-time PCR machine for a correct and precise melting analysis required to genotype the pathogens.
Embodiments described herein according to the present disclosure fully solve the above-mentioned issues of the tests and methods of the prior art, and further provide at least the following advantages:
These and other features, aspects and advantages of the present disclosure will become better understood with reference to the following description, the drawings and appended claims. The drawings, which are incorporated in and constitute a part of this specification, are used to illustrate embodiments of the present subject matter and, together with the description, serve to explain the principles of the disclosure.
The various aspects and features described in the present disclosure can be applied, individually, wherever possible. These individual aspects, for instance the aspects and features described in the attached dependent claims, can be made subject of divisional patent applications.
It is noted that anything found to be already known during the patenting process is understood not to be claimed and to be the subject of a disclaimer.
So that the manner in which the above recited features of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to embodiments. The accompanying drawings relate to embodiments of the disclosure and are described in the following:
Reference will now be made in detail to the various embodiments of the invention, using the attached figures. Generally, only the differences with respect to individual embodiments are described. Each example is provided by way of explanation of the invention and is not meant as a limitation of the invention. For example, features illustrated or described as part of one embodiment can be used on or in conjunction with other embodiments to yield yet a further embodiment. It is intended that the present invention includes such modifications and variations.
Before describing these embodiments, it shall be also clarified that the present description is not limited in its application to details of the construction and disposition of the components as described in the following description using the attached drawings. The present description can provide other embodiments and can be obtained or executed in various other ways. It shall also be clarified that the phraseology and terminology used here is for the purposes of description only, and cannot be considered as limitative.
All the percentages and ratios indicated refer to the weight of the total composition (for example indicated as % w/w), unless otherwise indicated. All the measurements are made, unless otherwise indicated, at 25° C. and atmospheric pressure. All the temperatures, unless otherwise indicated, are expressed in degrees Centigrade.
All the ranges reported here shall be understood to include the extremes, including those that report an interval “between” two values. Furthermore, all the ranges reported here shall be understood to include and describe the punctual values included therein, and also all the sub-intervals. Moreover, all the ranges are intended as such that the sum of the values comprised therein, in the final composition, gives 100%, in particular considering that the person of skill will know how to choose the values of the ranges so that the sum does not exceed 100%.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. In addition, it will be readily apparent to one of ordinary skill in the art in light of the teachings herein that certain changes and modifications may be made thereto without departing from the spirit and scope of the appended claims. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. To the extent such publications may set out definitions of a term that conflicts with the explicit or implicit definition of the present disclosure, the definition of the present disclosure controls. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
Embodiments of the present disclosure generally relate to molecular fingerprinting method to detect and characterize DNA targets in a sample through PCR is provided. The method of the present disclosure is able to discriminate and genotype different strains of the same pathogen, different pathogens belonging to separated genus and genetic variations in a single reaction. In one embodiment, the method includes:
According to embodiments, combinable with all embodiments described herein, the DNA target that can be detected and genotyped is a pathogen DNA target.
Advantageously, using the embodiments described herein, it is possible to detect and discriminate:
Advantageously, the sample can be a crude sample. The crude sample can be vaginal or cervical mucus, other bodily fluids, saliva, blood, urine, biopsies, formalin-fixed paraffin-embedded (FFPE) tissue, cells, fine needle aspiration biopsies or similar. The crude sample can be diluted prior to performing the PCR amplification and HRM analysis.
According to possible embodiments, combinable with all embodiments described herein, the signal variation between an input and an output signal can be detected in a circuit comprised in the reader, wherein said variation is a function of the presence, amount, genotype of different strains of the same pathogen, different pathogens belonging to separated genus and genetic variations present in the sample.
According to embodiments, combinable with all embodiments described herein, performing PCR amplification using said PCR reaction mixture includes amplifying the target purified nucleic acid using said PCR reaction mixture to generate an amplicon or amplification product.
According to embodiments, combinable with all embodiments described herein, the amplification primers are sufficiently complementary to the target nucleic acid to hybridize therewith and trigger polymerase-mediated synthesis.
In one specific embodiment, combinable with all embodiments described herein, the amplification primers are designed to amplify specifically pathogen DNA targets (e.g. HPV genotypes) producing corresponding amplicons, each from 38 to 1500 base pairs (bps).
In one further specific embodiment, combinable with all embodiments described herein, the amplification primers are designed to amplify an amplicon wherein the melting peak of the amplicon is between 68 and 95° C.
In still another embodiment, combinable with all embodiments described herein, each amplification primer is present in the PCR reaction mixture at a final concentration range from 50 to 1000 nanomolar (nM).
According to embodiments, combinable with all embodiments described herein, the PCR reaction mixture comprises the amplification primers and the amplification buffer.
According to embodiments, the amplification buffer is comprised in a diagnostic kit that is part of the present disclosure.
In one embodiment, combinable with all embodiments described herein, besides the above mentioned two or more pairs of amplification primers, the PCR reaction mixture comprises a DNA polymerase. The DNA polymerase is comprised in said amplification buffer. The DNA polymerase is an enzyme that polymerizes new DNA strands. For instance, heat resistant or heat stable polymerase can be used, since it is more likely to remain intact during the high-temperature DNA denaturation process. One example of heat resistant or heat stable polymerase that can be used in embodiments described herein is taq polymerase. Moreover, the polymerase that can be used in association with embodiments described herein is a hot-start polymerase. In a possible implementation, the hot-start polymerase can be (Hot Start) @Taq DNA Euroclone, (Hot Start) Phire Thermo Scientific, (Hot Start) Phusion Thermo Scientific, or (Hot Start) Gold Taq polymerase Sigma.
In one further embodiment, combinable with all embodiments described herein, the PCR reaction mixture may further comprise deoxynucleoside triphosphates (dNTPs) or analogues. dNTPs or analogues are comprised in said amplification buffer. dNTPs or analogues are used to provide the building blocks from which the DNA polymerase synthetizes a new DNA strand. dNTPs can be substituted by functional analogues like adenine, cytosine, guanine, tymine, uracil, orotidine, inositate, xanthylate.
As above described, the PCR reaction mixture comprises said intercalating molecule or compound, being incorporated into the double-stranded amplicon or amplification product and emitting fluorescence or any other detectable signal. The intercalating molecule or compound can be comprised in said amplification buffer.
In particular, according to embodiments, the intercalating molecule can be any sensor or reporter molecule emitting a signal that can be detected by a reader analysing an electric signal variation in terms of inductance, current, electric potential, in case of conductimetric, amperometric, voltammetric detection, or the presence of light at specific wavelengths, in case of a fluorescence/chemiluminescence detection, or light scattering and/or refraction/diffraction phenomena, in case of a plasmonic optical detection.
For instance, in some implementations the intercalating molecule or compound can be an intercalating dye emitting fluorescence. According to possible implementations, specific DNA intercalating dye, at a final concentration range from 1 to 8 μM, can be one or more of the following dyes: SYTO-9, SYTO-13, SYTO-16, SYTO-64, SYTO-82, YO-PRO-1, SYTO-60, SYTO-62, TOTO-3, POPO-3, BOBO-3, doxorubicin-conjugated quantum dot nanoparticles or similar.
In yet another embodiment, combinable with all embodiments described herein, the PCR reaction mixture may further comprise a buffer solution. The buffer solution is comprised in said amplification buffer. The buffer solution provides a suitable chemical environment for optimum activity and stability of DNA polymerase. For instance, the buffer solution may comprise water, in particular deionized water, TrisHCl and/or KCl and possibly in some cases MgCl2.
In one embodiment, combinable with all embodiments described herein, the PCR reaction mixture may further comprise a pH stabilizer.
In one further embodiment, combinable with all embodiments described herein, the PCR reaction mixture may further comprise preservatives.
In still another embodiment, combinable with all embodiments described herein, the PCR reaction mixture may further comprise water.
In yet another embodiment, combinable with all embodiments described herein, the PCR reaction mixture may further comprise a source of monovalent or bivalent cations. The source of monovalent or bivalent cations is comprised in said amplification buffer. For example, a chloride containing monovalent ion or bivalent ions can be used. As a source of monovalent cations, potassium ions can be used. K+ can be obtained from potassium salts, e.g. potassium chloride, in particular potassium chloride at a concentration of about 0.1 M. As source of bivalent cations magnesium or manganese ions can be used. Mg2+ can be obtained from magnesium salts, e.g. magnesium chloride.
In one further embodiment, combinable with all embodiments described herein, the PCR reaction mixture may further comprise bovine serum albumin (BSA). The BSA is comprised in said amplification buffer.
In one further embodiment, combinable with all embodiments described herein, the PCR reaction mixture further comprises one or more detergents. In possible implementations, said detergent can be Nonidet-P40 at a concentration of about 0.5%.
In still another embodiment, combinable with all embodiments described herein, the PCR reaction mixture may further comprise additives. The additives can be comprised in some embodiments of the above-mentioned amplification buffer. In possible implementations, the additives that can be used are selected among one, more or all of additives in a group comprising: NP40, DMSO, TMAC (Tetramethylammonium Cloride), Acetamide, Triton, Formamide, Betaine, E. coli ssDNA binding protein, Glycerol, L-Carnitine and Gelatine. Advantageously, in embodiments exploiting a fluorescence detection, the presence of additives can be important to avoid a high basal fluorescence background allowing an increased diagnostic sensitivity, specificity and accuracy (see
According to embodiments, combinable with all embodiments described herein, amplifying the target purified nucleic acid using said PCR reaction mixture, to generate an amplicon or amplification product, includes thermocycling by performing a ramp of temperature steps. In one possible implementation, a ramp of temperature includes performing the following temperature steps:
In possible implementations, the number of cycles of thermocycling is of at least 30 cycles, for instance between 30 and 50 cycles. One possible example is 35 cycles.
In one possible implementation, hot-start polymerase can be used. Hot-start PCR avoids a non-specific amplification of DNA by inactivating the polymerase at lower temperatures, for instance through antibodies interaction, chemical modification or aptamer technology. Typically, a specific inhibitor, such as an aptamer-based inhibitor or specific antibodies can be used to block the polymerase at lower temperatures. If hot-start polymerase is used, an initial incubation step which ranges from 95° C. to 98° C. for 1 second to 10 minutes is performed. This initial incubation step is necessary for activation of polymerase.
According to embodiments, combinable with all embodiments described herein, the method includes, during thermocycling in the PCR amplification, performing the monitoring emission signal changing, e.g. fluorescence, resulting from the temperature-induced denaturation of the double-stranded amplicons or amplification products into two single-stranded DNA, due to the releasing of the intercalating molecule or compound, i.e. intercalating dye. The intercalating molecule or compound binds to DNA in the double-strand configuration. At each amplification cycle, amplicons are generated in which the intercalating molecule or compound binds in the extension step, during which the signal (e.g. fluorescence) is acquired.
Advantageously, the PCR reaction can occur in a real-time PCR machine, that allows monitoring the change in the signal, e.g. fluorescence, emission at each amplification cycle in the PCR amplification, in turn allowing quantification of the presence of amplicons and quantification, therefore, of the viral DNA in the amplification phase.
In other embodiments, the PCR amplification may occur in a thermocycling machine able to acquire said signal emission each 0.1° C./second or less.
According to embodiments, combinable with all embodiments described herein, the HRM analysis includes performing a ramp of temperature on the PCR reaction mixture previously subjected to PCR amplification. In one possible implementation, a ramp of temperature includes performing the following temperature steps:
Advantageously, monitoring the change in the signal, e.g. fluorescence, emission in the HRM analysis, during which the large quantity of viral amplicons generated after the plurality of amplification thermocycling, allows to analyse the melting features of such amplicons at different temperatures. At low temperature, e.g. 60° C., amplicons are all double-stranded and the maximum level of signal, e.g. fluorescence, is detected. By slowly increasing temperature, however, amplicons start to denaturate up to complete separation into two single-stranded DNA and at this point the signal, e.g. fluorescence, will not be generated anymore. For instance, the shape and development of the Derivative Fluorescence vs. Temperature curves, as shown in
Therefore, according to advantageous embodiments using a real-time PCR machine, after monitoring the change in in the signal, e.g. fluorescence, emission at each amplification cycle in the real-time PCR, it will be possible to know if the analysed sample is infected or not by a pathogen or a group of pathogens, e.g. a virus, and also by which one of the possible genotypes that can be detected.
Moreover, according to the present disclosure, after monitoring the change in in the signal, e.g. fluorescence, emission in the HRM analysis it will be possible to know, in the case that a sample is infected, also exactly the specific genotype or strain of the pathogen infecting the sample, or which exact pathogen of the group of pathogens.
The overall result is therefore that it will be possible, in advantageous embodiments using the real-time PCR, to know, via the real-time PCR, if a patient if positive or not to a specific pathogen, or a group of pathogens and, when positive, to know, via HRM analysis, the genotype or strain of pathogen that is infecting the patient or to know the specific pathogen from the detected group of pathogens.
In still further embodiments described using
The stem-loop hybrid primers according to embodiments described herein act through two distinct mechanisms: a competitive mechanism that protects the primer from both aspecific interactions and self-interaction; and a DNA polymerase stop-system that allows to generate only the desired amplicon with the predicted melting features. The oligonucleotides of the stem-loop hybrid primers are structured in 4 domains: 1) a priming portion, 2) a loop portion, 3) a competitive palindromic portion and 4) a 5′ fidelity tail (see
According to embodiments, the priming portion 1) is a sequence present in the actual primer sequence that is complementary to the target sequence and it may range from 15 to 60 bases. The target sequence is a DNA sequence present in the pathogen genome that has at least 50% of identity with the priming portion.
According to embodiments, the loop portion 2) can contain from 30% to 90% of the priming portion bases and it forms a stable and unique hairpin structure. The loop portion contains a number of nucleotides (adenine, cytosine, timine, guanine, uracil, orotidine, inosinate, xanthylate) or DNA base analogue, in order to form a stable loop structure. Juxtaposed to the 5′ of the priming portion, the sequence of the loop portion contains a DNA polymerase stopping site (processivity blocker). This stopping site can be modulated by the presence of at least one 1,2-Dideoxyribose, or an equivalent abasic nucleotide, preceded or followed by at least one 1,2-Dideoxyribose or at least one polyethylen glycol. The stopping site can be formed even by only PEG (polyethylene glycol) molecules in a structure which has the space-filling of at least 2 nucleotides. In this embodiment, several members of PEG family are suitable, but the preferential molecule is triethylene glycol (TEG).
According to embodiments, the competitive palindromic portion 3) is complementary to the sequence of the priming portion. The competitive palindromic portion is at least one nucleotide shorter than the priming sequence. The 5′ end of the competitive palindromic portion contains a DNA polymerase stopping site (processivity blocker) composed as described above with reference to the loop portion 2).
According to embodiments, the 5′ fidelity tail length ranges from 1 to 1000 nucleotides, it contains bases or bases analogues (adenine, cytosine, timine, guanine, uracil, orotidine, inosinate, xanthylate) and is complementary or partially complementary to the downstream region of the target sequence.
In still further embodiments, in case a formation of primer dimers causes aspecific fluorescent background, decoy oligonucleotides can be employed in the PCR reaction mixture as above-described, in order to decrease the aspecific signal due to primer-dimer formations. Decoy oligonucleotides generally are oligonucleotide fragments able to bind to and block transcription and respectively translation factors. In these embodiments, decoy oligonucleotides range from 10 to 100 nucleotides, and are complementary for at least 50% to the specific amplification primers as above described. Decoys oligonucleotides are defined by a 3′ overhang on one side and by a 5′ blunt extremity on the other side. The difference between the melting temperature of each decoy oligonucleotide and the relative paired primer does not exceed 2° C.
Decoy oligonucleotides can be advantageous since they can prevent the amplification primers from aspecific interaction that create free 3′ OH groups, accessible by polymerase, because their sequence is complementary to the amplification primers, their melting temperature is slightly lower and they do not create structures that expose free 3′ OH groups.
According to embodiments, combinable with all embodiments described herein, PCR amplification can be for instance performed in a PCR thermocycler.
According to further embodiments, combinable with all embodiments described herein, PCR amplification can be typically performed in a real-time PCR machine, for instance a real-time PCR thermocycler.
Since, according to the present disclosure, the same sample is first subjected to the PCR amplification and then to HRM analysis, the whole method can be performed in a single apparatus, in particular a real-time PCR machine.
According to possible embodiments, the PCR amplification and detection can be performed simultaneously by means of Real Time PCR in any setup known in the art, including quantitative Real time PCR allowing assessment of the pathogenic load in the infected sample, followed by HRM analysis, performed in the same real-time PCR machine.
However, in other embodiments, the two operations, i.e. PCR amplification and HRM analysis, can also be performed in separate and distinct apparatuses coupled or associated each other, for instance a typical thermocycler for the PCR amplification and then a real-time PCR configured for HRM analysis.
For example, in possible implementations, the detection can be performed using a dedicated PCR device, also in portable format, containing a specific Peltier module coupled with a fluorescence optical reader or other appropriate reading device, able to perform HRM analysis.
In still further implementations, the detection via the PCR amplification can be performed using a dedicated PCR device containing for instance a specific Peltier module coupled to a read-out device different than a fluorescent read-out device, for instance a chemiluminescent or electrochemical read-out device, a conductimetric, amperometric, voltammetric read-out device, plasmonic optical red-out device or any other suitable read-out device.
Embodiments described herein can be used for diagnostic purposes. In particular, in the following, specific ranges of the reagents present in the two possible implementations of the PCR reaction mixture are described, that can be used for diagnostic purposes. Subsequently, specific ranges are described that can be used for specific detection of the HPV DNA.
In particular, the method and diagnostic kit containing the above-mentioned PCR reaction mixture according to the present disclosure can be used to detect clinical pathogens present in the sample, preferably blood borne pathogens including their genetic sequences.
In one embodiment, a possible first amplification buffer for diagnostic purposes comprises the dNTPs, the source of mono or bivalent cations, the buffer solution, the BSA, the Hot Start DNA polymerase, the intercalating molecule or compound. For instance, one specific implementation of the first amplification buffer comprises:
In another possible embodiment, a possible alternative second amplification buffer for diagnostic purposes is provided, that comprises the dNTPs, the source of mono or bivalent cations, the buffer solution, BSA, the hot start DNA polymerase, the intercalating molecule or compound and the above mentioned additives. By the addition of additives, the second amplification buffer can be used as a PCR enhancer buffer providing increased diagnostic sensitivity, specificity and accuracy as above discussed. For instance, one specific implementation of the alternative second amplification buffer comprises:
In the embodiments of methods and diagnostic kit described herein for diagnostic purposes, since different PCR machine, i.e. different thermocycler models, may generate different curves and in particular different melting peaks, a calibrator is provided to set each real-time PCR machine for a correct and precise melting analysis required to genotype the pathogen. Indeed, some variations might occur due to machine type, efficiency due to maintenance status or acquisition settings, and the calibrator allows the adjustment of the observed measurements in a specific machine. The calibrator can be composed by synthetic oligonucleotides corresponding to the amplicons generated by the specific primers of the PCR reaction mixture according to the present disclosure. Advantageously, a machine-specific calibrator can be loaded in PCR runs periodically to check the effective melting temperature of the amplicons of a particular thermocycler machine, and compare it with the expected melting temperature.
Further embodiments described herein for diagnostic purposes provide primers for obtaining combinations of melting temperature, in order to increase the number of targets detectable in the same assays. The limit of the number of targets simultaneously detectable in a single well of a PCR machine is generally defined by the capability of the system to distinguish and resolve two proximal peaks. However, according to embodiments described herein, it is possible to increase the number of targets detectable in the same assays, providing two or more sets of primers that are specific for the same DNA targets. According to embodiments, a first set of primers is present in a PCR reaction mixture in one well, at least a second set is present in another well. Each set of primers comprises primers each recognizing one specific DNA target. The DNA targets recognized by the first set of primers are the same as the DNA targets recognized by the second set of primers and the melting temperature of an amplicon generated by a primer of one set of primers recognizing a specific DNA target is different from the melting temperature of an amplicon generated by a primer of the other set of primers recognizing said specific DNA target.
As indicated in the table below as an example, 3 targets are identified by two primer sets: Target #1 is recognized by set A and B, Target #2 is recognized by set C and D, Target #3 is recognized by set E and F. The PCR reaction mixture aliquoted in the well 1 contains primer sets A, C and E; the PCR reaction mixture aliquoted in the well 2 contains primer sets B, D and F.
For each target DNA, HRM data will be collected for each well; hence each target DNA will be defined not by one single melting peak, but for several melting peaks, one for each well, as indicated in the table below as an example:
These embodiments allow the discrimination of more targets DNA thanks to the exponential increase in the number of possible combinations of the two or more melting temperatures that will be revealed, representing each of them a variable; thus, each target DNA will be precisely defined according multiple variables.
If x is the number of possible peaks distinguishable in a single well, the number of targets distinguishable in this embodiment is xa, where a is the number of sets of primers used for each target, and consequently the number of wells used for each sample.
This variant is particularly useful when the application aims at differentiating high number different targets (such as it occurs in HPV genotyping, where 40 genotypes can be simultaneously analysed and defined by the embodiments according to the present disclosure).
In the following, embodiments of methods and diagnostic kit according to the present disclosure are described for specific use for HPV diagnosis, using specific primers. In one embodiment, methods and diagnostic kit of the present disclosure are used to detect Human Papillomavirus DNA in clinical samples and to discriminate different genotypes by HRM analysis.
According to one embodiment, the methods and diagnostic kit, in addition to any of the afore-described combination of reagents required for carrying out DNA amplification by PCR, includes specific primers for each genotype of HPV are included from SEQ ID No. 20 to SEQ ID No. 616 and from SEQ ID No. 655 to SEQ ID No. 706 provided in the sequence listing attached hereto. The afore-mentioned specific primers for HPV diagnosis are characterized by the following features:
In yet further embodiments, one further set of normalizing primers can be provided, for the amplification of human genomic DNA, said amplification of human genomic DNA serving as an internal PCR validation control and/or control for normalization of the amplified pathogen, e.g. HPV, DNA obtained according to any of the embodiments described herein. An example of such pair of normalizing primers targeting a fragment from the human beta-globin gene is provided in SEQ ID No. 630 and SEQ ID No. 631 of the sequence listing attached hereto.
In a preferred embodiment of the diagnostic kit for HPV diagnosis, specific primers for the 13 most frequent high risk HPV serotypes are provided in the PCR reaction mixture of the present disclosure, that have the sequences SEQ ID NO. 591 to SEQ ID No. 616 of the sequence listing attached hereto.
In one embodiment, a specific amplification buffer for HPV diagnosis comprises the following reagents with concentration expressed as ranges:
One example of possible specific concentration values of the reagents is the following:
In another possible embodiment, a further alternative possible specific amplification buffer for HPV diagnosis, providing increased diagnostic sensitivity, specificity and accuracy comprises the following reagents with concentration expressed as ranges:
One example of possible specific concentration values of the reagents of this further alternative amplification buffer is the following:
Advantageously, the diagnostic HPV test based on the embodiments described herein can provide at least two important information about HPV testing and screening:
According to still further embodiments, combinable with all embodiments described herein, methods and diagnostic kit according to the present disclosure can be used to detect and discriminate further different pathogens responsible for other sexually transmitted diseases, such as chlamydia infection, syphilis infection, or gonorrhoea infection, by using specific amplification primers according to SEQ ID No. 1 to SEQ ID No. 19 and from SEQ ID No. 636 to SEQ ID No. 654 of the sequence listing attached hereto in the PCR reaction mixture as above described.
While the foregoing is directed to embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IT2017/000125 | 6/20/2017 | WO | 00 |
