Patent Application
20210155973
Embodiments of the present disclosure relate to methods for DNA targets detection directly in crude samples through PCR and genotyping via HRM analysis. In particular, embodiments of the present disclosure describe methods to detect several pathogenic strains responsible for sexually transmitted diseases, such as, but not limited to HR-HPV (High Risk Human Papilloma Virus).
More in details, the present disclosure allows, in one multiplex assay, the detection of pathogenic DNA directly from crude biological specimens, including for instance vaginal or cervical mucus, without a DNA extraction step.
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 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 each 0.1° C./sec or less.
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 automated. In particular, HRM requires highly pure extracted DNA because DNA prep contaminations cause unpredictable distortions of the melting curves.
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.
Indeed, there is a felt need to provide methods that can be used to apply HRM technique directly in crude samples, without the requirement of expensive and time-consuming DNA extraction steps, and without the use of expensive primers and probe pairs.
It is well known that HPV is a necessary cause of invasive cervical cancer worldwide.
This tumour is one of the 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 that allows 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 sensitivity5 and low negative predictive value.
Moreover, the use of endocervical brush can be painful and invasive, and this psychologically inhibits the access to the test by part of the population, especially in contexts where for social and religious reasons, women rarely undergo gynaecological visits.
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 specific 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 sets that lead to fluorescence a specific amplification signals (such as background derived from primer dimers formation). This can be limited thanks to fluorescently-labelled probes (e. g. Taqman probes, Life Technologies), that ensure a higher specificity compared to intercalating dyes that do not make distinction between amplicons. However, fluorescently-labelled probes are more expensive, affect the test robustness and the analysis requires more than one fluorescence detector.
There is a need to improve methods for DNA targets detection through PCR and genotyping via HRM, which overcome at least one of the drawbacks in the art.
In particular, one aim of the present disclosure is to provide methods for DNA targets detection directly from crude samples through PCE and genotyping via HRM analysis, while improving significantly the robustness of the PCR performance.
One further aim of the present disclosure is to provide methods that can work directly using crude samples such as, for instance, vaginal and cervical mucus, thus providing a test that has a better compliance with respect to the women population.
Still one further aim of the present disclosure is to avoid DNA extraction while improving significantly the robustness of the HRM performance.
According to embodiments, a method for DNA targets detection directly in crude samples through PCR and genotyping via HRM analysis is provided. According to one embodiment, the method includes:
providing a crude sample to be subjected to detection analysis;
diluting said crude sample using a processing buffer comprising a protein-digesting enzyme, wherein the crude sample is diluted up to a protein concentration ranging from 0.1 μg/μl to 10 μg/μl;
treating the diluted crude sample by performing a ramp of increasing and then decreasing temperature;
providing a PCR reaction mixture comprising two or more pairs of primers for a multiplex amplification approach of two or more target nucleic acids and an amplification buffer comprising an intercalating molecule or compound incorporated into the double-stranded amplicon for emitting a detectable signal;
performing PCR amplification using said PCR reaction mixture and said treated diluted crude sample;
performing, at the end of the PCR amplification, an HRM analysis on the PCR reaction mixture and said treated diluted crude sample previously subjected to PCR amplification;
wherein the method further comprises monitoring, during the HRM analysis, the change in the signal emission resulting from the temperature-induced denaturation of the double-stranded amplicons into two single-stranded DNA, due to the release of the intercalating molecule or compound,
wherein the method further comprises detecting DNA targets in the crude sample, through a reader analysing the signal variation and obtaining the result of the analysis through a graphic interface connected to said reader.
According to further embodiments, a diagnostic kit is provided comprising a processing buffer and a PCR reaction mixture that can be used for performing PCR amplification and a subsequent HRM analysis on the PCR reaction mixture previously subjected to real-time PCR. In one embodiment, the processing buffer comprises a protein-digesting enzyme and the PCR reaction mixture comprises two or more pairs of amplification primers for amplifying, in a multiplex approach, two or more target nucleic acids and an amplification buffer comprising an intercalating molecule or compound incorporated into the double-stranded amplicon and emitting a detectable signal.
According to yet further embodiments, an apparatus for performing DNA targets detection directly in crude samples through PCR and genotyping via HRM is provided. In one embodiment, the apparatus includes:
a processing buffer comprising a protein-digesting enzyme, for diluting said crude sample;
a heating/cooling device configured for subjecting the diluted crude sample to a treatment according to a ramp of increasing and then decreasing temperature;
a PCR reaction mixture comprising two or more pairs of amplification primers for amplifying in a multiplex approach two or more nucleic acids targets and an amplification buffer comprising an intercalating molecule or compound incorporated into the double-stranded amplicon and emitting a detectable signal;
a PCR amplification device configured for using said PCR reaction mixture and for subjecting the treated diluted crude sample to a PCR amplification;
an HRM device for performing HRM analysis on the PCR reaction mixture previously subjected to PCR amplification;
wherein the PCR reaction mixture comprises two or more pairs of amplification primers for amplifying in a multiplex approach two or more target nucleic acids,
monitoring means for monitoring, during the HRM analysis, the change in the signal emission resulting from the temperature-induced denaturation of the double-stranded amplicons into two single-stranded DNA, due to the release of the intercalating molecule or compound,
a reader analysing the signal variation for detecting DNA targets in the crude sample, so that the result of the analysis can be obtained through a graphic interface connected to said reader.
In advantageous embodiments, the methods according to the present disclosure have been applied to successfully improve the detection of HPV DNA, the main cause of cervical cancer.
According to the present disclosure, the methods described herein can work using for instance vaginal and cervical mucus, a biological sample that women can easily self-collect with no invasiveness and no pain, instead of endocervical brush that can be painful and invasive, thus psychologically inhibiting the access to the test by part of women population.
Moreover, the methods described herein work directly on crude samples: after a brief pre-treatment, the crude sample, e.g. vaginal or cervical mucus, is directly loaded in the reaction buffer. No DNA extraction is required, in contrast to the tests known in the prior art. The sample can be, for instance, vaginal or cervical mucus but also the common PreservCyt, ThinPrep (Hologic) or other unprocessed crude samples.
According to embodiments, combinable with all embodiments described herein, the DNA target that can be detected is a pathogen DNA target.
Moreover, according to the present disclosure, an innovative, fast and affordable multiplexing technology can be provided, enabling direct PCR detection, in particular real-time direct PCR, of multiple pathogenic, e.g. viral, DNA targets in a single reaction, using specific reaction mixture and conditions.
Embodiments described herein allow to detect, for example, HPV genotypes through a specific HRM analysis, by directly analysing crude samples.
Advantageously, embodiments described herein allow to detect up to 200 HPV types, Alpha-HPV, Beta-HPV, Gamma-HPV, Mu-HPV, Nupapillomavirus including the high risk and low risk HPV types 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). Advantageously, the methods according to the present disclosure can be performed with the most common real time PCR machines present on the market.
Other pathogens that can be detect according to the present disclosure include for instance other pathogens than HPV responsible for sexually transmissible 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 integrated HIV1 and HIV2 viral DNA.
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:
direct test: embodiments of the present disclosure describe a method to analyse and characterize DNA targets without DNA extraction steps, but directly in preprocessed crude samples, such as cervical and vaginal mucus, and other bodily fluids such as saliva, blood, urine, biopsies, formalin-fixed paraffin-embedded (FFPE) tissue cells, fine needle aspiration biopsies or similar biological samples;
affordability: embodiments of the present disclosure are affordable compared to the afore-mentioned tests and methods of the prior art, because they do not use dozens of expensive labelled-probes but a unique intercalating molecule or compound, e.g. an intercalating dye;
open-accessibility and versatility: embodiments of the present disclosure can be performed by any real-time PCR machine and do not require tailored and customized instruments;
single-channel: result of embodiments of the present disclosure can be provided through the analysis of a single signal, e.g. fluorescence, channel, in contrast to the afore-mentioned tests and methods of the prior art, where more than one channel is used. The selected channel can be chosen among those embedded in any real-time PCR machine and this makes embodiments of the present disclosure suitable for any real-time PCR machine.
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, as well as 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 a method that uses a processing buffer for pre-treating and diluting a crude sample prior to subjecting it to direct PCR amplification and HRM analysis.
Advantageously, the sample can be 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 biological samples.
The method of the present disclosure is able to detect DNA targets directly in crude samples through PCR and genotyping via HRM analysis. In one embodiment, the method includes:
providing a crude sample to be subjected to detection analysis;
diluting said crude sample using a processing buffer comprising a protein-digesting enzyme, wherein the crude sample is diluted up to a final protein concentration in a range between 0.1 μg/μl to 10 μg/μl;
treating the diluted crude sample by performing a ramp of increasing and then decreasing temperature;
providing a PCR reaction mixture comprising two or more pairs of amplification primers for amplifying in a multiplex approach two or more target nucleic acids and an amplification buffer comprising an intercalating molecule or compound incorporated into the double-stranded amplicon and eventually emitting a detectable signal;
performing PCR amplification using said PCR reaction mixture and said treated diluted crude sample;
performing, at the end of the PCR amplification, an HRM analysis on the PCR reaction mixture and said treated diluted crude sample previously subjected to PCR amplification;
wherein the method further comprises monitoring, during the HRM analysis, the change in the signal emission resulting from the temperature-induced denaturation of the double-stranded amplicons into two single-stranded DNA, due to the release of the intercalating molecule or compound,
wherein the method further comprises detecting DNA targets, through a reader analysing the signal variation and obtaining the result of the analysis through a graphic interface connected to said reader.
According to embodiments, combinable with all embodiments described herein, the DNA target that can be detected is a pathogen DNA target.
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 and type of DNA target, e.g. pathogen DNA target, present in the sample.
According to possible embodiments, combinable with all embodiments described herein, the protein-digesting enzyme can be selected from a group consisting of: endoprotease, proteinase K, pepsin, trypsin, α-chymotrypsin, subtilisin and endopeptidases.
According to possible embodiments, combinable with all embodiments described herein, the processing buffer further includes a hydrolase.
According to possible embodiments, combinable with all embodiments described herein, the processing buffer further includes a pH stabilizer.
According to possible embodiments, combinable with all embodiments described herein, the processing buffer further includes a preservative.
According to possible embodiments, the crude sample can be collected with a dedicated biological specimen collector, and possibly the processing buffer can be embedded inside the collector.
The complete pre-treatment with the processing buffer is crucial to obtain good amplification plots, decreasing the fluorescence background of the amplification signal and increasing fluorescence intensity of HRM analysis melting peaks; these are fundamental features that lead to an increased diagnostic sensitivity, specificity and accuracy of the analysis. In particular, in the case where the intercalating molecule or compound is an intercalating fluorescent dye, the initial high fluorescence background measured in the absence of processing buffer treatment is due to the complexity of the biological matrix, where proteins (mucins in case of mucus) together with other biological components entrap fluorescent dyes leading to a high fluorescent background. The treatment with the processing buffer according to the present disclosure has a pivotal role reducing the biological complexity of crude samples and avoiding structures able to retain fluorescent dyes that interfere with the PCR reaction and with the HRM analysis. The graph of
Different patterns of protein contents of vaginal mucus were observed by SDS-PAGE gel analysis followed by Coomassie staining (see
According to possible embodiments, the biological collector used to collect the crude sample, for instance vaginal or cervical mucus, can be suspended in the processing buffer according to the present disclosure, in order to achieve correct and required sample dilution.
According to the present disclosure, the final dilution of the crude sample, obtained for instance from the resuspension of biological collector, is crucial for the results of the analysis. As above discussed, in the final dilution, protein concentration advantageously ranges from 0.1 μg/μl to 10 μg/μl.
The graph of
The graph of
According to the present disclosure, the treatment of diluted crude sample occurs in the processing buffer containing at least the protein-digesting enzyme, and possibly a hydrolase, a pH stabilizer, a preservative, performing a ramp of temperature through a multistep process, wherein the temperature is first increased and then decreased. In particular, first the diluted crude sample in the processing buffer is heated, for example in a double step heating, and then it is cooled, for example in a single step cooling.
In possible implementations, a heating/cooling device can be used, configured for subjecting the diluted crude sample to a ramp of increasing and then decreasing temperature. As will be described more in detail herein below, the heating/cooling device can be a PCR amplification device or machine, a thermocycler, a thermocycler used for PCR reaction or the like embodying, including or embedding such heating/cooling device. In possible implementations, cooling can be obtained, for instance, by using specific Peltier cells or modules. According to possible embodiments, the ramp of temperature includes:
a first incubation step ranging from 5 to 60 minutes in a temperature range between 25° C. and 70° C., preferably between 50° and 60° C.,
a second incubation step ranging from 5 to 60 minutes in a temperature ranging from 90° C. to 100° C.,
a third incubation step ranging from 5 to 60 minutes in a temperature ranging from 0° C. to 10° C.
Advantageously, the protein-digesting enzyme contained in the processing buffer is crucial to obtain an efficient PCR amplification and a clear melting analysis, avoiding an excess of fluorescence background that can affect the sensitivity, specificity and accuracy of the final result.
The graph of
The graph of
According to embodiments, combinable with all embodiments described herein, performing PCR amplification using said PCR reaction mixture and said treated diluted crude sample includes amplifying the purified target 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.
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, the PCR reaction mixture further 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, or others.
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 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 between one, more or all of additives in a group comprising: NP40, DMSO, TMAC (Tetramethylammonium Chloride), Acetamide, Triton, Formamide, Betaine, E. coli ssDNA binding protein, Glycerol, L-Carnitine and Gelatin. 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
The use of additives according to embodiments described herein is advantageous to avoid a high basal fluorescence background in the PCR reaction, allowing an increased diagnostic sensitivity, specificity and accuracy. The graph of
According to embodiments, combinable with all embodiments described herein, amplifying the purified target 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:
denaturation ranging from 95° to 98° C. ranging from 1 to 30 seconds;
annealing in a ranging from 50° C. to 70° C. ranging from 1 to 60 seconds;
extension in a range between 60° C. and 75° C. ranging from 0 second to 5 minutes.
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 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. from 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 said monitoring the change in the signal, e.g. fluorescence, emission resulting from the temperature-induced denaturation of the double-stranded amplicons or amplification products into two single-stranded DNA, due to the release 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, performing 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:
incubation at 95° C. ranging from 1 seconds to 60 seconds
incubation at 60° C. ranging from 1 seconds to 2 minutes
ramping up to 95° C. increasing the temperature 0.1° C./second or less, and performing said monitoring the change in the signal, e.g. fluorescence, emission resulting from the temperature-induced denaturation of the double-stranded amplicons or amplification products into two single-stranded DNA, due to the release of the intercalating molecule or compound, i.e. intercalating dye.
The method according to the present disclosure as above described allows to genotype different pathogens through HRM analysis amplifying DNA directly from crude samples. Conversely, conventional PCR systems and methods do not allow to obtain clear melting curves starting without DNA extraction. The graph of
According to embodiments, combinable with all embodiments described herein, PCR amplification can be for instance performed in a PCR thermocycler. Such PCR thermocycler can embody, include or integrate the heating/cooling device for performing the treatment of the crude sample with the above described processing buffer.
According to further embodiments, combinable with all embodiments described herein, PCR amplification can be typically performed in a real-time PCR machine.
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.
According to possible embodiments, the PCR reaction can be performed using a container, e.g. a test tube or vial, possibly provided with a closure cap, or a well of a microplate, inside which the crude sample can be contained. In possible implementations, the crude sample can be treated using the above described processing buffer prior to be disposed inside said container, or alternatively the crude sample can be directly disposed into the container used for the PCR reaction and treated therein with the above processing buffer.
In still further embodiments described using
For instance, the processing buffer and the PCR mixture or amplification buffer can be characterized by different states of matter and disposed directly inside the container used for the PCR reaction.
In particular, in possible embodiments the container can comprise a first phase and a second phase, or upper phase.
In
As depicted in
In this embodiment, the solid/semi-solid/jellified state of the first phase 14 can be obtained by using a polysaccharide polymer material, such as agarose, in layer or as a diluent, and including the polymerase therein; or by using an air-bubble layer. In alternative, the polymerase can be embedded in microbeads, such as agarose microbeads or alginate microbeads.
The second phase, or upper phase, 16 is disposed on or above the first phase 14 and is liquid, e.g. aqueous. The processing buffer is comprised in said second phase 16.
In these embodiments, therefore, the pre-treatment using the processing buffer, the PCR reaction followed by the HRM analysis occurs in the same container 15. Advantageously, in possible implementations such three operations can be performed using the same specific container 15 according to the following protocol, involving the first step of sample treatment with protein-digesting enzyme and the second step of PCR amplification:
first incubation step ranging from 5 to 60 minutes in a range of temperature between 50° C. and 70° C.;
second incubation step ranging from 5 to 60 minutes in a range of temperature between 90° C. and 100° C.;
third incubation step ranging from 5 to 60 minutes in a range of temperature between 0° C. and 10° C.;
incubation at a temperature from 95° C. to 98° C. ranging from 5 second to 10 minutes (if hot-start polymerase is used);
thermocycling, e.g. at least 30 cycles of the following steps:
incubation at 95° C. ranging from 5 seconds to 60 seconds;
incubation at 60° C. ranging from 30 seconds to 2 minutes;
ramping up to 95° C. increasing the temperature 0.1° C./second or less.
According to such embodiment, the PCR mixture or amplification buffer of the first phase 14 present in the container 15 is advantageously protected during the pre-treatment with the second phase 16 comprising the processing buffer, and successively the content of the PCR mixture or amplification buffer is released at the end of the pre-treatment thanks to state changes of the two phases due to heating as above described.
In particular, as shown in
In the second step, as shown in
Embodiments described herein can be used for diagnostic purposes. In particular, in the following, specific ranges of the reagents present in the processing buffer and 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 processing buffer and 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, the processing buffer for diagnostic purposes comprises proteinase K and TrisHCl buffer solution. For instance, one specific implementation of the processing buffer comprises:
a) proteinase K (between 1 mg/mL and 1 μg/mL);
b) TrisHCl buffer solution (between 1 M and 10 mM; pH between 7.0 and 10.0).
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:
a) dNTPs (final concentration range: from 0.05 mM to 0.3 mM)
b) MgCl2 (final concentration range: from 0.3 mM to 4 mM)
c) TrisHCl buffer solution (final concentration range: from 10 mM to 50 mM; pH from 6.00 to 10.00)
d) KCl (final concentration range: from 10 mM to 50 mM)
e) BSA (final concentration range: from 0.005 to 0.05 mg/ml)
f) Hot Start polymerase
g) SYTO-9 (final concentration range: from 1 μM to 8 μM).
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, the 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:
a) dNTPs (final concentration range: from 0.05 mM to 0.5 mM)
b) MgCl2 (final concentration range: from 0.3 mM to 4 mM)
c) TrisHCl buffer solution (final concentration range: from 10 mM to 50 mM; pH from 6.00 to 10.00)
d) KCl (final concentration range: from 10 mM to 50 mM)
e) BSA (final concentration range: from 0.005 to 0.05 mg/ml)
f) Hot start polymerase
g) SYTO-9 (final concentration range: from 1 μM to 8 μM)
h) TMAC (Tetramethylammonium Chloride) (final concentration range: from 10 mM to 100 mM)
i) Acetamide (final concentration range: from 0% to 5%)
j) Formamide (final concentration range: from 0% to 5%)
k) Betaine (final concentration range: from 0 μM to 5M)
l) Gelatin (final concentration range: from 0.01 mg/ml to 1 mg/ml).
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, 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 comparing them with the expected melting temperature.
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.
In one embodiment, a specific processing buffer for HPV diagnosis, for example in the case of vaginal mucus sample, or PreservCyt-sample (or similar), or cervical swabs, or biopsies, formalin-fixed paraffin-embedded (FFPE) tissue or saliva, or urine or similar biological sample, comprises at least the following components:
proteinase K (between 1 mg/mL and 1 μg/mL)
TrisHCl buffer solution (between 1 M and 10 mM; pH from 7.0 to pH 10.0).
One example of possible specific concentration values of the reagents of this processing buffer for HPV diagnosis is the following:
proteinase K at a final concentration of 500 μg/mL;
TrisHCl buffer solution at a final concentration of 150 mM and pH 9.0.
In one embodiment, a specific amplification buffer for HPV diagnosis comprises the following reagents with concentration expressed as ranges:
a) dNTPs (final concentration range: from 0.05 mM to 0.5 mM)
b) MgCl2 (final concentration range: from 0.3 mM to 4 mM)
c) TrisHCl buffer solution (final concentration range: from 10 mM to 50 mM; pH from 7.00 to 10.00)
d) KCl (final concentration range: from 10 mM to 50 mM)
e) BSA (final concentration range: from 0.005 mg/ml to 0.05 mg/ml)
f) Hot Start polymerase
g) SYTO-9 (final concentration range: from 1 μM to 8 μM).
One example of possible specific concentration values of the reagents is the following:
a) dNTPs (final concentration 0.2 mM)
b) MgCl2 (final concentration 0.75 mM)
c) TrisHCl buffer solution (final concentration 30 mM: and pH 9.0)
d) KCl (final concentration 50 mM)
e) Bovise Serum Albumin (final concentration 10 μg/ml)
f) Hot start polymerase
g) SYTO-9 (final concentration 4 μM).
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:
a) dNTPs (final concentration range: from 0.05 mM to 0.3 mM)
b) MgCl2 (final concentration range: from 0.3 mM to 4 mM)
c) TrisHCl buffer solution (final concentration range: from 10 mM to 50 mM; pH from 7.00 to 10.00)
d) KCl (final concentration range: from 10 mM to 50 mM)
e) BSA (final concentration range: from 0.005 mg/ml to 0.05 mg/ml)
f) Hot start polymerase
g) SYTO-9 (final concentration range: from 1 μM to 8 μM)
h) TMAC (Tetramethylammonium Chloride) (final concentration range: from 10 mM to 100 mM)
i) Acetamide (final concentration range: from 0.5% to 5%)
j) Formamide (final concentration range: from 0.5% to 5%)
k) Betaine (final concentration range: from 100 μM to 5M)
l) Gelatin (final concentration range: from 0.01 mg/ml to 1 mg/ml). One example of possible specific concentration values of the reagents of this further alternative amplification buffer is the following:
a) dNTPs (final concentration 0.15 mM)
b) MgCl2 (final concentration 0.75 mM)
c) TrisHCl buffer solution (final concentration 30 mM; pH 9)
d) KCl (final concentration: 40 mM)
e) BSA (final concentration 10 μg/ml)
f) Hot start polymerase
g) SYTO-9 (final concentration 1 μM)
h) TMAC (Tetramethylammonium Chloride, final concentration of 75 mM)
i) Acetamide (final concentration 3%)
j) Formamide (final concentration 1.5%)
k) Betaine (final concentration 0.5M)
l) Gelatin (final concentration 0.1 mg/mL).
Advantageously, the diagnostic HPV test based on the embodiments described herein can provide at least an important diagnostic information, for example for HPV diagnostic, or other sexually transmissible diseases, like for instance Chlamydia infection, syphilis infection, or Gonorrhoea infection. For example, in the case of HPV diagnostic, the amplification curves obtained by the PCR allow the detection of the first 14 high risk genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68-Cit. IARC 2009) and thus give a diagnostic information. A sample is positive when the amplification curve occurs before 35 PCR cycles using a fluorescence threshold that range from 250.000 to 400.000. The present invention has been tested on positive samples (extracted DNA more than 200 HPV positive women with a CIN2+ cytology, according with Mejer guide lines, 2009). Clinical data showed that sensitivity of the diagnostic HPV test according to the embodiments described herein, intended as the sensitivity to detect High-Grade Squamous Intraepithelial Lesions (HSIL or CIN2/3 lesions, that are considered the clinical endpoint in the HPV diagnostics), is 98%. Sensitivity is the percentage of HPV-infected people with a diagnosed HSIL that are detected by the HPV test according to the embodiments described herein, divided by the total number of HPV-infected people with a diagnosed HSIL detected by conventional test (e.g. Pap test). The test specificity reaches almost the 100% and the test accuracy ranges from 0.93 to 0.98;
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/IT2017/000126 | 6/20/2017 | WO | 00 |
