METHOD FOR THE DETECTION AND QUANTIFICATION OF HUMAN CYTOMEGALOVIRUS BY MEANS OF VIRION RNAS

Abstract

A method for detection and/or quantification of a human cytomegalovirus (hCMV) virion mRNA in a sample obtained from a patient to identify and/or quantify hCMV infection includes: i) optionally removing from the sample any cellular component that might be present, obtaining a cell-free sample; ii) extracting RNA from a cell-free sample obtained from the patient or obtained in step i); iii) forming a reaction mixture by contacting said RNA extracted in step ii); iv) subjecting the reaction mixture to a reverse transcription process under conditions suitable to generate a cDNA corresponding to the hCMV virion mRNA; v) subjecting the cDNA to an amplification process under conditions adapted to generate an amplification product constituted by DNA; and vi) detecting a presence and/or quantifying the quantity of the amplification product constituted by DNA, as an indication of the presence and/or of the quantity of hCMV virion mRNA in the sample.

Claims

1-14. (canceled)

15. A method for detection and/or quantification of a human cytomegalovirus (hCMV) virion mRNA in a sample obtained from a patient treated with an antiviral drug capable of interfering with cleavage and encapsidation of hCMV DNA, comprising the steps of: i) optionally removing from the sample obtained from the patient any cellular component that might be present, obtaining a cell-free sample; ii) extracting RNA from a cell-free sample obtained from the patient or from the cell-free sample obtained in step i); iii) forming a reaction mixture by contacting said RNA extracted in step ii) with a solution comprising: a) one or more primer pairs specific for said hCMV virion mRNA; b) an RNA-dependent DNA polymerase; c) a DNA polymerase; iv) subjecting said reaction mixture to a reverse transcription process under conditions suitable to generate a cDNA corresponding to said hCMV virion mRNA; v) subjecting said cDNA to an amplification process under conditions adapted to generate an amplification product constituted by DNA; vi) detecting a presence and/or quantifying the quantity of said amplification product constituted by DNA, as an indication of the presence and/or of the quantity of hCMV virion mRNA in the sample obtained from the patient.

16. The method according to claim 15, wherein each of said one or more primer pairs is independently selected from: a primer pair from a first set (“set 1)”) wherein a first primer has a sequence selected from a group consisting of SEQ ID NOs: 1-12 and a second primer has a sequence selected from a group consisting of SEQ ID NOs: 13-45; or a primer pair from a second set (“set 2)”) wherein a first primer has a sequence selected from a group consisting of SEQ ID NOs: 50-71 and a second primer has a sequence selected from a group consisting of SEQ ID NOs: 72-84; or a primer pair from a third set (“set 3)”) wherein a first primer has a sequence selected from a group consisting of SEQ ID NOs: 92-105 and a second primer has a sequence selected from a group consisting of SEQ ID NOs: 106-111.

17. The method according to claim 16, wherein: a) at least one of the one or more primer pairs is a primer pair from set 1) and the mixture further comprises a probe having a sequence selected from a group consisting of SEQ ID NOs: 46-49; or b) at least one of the one or more primer pairs is a primer pair from set 2) and the mixture further comprises a probe having a sequence selected from a group consisting of SEQ ID NOs: 85-91; or c) at least one of the one or more primer pairs is a primer pair from set 3) and the mixture further comprises a probe having a sequence selected from a group consisting of SEQ ID NOs: 112-116.

18. The method according to claim 15, wherein said amplification process is polymerase chain reaction (PCR).

19. The method according to claim 15, wherein said antiviral drug capable of interfering with the cleavage and encapsidation of hCMV DNA is a viral terminase complex inhibitor.

20. The method according to claim 19, wherein said viral terminase complex inhibitor is letermovir or a pharmaceutically acceptable salt thereof.

21. The method according to claim 15, wherein said sample obtained from the patient is selected from a group consisting of plasma optionally containing an anticoagulant, serum, urine, saliva, oral swab supernatant, cerebrospinal fluid (CSF), bronchoalveolar lavages (BAL), nasopharyngeal lavages, nasopharyngeal aspirates, pharyngeal swab supernatant, tear fluid, vitreous humor, eye swab supernatant, fecal supernatant.

22. The method according to claim 15, wherein said hCMV virion mRNA is an mRNA of a gene or of a coding region (ORF or CDS) of hCMV selected from the group consisting of UL21.5 (SEQ ID NO: 117), UL65 (SEQ ID NO: 122), UL83 (SEQ ID NO: 123), UL106 (SEQ ID NO: 124), UL107 (SEQ ID NO: 125), UL108 (SEQ ID NO: 126), UL109 (SEQ ID NO: 127), UL110 (SEQ ID NO: 128), UL122 (SEQ ID NO: 129), UL123 (SEQ ID NO: 130), TRL/IRL2 (SEQ ID NO: 131), TRL/IRL3 (SEQ ID NO: 132), TRL/IRL4 (SEQ ID NO: 133), TRL/IRL5 (SEQ ID NO: 134), TRL/IRL7 (SEQ ID NO: 135), and TRL/IRL13 (SEQ ID NO: 136).

23. The method according to claim 15, wherein said hCMV virion mRNA is mRNA of the UL21.5 gene (SEQ ID NO: 117).

24. An oligonucleotide consisting of a sequence selected from a group consisting of SEQ ID NOs: 1-116.

25. The oligonucleotide according to claim 24, wherein said oligonucleotide is a primer having a sequence selected from a group consisting of SEQ ID NOs: 1-45, SEQ ID NOs: 50-84, and SEQ ID NOs: 92-111.

26. The oligonucleotide according to claim 24, wherein said oligonucleotide is a probe having a sequence selected from a group consisting of SEQ ID NOs: 46-49, SEQ ID NOs: 85-91, and SEQ ID NOs: 112-116.

27. A kit for detection and/or quantification of hCMV virion mRNA, comprising: a) at least one primer pair selected from: a primer pair from a first set (“set 1)”) wherein a first primer has a sequence selected from a group consisting of SEQ ID NOs: 1-12 and a second primer has a sequence selected from a group consisting of SEQ ID NOs: 13-45; or a primer pair from a second set (“set 2)”) wherein a first primer has a sequence selected from a group consisting of SEQ ID NOs: 50-71 and a second primer has a sequence selected from a group consisting of SEQ ID NOs: 72-84; or a primer pair from a third set (“set 3)”) wherein a first primer has a sequence selected from a group consisting of SEQ ID NOs: 92-105 and a second primer has a sequence selected from a group consisting of SEQ ID NOs: 106-111; and optionally b) an RNA-dependent DNA polymerase; and c) a DNA polymerase.

28. The kit according to claim 27, wherein: a) the at least one primer pair is a primer pair from set 1) and the kit further comprises a probe having a sequence selected from a group consisting of SEQ ID NOs: 46-49; or b) the at least one primer pair is a primer pair from set 2) and the kit further comprises a probe having a sequence selected from a group consisting of SEQ ID NOs: 85-91; or c) the at least one primer pair is a primer pair from set 3) and the kit further comprises a probe having a sequence selected from a group consisting of SEQ ID NOs: 112-116.

Description

DETAILED DESCRIPTION

[0035] The following definitions are applied in the description and in the claims.

[0036] An “amplification process” refers to any chemical reaction, including enzyme-based ones, that gives rise to an increase in copies of a DNA sequence. One method for DNA amplification is polymerase chain reaction (PCR), which is well-known to the person skilled in the art and is described for example in patent no. U.S. Pat. No. 4,683,202. Further amplification reactions comprise, among others, ligase chain reaction (LCR), polymerase and ligase chain reaction (PLCR), Gap-LCR, Strand Displacement Amplification (SDA), Rolling Circle Amplification (RCA) and loop-mediated isothermal amplification (LAMP).

[0037] The term “oligonucleotide” refers to molecules comprising two or more deoxyribonucleotides or ribonucleotides, such as for example primers, probes, fragments of nucleic acids to be detected and control nucleic acids. Oligonucleotides can be prepared by means of any suitable method known in the art, including for example cloning and restriction of appropriate sequences and direct chemical synthesis, such as the conventional and well-known chemistry of phosphoramidites. In the context of the present disclosure, the oligonucleotides can be chemically modified, i.e., the primer and/or the probe can comprise one or more modified nucleotides or non-nucleotide compounds.

[0038] The term “primer” is used here as known to the person skilled in the art and refers to natural or synthetic oligonucleotides capable of acting as a starting point for DNA synthesis in the conditions in which the synthesis of a primer extension product that is complementary to a nucleic acid filament is induced, i.e., in the presence of four different triphosphate nucleosides and of a polymerization agent (such as for example a DNA polymerase or reverse transcriptase enzyme) in an appropriate buffer and at a suitable temperature.

[0039] The term “probe” refers to natural or synthetic oligonucleotides capable of hybridizing, under the appropriate conditions, to an amplification product of a nucleic acid in order to detect that amplification product.

[0040] The term “sample” refers to a material obtained from a human patient, in particular a human patient treated with an antiviral drug capable of interfering with the cleavage and encapsidation of hCMV DNA, which is suspected of containing or potentially containing at least one human cytomegalovirus (hCMV) virion mRNA.

[0041] The term “cellular component” refers to the corpusculate part, constituted by cells or cell fragments, that can be present in a sample obtained from a patient.

[0042] In a first aspect, the present disclosure relates to a method for the detection and/or quantification of a human cytomegalovirus (hCMV) virion mRNA in a sample obtained from a patient treated with an antiviral drug capable of interfering with the cleavage and encapsidation of hCMV DNA, said method comprising the steps of:

[0043] i) optionally removing any cellular component that might be present from the sample obtained from the patient, obtaining a cell-free sample;

[0044] ii) extracting RNA from a cell-free sample obtained from the patient or from the cell-free sample obtained in step i);

[0045] iii) forming a reaction mixture by contacting said RNA extracted in step ii) with a solution comprising:

[0046] a) one or more primer pairs specific for said hCMV virion mRNA;

[0047] b) an RNA-dependent DNA polymerase;

[0048] c) a DNA polymerase;

[0049] iv) subjecting said reaction mixture to a reverse transcription process under conditions suitable to generate a cDNA corresponding to said hCMV virion mRNA;

[0050] v) subjecting said cDNA to an amplification process under conditions such as to generate an amplification product constituted by DNA;

[0051] vi) detecting the presence and/or quantifying the quantity of said at least one amplification product constituted by DNA, as an indication of the presence and/or of the quantity of hCMV virion mRNA in the sample obtained from the patient.

[0052] In one embodiment of the method according to the disclosure, the patient is immunodepressed or immunosuppressed.

[0053] If the sample obtained from patient is not cell-free at the source, the method according to the disclosure provides for a first step aimed at obtaining a cell-free sample, in order to be able to focus the analysis only on the virion mRNA transcribed by the hCMV genome and carried by the virions. The sample can therefore be a cell-free clinical sample or, for example in the case of whole blood, undergo a treatment (step (i) of the method according to the disclosure) to remove the cellular component. The methods for removing from clinical samples the cells that constitute the cellular component are known to the person skilled in the art and comprise, for example, whole blood centrifugation to obtain plasma, whole blood coagulation to obtain serum, centrifugation of bronchoalveolar lavages to obtain a supernatant, and others.

[0054] Preferably, the sample obtained from the patient is selected from the group consisting of plasma (optionally containing an anticoagulant), serum, urine, saliva, oral swab supernatant, cerebrospinal fluid (CSF), bronchoalveolar lavages (BAL), nasopharyngeal lavages, nasopharyngeal aspirates, pharyngeal swab supernatant, tear fluid, vitreous humor, eye swab supernatant, and fecal supernatant. [0055] Preferably, the hCMV virion mRNA is an mRNA of a gene or of a coding region (ORF or CDS) of hCMV selected from the group consisting of UL21.5 (SEQ ID NO: 117), UL65 (SEQ ID NO: 122), UL83 (SEQ ID NO: 123), UL106 (SEQ ID NO: 124), UL107 (SEQ ID NO: 125), UL108 (SEQ ID NO: 126), UL109 (SEQ ID NO: 127), UL110 (SEQ ID NO: 128), UL122 (SEQ ID NO: 129), UL123 (SEQ ID NO: 130), TRL/IRL2 (SEQ ID NO: 131), TRL/IRL3 (SEQ ID NO: 132), TRL/IRL4 (SEQ ID NO: 133), TRL/IRL5 (SEQ ID NO: 134), TRL/IRL7 (SEQ ID NO: 135), and TRL/IRL13 (SEQ ID NO: 136).

[0056] To avoid or at least limit reactivity with DNA present in the sample, the primers specific for hCMV virion mRNA are preferably designed with their 5′ region complementary to a mRNA exon and the 3′ region complementary to an adjacent exon. In this manner it is possible to obtain primers which, under the reaction conditions, can hybridize only to mature mRNAs, i.e., mRNA from which, after transcription, the introns were removed and the exons have been spliced, or to the corresponding cDNAs. As a non-limiting example, the inventors of the present disclosure have designed primers specific for the mRNA of the UL21.5 gene of hCMV (astride the junction region of exons 1 and 2).

[0057] In a preferred embodiment of the method according to the disclosure, each of said one or more primer pairs is selected independently from:

[0058] a primer pair from a first set (“set 1)”) wherein a first primer has a sequence selected from the group consisting of SEQ ID NOs: 1-12 and a second primer has a sequence selected from the group consisting of SEQ ID NOs: 13-45; or

[0059] a primer pair from a second set (“set 2)”) wherein a first primer has a sequence selected from the group consisting of SEQ ID NOs: 50-71 and a second primer has a sequence selected from the group consisting of SEQ ID NOs: 72-84.

[0060] In another embodiment of the method according to the disclosure, in order to limit reactivity with DNAs present in the sample, a selective extraction for RNA is performed. In this embodiment, each of said one or more primer pairs can be selected independently not only from sets 1) and 2) but also from a primer pair from a third set (“set 3)”) wherein a first primer has a sequence selected from the group consisting of SEQ ID NOs: 92-105 and a second primer has a sequence selected from the group consisting of SEQ ID NOs: 106-111.

[0061] In a preferred embodiment of the method according to the disclosure, the reaction mixture further comprises a probe, i.e., an oligonucleotide capable of hybridizing under the reaction conditions of the amplification process to the amplification product constituted by DNA, in order to allow its detection and/or quantification.

[0062] In a preferred embodiment of the method according to the disclosure:

[0063] a) at least one of the one or more primer pairs is a primer pair from set 1) and the mixture further comprises a probe having a sequence selected from the group consisting of SEQ ID NOs: 46-49; or

[0064] b) at least one of the one or more primer pairs is a primer pair from set 2) and the mixture further comprises a probe having a sequence selected from the group consisting of SEQ ID NOs: 85-91; or

[0065] c) at least one of the one or more primer pairs is a primer pair from set 3) and the mixture further comprises a probe having a sequence selected from the group consisting of SEQ ID NOs: 112-116.

[0066] The three sets of primer pairs described above with the corresponding three probe groups make it possible to detect three alternative regions of the sequence of the mRNA of the UL21.5 gene of hCMV. As it is evident to the person skilled in the art, each primer pair within a set is compatible with the probes that do not have sequence overlap with the primers on the same DNA filament or have overlap with the primers on the complementary filament for no more than 6 nucleotides.

[0067] In order to design a probe with ideal characteristics for PCR, such as sensitivity and specificity, one or more nucleotides of the probe can be replaced with corresponding nucleotide analogues. It is possible to use various nucleotide analogues to obtain the desired base pairing properties. These include, among others, peptide nucleic acids (PNA), morpholino, glycol nucleic acids (GNA), threose nucleic acids (TNA), xeno nucleic acids (XNA), and locked nucleic acids (LNA). Moreover, there are modifications aimed at stabilizing the probes by means of molecules that interact with the double strand of DNA.

[0068] The probes can also include modifications adapted to allow their detection directly (for example fluorophores, quenchers, metal complexes, etc.) or indirectly (for example biotin, digoxigenin, linker sequences, etc.).

[0069] In a preferred but nonlimiting embodiment of the present disclosure, the probes include two modifications, respectively a fluorophore in position 5′ and a quencher in position 3′. In a particularly preferred but nonlimiting embodiment of the disclosure, the fluorophore is 6-FAM and the quencher is TAMRA.

[0070] Preferably, the reaction mixture further comprises a primer pair and optionally a probe which are specific for a control sequence other than the hCMV mRNA target sequence. This control sequence is preferably added to the reaction mixture in the form of exogenous nucleic acid, for example a plasmid or a synthetic DNA or the genome of a virus, of a bacterium or of a bacteriophage, in order to constitute an “internal check” to evaluate the integrity of the nucleic acids and the absence of amplification inhibitors in the tested sample.

[0071] In a preferred embodiment of the method according to the disclosure, the amplification process is polymerase chain reaction (PCR).

[0072] Even more preferably, the amplification process is a quantitative PCR of the realtime type.

[0073] In a preferred embodiment of the method according to the disclosure, the antiviral drug capable of interfering with the cleavage and encapsidation of hCMV DNA is a viral terminase complex inhibitor.

[0074] In a more preferred embodiment of the method according to the disclosure, the viral terminase complex inhibitor is letermovir or a pharmaceutically acceptable salt thereof.

[0075] In another aspect, the present disclosure relates to an oligonucleotide, specific for mRNA of the UL21.5 gene of hCMV, consisting of a sequence selected from the group consisting of SEQ ID NOs: 1-116.

[0076] In a preferred embodiment, the oligonucleotide according to the disclosure is a primer having a sequence selected from the group consisting of SEQ ID NOs: 1-45, SEQ ID NOs: 50-84, and SEQ ID NOs: 92-111.

[0077] In a preferred embodiment, the oligonucleotide according to the disclosure is a probe having a sequence selected from the group consisting of SEQ ID NOs: 46-49, SEQ ID NOs: 85-91, and SEQ ID NOs: 112-116.

[0078] The present disclosure also relates to a kit for the detection and/or quantification of hCMV virion mRNA comprising:

[0079] a) at least one primer pair selected from: [0080] a primer pair from a first set (“set 1)”) wherein a first primer has a sequence selected from the group consisting of SEQ ID NOs: 1-12 and a second primer has a sequence selected from the group consisting of SEQ ID NOs: 13-45; or [0081] a primer pair from a second set (“set 2)”) wherein a first primer has a sequence selected from the group consisting of SEQ ID NOs: 50-71 and a second primer has a sequence selected from the group consisting of SEQ ID NOs: 72-84; or [0082] a primer pair from a third set (“set 3)”) wherein a first primer has a sequence selected from the group consisting of SEQ ID NOs: 92-105 and a second primer has a sequence selected from the group consisting of SEQ ID NOs: 106-111; and

[0083] optionally

[0084] b) an RNA-dependent DNA polymerase;

[0085] c) a DNA polymerase.

[0086] In a preferred embodiment, the kit according to the disclosure further comprises a probe. For example, in this embodiment of the kit:

[0087] a) the at least one primer pair is a primer pair from set 1) and the probe has a sequence selected from the group consisting of SEQ ID NOs: 46-49; or

[0088] b) the at least one primer pair is a primer pair from set 2) and the probe has a sequence selected from the group consisting of SEQ ID NOs: 85-91; or

[0089] c) the at least one primer pair is a primer pair from set 3) and the probe has a sequence selected from the group consisting of SEQ ID NOs: 112-116.

[0090] In a preferred embodiment, the kit according to the disclosure further comprises a primer pair and optionally a probe which are specific for a control sequence other than the hCMV mRNA target sequence.

[0091] In a preferred embodiment of the kit according to the disclosure, the control sequence other than the hCMV mRNA target sequence is that of the MS2 bacteriophage with RNA genome, such as for example the one listed in the EBI ENA sequence database with the identification code (ID) V00642 (version 1 of 21 Oct. 1996).

[0092] For example, the kit according to the disclosure can comprise a primer pair specific for the genomic RNA of the MS2 phage, having respectively the sequences of SEQ ID NO: 118 and SEQ ID NO: 119 and a probe having the sequence of SEQ ID NO: 120.

[0093] The disclosure will be described further with reference to the following nonlimiting example.

Example: Nonselective Extraction of Nucleic Acids (DNA+RNA) from 1 mL of Plasma and Selective Amplification for mRNA of the UL21.5 Gene of hCMV

[0094] This example describes a method for detecting and/or quantifying the mRNA of the UL21.5 gene of hCMV in a non-cellular sample as provided by the disclosure. The test was performed to verify the efficiency of reverse transcription and amplification, the specificity for mRNA of the UL21.5 gene of hCMV and the absence of interference by the corresponding sequences of hCMV genomic DNA.

[0095] The method consists in the extraction of the nucleic acids of the sample, reverse transcription/amplification/detection. All the steps of the method were performed automatically by using a model ELITe InGenius® integrated instrument (ELITechGroup S.p.A., code INT030).

[0096] Sample Preparation

[0097] Artificial samples simulating non-cellular clinical samples were prepared by serial dilution of an hCMV culture of the Merlin strain (ATCC® VR-1590TM), in a matrix constituted by plasma collected in EDTA from healthy donors and tested negative for hCMV DNA with a commercial molecular diagnostic assay (CMV ELITe MGB® Kit, ELITechGroup S.p.A., code RTK015PLD).

[0098] The hCMV culture had a concentration of 5×106 PFU (Plaque Forming Unit) of hCMV/mL. The same material was quantified with a commercial molecular diagnostic assay (CMV ELITe MGB® Kit, ELITechGroup S.p.A., code RTK015PLD) and was found to have a concentration, calculated on genomic DNA, of approximately 3×108 International Units (IU) of hCMV/mL.

[0099] This concentration in IU higher than that in PFU was expected, since PFU measurement only detects viral particles capable of infecting cells, whereas measurement of the genomic DNA copies also detects the viral particles that are not capable of infecting but contain the viral DNA, and the free viral DNA that is present in the culture supernatant, as occurs in plasma samples of patients with active hCMV infection being treated with antiviral drugs that inhibit the terminase complex of hCMV, such as letermovir.

[0100] The simulated samples were prepared by means of four serial dilutions of the hCMV culture having a concentration of 5×106 PFU/mL (Plaque Forming Unit) of hCMV in plasma: 1:100, 1:1,000, 1:10,000, 1:100,000. Each simulated sample was analyzed twice.

[0101] Extraction of the Nucleic Acids

[0102] For each analyzed simulated sample, 1 mL of sample was transferred into a test tube (Sonication Tube, supplied in the ELITe InGenius® SP 200 Consumable Set product, ELITechGroup S.p.A., code INT032CS) and loaded into the sample area of the ELITe InGenius® instrument.

[0103] At the beginning of the extraction step, 10 μL of the CPE-Internal Control product (ELITechGroup S.p.A., code CTRCPE), a stabilized solution that provides exogenous internal control of extraction and inhibition, were dispensed automatically into the extraction cartridge (supplied in the ELITe InGenius® SP 1000 product, ELITechGroup S.p.A., code INT033SP1000) of each sample. The internal control was then processed together with the sample for the entire procedure of extraction and reverse transcription and realtime PCR to demonstrate that the sample had been processed appropriately and that the result of the assay was valid. The target for the internal control is constituted by the genomic RNA of the MS2 bacteriophage, which is not correlated and does not have sequence homologies with hCMV. The formulation of the internal control is ready for use and only needs to be loaded by the operator into the reagent area of the ELITe InGenius® instrument.

[0104] The nucleic acids of the samples (DNA and RNA) were extracted automatically by the ELITe InGenius® instrument by using the ELITe InGenius® SP1000 Extraction Kit product (ELITechGroup S.p.A., code INT033SP1000), which uses chaotropic agents, proteases, magnetic balls, solutions of alcohol and water for molecular biology to lyse the sample, capture the nucleic acids, purify them and elute them. The nucleic acids were eluted in 100 μL of water for molecular biology.

Setup of the Reaction for Reverse Transcription and Realtime Amplification

[0105] In the step of reaction setup, the ELITe InGenius® instrument, for each sample, transferred 20 μL of “CMV RNA PCR Mix” mixture (see Table 1) into the well of the PCR cassette (supplied in the ELITe InGenius® PCR Cassette product, ELITechGroup S.p.A., code INT035PCR) placed in the thermal block.

[0106] In this example, the CMV RNA PCR Mix contains a primer pair specific for mRNA of the UL21.5 gene of hCMV selected from “set 2)” (SEQ ID NO: 52 and SEQ ID NO: 75) and the compatible probe having SEQ ID NO: 87 modified with 6-FAM in position 5′ and TAMRA in position 3′, since they are designed astride the splice between the exons of the mRNA of the UL21.5 gene of hCMV and are therefore selective for mRNA in an extracted sample in which both DNA and RNA are present. The CMV RNA PCR Mix also contains primers specific for the genomic RNA of the MS2 phage (SEQ ID NO 118 and SEQ ID NO 119) and the corresponding probe (SEQ ID NO 120) modified with 6-FAM in position 5′ and TAMRA in position 3′.

[0107] In addition to the primers and probes cited above, the CMV RNA PCR Mix also comprises triphosphate nucleosides, magnesium chloride and the following reagents:

[0108] Taq Buffer with KCl (Thermo Fisher Scientific, included in the product Ref EP0404) is a component that establishes the optimum ionic force (potassium chloride) and pH (TRIS-HCl) and provides the substances necessary for the activity of the enzymes (detergents).

[0109] RevertAid H Minus Reverse Transcriptase (Thermo Fisher Scientific, Ref EP0451) is a recombinant version of the enzyme of the Moloney Murine Leukemia Virus (M-MuLV). This enzyme has an RNA-dependent and DNA-dependent DNA polymerase activity without RNasi H activity. Thermal stability is increased and can be used for synthesis of the first filament of cDNA at temperatures from 42° C. up to 50° C., achieving a higher specificity.

[0110] Platinum® Taq DNA polymerase (Thermo Fisher Scientific, Ref EP0404) is a recombinant version of the DNA polymerase enzyme of the Thermus acquaticus bacterium complexed with an antibody which blocks 5′.fwdarw.3′ DNA polymerase activity at ambient temperature and without 3′.fwdarw.5′ exonucleasic activity to obtain a higher processivity (number of nucleotides added to each bond). DNA polymerase activity was restored with incubation at 95° C. (hot start), obtaining higher specificity and sensitivity.

[0111] The formulation of the CMV RNA PCR Mix mixture is presented in

TABLE-US-00001 TABLE 1 Formulation of the CMV RNA PCR Mix Component Final concentration in the PCR reaction CMV UL21.5 Forward Primer (SEQ ID NO 52)  0.5 μM CMV UL21.5 Reverse Primer (SEQ ID NO 75)  1.3 μM CMV UL21.5 probe (SEQ ID NO 87)  0.2 μM IC MS2 Forward Primer (SEQ ID NO 118) 0.05 μM IC MS2 Reverse Primer (SEQ ID NO 119) 0.13 μM IC MS2 Probe (SEQ ID NO 120)  0.2 μM dATP  0.2 mM dCTP  0.2 mM dGTP  0.2 mM dTTP  0.2 mM Magnesium chloride   4 mM Taq buffer with KCl 1X RevertAid H Minus Reverse Transcriptase   3 Units/reaction Platinum ® Taq DNA polymerase   3 Units/reaction

[0112] The ELITe InGenius® instrument delivers 10 μL of the nucleic acids purified from the sample in the well of the PCR cassette, mixes them with the CMV RNA PCR Mix so as to obtain the final 30 μL of the complete reaction and seals the well with the adapted clear stopper.

Reaction for Reverse Transcription and Realtime Amplification

[0113] The thermal block of the ELITe InGenius® instrument performs the reverse transcription and amplification cycle and the optical system of the instrument detects in real time the fluorescence in the well of the PCR cassette through the clear stopper.

[0114] The thermal cycle conditions of the assay for reverse transcription and amplification are listed in Table 2

TABLE-US-00002 TABLE 2 Conditions of the RT-PCR thermal cycle Description Cycles Temperature Duration Reverse transcription 1 50° C. 1200 sec DNApolymerase 1 95° C.  300 sec activation   Amplification 45 95° C.   10 sec (fluorescence reading) 60° C.   35 sec

Interpretation of the Results

[0115] At the end of the assay, the software of the ELITe InGenius® instrument automatically runs the interpretation of the results, following preset rules, and creates the reports.

[0116] The test required calculation of the standard calibration curve of the assay for the quantification of the samples with realtime PCR. Four serial dilutions of a plasmid DNA with known titer containing, cloned inside it, a sequence that corresponds to part of the cDNA of the UL21.5 gene of hCMV (SEQ ID NO:121) were used for standard curve calculation.

Results

[0117] The test yielded the results summarized in Table 3.

TABLE-US-00003 TABLE 3 Complete reaction mix Incomplete reaction mix (with reverse (without reverse Sample Parameter transcriptase) transcriptase) Linearity of the series of R.sup.2 0.9980 — dilutions Angular coeff. −3.2660 — Target dilution 1:100 Target copies/mL 172563 — Ct Target 25.13 Not determined (5 × 10.sup.4 PFU of hCMV/ mL) (3 × 10.sup.6 IU of hCMV/ mL) Target dilution 1:1,000 Target copies/mL 17566 — (5 × 10.sup.3 PFU of CMV/ Ct Target 28.27 Not determined mL) (3 × 10.sup.5 IU of hCMV/ mL) Target dilution 1:10,000 Target copies/mL 2130 — (5 × 10.sup.2 PFU of CMV/ Ct Target 31.17 Not determined mL) (3 × 10.sup.4 IU of hCMV/ mL) Target dilution 1:100,000 Target copies/mL 143 — (5 × 10.sup.1 PFU of CMV/ Ct Target 35.05 Not determined mL) (3 × 10.sup.3 IU of hCMV/ mL)

[0118] The data in Table 3 demonstrate that the method according to the disclosure makes it possible to detect and quantify virion mRNA of the UL21.5 gene of hCMV with the linear correlation that is typical of molecular assays based on quantification of genomic DNA commonly used to monitor hCMV infection (R.sup.2=0.998; angular coefficient=−3.266). The capability of the assay to detect and quantify the presence of mRNA of the UL21.5 gene in virions of hCMV by means of an efficient reaction of reverse transcription and realtime PCR has therefore been verified.

[0119] Moreover, absence of interference by the genomic DNA sequences that correspond to the mRNA of the UL21.5 gene that are present in the sample has been verified. At the dilutions examined, hCMV genomic DNA is approximately 15 times more concentrated than UL21.5 mRNA. However, in the controls in which reverse transcriptase was not used and therefore the RNA present in the extracted sample was not amplified, no signal was detected, even for the highest titers (˜3×106 IU of hCMV/mL).

[0120] Considering the titer in PFU of hCMV/mL of the reference material in the analysis of the results of this test, quantification of hCMV by means of the mRNA of the UL21.5 gene contained in virions is approximately 3 times higher than the value of the PFU. This result is as expected, since measurement of PFU detects only the viral particles capable of infecting the cells and not the particles that are present but not capable of infecting. Moreover, the virion of hCMV may carry multiple copies of the mRNA of the UL21.5 gene.

[0121] This example shows that the method according to the disclosure has provided a quantification of the hCMV contained in the simulated samples that is very similar to the concentration in PFU of hCMV/mL. The concentration in PFU of hCMV/mL is the measurement of viremia when performed with the culture method to identify the infecting viral particles that are present in the sample of the patient and is the most specific indicator of active infection with hCMV.

[0122] Taking into account the titer in copies of hCMV genomic DNA/mL of the reference material in the analysis of the results of this test, quantification of hCMV by means of mRNA of UL21.5 contained in virions is 15 times lower than that of copies of hCMV genomic DNA. This result is as expected, since measurement of the copies of genomic DNA also detects the viral genomic DNA that is present in the culture supernatant, as occurs in samples of plasma of patients with active hCMV infection being treated with antiviral drugs that inhibit the terminase complex of hCMV, such as letermovir.

[0123] Advantageously, the method according to the disclosure provides a quantification of hCMV that is not influenced by the presence of viral genomic DNA in the culture supernatant, DNA which is not contained in viral particles capable of infecting cells. In the dilutions examined, the concentration of IUs of hCMV is in fact approximately 60 times higher than that of PFUs of hCMV.

[0124] In practice it has been found that the method according to the disclosure, as well as the oligonucleotides and the kits useful in said method, fully achieve the intended aim, since they make it possible to perform a quantitative detection of hCMV in non-cellular samples by reverse transcription and realtime PCR of viral mRNAs contained in virions. Therefore, this method can be applied successfully for example in the case of immunodepressed or immunosuppressed patients monitored for the viral load of hCMV and treated with antivirals, including those that cause accumulation of viral DNA. The efficiency of the method is comparable to the one obtained with DNA-based methods and can meet the requirements of current guidelines for monitoring the viral load of hCMV in transplant patients (Kotton et al., Transplantation 2018; 102(6): 900-931, and Emery V et al., Br. J. Haematol. 2013; 162(1): 25-39). Moreover, monitoring of viral mRNA contained in viral particles of hCMV in the sample without the cellular fraction advantageously allows monitoring of the presence of circulating virions of hCMV.

[0125] The disclosures in Italian Patent Application No. 102020000007357 from which this application claims priority are incorporated herein by reference.