WO2023052798A1 - New virus transport medium - Google Patents
New virus transport medium Download PDFInfo
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- WO2023052798A1 WO2023052798A1 PCT/HU2021/050055 HU2021050055W WO2023052798A1 WO 2023052798 A1 WO2023052798 A1 WO 2023052798A1 HU 2021050055 W HU2021050055 W HU 2021050055W WO 2023052798 A1 WO2023052798 A1 WO 2023052798A1
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- WIPO (PCT)
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- weight
- azorubin
- transport medium
- salts
- alcohol
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- 239000006163 transport media Substances 0.000 title claims abstract description 129
- 241000700605 Viruses Species 0.000 title claims abstract description 72
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- XTUSEBKMEQERQV-UHFFFAOYSA-N propan-2-ol;hydrate Chemical compound O.CC(C)O XTUSEBKMEQERQV-UHFFFAOYSA-N 0.000 description 1
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- 238000010186 staining Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
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- 150000008163 sugars Chemical class 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
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- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-O vancomycin(1+) Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C([O-])=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)[NH2+]C)[C@H]1C[C@](C)([NH3+])[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-O 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Definitions
- the present invention relates to the improvement of the yield of a viral transport medium in which the virus is inactivated without damaging the DNA and/or RNA region to be tested.
- the present invention also relates to novel azorubin-containing viral transport medium solutions and their preparation and application.
- VTM The viral transport medium
- VTMs can be divided into two major groups, depending on the need to keep the pathogens intact for further breeding.
- noninactivating transport medium are used in which the microorganism retains its vital functions and remains infectious. Examples of such non-inactivating transport medium (according to RT_Flocked_Polyester_Swabs.pdf) are Copan UTM-RT transport medium. It consists of modified HBSS saline supplemented with bovine serum albumin, cysteine, gelatin, sucrose and glutamic acid. The pH is adjusted with HEPES buffer.
- Phenol red dye is used to indicate pH. Vancomycin, amphotericin B, and colistin inhibit the growth of competing bacteria and yeast. It also contains sucrose. Similar compositions of transport medium are described in US5545555. and US5702944 patents. Although UTM-RT is commonly used, its disadvantage, in addition to not killing the virus, does not open and does not bind RNA onto the silica bead used in the measurement. Eagle Minimum Essential Medium (E-MEM), also marketed by Sigma- Aldrich (https://www.sigmaaldrich.com/EN/en/technical-documents/technical- article/cell-culture-and-cell-culture-analysis I mammalian-cell-culture I memformulation) which has a very complex composition.
- E-MEM Eagle Minimum Essential Medium
- Transport medium 199 which is used to transport samples taken from animals, contains 0.5% calf albumin and 7 different antibiotics to stabilize the sample.
- Viral Transport Medium contains 2 ml Hanks Saline (HBSS), 20 mM HEPES- 1, 0.5% gelatin and 100 /g I ml gentamicin sulphate.
- HBSS Hanks Saline
- HEPES- 1 0.5% gelatin
- 100 /g I ml gentamicin sulphate 100 /g I ml gentamicin sulphate.
- EP2473596 discloses a lysis medium comprising a buffer and a nonionic surfactant. The disadvantage is that it does not contain a component that binds RNA to the beads and works at a very high pH, so the RNA decomposes in it.
- PCR I Polymerase chain reaction (PCR), LAMP, RCA, RPA, sequencing, etc. Some modem diagnostic methods are specialized to the detection of hereditary pathogens (PCR I Polymerase chain reaction (PCR), LAMP, RCA, RPA, sequencing, etc.), which can be used to specifically identify individual pathogens and can differentiate similar microbes that require different therapeutic approaches (e.g., SARS-CoV-19, influenza A, and B).
- the main feature of a good denaturing viral transport medium in addition to inactivating pathogens, preserves their DNA and RNA by inactivating enzymes that break down various nucleic acids until use at the diagnostic site.
- the nucleic acid-based diagnostic procedure starts with the isolation of the nucleic acid, which means that the hereditary material is bound to a silica-coated surface (membrane or magnetic bead in a column) from solution and then the nucleic acids washed into a clean tube after several washing steps from the silica-coated nano beads into a clear aqueous or buffered solution.
- the currently used reagent kits uses binding solutions for binding to the silica surface which are separately added independently from the used of the viral transport medium.
- a modem viral transport medium must be able to inactivate the virus so as not to damage the DNA or RNA of the virus, or even conserve it, and to help the DNA or RNA of the virus bind to it to the silica surface.
- GTC guanidine isothiocyanate
- GuHCI guanidine hydrochloride
- Contamination - the residue of viral transport medium e.g. the presence of a portion of the GTC - is a problem primarily in applications where a washing step carried with a large volume (300-1000 ul) of ethanol is not possible.
- One such application is lab-on- a-chip technology, where the solution containing the hereditary material flows in microfluidic systems (microliters).
- the specific ratio of silica-coated surfaces (typically magnetic beads) used here is extremely high, and therefore it is technically difficult to wash away the bound GTC, GuHCI or chaotropic salts.
- a further problem is that in many cases the sample taken contains very little virus, so if the medium does not contain an additive to improve the yield, the reliability of the test is reduced, i.e. the virus in it can no longer be detected at low concentrations. It is common practice to use so-called “carrier RNA” to eliminate this problem. This is advised by the authors of the article Analytica Chimica Acta (Volume 652, Issues 1 -2, 12 October 2009, Pages 231-233 Analytica Chimica Acta). DNA extraction was performed on silica-based monoliths within a microfluidic device.
- a solid phase DNA extraction method has been used in which the DNA binds to silica in the presence of a chaotropic salt such as guanidine hydrochloride and elutes in a solution having low ionic strength such as water.
- a chaotropic salt such as guanidine hydrochloride
- Addition of poly-A carrier RNA to chaotropic saline significantly increased the effective amount of recoverable DNA (25 ng) compared to the absence of RNA (5 ng) by using silica dioxide monolith.
- pone.0187005 found the use of yeast RNA and MS2 RNA to be beneficial in the detection of microRNAs.
- the authors of Scientific Reports volume 7, Article number: 45199 (2017) developed a nucleic acid co-extraction method based on magnetic beads from sputum. The method was optimized by evaluating influencing factors such as guanidinium thiocyanate (GTC) and dithiothreitol (DTT) concentration, amount of magnetic bead, incubation temperature, lysis buffer pH, and RNA carrier type.
- GTC guanidinium thiocyanate
- DTT dithiothreitol
- azorubin carbmoisine
- RNA carriers which require strong cooling used in viral transport medium to improve the yield.
- the present invention relates to viral transport medium solutions which contain, in addition to azorubin, a solvent and an antiviral agent.
- the solvent is preferably water
- the additional solvent and antiviral agent are preferably C1-C3 alcohol and a salt containing a monovalent cation.
- the azorubin-containing solution does not contain a chaotropic substance other than the C1-C3 alcohol.
- the present invention relates to the use of azorubin as a viral transport medium for yield enhancement. Surprisingly, we have found that staining the viral transport medium solution with the azorubin food dye increases the amount of DNA and/or RNA yields and makes the detection more sensitive. We have found that azorubin is effective even at very low concentrations. For use according to the invention, more than 10’ 5 % by weight, preferably 10’ 5 -5% by weight, more preferably 0.5 * 10’ 4 -3% by weight, most preferably 0.5 * 10’ 3 -0.01 % by weight of azorubin contains virus transport medium. The present invention also relates to a viral transport medium comprising azorubin.
- the viral transport medium according to the invention is more than 10’ 5 % by weight, preferably 10’ 5 -5% by weight, more preferably 0.5 * 10’ 4 -3% by weight, most preferably 0.5 * 10’ 3 -0.01 % by weight contains azorubin.
- azorubin increases the amount of RNA that can be extracted from the virus. For example, when with commercially available virus transport medium only 100 ng of viral RNA can be recovered from the sample, with the azorubincontaining virus transport medium of our invention, 150 ng can be recovered. Using the viral transport medium of the present invention, the purification steps work better and therefore there will be more RNA recovered at the end of the isolation. Thus, it has surprisingly been found that azorubin has improved RNA yield, so that yield can also be used as an enhancer.
- the present invention relates to the use of azorubin in a viral transport medium comprising, in addition to azorubin, a solvent, an antiviral agent and, optionally, additional excipients.
- the azorubin is preferably used as a solvent in a virus transport medium comprising water, an additional solvent, and an antiviral agent, preferably a C1-C3 alcohol and a salt containing a monovalent cation.
- the C1-C3 alcohol used is preferably methanol, ethanol, normal or isopropanol, preferably ethanol or isopropanol, most preferably ethanol.
- the densities of the alcohols and water are different, so that the composition is preferably given as a percentage by weight, but the person skilled in the art can also measure the C1-C3 alcohols by volume% by weight also.
- the concentration of the salts is mM, which is the number of millimoles dissolved in one liter, which can be converted by a person skilled in the art to a concentration of % by weight, if necessary.
- azorubin is used in a viral transport medium in which the monovalent cation-containing salt is an alkali metal salt, preferably a lithium, potassium or sodium salt, preferably an inorganic salt, more preferably a phosphate, sulphate, nitrate, halide salt, more preferably a chloride, most preferably sodium chloride.
- the monovalent cation-containing salt is an alkali metal salt, preferably a lithium, potassium or sodium salt, preferably an inorganic salt, more preferably a phosphate, sulphate, nitrate, halide salt, more preferably a chloride, most preferably sodium chloride.
- azorubin is using as a yield enhancing agent in a cvirus transport medium which comprises 30-95% by weight, preferably 35-75% by weight, most preferably 45-55% by weight of C1-C3 alcohol, 5- 70% by weight of water, preferably 25-65% by weight, most preferably 45-55% by weight of water, 50-500 mM, preferably 100-300 mM, more preferably 150-250 mM inorganic salt.
- the azorubin is used in a virus transport comprising more than 10’ 5 % by weight, preferably 10’ 5 -5% by weight, more preferably 0.5 * 10’ 4 -3% by weight, most preferably 0.5 * 10 -3 -0, 01 % by weight of azorubin, 30-95% by weight, preferably 35-75% by weight, most preferably 45-55% by weight of a C1-C3 alcohol, preferably ethanol or isopropanol, 5-70%, preferably 25-65% by weight, most preferably 45-55% by weight % by weight of water and an inorganic salt in a concentration of 50-500 mM, preferably 100-300 mM, more preferably 150- 250 mM, preferably monovalent cation salts, more preferably alkali metal salts, most preferably lithium, potassium or sodium salts, preferably inorganic salts, more preferably phosphate, sulfate, nitrate, halide salts, more
- azorubin is used most preferably in a virus transport medium which comprises azorubin in an amount of 0.5 * 10’ 3 -0.01 % by weight with 45-55% by weight of C1-C3 alcohol, 45-55% by weight of water and sodium chloride in a concentration of 150-250 mM.
- viral transport medium of the present invention the viral transport medium which does not contain a chaotropic substance or chaotropic salt other than C1-C3 alcohol, inorganic salt and azorubin.
- chaotropic substance or salt is meant a water-soluble molecule that can disrupt the hydrogen bonding network between water molecules (i.e., exerts chaotropic activity). This affects the stability of the native state of other molecules in solution, especially macromolecules (proteins, nucleic acids) by weakening the hydrophobic effect.
- a chaotropic agent reduces the order in the structure of the protein formed by water molecules, both in bulk and in hydrating shells around hydrophobic amino acids and can cause denaturation.
- chaotropic agents examples include n-butanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, sodium dodecyl sulfate, thiourea, urea, and the like.
- a preferred embodiment of the present invention is the use of azorubin in a viral transport medium consisting of in addition to azorubin, water, a C1-C3 alcohol and an inorganic salt, or a mixture of inorganic salts.
- azorubin is used in such a way that the viral transport medium in which it is used consists of than 10’ 5 % by weight, preferably 10’ 5 -5% by weight, more preferably 0.5 * 10’ 4 -3% by weight, most preferably 0.5 * 10’ 3 -0.01 % by weight of azorubin, 30-95% by weight, preferably 35-75% by weight, most preferably 45-55% by weight of a C1-C3 alcohol, preferably ethyl alcohol, isopropyl alcohol, or a mixture thereof, 5-70%, preferably 25-65% by weight, most preferably 45- 55% by weight of water and 50-500 mM, preferably 100-300 mM, more preferably 150- 250 mM of an inorganic salt, preferably monovalent cationic salts, more preferably alkali metal salts, most preferably lithium, potassium or sodium salts, preferably inorganic salts, more preferably phosphate, sulfate, nit
- the azorubin is most preferably used in a viral transport medium which consists of 0.5*10’ 3 -0.01 % by weight of azorubin, 45-55% by weight of a C1-C3 alcohol, preferably ethanol or isopropanol, or a mixture thereof and 45-55% by weight of water in a concentration of 150-250 mM sodium chloride.
- the present invention provides viral transport medium comprising azorubin. More particularly, the present invention provides a viral transport medium comprising a solvent, an antiviral agent and, optionally, additional excipients. In a particular embodiment of the present invention is a viral transport medium comprising, in addition to azorubin, preferably a solvent comprising water, preferably a C1-C3 alcohol, and a monovalent cation salt as an additional solvent and antiviral agent.
- the viral transport medium according to the present invention comprises more than 10’ 5 % by weight, preferably 10’ 5 -5% by weight, more preferably 0.5 * 10’ 4 - 3% by weight, most preferably 0.5 * 10’ 3 -0.01 % azorubin by weight based on the weight of the viral transport medium.
- the viral transport medium comprises 30-95% by weight, preferably 35-75% by weight, most preferably 45-55% by weight of C1-C3 of alcohol, 5-70% by weight of water, preferably 25-65% by weight, most preferably 45-55% by weight water, 50-500 mM, preferably 100-300 mM, more preferably 150-250 mM inorganic salt.
- methanol, ethanol, normal or isopropanol preferably ethanol or isopropanol, most preferably ethanol is used as C1-C3 alcohol.
- azorubin is used in a viral transport medium in which the monovalent cation-containing salt is an alkali metal salt, preferably a lithium, potassium or sodium salt, preferably an inorganic salt, more preferably a phosphate, sulphate, nitrate, halide, more preferably a bromide salt, or iodide salts, most preferably sodium chloride.
- the monovalent cation-containing salt is an alkali metal salt, preferably a lithium, potassium or sodium salt, preferably an inorganic salt, more preferably a phosphate, sulphate, nitrate, halide, more preferably a bromide salt, or iodide salts, most preferably sodium chloride.
- the viral transport medium comprises more than 10’ 5 % by weight, preferably 10’ 5 -5% by weight, more preferably 0.5 * 10’ 4 - 3% by weight, most preferably 0.5 * 10’ 3 -0.01 % by weight of azorubin, 30-95% by weight, preferably 35-75% by weight, most preferably 45-55% by weight of a C1-C3 alcohol, preferably ethanol or isopropyl alcohol, more preferably ethanol, 5-70% by weight, preferably 25-65% by weight, most preferably 45-55% by weight of water, in a concentration of 50-500 mM, preferably 100-300, mM, more preferably 150-250 mM of monovalent cation, preferably alkali metal, more preferably Na, K, Li salts, preferably they comprise phosphate, sulfate, nitrate, halide salts, more preferably chloride, bromide or iodide salts, most preferably sodium chlor
- the viral transport medium comprises 0.5*10’ 3 -0.01 % by weight of azorubin, 45-55% by weight of C1-C3 alcohol, preferably ethanol or isopropanol, or a mixture thereof, 45-55% by weight % of water, in a concentration of 150-250 mM of sodium chloride.
- the viral transport medium does not contain chaotropic substance other than C1-C3 alcohol, inorganic salt and azorubin.
- the viral transport medium is consisting of in addition to azorubin, solvents, preferably water, C1-C3 alcohol as an additional solvent and antiviral agent, or a mixture thereof, and a salt containing a monovalent cation, or a mixture of such salts.
- the viral transport medium in one embodiment, consists of more than 10’ 5 % by weight, preferably 10’ 5 -5% by weight, more preferably 0.5 * 10’ 4 -3% by weight, most preferably 0.5 * 10’ 3 -0.01 % by weight of azorubin, 30-95% by weight, preferably 35- 75% by weight, most preferably 45-55% by weight of a C1-C3 alcohol or a mixture of such alcohols 5-70% by weight, preferably 25-65% by weight, most preferably 45-55% by weight of water, based on the weight of the viral transport medium, 50-500 mM and preferably 100-300 mM, more preferably 150-250 mM inorganic salts.
- azorubin is used in a viral transport medium in which the monovalent cation-containing salt is an alkali metal salt, preferably a lithium, potassium or sodium salt, preferably an inorganic salt, more preferably a phosphate, sulphate, nitrate, halide, more preferably a bromide salt, or iodide salts, most preferably sodium chloride.
- the monovalent cation-containing salt is an alkali metal salt, preferably a lithium, potassium or sodium salt, preferably an inorganic salt, more preferably a phosphate, sulphate, nitrate, halide, more preferably a bromide salt, or iodide salts, most preferably sodium chloride.
- the viral transport medium consists of more than 10’ 5 % by weight, preferably 10’ 5 -5% by weight, more preferably 0.5 * 10’ 4 -3% by weight, most preferably 0.5 * 10’ 3 % -0.01 % by weight of azorubin, 30-95% by weight, preferably 35-75% by weight, most preferably 45-55% by weight of a C1-C3 alcohol or a mixture thereof, preferably ethanol or isopropyl alcohol, more preferably ethanol, 5-70% by weight, more preferably 25% -65% by weight, most preferably 45- 55% by weight of water, an inorganic salt or mixtures thereof containing a monovalent cation in a concentration of 50 to 500 mM, more preferably 150 to 250 mM, preferably an alkali metal, more preferably Na, K, Li salts, preferably their phosphate, sulfate, nitrate, halide salts, or mixtures thereof, more preferably chloride
- a viral transport medium consisting of azorubin in an amount of 0.5 * 10’ 3 -0.01 % by weight, 45-55% by weight of C1-C3 alcohol or a mixture thereof, preferably ethanol or isopropanol, or mixtures thereof, 44-55% by weight of water and sodium chloride in concentration of 150-250 mM.
- the virus transport medium according to the invention preferably comprises purified water as water, more preferably distilled deionized water.
- C1-C3 alcohols may be used as technical grade, analytical, or most preferably grade of pharmacopoeia alcohols or mixtures thereof, as well as aqueous mixtures of these alcohols to the extent that the amounts of C1-C3 alcohols in the viral transport medium is not less than 30% by weight.
- Another embodiment of the present invention is a method of producing a virus transport medium, comprising:
- - azorubin is dissolved in a solvent used to produce a viral transport medium, and then the solution is used to produce a viral transport medium.
- the process can also be carried out by mixing the C1-C3 alcohol or a mixture of C1-C3 alcohols, water, azorubin, inorganic salts of monovalent inorganic cations, preferably inorganic salts of alkali metals, preferably phosphate, sulfate, nitrate, halide salts, more preferably Na, K, Li iodide, chloride bromide salts, most preferably sodium chloride or a mixture thereof and optionally further excipients in any order.
- inorganic salts of monovalent inorganic cations preferably inorganic salts of alkali metals, preferably phosphate, sulfate, nitrate, halide salts, more preferably Na, K, Li iodide, chloride bromide salts, most preferably sodium chloride or a mixture thereof and optionally further excipients in any order.
- the azorubin can be carried out by dissolving the azorubin and, if necessaryry, the excipients, preferably inorganic salts, preferably alkali or alkaline earth metal salts, preferably Na, K, Li salts, in any order, and then the C1- C3 alcohol or a mixture of C1-C3 alcohols are added to the solution.
- the excipients preferably inorganic salts, preferably alkali or alkaline earth metal salts, preferably Na, K, Li salts
- the C1- C3 alcohol or a mixture of C1-C3 alcohols are added to the solution.
- methanol, ethyl alcohol, n-propanol, 2-propanol or a mixture thereof, preferably ethyl alcohol or 2-propanol, most preferably ethanol is used as C1- C3 alcohol.
- the materials used in our invention are commercially available. Azorubin E122 food dye.
- the virus transport medium solution of the present invention can be used for any assay for viral infections, e.g. for PCR tests, which is based on the detection of DNA or RNA of viruses and does not require culture of the virus.
- the method of use does not differ, only in that it is easier to apply because there is no need to wash away chaotropic substances.
- the viral transport medium of the present invention may also be used to stabilize the nucleic acids contained in the viral transport medium by placing a saliva sample taken from the patient into the viral transport medium.
- the sampler is sealed and the outer surface is disinfected with, for example, a disinfectant cloth.
- the sample can then be transported freely, even at room temperature, to the place of processing. Since the virus transport medium according to the invention sterilizes 100%, the processing site does not have to be a BSL-2 + rated site, which reduces the investment costs for the laboratory, since the sample placed in the virus transport medium is no longer infectious. This can be followed by RNA isolation and further testing from the sample using prior art methods and tools. The application of these tools and methods is part of the general knowledge of the person skilled in the art.
- the viral transport medium of the present invention can be used in any method based on qPCR or other RNA or DNA amplification to detect nucleic acids in viruses or bacteria, as well as in any method where DNA and/or RNA transportability is important and important to avoid infectious material in the viral transport medium.
- the viral transport medium of the present invention was compared to other commercially available denaturing viral transport medium with guanidium thiocyanate (GTC) as the major component.
- GTC is difficult to remove from a solution containing RNA and silica beads in multiple washing steps with large amounts of alcohol.
- GTC inhibits the action of polymerases even in very small amounts. This can be a problem especially if it is not possible to wash with large amounts of alcohol before the RNA sample enters the LAMP I PCR reaction.
- Such an application is e.g. detection method by amplification techniques following isolation on microfluidic devices (lab-on-a-chip, LOC).
- RNA isolation experiments were performed in a 150 pl closed epoxy-based microfluidic device (isolation chamber) connected to two 1 mm diameter channels (inlet and outlet).
- a VTM containing 500 pl of magnetic silica beads MagneticPrep® Silica Particles; Sigma-Aldrich; Cat. No. 1011930001
- a swab previously containing a pharyngeal swab was placed in the VTM for 1 -2 minutes and then removed. Due to the salt present in the VTM, the known copy number synthetic SARS-CoV-2 viral RNA segment present in the solution bound to the surface of the silica bead.
- the magnetic bead-RNA complex was trapped on the inner wall of the chamber due to a magnet located on the outer surface of the chamber wall.
- a pipette sucked out the liquid from the beads.
- the beads remained inside the chamber throughout.
- the beads were washed with 150 pl of 70% ethanol for 30 minutes and then the alcohol was sucked out.
- the step was repeated, and the beads were dried under vacuum for about 10 seconds.
- 150 pl of nuclease-free distilled water was pipetted into the chamber to elute RNA from the silica surface. After 15 seconds of incubation, the aqueous solution containing the RNA was sucked out from the chamber.
- the 3 parallel measurements with 6 technical parallels (18 RT-LAMP reactions in total) showed positivity in the interval of 12-18 minutes, while in the case of GTC-containing virus transport medium there were signal amplifications in 2 out of 18 reactions at intervals of 14-16 minutes.
- positive control reactions showed an increase in signal between 8 and 10 min, whereas no positive signal was obtained for the negative control.
- azorubin yield improves the measurement result significantly, because if the virus is present in the sample in an amount that is no longer effective, azorubin, especially in the composition of the present invention, the effect, i.e., the presence of virus, was detected from each of the positive samples.
- Figure 1.a shows that the azorubin-containing virus transport medium according to the invention shows an increase in the fluorescence characteristic of the positive samples in all cases in the case of a positive sample, while in the GTC-containing mixture in Figure 1 b show that only 3 samples show positive result, others show false negative result.
- Figure 2. a. shows that the virus transport medium according to the invention shows the presence of virus from all positive samples, while Figure 2.b. shows that it does not show false positive result if the sample does not contain virus sought. This can be very risky in an epidemic situation. This is because in early infections, where the virus concentration is lower, or in cases where few samples are submitted for testing due to a sampling error, it is a very serious source of error if the measurement is not sensitive enough.
- the viral transport medium of the present invention has surprisingly advantageous properties over commercially available GTC-containing viral transport medium during isolation in a small volume microfluidic device.
- the use of the azorubin yield in the VTM as a repair agent can replace the carrier RNA, the use of which requires special cooling.
- the washing off the carrier RNA in contrast to the fact that if the washing off the carrier RNA is not successful enough, it may inhibit the measurement.
- the residual amount of azorubin has no inhibitory/interfering effect, because of its small molecule size it does not block the binding of the target RNA as much as the carrier RNA.
- the carrier RNA cannot be separated from the RNA to be isolated, it can competitively block the binding of the target RNA on the silicon beads, thereby precisely inhibiting the measurement as the RNA to be measured is washed away.
- the carrier RNA is definitely included in the purified RNA solution and thus can cause off-target amplifications for detection PCRs.
- RNA and DNA can be used in any method based on the amplification of qPCR or other RNA or DNA to detect nucleic acids of viruses or bacteria, as well as in any method where the transportability of DNA and/or RNA is an important and important consideration for the non-infectious material entering the VTM.
- the virus transport medium we have developed allows for a one-step solution. This is because the substances in the viral transport medium of the present invention help to bind the RNA and DNA (i.e., nucleic acids) of the virus from the solution to the silica-coated surface.
- the measurement results shown in the figures show in particular that the azorubincontaining virus transfer medium according to the present invention clearly shows the presence of the virus even at low virus concentrations, where the measurement becomes uncertain using commercially available virus transport medium.
- RNA isolation experiments were performed in a 150 pl closed epoxy-based microfluidic device (isolation chamber) connected to two 1 mm diameter channels (inlet and outlet).
- a VTM containing 500 pl of magnetic silica beads MagneticPrep® Silica Particles; Sigma-Aldrich; Cat. No. 1011930001
- a swab previously containing a pharyngeal was placed in the VTM for 1 -2 minutes and then removed. Due to the salt present in the VTM, the known copy number synthetic SARS-CoV-2 viral RNA segment present in the solution bound to the surface of the silica bead.
- the magnetic bead-RNA complex was trapped on the inner wall of the chamber due to a magnet located on the outer surface of the chamber wall.
- a pipette we sucked off the liquid from the beads.
- the beads remained inside the chamber throughout.
- the beads were washed with 150 pl of 70% ethanol for 30 minutes and then the alcohol was sucked off.
- the step was repeated and then the beads were dried under vacuum for approx. For 10 seconds.
- 150 pl of nuclease-free distilled water was pipetted into the chamber to elute RNA from the silica surface. After 15 seconds of incubation, the aqueous solution containing the RNA was sucked off from the chamber.
- RT-LAMP measurement was measured with a Quantstudio 5 (ThermoFisher; Cat. No. A34322) for 35 minutes at 65 ° C. Samples were prepared for measurement as follows: RT-LAMP reactions were prepared in the ratio recommended by the manufacturer (WarmStart® LAMP Kit; New England Biolabs; Cat. No. E1700L) in a final volume of 15 pl as follows:
- RNA or positive control RNA 200,000 copies
- dH2O negative control
- the azorubin-containing virus transport medium according to the present invention shows the increase of fluorescence characteristic of the positive samples (Fig. 1.a.) in all cases, while in the case of the GTC-containing mixture shown in Fig. 1.b. only 3 positive samples show this results the others show false negative results.
- Figure 2. a. shows that the virus transport medium of the present invention shows the presence of virus from all positive samples, while Figure 2.b. shows that it does not give a false positive result if the sample does not contain the virus sought.
- Example 3 (50-50% by weight in a mixture of ethyl alcohol and water)
- the product of the present invention can be prepared in the following compositions:
- Example 4 (50-50% by volume in a mixture of ethyl alcohol and water)
- Sodium chloride (11 .7 g) and an aqueous solution of Azorubin (0.88 g 1 1000 g) (8 ml) were dissolved in water (598 ml, 598 g) at room temperature, and the resulting solution was mixed with ethyl alcohol (500 ml, 393.8 g).
- Example 5 (50-50% by weight in a methanol-water mixture)
- Example 6 (50-50% by weight in a 2-propanol-water mixture)
- Example 7 (50-50% by weight in a n-propanol-water mixture)
- Sodium chloride (11 .7 g) and azorubin (0.01 g) were dissolved in water (500 g) at room temperature, and the resulting solution was mixed with n-propanol (500 g).
- a test tube was charged with 3 ml of the VTM solution of Example 1 .
- a swab sample is taken from the patient's nose and then the sample is dissolved from the swab into the VTM solution.
- the tube is sealed with a suitable rubber closure, the outer surface of the tube is wiped with an isopropyl alcohol disinfectant, and the disinfected sample on the outside is transported to the test laboratory in a sample collection container. The sample is then subjected to a PCR test.
Abstract
The subject of our invention is the use of azorubin of formula (I) to improve the yield of viral transport medium. More particularly, the present invention relates to the enhancement of the yield of a virus transport to a medium in which the virus is inactivated without damaging the DNA and/or RNA region to be tested. The present invention also relates to novel azorubin-containing viral transport medium solutions and their preparation and application.
Description
New virus transport medium
The subject of our invention is the use of azorubin of formula
(I) to improve the yield of viral transport medium. More particularly, the present invention relates to the improvement of the yield of a viral transport medium in which the virus is inactivated without damaging the DNA and/or RNA region to be tested. The present invention also relates to novel azorubin-containing viral transport medium solutions and their preparation and application.
BACKGROUND OF THE INVENTION:
The viral transport medium (hereinafter VTM) is used to deliver a sample containing potential pathogens from the sampling site to the diagnostic site after sampling from patients. VTMs can be divided into two major groups, depending on the need to keep the pathogens intact for further breeding. If the purpose is further culture, noninactivating transport medium are used in which the microorganism retains its vital functions and remains infectious. Examples of such non-inactivating transport medium (according to RT_Flocked_Polyester_Swabs.pdf) are Copan UTM-RT transport medium. It consists of modified HBSS saline supplemented with bovine serum albumin, cysteine, gelatin, sucrose and glutamic acid. The pH is adjusted with HEPES buffer. Phenol red dye is used to indicate pH. Vancomycin, amphotericin B, and colistin inhibit the growth of competing bacteria and yeast. It also contains sucrose. Similar compositions of transport medium are described in US5545555. and US5702944 patents. Although UTM-RT is commonly used, its disadvantage, in addition to not killing the virus, does not open and does not bind RNA onto the silica bead used in the measurement. Eagle Minimum Essential Medium (E-MEM), also marketed by Sigma-
Aldrich (https://www.sigmaaldrich.com/EN/en/technical-documents/technical- article/cell-culture-and-cell-culture-analysis I mammalian-cell-culture I memformulation) which has a very complex composition. It contains a buffer mixture of many salts and a mixture of more than 20 different amino acids, sugars, sugar alcohols, vitamins and other excipients, and for the preparation of the product all of these compounds appear necessary. Transport medium 199, which is used to transport samples taken from animals, contains 0.5% calf albumin and 7 different antibiotics to stabilize the sample. APBD-Glycerol transport medium consists of a 1 : 1 mixture of a phosphate buffer and sterile glycerol, which also contains 7 different antibiotics. According to the description (https://www.who. int/ihr/publications/Annex8.pdf?ua=1 ) the finished transport medium has to be kept at -20°C until use. It can only be stored at room temperature for a short time, 1 -2 days.
According to the information in the BD Cellmatics TM shipping package (https://legacy.bd.com/ds/technicalCenter/inserts/S1059(0502). qxd. pdf), Viral Transport Medium contains 2 ml Hanks Saline (HBSS), 20 mM HEPES- 1, 0.5% gelatin and 100 /g I ml gentamicin sulphate. These also have the disadvantage that they do not kill the virus, do not open it and do not bind the RNA on the bead. EP2473596 discloses a lysis medium comprising a buffer and a nonionic surfactant. The disadvantage is that it does not contain a component that binds RNA to the beads and works at a very high pH, so the RNA decomposes in it.
If it is not the aim of further cultivation, but only one of its components (eg. hereditary material) is to be subjected to further testing, then - also for security reasons - it is advisable to use transport medium in which a denaturing agent is added to the transport medium which drastically reduces the infectivity of the pathogen but intactly preserves the component to be tested, such as DNA, RNA, or protein fragments. Some modem diagnostic methods are specialized to the detection of hereditary pathogens (PCR I Polymerase chain reaction (PCR), LAMP, RCA, RPA, sequencing, etc.), which can be used to specifically identify individual pathogens and can differentiate similar microbes that require different therapeutic approaches (e.g., SARS-CoV-19, influenza A, and B). Thus, the main feature of a good denaturing viral transport medium, in addition to inactivating pathogens, preserves their DNA and RNA by inactivating enzymes that break down various nucleic acids until use at the diagnostic site.
In the vast majority of cases the nucleic acid-based diagnostic procedure starts with the isolation of the nucleic acid, which means that the hereditary material is bound to a silica-coated surface (membrane or magnetic bead in a column) from solution and then the nucleic acids washed into a clean tube after several washing steps from the silica-coated nano beads into a clear aqueous or buffered solution. The currently used reagent kits uses binding solutions for binding to the silica surface which are separately added independently from the used of the viral transport medium.
In summary, a modem viral transport medium must be able to inactivate the virus so as not to damage the DNA or RNA of the virus, or even conserve it, and to help the DNA or RNA of the virus bind to it to the silica surface.
Current VTM technologies are almost exclusively aqueous-based buffer solutions in which the active ingredient is guanidine isothiocyanate (GTC), possibly guanidine hydrochloride (GuHCI), or, less commonly, other chaotropic salts. In these commercially available viral transport medium, GTC is used in concentrations of 4-6M, GuHCI in 4M, or other very concentrated concentrations to neutralize the virus, bind RNA and DNA degrading enzymes, and bind the released nucleic acids to the silica surface during the isolation step. An example is the viral transport medium in the DNA I RNA Shield Collection Tube (w I Swab) of Zymo Research Corporation, catalog number R1107, which is described as containing less than 6M GTC. The problem is that these solutions must be removed from silica beads before measurement, which is very difficult at such a high concentration. Therefore, several washing steps (usually with 70-100% ethanol) must be carried out and after the elution there can be some residue of the transport medium, e.g. and GTC. This can be a serious problem, as all chaotropic salts, but GTC is particularly potent in inhibiting the PCR reaction, even resulting in a false-negative diagnosis.
Contamination - the residue of viral transport medium, e.g. the presence of a portion of the GTC - is a problem primarily in applications where a washing step carried with a large volume (300-1000 ul) of ethanol is not possible. One such application is lab-on- a-chip technology, where the solution containing the hereditary material flows in microfluidic systems (microliters). The specific ratio of silica-coated surfaces (typically magnetic beads) used here is extremely high, and therefore it is technically difficult to wash away the bound GTC, GuHCI or chaotropic salts.
Thus, based on the facts above, most prior art viral transport medium solutions do not inactivate the virus, or even if neutralize, a high concentration of GTC, GuHCI, are used during the isolation step to neutralize the virus and RNA and DNA degrading enzymes. The problem is that very difficult to remove these concentrated virus transport medium solutions from the system. However, the presence of these chaotropic salts strongly inhibits the analysis, e.g. the PCR reaction and thus the contaminants remaining from the virus transport medium hinder the detection of the virus.
Thus, prior art solutions do not aid in the binding of DNA or RNA segments from the sample to the silica. An additional problem is that when non-deactivating virus transport medium are used, transport and processing can only be performed with appropriate safeguards. For example, the processing location must have a BSL-2 + rating.
A further problem is that in many cases the sample taken contains very little virus, so if the medium does not contain an additive to improve the yield, the reliability of the test is reduced, i.e. the virus in it can no longer be detected at low concentrations. It is common practice to use so-called “carrier RNA” to eliminate this problem. This is advised by the authors of the article Analytica Chimica Acta (Volume 652, Issues 1 -2, 12 October 2009, Pages 231-233 Analytica Chimica Acta). DNA extraction was performed on silica-based monoliths within a microfluidic device. A solid phase DNA extraction method has been used in which the DNA binds to silica in the presence of a chaotropic salt such as guanidine hydrochloride and elutes in a solution having low ionic strength such as water. Addition of poly-A carrier RNA to chaotropic saline significantly increased the effective amount of recoverable DNA (25 ng) compared to the absence of RNA (5 ng) by using silica dioxide monolith. These results confirmed that techniques using nucleic acid carrier molecules can improve DNA extraction methods in microfluidic applications. Similarly, PLoS One. 2017; 12 (10): e0187005. (Published online 2017 Oct 27. doi: 10.1371 / journal. pone.0187005) found the use of yeast RNA and MS2 RNA to be beneficial in the detection of microRNAs. The authors of Scientific Reports volume 7, Article number: 45199 (2017) developed a nucleic acid co-extraction method based on magnetic beads from sputum. The method was optimized by evaluating influencing factors such as guanidinium thiocyanate (GTC) and dithiothreitol (DTT) concentration, amount of magnetic bead, incubation temperature, lysis buffer pH, and RNA carrier type.
However, it is difficult from a practical point of view, for example in the case of an acute epidemic situation, to use a carrier RNA because the RNA is easily degraded and thus has to be stored at -80°C. This severely limits the use of RNA carriers.
Thus, it has been necessary to find an additive with which the yield can be improved in such a way that it does not require special storage, suitable to remain stable in virus transport medium solutions at room temperature for a long time. In addition, it has become necessary to develop a virus transport medium solution that contains a stable, room-temperature-enhancing additive, neutralizes the virus, assists in the binding of collected DNA and/or RNA on silica beads, and does not inhibit measurement as a contaminant, success of the process. It is particularly important to use such viral transport medium, for example, for lab-on-a-chip technology, which uses very small sample solutions.
BRIEF DESCRIPTION OF THE INVENTION
The above objects were achieved by using a very stable food color, azorubin (carmoisine) of formula (I) in viral transport medium solutions, instead of the highly sensitive RNA carriers which require strong cooling used in viral transport medium to improve the yield. We have found surprisingly, when the VTM solution is stained with azorubin, it is not necessary to use RNA carriers. It has also been found that when azorubin is used, the measurement is not inhibited by the fact that some azorubin remains in the sample. In particular, the present invention relates to viral transport medium solutions which contain, in addition to azorubin, a solvent and an antiviral agent. The solvent is preferably water, the additional solvent and antiviral agent are preferably C1-C3 alcohol and a salt containing a monovalent cation. This eliminates the additional disadvantages of the prior art viral transport medium listed above, namely that alcohol inactivates the viruses and does not prevent the DNA and/or RNA content of the sample from binding to the silica beads. In a highly preferred embodiment of the invention, the azorubin-containing solution does not contain a chaotropic substance other than the C1-C3 alcohol.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the use of azorubin as a viral transport medium for yield enhancement. Surprisingly, we have found that staining the viral transport medium solution with the azorubin food dye increases the amount of DNA and/or RNA yields and makes the detection more sensitive. We have found that azorubin is effective even at very low concentrations. For use according to the invention, more than 10’5% by weight, preferably 10’5-5% by weight, more preferably 0.5 * 10’4-3% by weight, most preferably 0.5 * 10’3-0.01 % by weight of azorubin contains virus transport medium. The present invention also relates to a viral transport medium comprising azorubin. In particular, the viral transport medium according to the invention is more than 10’5% by weight, preferably 10’5-5% by weight, more preferably 0.5 * 10’4-3% by weight, most preferably 0.5 * 10’3-0.01 % by weight contains azorubin.
Indeed, we have found that azorubin increases the amount of RNA that can be extracted from the virus. For example, when with commercially available virus transport medium only 100 ng of viral RNA can be recovered from the sample, with the azorubincontaining virus transport medium of our invention, 150 ng can be recovered. Using the viral transport medium of the present invention, the purification steps work better and therefore there will be more RNA recovered at the end of the isolation. Thus, it has surprisingly been found that azorubin has improved RNA yield, so that yield can also be used as an enhancer.
More particularly, the present invention relates to the use of azorubin in a viral transport medium comprising, in addition to azorubin, a solvent, an antiviral agent and, optionally, additional excipients.
In a highly preferred embodiment of the invention, the azorubin is preferably used as a solvent in a virus transport medium comprising water, an additional solvent, and an antiviral agent, preferably a C1-C3 alcohol and a salt containing a monovalent cation. In the viral transport medium according to the invention, the C1-C3 alcohol used is preferably methanol, ethanol, normal or isopropanol, preferably ethanol or isopropanol, most preferably ethanol.
The densities of the alcohols and water are different, so that the composition is preferably given as a percentage by weight, but the person skilled in the art can also measure the C1-C3 alcohols by volume% by weight also. The concentration of the salts
is mM, which is the number of millimoles dissolved in one liter, which can be converted by a person skilled in the art to a concentration of % by weight, if necessary.
In a very preferred embodiment of the invention, azorubin is used in a viral transport medium in which the monovalent cation-containing salt is an alkali metal salt, preferably a lithium, potassium or sodium salt, preferably an inorganic salt, more preferably a phosphate, sulphate, nitrate, halide salt, more preferably a chloride, most preferably sodium chloride.
In a highly preferred embodiment of the invention, azorubin is using as a yield enhancing agent in a cvirus transport medium which comprises 30-95% by weight, preferably 35-75% by weight, most preferably 45-55% by weight of C1-C3 alcohol, 5- 70% by weight of water, preferably 25-65% by weight, most preferably 45-55% by weight of water, 50-500 mM, preferably 100-300 mM, more preferably 150-250 mM inorganic salt.
According to a preferred embodiment of the invention, the azorubin is used in a virus transport comprising more than 10’5% by weight, preferably 10’5-5% by weight, more preferably 0.5 * 10’4-3% by weight, most preferably 0.5 * 10-3-0, 01 % by weight of azorubin, 30-95% by weight, preferably 35-75% by weight, most preferably 45-55% by weight of a C1-C3 alcohol, preferably ethanol or isopropanol, 5-70%, preferably 25-65% by weight, most preferably 45-55% by weight % by weight of water and an inorganic salt in a concentration of 50-500 mM, preferably 100-300 mM, more preferably 150- 250 mM, preferably monovalent cation salts, more preferably alkali metal salts, most preferably lithium, potassium or sodium salts, preferably inorganic salts, more preferably phosphate, sulfate, nitrate, halide salts, more preferably chloride, bromide or iodide salts, most preferably sodium chloride.
According to a most preferred embodiment of the invention, azorubin is used most preferably in a virus transport medium which comprises azorubin in an amount of 0.5 * 10’3-0.01 % by weight with 45-55% by weight of C1-C3 alcohol, 45-55% by weight of water and sodium chloride in a concentration of 150-250 mM.
In a highly preferred embodiment viral transport medium of the present invention the viral transport medium which does not contain a chaotropic substance or chaotropic salt other than C1-C3 alcohol, inorganic salt and azorubin. By chaotropic substance or
salt is meant a water-soluble molecule that can disrupt the hydrogen bonding network between water molecules (i.e., exerts chaotropic activity). This affects the stability of the native state of other molecules in solution, especially macromolecules (proteins, nucleic acids) by weakening the hydrophobic effect. For example, a chaotropic agent reduces the order in the structure of the protein formed by water molecules, both in bulk and in hydrating shells around hydrophobic amino acids and can cause denaturation. Examples of such chaotropic agents include n-butanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, sodium dodecyl sulfate, thiourea, urea, and the like.
Thus, a preferred embodiment of the present invention is the use of azorubin in a viral transport medium consisting of in addition to azorubin, water, a C1-C3 alcohol and an inorganic salt, or a mixture of inorganic salts.
In this embodiment of the present invention, azorubin is used in such a way that the viral transport medium in which it is used consists of than 10’5% by weight, preferably 10’5-5% by weight, more preferably 0.5 * 10’4-3% by weight, most preferably 0.5 * 10’ 3-0.01 % by weight of azorubin, 30-95% by weight, preferably 35-75% by weight, most preferably 45-55% by weight of a C1-C3 alcohol, preferably ethyl alcohol, isopropyl alcohol, or a mixture thereof, 5-70%, preferably 25-65% by weight, most preferably 45- 55% by weight of water and 50-500 mM, preferably 100-300 mM, more preferably 150- 250 mM of an inorganic salt, preferably monovalent cationic salts, more preferably alkali metal salts, most preferably lithium, potassium or sodium salts, preferably inorganic salts, more preferably phosphate, sulfate, nitrate, halide salts, even more preferably chloride, bromide or iodide salts, most preferably sodium chloride.
According to a most preferred embodiment of the invention, the azorubin is most preferably used in a viral transport medium which consists of 0.5*10’3-0.01 % by weight of azorubin, 45-55% by weight of a C1-C3 alcohol, preferably ethanol or isopropanol, or a mixture thereof and 45-55% by weight of water in a concentration of 150-250 mM sodium chloride.
In another aspect, the present invention provides viral transport medium comprising azorubin. More particularly, the present invention provides a viral transport medium comprising a solvent, an antiviral agent and, optionally, additional excipients. In a particular embodiment of the present invention is a viral transport medium comprising,
in addition to azorubin, preferably a solvent comprising water, preferably a C1-C3 alcohol, and a monovalent cation salt as an additional solvent and antiviral agent. In particular, the viral transport medium according to the present invention comprises more than 10’5% by weight, preferably 10’5-5% by weight, more preferably 0.5 * 10’4- 3% by weight, most preferably 0.5 * 10’3-0.01 % azorubin by weight based on the weight of the viral transport medium. In a preferred embodiment of the invention, the viral transport medium comprises 30-95% by weight, preferably 35-75% by weight, most preferably 45-55% by weight of C1-C3 of alcohol, 5-70% by weight of water, preferably 25-65% by weight, most preferably 45-55% by weight water, 50-500 mM, preferably 100-300 mM, more preferably 150-250 mM inorganic salt.
In the viral transport medium according to the present invention, preferably methanol, ethanol, normal or isopropanol, preferably ethanol or isopropanol, most preferably ethanol is used as C1-C3 alcohol.
In a highly preferred embodiment of the invention, azorubin is used in a viral transport medium in which the monovalent cation-containing salt is an alkali metal salt, preferably a lithium, potassium or sodium salt, preferably an inorganic salt, more preferably a phosphate, sulphate, nitrate, halide, more preferably a bromide salt, or iodide salts, most preferably sodium chloride.
Thus, in a preferred embodiment of the invention, the viral transport medium comprises more than 10’5% by weight, preferably 10’5-5% by weight, more preferably 0.5 * 10’4- 3% by weight, most preferably 0.5 * 10’3-0.01 % by weight of azorubin, 30-95% by weight, preferably 35-75% by weight, most preferably 45-55% by weight of a C1-C3 alcohol, preferably ethanol or isopropyl alcohol, more preferably ethanol, 5-70% by weight, preferably 25-65% by weight, most preferably 45-55% by weight of water, in a concentration of 50-500 mM, preferably 100-300, mM, more preferably 150-250 mM of monovalent cation, preferably alkali metal, more preferably Na, K, Li salts, preferably they comprise phosphate, sulfate, nitrate, halide salts, more preferably chloride, bromide or iodide salts, most preferably sodium chloride.
According to a most preferred embodiment of the present invention, the viral transport medium comprises 0.5*10’3-0.01 % by weight of azorubin, 45-55% by weight of C1-C3 alcohol, preferably ethanol or isopropanol, or a mixture thereof, 45-55% by weight % of water, in a concentration of 150-250 mM of sodium chloride.
A highly preferred embodiment of the present invention the viral transport medium does not contain chaotropic substance other than C1-C3 alcohol, inorganic salt and azorubin.
In particular, another embodiment of the present invention the viral transport medium is consisting of in addition to azorubin, solvents, preferably water, C1-C3 alcohol as an additional solvent and antiviral agent, or a mixture thereof, and a salt containing a monovalent cation, or a mixture of such salts. In particular, in one embodiment of the viral transport medium according to the present invention, consists of more than 10’5% by weight, preferably 10’5-5% by weight, more preferably 0.5 * 10’4-3% by weight, most preferably 0.5 * 10’3-0.01 % by weight of azorubin, 30-95% by weight, preferably 35- 75% by weight, most preferably 45-55% by weight of a C1-C3 alcohol or a mixture of such alcohols 5-70% by weight, preferably 25-65% by weight, most preferably 45-55% by weight of water, based on the weight of the viral transport medium, 50-500 mM and preferably 100-300 mM, more preferably 150-250 mM inorganic salts.
In a highly preferred embodiment of the invention, azorubin is used in a viral transport medium in which the monovalent cation-containing salt is an alkali metal salt, preferably a lithium, potassium or sodium salt, preferably an inorganic salt, more preferably a phosphate, sulphate, nitrate, halide, more preferably a bromide salt, or iodide salts, most preferably sodium chloride.
Thus, in a preferred embodiment of the invention, the viral transport medium consists of more than 10’5% by weight, preferably 10’5-5% by weight, more preferably 0.5 * 10’ 4-3% by weight, most preferably 0.5 * 10’3% -0.01 % by weight of azorubin, 30-95% by weight, preferably 35-75% by weight, most preferably 45-55% by weight of a C1-C3 alcohol or a mixture thereof, preferably ethanol or isopropyl alcohol, more preferably ethanol, 5-70% by weight, more preferably 25% -65% by weight, most preferably 45- 55% by weight of water, an inorganic salt or mixtures thereof containing a monovalent cation in a concentration of 50 to 500 mM, more preferably 150 to 250 mM, preferably an alkali metal, more preferably Na, K, Li salts, preferably their phosphate, sulfate, nitrate, halide salts, or mixtures thereof, more preferably chloride, bromide or iodide salts, most preferably sodium chloride. According to a most preferred embodiment of the invention is a viral transport medium consisting of azorubin in an amount of 0.5 * 10’3-0.01 % by weight, 45-55% by weight of C1-C3 alcohol or a mixture thereof,
preferably ethanol or isopropanol, or mixtures thereof, 44-55% by weight of water and sodium chloride in concentration of 150-250 mM.
The virus transport medium according to the invention preferably comprises purified water as water, more preferably distilled deionized water. As the alcohol, C1-C3 alcohols may be used as technical grade, analytical, or most preferably grade of pharmacopoeia alcohols or mixtures thereof, as well as aqueous mixtures of these alcohols to the extent that the amounts of C1-C3 alcohols in the viral transport medium is not less than 30% by weight.
Another embodiment of the present invention is a method of producing a virus transport medium, comprising:
- dissolving azorubin in a virus transport medium, or
- azorubin is dissolved in a solvent used to produce a viral transport medium, and then the solution is used to produce a viral transport medium.
According to another embodiment of the invention, the process can also be carried out by mixing the C1-C3 alcohol or a mixture of C1-C3 alcohols, water, azorubin, inorganic salts of monovalent inorganic cations, preferably inorganic salts of alkali metals, preferably phosphate, sulfate, nitrate, halide salts, more preferably Na, K, Li iodide, chloride bromide salts, most preferably sodium chloride or a mixture thereof and optionally further excipients in any order. It can be carried out by dissolving the azorubin and, if necesarry, the excipients, preferably inorganic salts, preferably alkali or alkaline earth metal salts, preferably Na, K, Li salts, in any order, and then the C1- C3 alcohol or a mixture of C1-C3 alcohols are added to the solution. In the process according to the invention, methanol, ethyl alcohol, n-propanol, 2-propanol or a mixture thereof, preferably ethyl alcohol or 2-propanol, most preferably ethanol is used as C1- C3 alcohol. More particularly, it can be carried out by using 30-95% by weight, preferably 35-75% by weight, most preferably 45-55% by weight of C1-C3 alcohol, or a mixture thereof, 5-70% by weight of water, preferably 25%-65% by weight, most preferably 45-55% by weight of water and 10’5-5% by weight, preferably 0.5 * 10’4-3% by weight, most preferably 0.5 * 10’3-0.01 % by weight of azorubin by weight of the finished virus transport medium and optionally further excipients, preferably 50-500 mM, preferably 100-300 mM, more preferably 150-250 mM inorganic salts, preferably
lithium, potassium or sodium, phosphate, sulphate, nitrate, halide salts, preferably chloride, bromide , iodide salts are more preferably sodium chloride.
The materials used in our invention are commercially available. Azorubin E122 food dye.
The operations, the dissolution and mixing of the substances, the handling of the substances is within the general knowledge of the person skilled in the art. The preparation of the VTM solution according to the invention does not require any equipment other than that generally used in the chemical industry. The handling of these and the execution of the production processes are also part of the general knowledge of the person skilled in the art.
The virus transport medium solution of the present invention can be used for any assay for viral infections, e.g. for PCR tests, which is based on the detection of DNA or RNA of viruses and does not require culture of the virus. In essence, the method of use does not differ, only in that it is easier to apply because there is no need to wash away chaotropic substances.
The viral transport medium of the present invention may also be used to stabilize the nucleic acids contained in the viral transport medium by placing a saliva sample taken from the patient into the viral transport medium. The sampler is sealed and the outer surface is disinfected with, for example, a disinfectant cloth. The sample can then be transported freely, even at room temperature, to the place of processing. Since the virus transport medium according to the invention sterilizes 100%, the processing site does not have to be a BSL-2 + rated site, which reduces the investment costs for the laboratory, since the sample placed in the virus transport medium is no longer infectious. This can be followed by RNA isolation and further testing from the sample using prior art methods and tools. The application of these tools and methods is part of the general knowledge of the person skilled in the art.
The viral transport medium of the present invention can be used in any method based on qPCR or other RNA or DNA amplification to detect nucleic acids in viruses or bacteria, as well as in any method where DNA and/or RNA transportability is important and important to avoid infectious material in the viral transport medium.
The viral transport medium of the present invention was compared to other commercially available denaturing viral transport medium with guanidium thiocyanate (GTC) as the major component. GTC is difficult to remove from a solution containing RNA and silica beads in multiple washing steps with large amounts of alcohol. GTC inhibits the action of polymerases even in very small amounts. This can be a problem especially if it is not possible to wash with large amounts of alcohol before the RNA sample enters the LAMP I PCR reaction. Such an application is e.g. detection method by amplification techniques following isolation on microfluidic devices (lab-on-a-chip, LOC).
Our RNA isolation experiments were performed in a 150 pl closed epoxy-based microfluidic device (isolation chamber) connected to two 1 mm diameter channels (inlet and outlet). A VTM containing 500 pl of magnetic silica beads (MagPrep® Silica Particles; Sigma-Aldrich; Cat. No. 1011930001 ) was pipetted into the inlet channel. A swab previously containing a pharyngeal swab was placed in the VTM for 1 -2 minutes and then removed. Due to the salt present in the VTM, the known copy number synthetic SARS-CoV-2 viral RNA segment present in the solution bound to the surface of the silica bead. After pipetting into the chamber, the magnetic bead-RNA complex was trapped on the inner wall of the chamber due to a magnet located on the outer surface of the chamber wall. Using a pipette, sucked out the liquid from the beads. The beads remained inside the chamber throughout. The beads were washed with 150 pl of 70% ethanol for 30 minutes and then the alcohol was sucked out. The step was repeated, and the beads were dried under vacuum for about 10 seconds. Finally, 150 pl of nuclease-free distilled water was pipetted into the chamber to elute RNA from the silica surface. After 15 seconds of incubation, the aqueous solution containing the RNA was sucked out from the chamber. This procedure was performed in three parallel measurements with the composition of the present invention containing 50% by volume of H2O according to the invention in 50% by volume of ethanol, 200 mM NaCI and 0.001 % by weight of Azorubin, and in three parallel measurements with GTC- containing virus transport medium (Zymo Research Corporation catalog number: R1107) The success of the isolation was measured in RT-LAMP reaction (with 6 technical parallels).
In the case of using the virus transport medium according to the invention, the 3 parallel measurements with 6 technical parallels (18 RT-LAMP reactions in total) showed
positivity in the interval of 12-18 minutes, while in the case of GTC-containing virus transport medium there were signal amplifications in 2 out of 18 reactions at intervals of 14-16 minutes. This means that positive control reactions showed an increase in signal between 8 and 10 min, whereas no positive signal was obtained for the negative control. Thus, it can be clearly shown that the use of azorubin yield improves the measurement result significantly, because if the virus is present in the sample in an amount that is no longer effective, azorubin, especially in the composition of the present invention, the effect, i.e., the presence of virus, was detected from each of the positive samples. The results of the measurements are compared with the measurement results in Figure 1. Figure 1.a. shows that the azorubin-containing virus transport medium according to the invention shows an increase in the fluorescence characteristic of the positive samples in all cases in the case of a positive sample, while in the GTC-containing mixture in Figure 1 b show that only 3 samples show positive result, others show false negative result. Figure 2. a. shows that the virus transport medium according to the invention shows the presence of virus from all positive samples, while Figure 2.b. shows that it does not show false positive result if the sample does not contain virus sought. This can be very risky in an epidemic situation. This is because in early infections, where the virus concentration is lower, or in cases where few samples are submitted for testing due to a sampling error, it is a very serious source of error if the measurement is not sensitive enough.
In summary, the viral transport medium of the present invention has surprisingly advantageous properties over commercially available GTC-containing viral transport medium during isolation in a small volume microfluidic device.
List of figures:
1.a. LAMP test result of the viral transport medium containing 50% by volume H2O, 50% by volume of ethanol, 200 mM of NaCI and 0.001 % % by weight of Azorubin in the case of a positive sample I 3 parallel measurements with 6 technical parallels (18 RT in total-LAMP reaction)/.
1.b. State-of-the-art virus transport medium LAMP test result in case of a positive sample 13 parallel measurements with 6 technical parallels (18 RT-LAMP reactions in total) /.
2. a. 50% by volume H2O according to the invention 50% by volume ethanol, 200 mM NaCI and 0.001 % by weight of Azorubin virus transport medium LAMP test result in case of positive sample I 3 parallel measurements with 6 technical parallels (18 RT- LAMP reactions in total)/.
2.b 50% by volume H2O according to the invention 50% by volume ethanol, 200 mM NaCI and 0.001 % by weight of Azorubin virus transport medium LAMP test result in case of negative sample I 3 parallel measurements with 6 technical parallels (total 18 RT -LAMP reaction)/.
Advantages of the present invention over the prior art:
- the use of the azorubin yield in the VTM as a repair agent can replace the carrier RNA, the use of which requires special cooling.
- In addition, in contrast to the fact that if the washing off the carrier RNA is not successful enough, it may inhibit the measurement. The residual amount of azorubin has no inhibitory/interfering effect, because of its small molecule size it does not block the binding of the target RNA as much as the carrier RNA.
- While the carrier RNA cannot be separated from the RNA to be isolated, it can competitively block the binding of the target RNA on the silicon beads, thereby precisely inhibiting the measurement as the RNA to be measured is washed away.
- Furthermore, alcoholic washing of carrier RNA from silica beads is difficult contrary to azorubin which is easily washed away due to its excellent solubility and does not enter the purified RNA.
- The carrier RNA is definitely included in the purified RNA solution and thus can cause off-target amplifications for detection PCRs.
- A preparation that is stable at room temperature and contains a yield enhancing component, the stability of which does not need to be demonstrated separately because, as a food coloring, it has already been demonstrated in both aqueous and alcoholic medium.
- A simple composition that does not require the addition of antibiotics to stabilize the sample, as alcohol kills any over-infectious bacteria.
- Can be stored for a long time at room temperature either after use or with the sample.
- Its production does not require disinfection steps.
- Its production does not require special tools.
- Samples taken are non-infectious and the testing laboratory does not need to be BSL- 2 + certified.
- It can be used in any method based on the amplification of qPCR or other RNA or DNA to detect nucleic acids of viruses or bacteria, as well as in any method where the transportability of DNA and/or RNA is an important and important consideration for the non-infectious material entering the VTM.
- During the processing with sample, it is not necessary to remove or wash the virus transport medium according to the invention from the samples, because the medium itself - by drying the beads, does not interfere with the measurement. Thus, the virus transport medium we have developed allows for a one-step solution. This is because the substances in the viral transport medium of the present invention help to bind the RNA and DNA (i.e., nucleic acids) of the virus from the solution to the silica-coated surface.
- The measurement results shown in the figures show in particular that the azorubincontaining virus transfer medium according to the present invention clearly shows the presence of the virus even at low virus concentrations, where the measurement becomes uncertain using commercially available virus transport medium.
The invention is illustrated in detail by the following examples, without limiting our claims to the following examples:
Example 1. RNA isolation experiments:
Our RNA isolation experiments were performed in a 150 pl closed epoxy-based microfluidic device (isolation chamber) connected to two 1 mm diameter channels (inlet and outlet). A VTM containing 500 pl of magnetic silica beads (MagPrep® Silica Particles; Sigma-Aldrich; Cat. No. 1011930001 ) was pipetted into the inlet channel. A swab previously containing a pharyngeal was placed in the VTM for 1 -2 minutes and then removed. Due to the salt present in the VTM, the known copy number synthetic
SARS-CoV-2 viral RNA segment present in the solution bound to the surface of the silica bead. After pipetting into the chamber, the magnetic bead-RNA complex was trapped on the inner wall of the chamber due to a magnet located on the outer surface of the chamber wall. Using a pipette, we sucked off the liquid from the beads. The beads remained inside the chamber throughout. The beads were washed with 150 pl of 70% ethanol for 30 minutes and then the alcohol was sucked off. The step was repeated and then the beads were dried under vacuum for approx. For 10 seconds. Finally, 150 pl of nuclease-free distilled water was pipetted into the chamber to elute RNA from the silica surface. After 15 seconds of incubation, the aqueous solution containing the RNA was sucked off from the chamber. This procedure was performed in three parallel measurements with our developed VTM, and in three parallel measurements with a GTC-containing VTM (beads (MagPrep® Silica Particles; Sigma- Aldrich; cat. No. 1011930001 )), and finally in an RT-LAMP reaction, isolation success (with 6 technical parallels).
Example 2. Measurement of RT-LAMP reaction
Equipment and settings:
RT-LAMP measurement was measured with a Quantstudio 5 (ThermoFisher; Cat. No. A34322) for 35 minutes at 65 ° C. Samples were prepared for measurement as follows: RT-LAMP reactions were prepared in the ratio recommended by the manufacturer (WarmStart® LAMP Kit; New England Biolabs; Cat. No. E1700L) in a final volume of 15 pl as follows:
7.5 pl WarmStart LAMP 2X Master Mix
0,3 pl Fluorescent dye (50X)
1.5 pl LAMP Primer Mix (1 OX)
0,7 pl dH2O
5 pl of isolated RNA or positive control RNA (200,000 copies) or dH2O (negative control)
Object and results of the measurements:
- LAMP test result of the viral transport medium according to the present invention in the case of a positive sample I 3 parallel measurements with 6 technical parallels (18 RT-LAMP reactions in total) /: Figure 1.a.
- LAMP test result of prior art virus transport medium in case of a positive sample I 3 parallel measurements with 6 technical parallels (18 RT-LAMP reactions in total) /. Figure 1.b.
- LAMP test result of the viral transport medium according to the present invention in case of a positive sample 1 3 parallel measurements with 6 technical parallels (18 RT- LAMP reactions in total) /. Figure 2. a.
- LAMP test result of the viral transport medium according to the present invention in case of a negative sample 13 parallel measurements with 6 technical parallels (18 RT- LAMP reactions in total) /. Figure 2.b.
According to the results of the measurements, the azorubin-containing virus transport medium according to the present invention shows the increase of fluorescence characteristic of the positive samples (Fig. 1.a.) in all cases, while in the case of the GTC-containing mixture shown in Fig. 1.b. only 3 positive samples show this results the others show false negative results. Figure 2. a. shows that the virus transport medium of the present invention shows the presence of virus from all positive samples, while Figure 2.b. shows that it does not give a false positive result if the sample does not contain the virus sought.
Example 3: (50-50% by weight in a mixture of ethyl alcohol and water)
Sodium chloride (11.7 g) and azorubin (0.01 g) were dissolved in water (500 g, 500 ml) at room temperature and the resulting solution was mixed with ethyl alcohol (500 g, 633 ml). In a similar manner, the product of the present invention can be prepared in the following compositions:
Example 4: (50-50% by volume in a mixture of ethyl alcohol and water)
Sodium chloride (11 .7 g) and an aqueous solution of Azorubin (0.88 g 1 1000 g) (8 ml) were dissolved in water (598 ml, 598 g) at room temperature, and the resulting solution was mixed with ethyl alcohol (500 ml, 393.8 g).
Example 5: (50-50% by weight in a methanol-water mixture)
Sodium chloride (11.25 g) and azorubin (0.01 g) were dissolved in water (500 g) at room temperature and the resulting solution was mixed with methanol (500 g).
Example 6: (50-50% by weight in a 2-propanol-water mixture)
Sodium chloride (11 .7 g) and azorubin (0.01 g) were dissolved in water (500 g) at room temperature, and the resulting solution was mixed with 2-propanol (500 g).
Example 7: (50-50% by weight in a n-propanol-water mixture)
Sodium chloride (11 .7 g) and azorubin (0.01 g) were dissolved in water (500 g) at room temperature, and the resulting solution was mixed with n-propanol (500 g).
Example 8: Application
A test tube was charged with 3 ml of the VTM solution of Example 1 . With a swab with a tissue suitable for removing pharyngeal secretion at the end, a swab sample is taken from the patient's nose and then the sample is dissolved from the swab into the VTM solution. The tube is sealed with a suitable rubber closure, the outer surface of the tube is wiped with an isopropyl alcohol disinfectant, and the disinfected sample on the outside is transported to the test laboratory in a sample collection container. The sample is then subjected to a PCR test.
Claims
1 . The Use of azorubin of formula
(I) as a yield enhancement agent in a viral transport medium.
2. The use of azorubin according to Claim 1 , characterized in that it is used in a mixture comprising azorubin, a solvent, an antiviral agent and, if necessary, further excipients.
3. The use of azorubin of formula (I) according to claim 1 or 2, characterized in that the azorubin is used as a solvent in a virus transport medium medium comprising preferably water, as a further solvent and antiviral agent preferably a C1-C3 alcohol and a salt containing a monovalent cation.
4. The use of azorubin of formula (I) according to any one of claims 1 -3 characterized in that azorubin is used more than 10’5% by weight, preferably 10’5-5% by weight, more preferably 0.5 * 10’4-3% by weight, most preferably 0.5*10’3-0.01 %% by weight based on the weight of the viral transport medium.
5. The use of azorubin of formula (I) according to any one of claims 1 -4, characterized in that the azorubin is us used in a medium comprising 30-95% by weight, preferably 35-75% by weight, most preferably 45-55% by weight of Ci -C3 alcohol, 5-70% by weight of water, preferably 25-65% by weight, most preferably 45-55% by weight of water, 50-500 mM, preferably 100-300 mM, more preferably 150-250 mM inorganic salt.
6. The use of azorubin of formula (I) according to any one of claims 1 -5, characterized in that the azorubin is used in a virus transport medium which comprises as alkali metal cation comprising alkali metal salts, preferably lithium, potassium or sodium salts,
preferably inorganic salts, more preferably phosphate, sulphate, nitrate, halide salts, more preferably chloride, bromide or iodide salts, most preferably sodium chloride.
7. Viral transport medium comprising azorubin.
8. The viral transport medium comprising azorubin according to claim 7, characterized in that it comprises a solvent, an antiviral agent and, if necessary, further excipients.
9. The viral transport medium comprising azorubin according to Claim 7 or 8, characterized in that, in addition to the azorubin, the solvent is preferably water, the additional solvent and antiviral agent is preferably a C1-C3 alcohol and a salt containing a monovalent cation.
10. The viral transport medium comprising azorubin according to Claim 7-9. characterized in that it comprises azorubin more than 10’5% by weight, preferably 10’ 5-5% by weight, more preferably 0.5 * 10’4-3% by weight, most preferably 0.5*10’3- 0.01 % by weight based on the weight of the viral transport medium.
11. The viral transport medium containing azorubin of the formula (I) according to Claim 7-10. characterized in that viral transport medium comprises 30-95% by weight, preferably 35-75% by weight, most preferably 45-55% by weight of C1-C3 alcohol, 5- 70% by weight of water, preferably 25-65% by weight, most preferably 45-55% by weight of water, 50-500 mM, preferably 100-300 mM, more preferably 150-250 mM of inorganic salt.
12. Viral transport medium comprising azorubin of formula (I) according to any one of claims 7-11. characterized in that the viral transport medium is alkali metal salts, preferably lithium, potassium or sodium salts, preferably inorganic salts, more preferably phosphate sulphate, nitrate, halide salts, more preferably chloride, bromide, bromide, monovalent cation, salts, most preferably sodium chloride.
13. Process for the preparation of virus transport medium according to claim 7, characterized in that
- dissolving azorubin in a virus transport medium, or
- azorubin is dissolved in a solvent used to produce a viral transport medium, and then the solution is used for producing a viral transport medium.
14. Process for the preparation of virus transport medium according to Claim 13, characterized in that the C1-C3 alcohol or a mixture of CI -C3 alcohols, water, azorubin, inorganic salts of monovalent inorganic cations, preferably inorganic salts of alkali metals, preferably phosphate, sulphate, nitrate, halide salts, more preferably Na, K, Li iodide, chloride bromide salts, most preferably sodium chloride or a mixture thereof is mixed in any order.
15. Process according to Claim 14, characterized in that the azorubin and optionally excipients, preferably inorganic salts, preferably alkali or alkaline earth metal salts, preferably Na, K, Li salts, are dissolved in any order and then the C1-C3 alcohol, or a mixture of C1-C3 alcohols are added to the solution.
16. Process according to Claim 14 or 15, characterized in that methanol, ethyl alcohol, n-propanol, 2-propanol or a mixture thereof, preferably ethyl alcohol or 2-propanol, most preferably ethanol are used as the C1-C3 alcohol.
17. Process according to Claim 16, characterized in that 30-95% by weight, preferably 35-75% by weight, most preferably 45-55% by weight of Ci -C3 alcohol, 5-70% by weight of water, preferably 25-65% by weight, most preferably 45%-55% by weight of water and 10’5-5% by weight, preferably 0.5 * 10’4-3% by weight, most preferably 0.5 * 10’3-0.01 % by weight of azorubin and optionally further excipients, preferably which 50-500 mM, preferably 100-300, mM, more preferably 150-250 mM inorganic salt, preferably lithium, potassium or sodium chloride, most preferably sodium chloride is used.
18. Use of a viral transport medium according to claim 7-13 in a method based on RNA or DNA amplification for detecting nucleic acids of viruses or bacteria, preferably in qPCR method.
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|---|---|---|---|---|
| WO2017011913A1 (en) * | 2015-07-21 | 2017-01-26 | Klox Technologies Inc. | Compositions and methods for the treatment of mucositis associated with treatment-induced neutropenia |
| WO2019216829A1 (en) * | 2018-05-10 | 2019-11-14 | Temasek Life Sciences Laboratory Limited | Application of a food azo dye, brilliant black bn, on inhibition of infectivity of human enteroviruses causing hand foot and mouth diseases |
| CN111363729A (en) * | 2020-03-12 | 2020-07-03 | 苏州白垩纪生物科技有限公司 | RNA virus inactivation preservation solution and application thereof |
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2021
- 2021-09-29 HU HU2100341A patent/HUP2100341A1/en unknown
- 2021-09-30 WO PCT/HU2021/050055 patent/WO2023052798A1/en unknown
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017011913A1 (en) * | 2015-07-21 | 2017-01-26 | Klox Technologies Inc. | Compositions and methods for the treatment of mucositis associated with treatment-induced neutropenia |
| WO2019216829A1 (en) * | 2018-05-10 | 2019-11-14 | Temasek Life Sciences Laboratory Limited | Application of a food azo dye, brilliant black bn, on inhibition of infectivity of human enteroviruses causing hand foot and mouth diseases |
| CN111363729A (en) * | 2020-03-12 | 2020-07-03 | 苏州白垩纪生物科技有限公司 | RNA virus inactivation preservation solution and application thereof |
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| GARNETT LAUREN ET AL: "Comparison analysis of different swabs and transport mediums suitable for SARS-CoV-2 testing following shortages", JOURNAL OF VIROLOGICAL METHODS, ELSEVIER BV, NL, vol. 285, 8 August 2020 (2020-08-08), XP086276826, ISSN: 0166-0934, [retrieved on 20200808], DOI: 10.1016/J.JVIROMET.2020.113947 * |
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