• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The present invention relates to a container for saliva, a coronavirus-deactivating composition in solid form being present within the container, the use of the container for collecting saliva, a kit for collecting saliva samples comprising the container, the use of the Kits for collecting saliva, in particular saliva comprising viral RNA and a method for detecting RNA or DNA.
    公开号:CH717637B1
    申请号:CH00285/21
    申请日:2021-03-17
    公开日:2022-01-31
    发明作者:Wallerstorfer Daniel；Önder Kamil
    申请人:Procomcure Biotech Gmbh；
    IPC主号:
    专利说明:

    The present invention relates to a container for saliva, a coronavirus-deactivating composition in solid form being present within the container, the use of the container for collecting saliva, a kit for collecting saliva samples comprising the container, the use of the kit for collecting saliva, in particular saliva comprising viral RNA, and a method for detecting RNA or DNA.
    [0002] Pandemic situations are critical and a challenge to healthcare systems worldwide. The effects of a pandemic can quickly overwhelm health and care systems around the world. Finding adequate staff, availability of medication and supplies, and alternative care resources must be incorporated into detailed plans that are tested and practiced in a timely manner. Accurate information about the disease must be made available to caregivers and the public to reduce anxiety. Gathering information and testing people has become of utmost importance to get the pandemic under control and safely reopen the economy. This is especially true for highly contagious diseases, such as diseases caused by coronavirus. According to the Centers of Disease Control and Prevention, coronavirus disease 2019 or "COVID-19" is a respiratory disease caused by SARS coronavirus 2 or "SARS-CoV-2" (SARS = severe acute respiratory syndrome). This disease spreads via droplets produced when an infected individual sneezes or coughs. Symptoms include fever, cough and shortness of breath. Serious complications include pneumonia, multiple organ failure and, in some cases, death.
    Viral tests, which detect active COVID-19 infections, are the primary type of testing, although antibody tests are also available, which indicate past infections based on the presence of COVID-19 antibodies.
    [0004] There are several reasons that testing humans for active COVID-19 infections is important. SARS-CoV-2 is particularly contagious as this is the first time this particular coronavirus has been transmitted among humans. Without a vaccine or herd immunity, the only way to prevent transmission of COVID-19 is to isolate those who are infected from those who are not. People who receive a positive diagnosis should self-isolate to prevent further spread of COVID-19. When people know for sure that they are infected with COVID-19, they are more likely to take appropriate precautionary measures including isolation, social distancing and mask wearing. This information is also useful for those around infected individuals, as they may avoid contact at all or, when in the vicinity of the infected individual, exercise more caution.
    [0005] This is particularly important as there is evidence that a significant number of people with COVID-19 are asymptomatic, meaning they never show any of the usual signs of illness or have symptoms mild enough to cause COVID-19 can be mistaken for another illness, such as a cold. Studies suggest that approximately 40% to 75% of people with COVID-19 are asymptomatic. Also, as more people are being treated for confirmed cases of COVID-19, more data is available for doctors, epidemiologists and researchers to better understand the disease, its effects and possible treatments.
    Understanding who is infected with COVID-19 and where outbreaks are likely is a critical factor in dealing with the pandemic, from preparing hospitals for potential spikes to safely reopening businesses, schools, churches and other places , where people gather. As previously mentioned, because the virus is so contagious, people need to know with relative certainty whether they currently have the disease before engaging with others at work, meeting friends and acquaintances, or in other public spaces . Otherwise, they run the risk of spreading the disease throughout their social environment.
    During the extended lockdowns, health experts warned that lifting lockdowns and returning to regular work and social habits without extensive testing would likely lead to a sharp rise in infections. These experts emphasized that effective widespread testing to prevent outbreaks is the key to successfully restarting the economy. However, increasing COVID-19 testing to the recommended level is not that easy. Testing has become a problem due to a lack of supplies and a backlog at the labs processing the diagnostic test. Furthermore, clinics and laboratories must provide specialist staff to take suitable body samples. Compared to classic nasal swab sampling, taking a saliva sample is easier, non-invasive and not as uncomfortable for the person being tested. Thanks to the user-friendly aspects, the use of saliva samples can be an advantage, for example during a SARS-CoV-2 outbreak in a school where children need to be tested. There is also less risk for caregivers when taking a saliva sample as patients can collect it themselves and there is less risk of coughing or sneezing than taking a sample with a cotton swab.
    In view of the problems associated with testing systems, there is a need for a sampling system that allows sampling from subjects to be tested at home. However, this requires reliable and safe handling of the extraction process, which is a difficult task. Furthermore, home sampling continues to require safe and stable storage of the collected samples in such a manner that the samples can be safely transported by postal services to laboratories performing sample analysis. In particular, it is important that DNA and RNA, such as viral RNA, are not destroyed or damaged.
    The above problems are solved by the embodiments reflected in the present invention.
    An object of the present invention is to provide a container for collecting saliva, which allows safe and stable storage of the collected samples and provides sufficient security for the person to be tested. Thus, the container of the present invention is suitable for self-collection of saliva by the subject himself, e.g., at home.
    [0011] One embodiment of the present invention is a container for saliva, wherein a solid-form coronavirus deactivating composition is present within the container.
    [0012] Containers for saliva within the meaning of the present invention are generally considered to be in vitro diagnostic medical devices. The containers are particularly suitable for containing and preserving samples originating from the human body, in particular saliva, for the purpose of in vitro diagnostic testing. Preferably, the container of the present invention is a test tube or vial. In one embodiment of the invention, the container may have a liquid capacity of 0.5 ml to 1 liter, preferably in the range of 1 ml to 200 ml, more preferably in the range of 1.5 ml to 20 ml or 2 ml to 10 ml .
    In a further embodiment, the container has a sealable opening and is preferably a sealable sample tube.
    In a further embodiment, the container can advantageously be closed in a liquid-tight manner, particularly preferably closed in an air-tight manner. Airtight sealing or liquid-tight sealing is advantageous for transporting the samples in order to avoid contamination.
    Suitable containers that can be easily and securely closed by the customer or generally by persons to be tested are particularly preferred.
    Therefore, in a further embodiment of the present invention, the container of the invention comprises a closure, preferably a screw closure.
    The container of the present invention can generally be made of any material suitable for contact with saliva and/or biological material. Non-limiting examples are glass or organic polymers.
    [0018] Organic polymers are preferred due to the improved crush and impact resistance that allows subjects to be tested to handle them safely.
    [0019] Particularly good results have been achieved with a container which comprises or consists of polystyrene or polyolefins, preferably polyolefins such as polypropylene and/or polyethylene.
    In a preferred embodiment, the container consists essentially of an organic polymer having a melting point greater than 120°C, preferably greater than 140°C, and most preferably a melting point greater than 150°C. for example between 150 and 200 °C. A higher melting point is advantageous for the container of the present invention because in a preferred embodiment the coronavirus-deactivating composition is precipitated onto the inner surface of the container by evaporating water from a composition comprising the coronavirus-deactivating composition, preferably at higher ones Temperatures such as higher than 80°C or higher than 90°C.
    In one embodiment of the invention, the container consists essentially of high density polyolefins. In a further embodiment, the material of the container can be different from the material of the seal, such as the closure.
    In one embodiment, the container of the invention comprises or consists essentially of polypropylene and the closure comprises or consists essentially of polyethylene.
    Preferably the container is in the form of a collection tube.
    The container of the present invention has an inner surface adapted to be contacted with saliva and, when the container is used and filled, is in contact with the collected saliva. Within the container of the invention is a solid coronavirus deactivation composition. The solid coronavirus deactivation composition is water soluble at 20°C.
    Usually, the composition can be placed in the container in solid form, preferably in powder form. In one embodiment, the interior surface of the container is at least partially coated with the coronavirus deactivating composition. Advantageously, the composition adheres at least partially, preferably fully, to the interior surface of the container.
    In a particularly preferred embodiment of the invention, the coronavirus-deactivating composition is deposited on the interior surface of the container by precipitation. The precipitation is carried out according to a preferred embodiment by evaporating the solvent, preferably water, from a solution or dispersion comprising the coronavirus-deactivating composition in the container of the invention.
    A container containing a solid composition of coronavirus has been shown to be safer for subjects to be tested. In particular, when the container is used in a sample collection kit, preferably a kit comprising a second container containing a gargle composition, the user can be prevented from accidentally taking the container containing the coronavirus composition as a gargle composition.
    The deactivation of corona viruses is known in the literature (Evaluation of Chemical Protocols for Inactivating SARS-CoV-2 Infectious Samples; Viruses 2020, 12, 624).
    It has been shown that corona viruses, in particular SARS-CoV-2, can be deactivated by chaotropic agents. Certain embodiments described herein may include at least one chaotropic agent in the composition for substantially stable storage of nucleic acid and/or polypeptide molecules in a biological sample. Several chaotropic agents are known in the art that disrupt the secondary, tertiary and/or quaternary structure of biological macromolecules such as polypeptides, proteins and nucleic acids including DNA and RNA. Non-limiting examples of such chaotropic agents contemplated for use in certain of the compositions disclosed herein are guanidinium salts, guanidinium chloride, guanidinium thiocyanate, potassium thiocyanate, sodium thiocyanate, and urea. Certain contemplated embodiments, including those that may relate to specific types of biological samples, preclude the presence of a non-chaotropic agent when a chelating agent is also present, and in particular a chaotropic agent at a concentration sufficient to produce a protein, to denature a polypeptide or nucleic acid molecule. Certain embodiments contemplate including, but not limited to, the inclusion of a chaotropic agent at a concentration of about 0.05, 0.1, 0.5, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.4, 3.6, 3.8 or 4, 0 M into consideration, where "about" is to be understood as representing a quantitative variation that is less than 50%, preferably less than 40%, more preferably less than 30% and more preferably less than 20%, 15% , 10% or 5% greater or less than said amount.
    In a preferred embodiment, the guanidinium salt, in particular the guanidinium thiocyanate, can be used in a concentration of 1M to 6M, preferably 2M to 5M, such as 4M. Usually, the solutions are poured into the container and the solvent is evaporated. The saliva poured into the container then dissolves the solidified coronavirus-inactivating composition, again resulting in sufficient concentrations to inactivate the coronavirus.
    A major reason for the instability of nucleic acid in biological samples is the presence of deoxyribonucleases and ribonucleases. Deoxyribonucleases and ribonucleases are enzymes that break down DNA and RNA, respectively. Their main source in the digestive tract is secretions of the pancreas, although these enzymes may also be present in secretions and cells of the salivary gland and buccal mucosa. In addition, microorganisms living in the mouth or those from recently ingested foods may release deoxyribonucleases or ribonucleases. A nucleic acid within a biological sample (e.g., saliva) stored in water would be expected to degrade or degrade over time.
    Guanidinium salts, in particular guanidinium thiocyanate and/or guanidinium chloride, are also known to inhibit deoxyribonucleases and ribonucleases (Methods in Enzymology; Vol. 502, 2012, pages 273-290).
    According to a preferred embodiment, the coronavirus-deactivating composition comprises or consists of a guanidinium salt in solid form.
    In another embodiment of the present invention, the coronavirus-deactivating composition may comprise a solidified buffer.
    Non-limiting examples of suitable buffering agents are sodium cyclohexanediaminetetraacetate (CDTA), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 4,(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), acetic acid or Acetate (e.g. sodium acetate), citric acid or citrate, malic acid, phthalic acid, succinic acid, histidine, diphosphoric acid, maleic acid, cacodylic acid, BB'-dimethylglutaric acid, carbonic acid or carbonate, 5(4)-hydroxymethylimidazole, glycerol-2-phosphoric acid, ethylenediamine, Imidazole, arsenic acid, phosphoric acid or phosphate, sodium acetate, 2:4:6 collidine, 5(4)-methylimidazole, N-ethylmorpholine, triethanolamine, diethylbarbituric acid, tris(hydroxymethyl)aminomethane (Tris), 3-(N-morpholino)propanesulfonic acid ; 4-morpholinepropanesulfonic acid (MOPS), 2-morpholinoethanesulfonic acid (MES), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), N-[tris(hydroxymethyl)methyl)-2-aminoethanesulfonic acid (TES), 4-( 2-Hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), or combinations thereof. Other examples are phosphate, carbonate, ethylenediamine or imidazole buffers. Additional non-limiting examples of a suitable buffering agent include buffering agents having a pKa at 25°C from about 4.7 to about 8.0.
    It has proven to be advantageous and improves the storage stability of the samples taken if the coronavirus-deactivating composition comprises tris(hydroxymethyl)aminomethane and/or ethylenediaminetetraacetic acid.
    In a preferred embodiment, the coronavirus deactivating composition comprises tris(hydroxymethyl)aminomethane and a guanidinium salt in a molar ratio in the range of 1:1000 to 1:10, more preferably 1:500 to 1:20, most preferably 1: 150 to 1:50.
    In another preferred embodiment, the coronavirus-deactivating composition comprises ethylenediaminetetraacetic acid and a guanidinium salt in a molar ratio in the range of 1:1000 to 1:20, preferably 1:600 to 1:50, more preferably 1:300 to 1:100 .
    In another preferred embodiment of the present invention, the coronavirus deactivating composition comprises tris(hydroxymethyl)aminomethane and ethylenediaminetetraacetic acid in a molar ratio in the range of 20:1 to 0.5:1, preferably 10:1 to 0.8:1 and most preferably 5:1 to 1:1.
    In another embodiment of the present invention, the guanidinium salt is selected from the group consisting of guanidinium thiocyanate, guanidinium chloride, guanidinium isothiocyanate, and mixtures thereof.
    Most preferably, the guanidinium salt is guanidinium thiocyanate and/or guanidinium chloride.
    The container of the present invention is suitable for collecting saliva.
    The saliva can dissolve the solidified coronavirus-deactivating composition in the container of the invention. The volume of the container is usually adjusted to obtain a sufficient concentration of the coronavirus-deactivating composition when completely filled with saliva.
    The term "saliva" as used herein refers to the secretion or combination of secretions from any of the salivary glands including the parotid, submandibular and sublingual glands, optionally mixed with the secretions from the numerous small lip , cheek and palate glands lining the mouth.
    Benefits to the patient of providing a saliva or nasal, anterior nasal, and/or nasopharyngeal sample rather than a blood sample as a source of ribonucleic acid are that patients typically prefer to avoid the discomfort, pain, and pain associated with blood collection to avoid fears. In addition, while using a needle prick to obtain a drop of blood is sufficient to obtain a usable amount of DNA, the expected amount of RNA is too small to be usable for most purposes. Saliva, sputum, nasal, anterior nasal and/or nasopharyngeal sampling have the further advantage that they do not require skilled personnel for collection, thereby reducing costs where bulk sampling is performed (e.g. during an epidemic/pandemic). However, those skilled in the art will appreciate that while saliva is a source of RNA, other body fluids, including blood, can also be used. In order to collect saliva from a patient, the mouth is preferably rinsed prior to sampling. Food particles can introduce foreign RNA, and kissing-borne saliva can be a source of foreign human RNA or viral RNA. The mouth can be rinsed with about 50 ml of water by vigorous rinsing or brushing with a toothbrush without toothpaste. Unstimulated saliva is usually of the mucous type and is slowly secreted. Stimulated saliva (anticipation of tasty food, sweet or sour candy) is of the serous (watery) type and is secreted faster. After rinsing the mouth and waiting about 5 minutes until the mouth is water-free, the patient may spit a volume (e.g. about 1-2 ml) of saliva, preferably stimulated saliva, into the container of the present invention. Salivation can be conveniently stimulated with a few grains/pinch of table sugar placed on the tongue, or any other such salivary-stimulating substance that does not interfere with RNA stability or subsequent amplification. Saliva can also be obtained from patients such as infants, young children, and people with disabilities and/or illness who may not be able to spit directly into a collection device. In this case, a utensil (such as a cotton swab, etc.) is used to collect saliva.
    [0046] Saliva can also be obtained from non-human animals, such as livestock, pets, and the like, which may be unable or unwilling to spit directly into a collection device. In this case, a utensil (such as a cotton swab, etc.) can be used to collect saliva. A variety of tools can be used to obtain an anterior nasal or nasopharyngeal sample from a patient. Mucosal cells can be scraped using hard or flexible brushes, cotton swabs, or plastic/wooden scrapers, and cells can be flushed from the nasal cavity by introducing a fluid (e.g., saline) and retrieving the fluid. For example, a stiff cotton swab/brush can be placed in the front of the nose and a flexible cotton swab/brush in the back of the nasopharynx and used to collect mucosal secretions and gently rub cells from the mucosa. Samples taken with the liquid and/or tools can be placed in the container of the invention.
    The use of the container of the present invention is for the collection of saliva, particularly saliva that forms a mixture with a gargling composition, e.g., water.
    A mixture of saliva and gargle composition has been shown to result in samples with a higher viral load, particularly in patients infected with coronavirus such as SARS-CoV-2 virus.
    It has been shown that coronavirus infections can lead to severe acute respiratory syndrome and that gargling provides sufficient viral load for detection of viral RNA, e.g., by PCR techniques.
    Therefore, another embodiment of the present invention is a saliva sampling kit comprising: a) a first container for saliva according to the present invention; b) a second container comprising a gargling composition; and c) optionally a filling device, preferably a funnel.
    The kit is suitable for taking a human saliva sample and stabilizing it for transport. The downstream procedure is the extraction of DNA and RNA and subsequent PCR analysis. The kit of the invention has been found to perform extremely well in taking saliva samples and stabilizing the sample for transport at ambient temperatures by normal postal or courier services.
    The kit of the invention may additionally comprise or consist of one or more of the following components: a container for transporting the container of the invention filled with the saliva sample; the container may be a sealable plastic bag; an adhesive label for labeling the samples; and absorbent material.
    The kit consists of a container of the invention and preferably a funnel to collect human gargle composition. This kit allows for the collection of cells from the back of the throat and saliva at home. The sample is stabilized, protein is denatured and prepared for transport at ambient temperatures. The collection tubes ensure that the samples are not contaminated during transport.
    The samples can then be used in specially equipped facilities to extract the DNA or RNA from the stabilized cells or saliva. The downstream application of the sample is an analysis of DNA or RNA. The purpose of this analysis can be medicinal for virology or it can have its origins in non-medical lifestyle and nutritional purposes.
    In a preferred embodiment, the kit of the invention comprises a container of the invention in the form of a sample tube having a closable opening, preferably a closure.
    The user removes the cap from the collection tube and inserts the filling device, preferably the funnel, into the opening of the tube. The user then opens a second container comprising a gargle composition and places the gargle composition, preferably water, in the mouth. The gargling composition is used to gargle at the back of the throat for a sufficient time, e.g., 10 seconds. The user transfers the liquid into the first container using the funnel. The funnel is discarded and the tube is closed with the cap provided. The tube is returned in the plastic bag for transport. The user then sends the samples to the facility for analysis.
    According to a preferred embodiment of the present invention, the kit comprises a container for saliva which is a sealable test tube comprising or consisting of polypropylene or polyethylene or polystyrene.
    In a further embodiment of the present invention, the second container of the saliva collection kit according to the present invention is a test tube or ampoule, the second container preferably comprising or consisting of polypropylene or polyethylene or polystyrene.
    In one embodiment of the present invention, the gargling composition present in the second container comprises or consists of water or buffered saline.
    Another embodiment of the present invention is the use of a kit of the present invention for collecting saliva, in particular saliva comprising viral RNA, in particular RNA from coronavirus such as RNA from SARS-CoV-2 virus.
    Another embodiment of the present invention is a method for detecting RNA or DNA, comprising the steps of: i) providing a sample of saliva or a mixture of saliva and gargle composition; ii) filling the saliva sample into a container according to one or more of claims 1 to 15; and iii) analyzing the sample,
    In a preferred embodiment of the invention, the saliva sample is obtained by gargling with a gargling composition.
    Methods of the invention are conveniently practiced by providing the compositions used in such a method in kit form, e.g., the kit of the invention. Such a kit preferably contains a container of the invention and appropriate collection devices, such as cotton swabs, to facilitate sample collection. At least one type of positive control or standard may be provided, which may be a nucleic acid (DNA or RNA) template, to demonstrate the suitability of the sample to detect a target gene or nucleic acid sequence (eg, a transcript). . Desirably, the container facilitates sampling in the field without requiring a clinic or hospital and is large enough to be mailed to a collection and/or analysis site.
    In a more preferred embodiment, the present invention relates to the extraction of viral RNA, in particular RNA from coronavirus, such as RNA from SARS-CoV-2 virus.
    The detection of RNA or DNA is preferably the detection of viral RNA, in particular RNA from coronavirus, such as RNA from SARS-CoV-2 virus.
    In a particularly preferred embodiment of the method according to the present invention, the sample is analyzed using PCR techniques such as reverse transcription polymerase chain reaction (RT-PCR) or RT-qPCR (real-time quantitative PCR) or RNA/DNA sequencing or RNA/DNA hybridization.
    Examples:
    The container of the invention was prepared in that a sample tube made of polypropylene was filled with 1.5 ml of an aqueous composition having a concentration of 4 M guanidinium thiocyanate, 55 mM tris(hydroxymethyl)aminomethane (Tris) and ethylenediaminetetraacetic acid (EDTA ) has been filled. The solution was incubated at 90°C for 15 hours. The sample tube containing the solidified coronavirus-inactivating composition was fitted with a screw cap made of polyethylene.
    The container is intended for sample stabilization for downstream genetic analysis. Genetic analyzes can be performed on DNA or RNA. Since RNA is considered to be significantly less stable than DNA, the stability validation was performed on RNA. Thus, if RNA is stable, it is believed that DNA will be even more stable. In addition, viral RNA is considered even more unstable, so this validation explores two aspects of stability: • Stability of human RNA over time, measuring RNase P RNA in the samples; • Viral (SARS-CoV-2) RNA over time
    sample preparation
    The aim of this experiment was to evaluate the stability of human and viral RNA over long periods of time at 0°C and at 45°C. The typical time it takes for a sample to be collected and transported to the laboratory is less than 24 hours but can take up to 72 hours. In order to evaluate the stability of these samples, the following experiment was set up: Saliva samples from 4 individuals were taken with the kit of the invention comprising the above container of the invention and a second container consisting of a sample tube containing 1 ml of water. The test subjects used the gargling composition and gargled for 10 seconds. Then the gargle samples were collected. These individuals were previously tested for the presence of SARS-CoV-2 virus infection and found negative. The samples were divided into 4 equal aliquots. Each aliquot was infused with genuine SARS-CoV-2 virus from a clinical specimen previously analyzed as positive and carrying a high viral load. The CT value of the positive sample was 24.27, indicating a crude viral load of 250,000 virus copies per reaction. Since the container of the invention should be able to preserve lower viral loads, the negative saliva samples were spiked with the positive viral material at a dilution of 1:500. This results in a sample having a factor of 500 less viral material than a highly infectious individual, approximately 500 copies per reaction. (The detection limit of only 2.5 copies will be determined at a later stage of this application).
    1. Sample stability of human RNA
    The presence of human RNA was measured in quadruplicate at all time points. The mean of the quadruplicate determination was taken and used for calculations. All amplification curves were displayed as an rtPCR exponential amplification curve and the CT value was determined. The curves are shown in FIG.
    Result
    Human RNA was present in approximately equal amounts in all samples (which is to be expected) since dilutions were made from negative human saliva samples.
    The NTC, sample without human material, showed no amplification, which was to be expected.
    2. Sample stability of human RNA
    The presence of human RNA was measured in quadruplicate at all time points. The mean of the quadruplicate determination was taken and used for calculations. All amplification curves were displayed as an rtPCR exponential amplification curve and the CT value was determined. The curves are shown in FIG.
    Result
    Human RNA was also stable at 45°C. There was no increase in CT, which is a measure of degradation.
    3. Sample stability of human RNA
    The presence of human RNA was measured in quadruplicate at all time points. The mean of the quadruplicate determination was taken and used for calculations. All amplification curves were displayed as an rtPCR exponential amplification curve and the CT value was determined. The curves are shown in FIG.
    Result
    Human RNA was also stable at 45°C. There was no increase in CT, which is a measure of degradation.
    4. Sample Stability of Human RNA
    The presence of human RNA was measured in quadruplicate at all time points. The mean of the quadruplicate determination was taken and used for calculations. All amplification curves were displayed as an rtPCR exponential amplification curve and the CT value was determined. The curves are shown in FIG. Individual 1 30.46 28.89 32.13 31.22 30.66 32.16 31.45 Individual 2 28.28 28.38 29.03 28.20 28.88 29.14 28.81 Individual 3 31, 98 29.05 29.92 28.92 28.85 29.88 29.02 individual 4 27.55 28.36 27.99 27.81 27.70 27.96 27.54 mean CT 29.57 28, 67 29.77 29.04 29.02 29.78 29.20
    results
    The CT value was stable at both temperatures, showing a high degree of stability. A temperature of 45 °C simulates accelerated aging by a factor of 4.5 compared to room temperature. This means that a period of 72 hours at this temperature is the equivalent of 72 x 4.5 = 324 hours or 13.5 days. Thus, human RNA is stable at room temperature for at least 2 weeks.
    5. Sample stability of viral RNA
    The presence of viral RNA was measured in triplicate at all time points. The mean of the triplicate determination was taken and used for calculations. All amplification curves were displayed as an rtPCR exponential amplification curve and the CT value was determined. The curves are shown in FIG.
    6. Sample Stability of Viral RNA
    The presence of viral RNA was measured in triplicate at all time points. The mean of the triplicate determination was taken and used for calculations. All amplification curves were displayed as an rtPCR exponential amplification curve and the CT value was determined. The curves are shown in FIG.
    Result
    [0081] Viral RNA was very stable at 0°C with no observable degradation.
    7. Sample Stability of Viral RNA
    The presence of viral RNA was measured in triplicate at all time points. The mean of the triplicate determination was taken and used for calculations. All amplification curves were displayed as an rtPCR exponential amplification curve and the CT value was determined. The curves are shown in FIG.
    Result
    Viral RNA was also stable at 45°C. There was only a marginal increase in CT, which is a measure of degradation.
    8. Sample stability of viral RNA
    The presence of viral RNA was measured in triplicate at all time points. The mean of the triplicate determination was taken and used for calculations. All amplification curves were displayed as an rtPCR exponential amplification curve and the CT value was determined. The curves are shown in FIG. Individual 1 29.28 30.92 29.62 27.59 30.11 33.07 32.64 Individual 2 29.19 27.98 28.63 26.82 30.40 30.89 31.16 Individual 3 30, 92 28.71 29.11 27.40 30.21 31.78 31.15 Individual 4 28.36 28.06 27.51 26.06 30.09 32.43 32.00 Mean CT 29.44 28, 92 28.72 26.97 30.20 32.04 31.74
    results
    The CT value was stable at both temperatures, showing a high degree of stability. A temperature of 45 °C simulates accelerated aging by a factor of 4.5 compared to room temperature. This means that a period of 72 hours at this temperature is the equivalent of 72 x 4.5 = 324 hours or 13.5 days. Thus, viral RNA is stable for at least 2 weeks at room temperature or 3 days at 45°C.
    9.Multiple freeze-thaw cycles
    [0086] Freezing and thawing are thought to affect sample quality and lead to degradation of DNA and RNA. To assess whether repeated freeze-thaw cycles affect sample quality, a sample was frozen at -20°C, then thawed at room temperature, measured, and then refrozen and thawed again and measured a total of five times. The curves are shown in FIG. Mean HEC-CT without thawing 28.67 1x freeze-thaw 29.30 2x freeze-thaw 28.87 3x freeze-thaw 29.14 4x freeze-thaw 28.70 5x freeze-thaw 29.34 mean SARS-CoV-2 -CT without thawing 30.62 1x freeze-thaw 31.30 2x freeze-thaw 30.84 3x freeze-thaw 30.81 4x freeze-thaw 30.63 5x freeze-thaw 31.55
    Result
    [0087] Even 5 cycles of freezing and thawing do not affect the stability of either the viral or the human RNA.
    10. Sample stability of viral RNA
    A dilution series of viral copies was made from samples from negative individuals. The estimated viral copy number is shown in the graph (Figure 10).
    Result
    The container of the invention allows accurate detection of up to (lower limit) 2.5 copies of viral RNA per reaction.
    权利要求:
    Claims (11)
    [1]
    A kit for collecting saliva samples for the detection of SARS-CoV-2 virus viral RNA in saliva, comprising:a) a first container for receiving a saliva sample, wherein a solid form of a coronavirus-deactivating composition is present within the container; wherein the coronavirus-deactivating composition comprises at least one chaotropic agent,b) a second container comprising a gargling composition containing water,
    [2]
    2. Kit according to claim 1, characterized in that the kit also comprises a filling device, preferably a funnel.
    [3]
    3. Kit according to claim 1 or 2, characterized in that the solid coronavirus-activating composition adheres at least partially, preferably completely, to an inner surface of the first container or the inner surface is at least partially coated with the solid coronavirus-activating composition.
    [4]
    4. Kit according to one of claims 1 to 3, wherein the first container for saliva is a tightly sealable sample tube which comprises or consists of an organic polymer, in particular polypropylene or polyethylene or polystyrene.
    [5]
    5. Kit according to any one of claims 1 to 4, wherein the coronavirus-deactivating composition is water-soluble at 20°C.
    [6]
    The kit of any one of claims 1 to 5, wherein the coronavirus-deactivating composition comprises or consists of a guanidinium salt in solid form, preferably wherein the guanidinium salt is selected from the group consisting of guanidinium thiocyanate, guanidinium chloride, guanidinium isothiocyanate, and mixtures thereof.
    [7]
    7. The kit according to any one of claims 1 to 6, wherein the coronavirus deactivating composition comprises a solidified buffer.
    [8]
    A kit according to any one of claims 1 to 7, wherein the gargling composition comprises or consists of water or buffered saline.
    [9]
    9. Kit according to any one of claims 1 to 8, wherein the coronavirus deactivating composition tris(hydroxymethyl)aminomethane and a guanidinium salt in a molar ratio in the range of 1:1000 to 1:10, preferably 1:500 to 1:20, particularly preferred 1:150 to 1:50.
    [10]
    10. Kit according to any one of claims 1 to 9, wherein the coronavirus-deactivating composition ethylenediaminetetraacetic acid and a guanidinium salt in a molar ratio in the range of 1:1000 to 1:20, preferably 1:600 to 1:50, more preferably 1:300 to 1:100 includes.
    [11]
    The kit according to any one of claims 1 to 10, wherein the coronavirus deactivating composition comprises tris(hydroxymethyl)aminomethane and ethylenediaminetetraacetic acid in a molar ratio in the range of 20:1 to 0.5:1, preferably 10:1 to 0.8:1 , particularly preferably 5:1 to 1:1.
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    EP20076183|2020-09-18|
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