DE202021103096U1 - Kit for collecting saliva samples - Google Patents

Abstract

Kit for taking saliva samples for the detection of SARS-CoV-2 virus in saliva, characterized in that the kit comprises:
a) a first container for receiving the saliva sample, a coronavirus-deactivating composition being present in solid form within the container;
b) a second container comprising a gargle composition; and
c) optionally a filling device, preferably a funnel.

Description

The present invention relates to a kit for taking saliva samples for the detection of SARS-CoV-2 virus in saliva, the use of the kit for taking saliva, in particular saliva, which comprises viral RNA from SARS-CoV-2 virus, and a method for the detection of viral RNA from SARS-COV-2.

Pandemic situations are critical and a challenge for health systems around the world. The effects of a pandemic can quickly overwhelm health and care systems around the world. The search for sufficient staff, the availability of medicines and supplies and alternative care products must be incorporated into detailed plans that are tested and practiced in good time. Accurate information about the disease needs to be made available to caregivers and the public to reduce anxiety. Gathering information and testing people has become of paramount importance in getting the pandemic under control and safely reopening 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 through droplets that are 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.

Virus tests that detect active COVID-19 infections are the primary type of testing, although antibody tests are also available that show past infections based on the presence of COVID-19 antibodies.

There are several reasons that testing people for active COVID-19 infections is important. SARS-CoV-2 is particularly contagious as it 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 segregate the infected from the uninfected. People who receive a positive diagnosis should self-isolate to prevent the further spread of COVID-19. If people know for sure that they are infected with COVID-19, they are more likely to take appropriate preventive measures including isolation, keeping their distance, and wearing masks. This information is also useful for those around the infected, as they can avoid contact at all or, if they are around the infected person, be more careful.

This is especially important as there is evidence that a significant number of people with COVID-19 are asymptomatic, which means they never show any of the usual signs of illness or have symptoms so mild that COVID-19 does for another Illness, like a cold, can be kept. Studies suggest that approximately 40% to 75% of people with COVID-19 are asymptomatic. Also, the more people there are being treated for confirmed cases of COVID-19, the more data doctors, epidemiologists, and researchers will have to better understand the disease, its effects, and possible treatments.

Understanding who is infected with COVID-19 and where outbreaks are likely is a crucial factor in managing the pandemic, from preparing hospitals for potential spikes to safely reopening businesses, schools, churches, and other places where people get getting together. As mentioned earlier, because the virus is so contagious, people need to know with relative certainty whether they are currently infected with the disease before coming into contact 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 curfews 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 extensive testing to prevent outbreaks is the key factor in successfully rebooting the economy. However, it's not that easy to get COVID-19 tests up to the recommended level. Testing has become a problem due to a lack of supplies and a backlog in the laboratories that process the diagnostic test. Furthermore, clinics and Provide laboratories with qualified personnel to take appropriate body samples. Compared to traditional sampling via a nasal swab, taking a saliva sample is easier, non-invasive and not so uncomfortable for the person to be 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 a lower risk for caregivers when they collect a saliva sample as the patient can collect it themselves and there is less chance of coughing or sneezing than if they were to collect a sample with a cotton swab.

In view of the problems associated with test systems, there is a need for a sampling system that allows samples to be taken from individuals to be tested at home. However, this requires reliable and safe handling of the extraction process, which is a difficult task. Furthermore, taking samples at home still requires safe and stable storage of the samples taken in such a way that the samples can be safely transported by postal services to laboratories performing the sample analysis. In particular, it is important that DNA and RNA, like viral RNA, not be destroyed or damaged.

The above-mentioned problems are solved by means of a kit for taking saliva samples for the detection of SARS-CoV-2 virus in saliva, which is characterized according to the invention by the features according to claim 1, and by the embodiments as they are in the present invention reflect.

One aim of the present invention is to provide a kit for taking saliva samples which enables the samples taken to be stored safely and stably and which ensures sufficient safety for the person to be tested. Thus, the kit of the present invention is suitable for self-withdrawal of body fluids by the test subject himself, e.g., at home.

According to the invention, the kit comprises a first container for receiving saliva samples, a coronavirus-deactivating composition being present in solid form within the first container.

Containers for body fluids such as saliva samples for the purposes of the present invention are generally viewed as in vitro diagnostic medical devices. The containers are particularly suitable for containing and preserving samples that originate from the human body, in particular body fluids, for the purpose of in vitro diagnostic testing. Preferably the first container of the present invention is a test tube or a puncture vial. In one aspect of the invention, the first container can have a capacity for liquids from 0.5 ml to 1 l, preferably in the range from 1 ml to 200 ml, in particular in the range from 1.5 ml to 20 ml or 2 ml to 10 ml, exhibit.

In a further aspect, the first container has a tightly sealable opening and is preferably a tightly sealable sample tube.

In a further aspect, the first container can advantageously be closed in a liquid-tight manner, particularly preferably closed in an airtight manner. The airtight sealing or the liquid-tight sealing is advantageous for the transport of the samples in order to avoid contamination.

Suitable containers that can be easily and securely closed by the customer or generally by people to be tested are particularly preferred.

Therefore, in a further aspect of the present invention, the first container of the kit according to the invention has a closure, preferably a screw closure.

The first container of the kit according to the present invention can generally consist of any material which is suitable for contact with body fluids and / or biological material. Non-limiting examples are glass or organic polymers. Organic polymers are preferred because of their improved resistance to breakage and impact, which enables the subjects to be tested to be handled safely.

Particularly good results could be achieved with a container which comprises or consists of polystyrene or polyolefins, preferably polyolefins such as polypropylene and / or polyethylene.

In a preferred aspect, the first container consists essentially of an organic polymer having a melting point higher than 120 ° C, preferably higher than 140 ° C, and most preferably a melting point higher than 150 ° C, for example between 150 and 200 ° C. A higher melting point is advantageous for the first container of the present invention because, in a preferred aspect, the coronavirus-deactivating composition is precipitated on the inner surface of the container by evaporating water from a composition comprising the coronavirus-deactivating composition, preferably at higher temperatures, such as higher than 80 ° C or higher than 90 ° C.

In one aspect of the invention, the first container consists essentially of high density polyolefins. In a further aspect, the material of the first container can be different from the material of the seal, such as the closure.

In one aspect, the first container of the kit according to the invention comprises or consists essentially of polypropylene and the closure comprises or essentially consists of polyethylene.

The first container is preferably in the form of a collecting tube.

The first container of the kit according to the present invention has an inner surface which is suitable for being brought into contact with body fluids and which, when the container is used and filled, is in contact with the collected body fluid. Inside the first container of the kit according to the invention there is a solid coronavirus-deactivating composition. The solid coronavirus-deactivating composition is water-soluble at 20 ° C.

Usually the composition can be added to the first container in solid form, preferably powder form. In one aspect, the inner surface of the first container is at least partially coated with the coronavirus deactivating composition. Advantageously, the composition adheres at least partially, preferably completely, to the inner surface of the first container.

In a particularly preferred embodiment of the invention, the coronavirus-deactivating composition is deposited on the inner surface of the first container by precipitation. According to a preferred aspect, the precipitation takes place by evaporating the solvent, preferably water, from a solution or dispersion which comprises the coronavirus-deactivating composition in the first container of the kit according to the invention.

It has been shown that a container containing a solid coronavirus composition is safer for people to be tested. In particular, if the first container is used in a kit according to the invention for sampling, which comprises a second container with a gargle preparation, it can be avoided that the user accidentally takes the first container, which contains the coronavirus composition, as a gargle preparation.

The deactivation of coronaviruses 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 coronaviruses, in particular SARS-CoV-2, can be deactivated by chao-tropic 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 as 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, expressly exclude the presence of a chaotropic agent when a chelating agent is also present, and particularly a chaotropic agent in sufficient concentration to produce a protein, Denature polypeptide or nucleic acid molecule, while certain other contemplated embodiments are not so limited. Certain embodiments include, but are not limited to, the use of a chaotropic agent in 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 contemplated, where “about” is to be understood to represent a quantitative variation that is less than 50%, preferably less than 40%, particularly preferably less than 30% and particularly preferably less than 20%, 15%, 10% or 5% larger or smaller than the stated 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 filled into the container and the solvent is evaporated. The body fluids (herein, saliva samples) that are filled into the first container then dissolve the solidified coronavirus-deactivating composition and in turn lead to concentrations sufficient to deactivate 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 from the pancreas, although these enzymes can also be present in secretions and cells of the salivary gland and buccal lining. In addition, microorganisms living in the mouth or from recently ingested foods can release deoxyribonucleases or ribonucleases. It would be expected that a nucleic acid within a biological sample (e.g. saliva) kept in water would degrade or degrade over time.

Guanidinium salts, especially guanidinium thiocyanate and / or guanidinium chloride, are also known to inhibit deoxyribonucleases and ribonucleases ( Methods in Enzymology; Volume 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 aspect of the present invention, the coronavirus deactivating composition can comprise a solidified buffer.

Non-limiting examples of suitable buffering agents are sodium cyclohexanediamine tetraacetate (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, imidazylenediic 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-morpholine propanesulfonic 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. Other non-limiting examples of suitable buffering agents are buffering agents having a pKa at 25 ° C of 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 from 1: 1000 to 1:10, particularly preferably 1: 500 to 1:20, most preferably 1: 150 to 1 : 50.

In a further preferred embodiment, the coronavirus-deactivating composition comprises ethylenediaminetetraacetic acid and a guanidinium salt in a molar ratio in the range from 1: 1000 to 1:20, preferably 1: 600 to 1:50, particularly preferably 1: 300 to 1: 100.

In a further preferred aspect of the present invention, the coronavirus-deactivating composition comprises tris (hydroxymethyl) aminomethane and ethylenediaminetetraacetic acid in a molar ratio in the range from 20: 1 to 0.5: 1, preferably 10: 1 to 0.8: 1 and most preferably 5: 1 to 1: 1.

In a further aspect 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 kit of the present invention is suitable for collecting saliva samples.

The saliva sample can dissolve the solidified coronavirus deactivating composition in the first container of the invention. The volume of the first container is usually adjusted to provide a sufficient concentration of the coronavirus deactivating composition when it is completely filled with the saliva sample.

As used herein, the term "saliva" refers to the secretion or a combination of secretions from one of the salivary glands including the parotid, submandibular and sublingual glands, optionally mixed with the secretions from the numerous small lips, cheeks and palatine glands that line the mouth.

Advantages for the patient of providing a saliva sample or a nasal, anterior nasal and / or nasopharyngeal sample instead of a blood sample as a source of ribonucleic acid are that patients typically prefer to avoid the discomfort, pain and anxiety associated with a blood draw . In addition, while the use of 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 samples have the further advantage that they do not require specialist personnel for collection, which reduces costs where mass sampling is carried out (e.g. during an epidemic / pandemic). In order to collect saliva from a patient, the mouth is preferably rinsed before sampling. Food particles can introduce foreign RNA, and kissable saliva can be a source of foreign human RNA or viral RNA. The mouth can be rinsed out with about 50 ml of water by rinsing vigorously or brushing with a toothbrush without toothpaste. Unstimulated saliva is usually of the mucous type and is slowly secreted. Stimulated saliva (expectation of tasty food, sweet or sour candy) is of the serous (watery) type and is secreted more quickly. After rinsing the mouth and waiting for about 5 minutes until there is no water in the mouth, the patient may spit a volume (e.g. about 1-2 ml) of saliva, preferably stimulated saliva, into the first container of the kit of the present invention. The flow of saliva can conveniently be stimulated with a few grains / pinch of table sugar placed on the tongue or any other such saliva-stimulating substance that does not affect RNA stability or subsequent amplification.

In examples not forming part of the invention, 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 (e.g. a cotton swab, etc.) is used to collect saliva. Saliva can also be obtained from non-human animals, such as livestock, domestic animals, and the like, that may not be able or willing to spit directly into a collection device. In this case, a utensil (e.g. 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 off with the help of hard or flexible brushes, cotton swabs, or plastic / wooden scrapers, and cells can be flushed from the nasal cavity by introducing a liquid (e.g. saline solution) and recovering the liquid. 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 mucous membrane. Samples taken with the liquid and / or tools can be placed in the first container.

According to the invention, the first container of the kit of the present invention is used to collect saliva obtained with a gargle composition such as water.

In the context of the present invention, the term “saliva” also includes a gargle composition that includes saliva. It has been shown that obtained with a gargle composition Saliva and thus a mixture of saliva and gargle composition leads to samples with a higher viral load, especially in patients infected with coronavirus, such as SARS-CoV-2 virus.

It has been shown that infections by the coronavirus can lead to severe acute respiratory syndromes and that gargling provides a sufficient viral load for the detection of viral RNA, e.g. by PCR techniques.

The kit according to the invention is suitable for taking a human saliva sample and stabilizing it for transport. The downstream process is the extraction of DNA and RNA and subsequent PCR analysis. It has been found that the kit of the invention is excellently suited for taking saliva samples and that it stabilizes the sample so that it can be transported by normal postal or courier services at ambient temperatures.

• ein Behältnis für den Transport des Behälters der Erfindung, der mit der Körperflüssigkeitsprobe, z.B. der Speichelprobe, gefüllt ist; das Behältnis kann ein dicht verschließbarer Kunststoffbeutel sein;
• ein Klebeetikett zum Etikettieren der Proben; und
• saugfähiges Material.

The kit of the invention can additionally comprise or consist of one or more of the following components:

• a container for transporting the container of the invention filled with the body fluid sample, for example the saliva sample; the container can be a tightly sealable plastic bag;
• an adhesive label for labeling the samples; and
• absorbent material.

The kit consists of the first container and preferably a funnel to collect human gargle solution. 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 the DNA or RNA. The purpose of this analysis can be medical in nature for virology or originate in the non-medical way of life and for nutritional purposes.

In a preferred aspect, the kit of the invention comprises a first container in the form of a sample tube with a closable opening, preferably a closure.

The user removes the closure of the collecting tube and puts the filling device, preferably the funnel, into the opening of the tube. Then the user opens the second container, which contains a gargle composition, and puts the gargle composition, preferably water, in the mouth. The gargle composition is used to gargle the back of the throat for a sufficient time, e.g., 10 seconds. The user transfers the liquid into the first container with the aid of the funnel. The funnel is discarded and the tube is closed with the enclosed cap. The tube is returned to 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 first container for body fluids, which is a sealable sample tube which comprises or consists of polypropylene or polyethylene or polystyrene.

In a further embodiment of the present invention, the second container of the kit for collecting saliva according to the present invention is a sample tube or an ampoule, the second container preferably comprising or consisting of polypropylene or polyethylene or polystyrene.

In one aspect of the present invention, the gargle 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 removing saliva, in particular saliva, which comprises viral RNA, in particular RNA from coronavirus, such as RNA from SARS-CoV-2 virus.

1. i) Bereitstellen einer Probe von Speichel, die durch Gurgeln mit einer Gurgelzusammensetzung erhalten wird;
2. ii) Einfüllen der Speichelprobe in einen Behälter, wobei innerhalb des Behälters eine Coronavirus-deaktivierende Zusammensetzung in fester Form vorhanden ist; und
3. iii) Analysieren der Probe mit PCR-Techniken.

Another embodiment of the present invention is a method for the detection of viral RNA from SARS-CoV-2 virus in a saliva sample, which comprises the following steps:

1. i) providing a sample of saliva obtained by gargling with a gargle composition;
2. ii) filling the saliva sample into a container, a coronavirus-deactivating composition being present in solid form within the container; and
3. iii) analyzing the sample using PCR techniques.

According to the invention, the saliva sample is obtained by gargling with a gargle 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. At least one type of positive control or standard can be provided, which can be a nucleic acid (DNA or RNA) template to demonstrate the suitability of the sample for the detection of a target gene or nucleic acid sequence (e.g., a transcript) . Desirably, the container facilitates sampling in the field without the need for a clinic or hospital and is large enough to be mailed to a collection and / or analysis site.

In a further preferred embodiment, the present invention relates to the extraction of viral 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 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) . The solution was incubated at 90 ° C. for 15 hours. The sample tube with the solidified coronavirus-deactivating composition was fitted with a screw cap made of polyethylene.

• • Stabilität humaner RNA über die Zeit, wobei RNAse-P-RNA in den Proben gemessen wird;
• • Virale (SARS-CoV-2) RNA mit der Zeit

The container is intended for sample stabilization for downstream genetic analyzes. Genetic analyzes can be performed on DNA or RNA. Since RNA is considered to be considerably less stable than DNA, the stability validation was carried out on RNA. Thus, it is believed that if RNA is stable, DNA will be even more stable. In addition, viral RNA is considered to be even more unstable, and so this validation explores two aspects of stability:

• • Stability of human RNA over time, where RNAse-P-RNA is measured in the samples;
• • Viral (SARS-CoV-2) RNA over time

Sample preparation

• Speichelproben von 4 Individuen wurden mit dem Kit der Erfindung entnommen, der den oben genannten Behälter der Erfindung und einen zweiten Behälter, der aus einem 1 ml Wasser enthaltenden Probenröhrchen bestand, umfasste. Die Testindividuen verwendeten die Gurgellösung und gurgelten 10 Sekunden lang. Anschließend wurden die Gurgelproben gesammelt. Diese Individuen wurden zuvor auf die Anwesenheit einer SARS-CoV-2-Virusinfektion getestet und erwiesen sich als negativ. Die Proben wurden in 4 gleiche Aliquote aufgeteilt. Jedes Aliquot wurde mit echtem SARS-CoV-2-Virus aus einer klinischen Probe infundiert, die zuvor als positiv und eine hohe Viruslast tragend analysiert wurde. Der CT-Wert der positiven Probe betrug 24,27, was eine grobe virale Last von 250 000 Viruskopien pro Reaktion anzeigt. Da der Behälter der Erfindung in der Lage sein sollte, geringere Viruslasten zu konservieren, wurden die negativen Speichelproben mit dem positiven viralen Material in einer Verdünnung von 1:500 versetzt. Dies führt zu einer Probe, die um einen Faktor 500 weniger virales Material aufweist als ein stark infektiöses Individuum, ungefähr 500 Kopien pro Reaktion. (Die Nachweisgrenze von nur 2,5 Kopien wird in einem späteren Stadium dieser Anmeldung bestimmt).

The aim of this experiment was to assess the stability of human and viral RNA over extended periods of time at 0 ° C and at 45 ° C. The typical time in which a sample is taken and transported to the laboratory is less than 24 hours, but it can also be up to 72 hours. 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, which included the above-mentioned container of the invention and a second container consisting of a sample tube containing 1 ml of water. The test subjects used the gargle solution and gargled for 10 seconds. The gargle samples were then 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 real SARS-CoV-2 virus from a clinical sample that had previously been analyzed as positive and carrying a high viral load. The CT value of the positive sample was 24.27, indicating a gross 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 are mixed with the positive viral material in a dilution of 1: 500. This results in a sample that has a factor of 500 less viral material than a highly infectious individual, approximately 500 copies per reaction. (The limit of detection of only 2.5 copies will be determined at a later stage of this application).

Sample stability of human RNA

The presence of human RNA was measured in quadruple at all times. The mean of the quadruple determination was taken and used for calculations. All amplification curves were displayed as the rtPCR exponential amplification curve and the CT value was determined. The curves are in 1 reproduced. Medium HEC CT Individual 1 30.46 Individual 2 28.28 Average CT: Individual 3 31.98 0 h at 0 ° C, CT = 29.59 Individual 4 27.55 Pool of positive samples diluted 1:10 29.69

Result

Approximately the same amount of human RNA was present in all samples (which was to be expected) since dilutions were made on negative human saliva samples.

The NTC, sample without human material, showed no amplification, which was to be expected.

Sample stability of human RNA

The presence of human RNA was measured in quadruple at all times. The mean of the quadruple determination was taken and used for calculations. All amplification curves were displayed as the rtPCR exponential amplification curve and the CT value was determined. The curves are in 2 reproduced. Medium HEC CT Individual 1 31.22 Individual 2 28.20 Average CT: Individual 3 28.92 0 h at 0 ° C, CT = 29.59 Individual 4 27.81 72 h at 0 ° C, CT = 29.22 Pool of positive samples diluted 1:10 29.92

Result

Human RNA was also stable at 45 ° C. There was no increase in CT, which is a measure of the degradation.

Sample stability of human RNA

The presence of human RNA was measured in quadruple at all times. The mean of the quadruple determination was taken and used for calculations. All amplification curves were displayed as the rtPCR exponential amplification curve and the CT value was determined. The curves are in 3 reproduced. Medium HEC CT Individual 1 31.45 Individual 2 28.81 Average CT: Individual 3 29.02 0 h at 0 ° C, CT = 29.59 Individual 4 27.54 72 h at 0 ° C, CT = 29.22 Pool of positive samples diluted 1:10 30.92 72 h at 45 ° C, CT = 29.55

Result

Human RNA was also stable at 45 ° C. There was no increase in CT, which is a measure of the degradation.

Sample stability of human RNA

The presence of human RNA was measured in quadruple at all times. The mean of the quadruple determination was taken and used for calculations. All amplification curves were displayed as the rtPCR exponential amplification curve and the CT value was determined. The curves are in 4th reproduced. Human control (mean CT) begin 0 ° C 24 0 ° C 48 h 0 ° C 72 45 ° C 45 ° C 45 ° C 72 h 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 Average CT 29.57 28.67 29.77 29.04 29.02 29.78 29.20

Results

The CT value was stable at both temperatures, indicating a high degree of stability. A temperature of 45 ° C simulates aging accelerated 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 for at least 2 weeks at room temperature.

Sample stability of viral RNA

The presence of viral RNA was measured in triplicate at all time points. The mean of the triplicate was taken and used for calculations. All Amplification curves were displayed as rtPCR exponential amplification curve and the CT value was determined. The curves are in 5 reproduced. Medium SARS-CoV-2-CT Individual 1 29.28 Individual 2 29.19 Average CT: Individual 3 30.92 0 h at 0 ° C, CT = 29.44 Individual 4 28.36 Pool of positive samples diluted 1:10 24.27

Sample stability of viral RNA

The presence of viral RNA was measured in triplicate at all time points. The mean of the triplicate was taken and used for calculations. All amplification curves were displayed as the rtPCR exponential amplification curve and the CT value was determined. The curves are in 6th reproduced. Medium SARS-CoV-2-CT Individual 1 27.59 Individual 2 26.82 Average CT: Individual 3 27.40 0 h at 0 ° C, CT = 29.44 Individual 4 26.06 72 h at 0 ° C, CT = 26.97 Pool of positive samples diluted 1:10 23.26

Result

Viral RNA was very stable at 0 ° C with no observable degradation.

Sample stability of viral RNA

The presence of viral RNA was measured in triplicate at all time points. The mean of the triplicate was taken and used for calculations. All amplification curves were displayed as the rtPCR exponential amplification curve and the CT value was determined. The curves are in 7th reproduced. Medium SARS-CoV-2-CT Individual 1 32.64 Individual 2 31.16 Average CT: Individual 3 31.15 0 h at 0 ° C, CT = 29.44 Individual 4 32.00 72 h at 0 ° C, CT = 29.97 Pool of positive samples diluted 1:10 30.36 72 h at 45 ° C, CT = 31.74

Result

Viral RNA was stable even at 45 ° C. There was only a marginal increase in CT, which is a measure of the degradation.

Sample stability of viral RNA

The presence of viral RNA was measured in triplicate at all time points. The mean of the triplicate was taken and used for calculations. All amplification curves were displayed as the rtPCR exponential amplification curve and the CT value was determined. The curves are in 8th reproduced. Viral RNA (mean CT) begin 0 ° C 24 0 ° C 48 h 0 ° C 72 45 ° C 45 ° C 45 ° C 72 h 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 Average CT 29.44 28.92 28.72 26.97 30.20 32.04 31.74

Results

The CT value was stable at both temperatures, indicating a high degree of stability. A temperature of 45 ° C simulates aging accelerated 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 for 3 days at 45 ° C.

Multiple freeze-thaw cycles

It is assumed that freezing and thawing impair the sample quality and lead to a breakdown of DNA and RNA. To evaluate whether repeated freeze-thaw cycles impaired sample quality, a sample was allowed to freeze a total of five times at -20 ° C, then thawed at room temperature, measured, and then re-freeze and thawed and measured. The curves are in 9 reproduced. Mean HEC-CT without thawing 28.67 1x frozen-thawed 29.30 Frozen-thawed twice 28.87 3x frozen-thawed 29.14 Frozen-thawed 4x 28.70 5x frozen-thawed 29.34 Mean SARS-CoV-2-CT without thawing 30.62 1x frozen-thawed 31.30 Frozen-thawed twice 30.84 3x frozen-thawed 30.81 Frozen-thawed 4x 30.63 5x frozen-thawed 31.55

Result

Even 5 cycles of freezing and thawing affect the stability of neither the viral nor the human RNA.

Sample stability of viral RNA

A serial dilution of viral copies was made on samples from negative individuals. The estimated number of viral copies is shown in the graph ( 10 ).

Result

The container of the invention enables accurate detection of up to (lower limit) 2.5 copies of viral RNA per reaction.

QUOTES INCLUDED IN THE DESCRIPTION

This list of the documents listed by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Non-patent literature cited

• The deactivation of coronaviruses is known in the literature (Evaluation of Chemical Protocols for Inactivating SARS-CoV-2 Infectious Samples; Viruses 2020, 12, 624) [0027]
• Methods in Enzymology; Volume 502, 2012, pages 273-290 [0031]

Claims (22)

Kit for taking saliva samples for the detection of SARS-CoV-2 virus in saliva, characterized in that the kit comprises: a) a first container for receiving the saliva sample, a coronavirus-deactivating composition being present in solid form within the container ; b) a second container comprising a gargle composition; and c) optionally a filling device, preferably a funnel. Kit according to Claim 1 , characterized in that the solid coronavirus-deactivating 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-deactivating composition. Kit according to Claim 1 or 2 , characterized in that the coronavirus-deactivating composition is water-soluble at 20 ° C. Kit according to one or more of the preceding claims, characterized in that the first container has a capacity for liquids of 1.5 ml to 20 ml. Kit according to one or more of the preceding claims, characterized in that the kit further comprises the following components: i) a container for the transport of the first container; ii) an adhesive label for labeling the first container; iii) absorbent material. Kit according to one or more of the preceding claims, characterized in that the kit comprises a filling device. Kit according to one or more of the preceding claims, characterized in that the kit comprises a funnel. Kit according to one or more of the preceding claims, characterized in that the first container is a tightly sealable sample tube which comprises or consists of an organic polymer, in particular polypropylene or polyethylene or polystyrene, preferably with a melting point between 150 and 200 ° C. Kit according to one or more of the preceding claims, characterized in that the second container is a sample tube or an ampoule, the second container preferably comprising or consisting of polypropylene or polyethylene or polystyrene. Kit according to one or more of the preceding claims, characterized in that the gargle composition comprises or consists of water or buffered saline solution. Kit according to one or more of the preceding claims, characterized in that the coronavirus-deactivating composition is deposited on the inner surface of the first container by precipitation. Kit according to one or more of the preceding claims, characterized in that the coronavirus-deactivating composition comprises a solidified buffer. Kit according to one or more of the preceding claims, characterized in that the coronavirus-deactivating composition comprises at least one chaotropic agent. Kit according to one or more of the preceding claims, characterized in that the coronavirus-deactivating composition comprises or consists of a guanidinium salt in solid form. Kit according to one or more of the preceding claims, characterized in that the coronavirus-deactivating composition comprises tris (hydroxymethyl) aminomethane and / or ethylenediaminetetraacetic acid. Kit according to one or more of the preceding claims, characterized in that the coronavirus-deactivating composition tris (hydroxymethyl) aminomethane and a guanidinium salt in a molar ratio in the range from 1: 1000 to 1:10, preferably 1: 500 to 1:20, especially preferably 1: 150 to 1:50. Kit according to one or more of the preceding claims, characterized in that the coronavirus-deactivating composition ethylenediaminetetraacetic acid and a guanidinium salt in a molar ratio in the range from 1: 1000 to 1:20, preferably 1: 600 to 1:50, particularly preferably 1: 300 up to 1: 100. Kit according to one or more of the preceding claims, characterized in that the coronavirus-deactivating composition tris (hydroxymethyl) aminomethane and ethylenediaminetetraacetic acid in a molar ratio in the range from 20: 1 to 0.5: 1, preferably 10: 1 to 0.8: 1, particularly preferably 5: 1 to 1: 1. Kit according to one or more of the preceding claims, characterized in that the guanidinium salt is selected from the group consisting of guanidinium thiocyanate, guanidinium chloride, guanidinium isothiocyanate and mixtures thereof. Kit according to one or more of the preceding claims, characterized in that the guanidinium salt is guanidinium thiocyanate and / or guanidinium chloride. Kit according to one or more of the preceding claims, characterized in that the first container has a closure, preferably a screw closure. Use of a kit according to one or more of Claims 1 until 21 for the removal of saliva, in particular saliva, which comprises viral RNA, in particular RNA from coronavirus, such as RNA from SARS-CoV-2 virus.

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