Patent Application
20200347465
The present invention relates to a method for the detection of SARS-CoV-2 in a plurality of biological samples of living beings and to a kit for carrying out said method.
The invention relates to the field of molecular biology, more particular to the detection of viral material in a biological sample.
At the end of the last century the HIV AIDS scandal [13,22,39] enables the integration of a new technology into blood donor screening by the investigation of molecular parameters by nucleic acid technologies (NAT) [1,6,11,16,29,31-34]. In the beginning only a few medical laboratories were able to implement this technology in the late 90ies into blood donor screening. For NAT it was strictly necessary to have separate rooms (pre-NAT room, NAT room and post NAT room). Staff were trained to work in an one-way-direction and it was strictly forbidden to go back to the NAT room after entry the post-NAT room on the same day. The detection of RNA and DNA viruses was a further challenge to screening tests. In the beginning samples tubes have to be open again after the reverse transcription step to change the enzyme from a reverse transcriptase into a DNA polymerase. But the technique developed rapidly and complete NAT robot systems are now available for blood donor screening [7,12,15] as well as for virus detection in clinical laboratories.
Current screening NAT robot systems (e.g. Roche Cobas 8800 or Grifols Panther) [14] have implemented different chambers to separate the pre-NAT room, from the NAT room and the post-NAT room. Working with these “all in one NAT systems” needs only trained staff, input of individual samples or mini-pool samples, reagents and disposables.
In 2019, a new Corona virus was first discovered in China named SARS-CoV-2. The new virus spread quickly from person to person. Due to intensive human travel activities, SARS-CoV-2 infected people worldwide. In March 2020 the WHO declared all criteria of a pandemic were fulfilled. In young people SARS-CoV-2 induce flu-like symptoms such as cough, sore throat, diarrhea or fever. However, severe pneumonia with fatalities also occurs in some cases especially in immunosuppressed people, old people or people with lung diseases like bronchial asthma. Up to now no suitable therapy is available. Therefore, the global community is relying on an early diagnosis with subsequent isolation of the infected people in order to slow down the further spread of SARS-CoV-2. As a result, since March 2020, many countries have introduced special measures as part of their national pandemic plans that restrict people's freedom of movement. Early diagnosis is possible using the nucleic acid amplification technique (NAT). This enables the detection of the virus directly. However, due to the exponentially increasing number of infected people, there is also a shortage of reagents with this detection method, so that not all people who are first contact persons to SARS-CoV-2 infected people or have mild flu-like symptoms can receive a NAT examination.
Against this background it is an object underlying the invention to pro-vide a method for the detection of SARS-CoV-2 which overcomes the disadvantages in the art. Preferably such a method should be provided which allows acceleration of the test throughput and an increase of the test capacity over the current detection methods. Furthermore, such a detection method should be provided which requires less reagents, process steps, test equipment and utensils.
The present invention satisfies these and other needs.
The present invention provides a method for the detection of SARS-CoV-2 in a plurality of biological samples of living beings comprising the following steps:
The object underlying the invention is herewith completely solved. By the simultaneous testing of at least two biological samples, i.e. biological samples from at least two living beings, e.g. humans or human patients, respectively, the performance of testing is significantly increased. Furthermore, the available test resources are employed in a more efficient manner by—in case of a negative outcome in step (4)—obtaining a test result for more than one living being in a single round of the method.
According to the invention, “at least a second living being” means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, . . . 49, . . . , 99, . . . , 199, . . . , 299, . . . 399, . . . 499 etc. additional biological sample or additional living beings to be tested by the method according to the invention.
According to the invention “SARS-CoV-2 derived material” refers to structures, molecules or features being part of or originating from SARS-CoV-2 being suitable of indicating the presence of SARS-CoV-2 in the biological sample. “SARS-CoV-2 derived material” includes, e.g., nucleic acid, proteins, peptides and fragments thereof.
In an embodiment of the invention said first and said at least second living being represent a group of living beings, said group consisting of a number of living beings which is selected from an integer of 2-500, preferably of 3-100, further preferably of 5-50, and further preferably of 10.
This embodiment has the advantage that by the method according to the invention biological samples of up to 500 living beings, e.g. humans or human patients, respectively, can be rather simultaneously tested in one round of the method.
In an embodiment of the method according to the invention said biological samples are swaps, preferably swaps from the throat.
This measure has the advantage that such kind of sample provision is employed which ensures, due to the high concentration of the virus in the throat area, that best possible biological source material in subjected to the method according to the invention. “Swap” is to be understood as referring to the wiped-off biological material itself and, alternatively, to the stick or cotton stick comprising the biological material.
In an embodiment of the method according to the invention said SARS-CoV-2 derived material is SARS-CoV-2 nucleic acid.
This measure has the advantage that the detection can be carried out via standard laboratory methods allowing the direct detection of the virus or material derived therefrom in a reliable manner.
In another embodiment of the method according to the invention said testing of the samples for the presence of SARS-CoV-2 and/or SARS-CoV-2 derived material is made by nucleic amplification technique (NAT), preferably by polymerase chain reaction (PCR).
This measure takes the advantage of using well-established and highly reliably detection methods, rendering the method according to the invention very accurate.
Another subject-matter according to the invention relates to a method for the detection of SARS-CoV-2 in a plurality of biological samples of living beings, preferably such a method as disclosed in the preceding paragraphs, said method comprises the following steps:
Such embodiment of the method according to the invention is also referred to as “dilution method”. The first biological sample, e.g. a first swap from a first individual, is provided and placed into a tube of buffer (primary sample or tube) for a time sufficient to dispense SARS-CoV2 or material derived therefrom into the buffer. In a next step a small volume of the primary sample or tube is diluted and transferred into the pool tube or pool sample, respectively. For two biological samples or swaps/beings (n=2) a dilution of 1:2 is used for other numbers of biological samples or swaps/beings other dilutions were used, i.e. n=3 a dilution of 1:3, n=4 a dilution of 1:4, n=5 a dilution of 1:5 etc. is used.
This embodiment has the advantage that an optimum test volume for the subsequent testing step is achieved. This is important because the test apparatus, such as the PCR device has a certain maximum sample volume which must not be exceeded.
The features, elements, and advantages disclosed for the method at the outset apply to the “dilution method” likewise and vice versa.
In an embodiment of the “dilution method” according to the invention in step (1) each of said n biological samples is provided via a swab, preferably a throat swap.
This measure has the advantage that such kind of sample provision is employed which ensures, due to the high concentration of the virus in the throat area, that best possible biological source material in subjected to the method according to the invention.
Another subject-matter according to the invention relates to a method for the detection of SARS-CoV-2 in a plurality of biological samples of living beings, preferably such a method as disclosed in the preceding paragraphs, said method comprises the following steps:
Such embodiment according to the invention is also referred to as “dual swap method”. Providing “2n biological samples” means that per living being or individual out of n samples or individuals two samples or a “double sample” are provided, e.g. two throat swaps for each being are provided. One of the samples or swamps is placed into the archive sample or tube, respectively. The other sample or swamp is placed into the pool sample or tube. After the dispensing time the pool sample is analyzed.
The features, elements, and advantages disclosed for the method at the outset or the “dilution method” apply to the “dual swap method” likewise and vice versa.
In an embodiment of the “dual swap method” in step (1) each of said 2n biological samples (double samples) is provided via 2 swabs.
This measure has the advantage that such kind of sample provision is employed which ensures, due to the high concentration of the virus in the throat area, that best possible biological source material in subjected to the method according to the invention.
Another subject-matter according to the invention relates to a method for the detection of SARS-CoV-2 in a plurality of biological samples of living beings, preferably such a method as disclosed in the preceding paragraphs, said method comprises the following steps:
Such embodiment according to the invention is also referred to as “single swap method”. The first or original sample or swap is placed first into an archive sample of tube, respectively. After the dispensing time which displaces a part of the virus or virus material from the sample or swap, however still leaves virus or virus material in the sample or swap sufficient for a later detection, the sample or swamp is placed into the pool sample or tube. This procedure is repeated for further samples or swamps out of n samples or swamps, respectively.
The features, elements, and advantages disclosed for the method at the outset, the “dilution method” or the “dual swap method” apply to the “single swap method” likewise and vice versa.
In an embodiment of the “single swap method” in step (1) each of said n biological samples is provided via a swab.
This measure has the advantage that such kind of sample provision is employed which ensures, due to the high concentration of the virus in the throat area, that best possible biological source material in subjected to the method according to the invention.
In an embodiment of the method according to the invention, in particular of any of any of the “dilution method” or the “dual swap method” or the “single swap method” said incubation time span is in the range of approx. 1 second to approx. 2 hours.
Such measure has the advantage that such an incubation time is chosen which ensured the sufficient displacement of virus or virus material from the sample and its transfer into the buffer.
In an embodiment of the method according to the invention, in particular of any of any of the “dilution method” or the “dual swap method” or the “single swap method” said buffer is conventional PCR medium.
By such measure a medium is used which allows a direct processing of the sample by conventional detection methods such as PCR.
Another subject-matter of the invention relates to a kit for the detection of SARS-CoV-2 in a plurality of biological samples of living beings, comprising swabs, test tubes, buffer solutions and a manual of the method according to the invention.
The features, characteristics, advantages and embodiments disclosed for the method according to the invention apply likewise to the kit according to the invention.
A “kit” is a combination of individual elements useful for carrying out the method of the invention, wherein the elements are optimized for use together in the methods. The kit may also contain additional reagents, chemicals, buffers, reaction vials etc. which may be useful for carrying out the method according to the invention. Such a kit unifies all essential elements required to work the method according to the invention, thus minimizing the risk of errors. Therefore, such kits also allow semi-skilled laboratory staff to perform the method according to the invention.
It is to be understood that the before-mentioned features and those to be mentioned in the following cannot only be used in the combination indicated in the respec-tive case, but also in other combinations or in an isolated manner without departing from the scope of the invention.
The invention is now described and explained in further detail by referring to the following non-limiting examples and drawings.
The following three different independent embodiments of the methods according to the invention are illustrated which perform NAT detection in a mini-pool format in order to be able to examine more people without a significant reducing of the diagnostic NAT sensitivity.
The aim of the current validation was the comparison of an individual SARS-CoV-2 NAT detection with different mini-pool NAT formats.
In the first embodiment of the pooling method (dilution method), the swabs are taken up in first step in a buffer and then different volumes are pipetted together depending on the pool size. The primary tubes in which the viruses were removed from the swab remain intact for a pool resolution. The validation results show comparable cycle threshold (CT) values up to a mini pool size of 3 (primary tube CT value of 24.33, mini-pool of 3 CT of 26.65).
The second embodiment (dual swap method), 2 swabs were removed from each person. One swab is placed in the archive tube and the other swab is placed in the mini-pool tube. Since both swabs were removed at the same time, the virus concentration is comparable (CT value archive tube 24.87; CT value mini-pool tube 24.72). Comparable CT values between the swabs are shown in the validation. The method is also suitable for min-pool sizes between 2 and 100 samples.
The third embodiment (single swap method) is a method that works with a single swab. The swab is first placed in an archive tube which can be used for mini-pool deconstruction. After a suitable incubation period, the swab is transferred to the pool tube.
In the validation, comparable CT values are shown between the archive tube (CT value 24.47) and the mini-pool tube (CT value 24.93). This method is suitable for mini-pool sizes between 2 and 100 samples.
The three embodiments of pooling methods described are suitable for screening SARS CoV-2 in a mini-pool format to save limited resources of NAT reagents without losing significantly diagnostic NAT sensitivity. The Mini-Pool NAT procedure is particularly suitable for SARS-CoV-2 screening asymptomatic people who have little suspicion of a positive result.
All conventional swaps are possible (e.g. Sarstedt forensic swap xl)
All conventional reagents for virus displacement from the swaps are possible (e.g. Roche PCR media)
All NAT tests were performed on the Roche Cobas 6800/8800 instru-ments with the Roche SARS-CoV-2 assay.
Due to the fact that SARS-CoV-2 infected primarily the nasopharynx area, samples were taken with dry swaps.
In the next step the swaps were placed in a displacement solution (Guanidinium thiocyanate; GTC or PBS, or plasma, or other solutions). Then the RNA of SARS-CoV-2 is extracted manually of by robotic systems followed by a NAT method.
The original swap was placed into the displacement buffer for approxi-mately 5 minutes to dispense SARS-CoV-2 from the swap into the buffer (all buffers are possible e. g. Roche PCR media). The dispensing time can be in a range between 1 second and 2 hours depending on the buffer used. In the next step a small volume of primary tube were transferred into the mini-pool tube. For two swaps mini-pools a dilution 1:2 is used. For other mini-pool formats other dilutions were taken (mini-pools of 3 a dilution 1:3; mini-pools of 4 a dilution 1:4; mini-pools of 5 a dilution 1:5).
The CT-value of individual swaps (24.33) were not significantly different to mini-pools of 2 swaps (25.82) or mini-pools of 3 swaps (26.65) and slightly increased in mini-pools of 5 (27.05) or mini-pools of 10 (27.40).
Two swaps were taken from each patient. One of the swaps was placed into the archive sample tube. The other swap was placed into a mini-pool tube. After the dispensing time the mini-pool tube was analyzed by the NAT system first. In case of a negative test result of the mini-pool test all sample included in the mini-pool are negative.
In case of a positive mini-pool result, all archived samples of the mini-pool have to be investigated individually.
The CT-value of archive swaps (24.87) were not significantly different to mini-pools swaps (24.72). The dual swap mini-pool method is possible for mini-pools between 2 swaps and 100 swaps (preferably mini-pools of 5).
For the single swap method the original swap is placed first into an archive tube. After the dispensing time (range between 1 second and two hours, preferably 5 minutes) the swap was placed into the mini-pool tube starting a second dispensing time (range between 1 second and two hours, preferably 5 minutes). This procedure will be repeat for further swaps. In case of a negative test result of the mini-pool test all sample included in the mini-pool are negative. In case of a positive mini-pool result, all archived samples of the mini-pool have to be investigated individually.
The CT-value of archive swaps (24.47) were not significantly different to mini-pools swaps (24.93). The single swap mini-pool method is possible for mini-pools between 2 swaps and 100 swaps (preferably mini-pools of 5).
In summary, pooling of swap samples for SARS-CoV-2 detection is possible as embodied by three different variants of methods according to the invention without losing diagnostic sensitivity of the NAT detection method. The mini pool NAT procedure is required in a pandemic in order to optimally use of existing limited resources so that as many people as possible can benefit from the early detection of a SARS infection.
| Number | Date | Country | |
|---|---|---|---|
| 62994023 | Mar 2020 | US |
