Description:Technical field
The present disclosure relates to a kit for detecting a virus particle in a given sample. More particularly, the present disclosure provides a kit for rapidly detecting COVID-19 or corona virus sample in a subject who is in need thereof.
Background
During the fag end of 2019, Wuhan – the most densely populated city and capital of Hubei Province, China was witnessing pneumonia whose etiology was unknown. In the beginning of 2020, the pathogen was successfully identified and was named as 2019 novel Coronavirus (2019-nCoV). Later, on 20th February 2020, the International Committee on Taxonomy of Viruses (ICTV) named it as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a new brother of SARS-CoV, and the disease caused was Coronavirus Disease 2019 (COVID-19), 2019 being the year. Since, then the whole world is on ‘High Alert’ due to new viral threat, COVID -19. It has almost impacted anyone and everyone either directly or indirectly. Millions of people have been quarantined so as to prevent it from spreading. Nonetheless, the number of people becoming corona victims is still on the rise.
Early detection of the virus is the key for combating this dreadful virus. Therefore, availability and accuracy in rapidly testing this virus is of immense importance in unraveling the arduous complexities involved in COVID-19 infection. Towards this aspect, scientists across the globe are racing to discover, develop and deliver the critically needed test kits. One of the many challenges for containing the spread of COVID-19 is the ability to identify asymptomatic cases that result in spreading of the virus to close contacts. Since this dangerous virus exhibits a range of clinical manifestations, from mild flu-like symptoms to life-threatening conditions, it is important to have efficient testing during the early stages of infection to identify COVID-19 subjects from those with other illnesses. Early diagnosis permits physicians to provide prompt intervention for subjects who are at higher risk for developing more serious complications from COVID-19 illness.
Generally speaking, commercially available COVID-19 detection tests broadly fall into two major categories. The first being the molecular assays for detection of SARS-CoV-2 viral RNA using polymerase chain reaction (PCR)-based techniques or nucleic acid hybridization-related strategies. The second category includes serological and immunological assays that largely rely on detecting antibodies produced by individuals as a result of exposure to the virus or on detection of antigenic proteins in infected individuals. Nonetheless, the existing devices for in-vitro qualitative determination/ detection suffer with various limitations ranging from sample quantity to time taken for performing the tests. Usually the existing kits take time something between 15 to 20 minutes. Abbott has recently introduced a kit that takes five minutes to test or detect the corona virus. Nonetheless, there exists a strong need to have a simple, rapid and yet economical test to rapidly detect corona virus in less than five minutes. Additionally, the information disclosed in this background section is only for enhancement of understanding of the general background of the disclosure and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Objectives
First and foremost objective of the present disclosure is to provide a kit for rapidly detecting COVID-19 or corona virus in a subject who is in need thereof.
Second objective of the present disclosure is to provide a method for rapidly detecting COVID-19 or corona virus in a subject who is in need thereof.
Summary
One or more shortcomings of conventional kit for detecting COVID-19 or corona virus are overcome and additional advantages are provided through the rapid detection kit for detecting COVID-19 as claimed in the present disclosure. Additional features and advantages are realized through the techniques of the present disclosure. Other embodiments and aspects of the disclosure are described in detail herein and are considered a part of the claimed disclosure.
The present disclosure provides a kit comprising of gold conjugate comprising of colloidal gold and a secondary monoclonal antibody attached to it; bottle with extraction buffer solution comprising of sodium dodecyl sulphate at a concentration of 0.01% w/w, sodium azide at a concentration of 0.1% w/w, bovine serum albumin at a concentration of 0.2 ± 0.1% w/w in 10mM phosphate buffer having pH of 7.0 ± 0.2; bottle with wash buffer comprising of sodium dodecyl sulphate at a concentration of 0.1% w/w, tween 20 at a concentration of 0.05% w/w, sodium chloride at a concentration of 0.8% w/w, sodium azide at a concentration of 0.1% w/w, non-ionic surfactant with hydrophilic polyethylene oxide chain and an aromatic hydrocarbon group 4-(1,1,3,3-tetramethylbutyl)-phenyl group at a concentration of 2% w/w, polyvinylpyrrolidone at a concentration of 0.5% w/w, hydrophobic-Immunoglobulin interaction eliminating reagent at a concentration of 0.2 mg/ml in 10mM of EDTA buffer having pH of 8.0; antibody coated device comprising of a top plastic cassette configured to hold nitrocellulose membrane in a specific grid on the top plastic cassette, covering the nitrocellulose membrane with a first rigid plastic sheet, second absorption sheet and a third absorption pad and close the back plastic cassette by using an adhesive agent; at least one sterile nasal swab stick; a desiccant, a disposable bag and an instruction manual having instructions to use the kit; and is also disclosed is an in-vitro method for identifying a virus particle in a sample using a kit as explained above, comprising steps of transferring the swab sample to filter cups with extraction buffer to obtain extracted sample with lysed antigen sample components; incubating the extracted sample by adding it onto an antibody coated device comprising of a top plastic cassette configured to hold nitrocellulose membrane in a specific grid on the top plastic cassette, covering the nitrocellulose membrane with a first rigid plastic sheet, second absorption sheet and a third absorption pad and close the back plastic cassette by using an adhesive agent; and waiting for a time period of one minute and interpreting the result based on appearance or non-appearance of red/pink spot to confirm the presence or absence of virus particle in a sample.
The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.
Brief description of the accompanying drawings:
The features of the present disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments in accordance with the disclosure and are therefore not to be considered limiting of its scope the disclosure will be described with additional specificity and detail through use of the accompanying drawings.
Figure 1: illustrates the antibody coated device with layers of the cassette setup, in accordance with an exemplary embodiment of the present disclosure.
Figure 2: illustrates the kit for rapidly detecting COVID-19 or corona virus in a subject in need thereof, in accordance with an exemplary embodiment of the present disclosure.
Figure 3: illustrates interpretation of the test result (a) positive (b) negative (c) invalid, in accordance with an exemplary embodiment of the present disclosure.
The figures depict embodiments of the disclosure for purposes of illustration only. One skilled in the art will readily recognize from the following description that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the disclosure described herein.
Detailed Description
Before explaining any one embodiment of the present disclosure by way of drawings, experimentation, results, and pertinent procedures, it is to be understood that the disclosure is not limited in its application to the details as explained in below embodiments set forth in the following description or illustrated in the drawings, experimentation and/or results. The disclosure is further capable of other embodiments which can be practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary--not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
Definitions:
The features and advantages of the invention may be more readily understood by those of ordinary skill in the art upon reading the following detailed description. It is to be appreciated that certain features of the invention that are, for clarity reasons, described above and below in the context of separate embodiments, may also be combined to form a single embodiment. Conversely, various features of the invention that are, for brevity reasons, described in the context of a single embodiment, may also be combined so as to form sub-combinations thereof. Embodiments identified herein as exemplary or preferred are intended to be illustrative and not limiting.
Unless specifically stated otherwise herein, references made in the singular may also include the plural. For example, "a" and "an" may refer to either one, or one or more.
The present disclosure is in relation to a rapidly detecting kit for detecting COVID-19 or corona virus in a subject who is in need thereof. First and foremost, the kit comprises of a gold conjugate comprising of colloidal gold and a secondary monoclonal antibody attached to it. Secondly, it comprises of a bottle with extraction buffer solution comprising of sodium dodecyl sulphate at a concentration of 0.01% w/w, sodium azide at a concentration of 0.1% w/w, bovine serum albumin at a concentration of 0.2 ± 0.1% w/w in 10mM phosphate buffer having pH of 7.0 ± 0.2. Finally, the extraction buffer in the bottle or plastic tubes with integrated dropper cap is having a pH is 7.4 ± 0.2. Thirdly, it comprises of a bottle with wash buffer comprising of sodium dodecyl sulphate at a concentration of 0.1% w/w, tween 20 at a concentration of 0.05% w/w, sodium chloride at a concentration of 0.8% w/w, sodium azide at a concentration of 0.1% w/w, non-ionic surfactant with hydrophilic polyethylene oxide chain and an aromatic hydrocarbon group 4-(1,1,3,3-tetramethylbutyl)-phenyl group at a concentration of 2% w/w, polyvinylpyrrolidone at a concentration of 0.5% w/w, hydrophobic-Immunoglobulin interaction eliminating reagent at a concentration of 0.2 mg/ml in 10mM of EDTA buffer having pH of 8.0. More particularly, the kit comprises four drops of wash buffer in the bottle or plastic tubes with integrated dropper cap having pH of 8.0±0.2. Further, the device comprises an antibody coated device comprising of a top plastic cassette configured to hold nitrocellulose membrane in a specific grid on the top plastic cassette, covering the nitrocellulose membrane with a first rigid plastic sheet, second absorption sheet and a third absorption pad and close the back plastic cassette by using an adhesive agent. The kit also contains at least one nasal swab stick. Lastly, the kit comprises of an instruction manual having instructions to use the kit.
In another embodiment of the present disclosure it provides a colloidal gold is either a solution or as a dispersion in water and is bright red spherical particles with particle size of less than 100 nm, preferably of 40 nm or blue/ purple spherical nanorods with particle size of more than 100 nm.
In another embodiment of the present disclosure it provides a kit for rapidly detecting COVID-19 antigen in one minute and the said kit is stored at a temperature ranging from 2℃to 8℃.
The present disclosure provides an in-vitro method for identifying a virus particle in a sample using a kit as explained in the above embodiments comprising following broad steps. First and foremost the aluminium pouch having the kit of the present disclosure is opened to take out the sterile nasal swab stick followed by collecting the nasopharyngeal swab specimen from a subject and inserting it into the extraction buffer stored in the bottle. The nasal swab is rotated 3 to 4 times and squeezed to remove the extraction buffer absorbed to the swab. Discard the nasal swab and transfer the sample onto the antibody coated device. It is pertinent to state that the specimen collected must be tested immediately after collection. Alternately, the specimen can be stored at room temperature of 2 to 8℃for four hours or at room temperature for one hour. After the specimen sample (five drops) is added, the device is incubated at room temperature for 50 to 60 seconds and allows the sample to get absorbed onto the membrane. Thereafter, four drops of gold conjugate is added and allowed it to get absorbed. Following this, wash buffer is added and allowed the buffer to soak in the membrane completely. Read the result immediately and discard the device and sample extraction buffer.
In another embodiment of the present disclosure, it provides a transferring swab the sample to filter cups with extraction buffer to obtain extracted sample with lysed antigen sample components, incubating the extracted sample by adding it onto an antibody coated device comprising of a top plastic cassette configured to hold nitrocellulose membrane in a specific grid on the top plastic cassette, covering the nitrocellulose membrane with a first rigid plastic sheet, second absorption sheet and a third absorption pad and close the back plastic cassette by using an adhesive agent; and waiting for a time period of one minute and interpreting the result based on appearance or non-appearance of red/pink spot to confirm the presence or absence of virus particle in a sample.
In yet another embodiment of the present disclosure, it provides a kit for testing virus particle including but not limited to corona virus, and influenza virus type – A and B.
In still another embodiment of the present disclosure, the result interpretation using spot includes control spot and test spot, appearance of both the spots confirm positive and appearance of only control and absence of test spot confirms negative test or absence of virus particle in the sample. In some instances, the control spot fails to appear on the membrane of the device and is probably due to kit contamination, particularly contamination of gold conjugate, wash or extraction buffer due to improper handling.
Additionally, the disclosure is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope of the present invention. On the contrary, it is to be clearly understood that various other embodiments, modifications, and equivalents thereof, after reading the description herein in conjunction with the drawings and appended claims, may suggest themselves to those skilled in the art without departing from the spirit and scope of the presently disclosed and claimed invention.
Example 1: Preparation of extraction buffer
Extraction buffer is employed in the kit to dissolve the sample collected from nasopharyngeal area using a sterile nasal swab. It is used to extract the components from the sample cells. More particularly, the buffer helps in the lysis of cell sample antigens and extracts them for the testing. The extraction buffer is prepared using sodium dodecyl sulphate (SDS) at a concentration of 0.01% w/w, sodium azide at a concentration of 0.1 % w/w and bovine serum albumin at a concentration of 0.02% in 10mM phosphate buffer having pH of 7.0 ± 0.2 which constitutes the remaining percentage of the portion of the extraction buffer. Each ingredient of the extraction buffer has a particular role in effectively detecting the virus particle in question. For instance, SDS acts as an anionic deterging agent wherein it helps the sample to get negatively charged. Secondly, sodium azide acts as a bacteriostatic. Thirdly, bovine serum albumin helps to prevent nonspecific binding where it binds to the remaining SDS and not let the remaining SDS bind with antigens during lysis of the cell sample. The extraction buffer is stored in a dropper bottle or in a plastic tube with integrated dropper cap.
Example 2: Preparation of wash buffer
Wash buffer is employed in the kit to wash out the remaining unbounded antigens or reagents after binding of Ab-Ag-Ab complex to give a coloured spot. Generally speaking, it is pertinent to state that the step of washing using washing buffer is required in an immunoassay technique to eliminate unbound and excessive components that could interfere with the assay. Particularly, the wash buffer clears the background of the device (100) with membrane so that the spot is visible to the naked eye with utmost clarity. The wash buffer contains 0.1 % w/w of SDS, 0.05% w/w of Tween 20, 0.8% w/w of sodium chloride, 0.1% w/w of sodium azide, 2% w/w of TritonX100 (neutral detergent), 0.5% w/w of polyvinylpyrrolidone 30, 0.2 mg/ml of hydrophobic-immunoglobulin interaction eliminating reagent in 10mM of EDTA buffer having pH of 8.0. The wash buffer is stored in a dropper bottle or in a plastic tube with integrated dropper cap.
Example 3: Preparation of gold conjugate
The process involved in preparation of gold conjugate is twofold in nature. First and foremost, it begins by preparing the gold colloid and secondly the conjugation of gold colloid with monoclonal antibody.
Example 3a: Preparation of gold colloid
In general, colloidal gold is a sol or a dispersion of gold nanoparticles in a fluid, usually water. The gold colloid is usually in bright red (for spherical particles less than 100 nm) or blue/purple (for particles greater than 100 nm) in color (for larger spherical particles or nanorods). The process involves pouring predetermined amount of distilled water in an amber colored flask followed by addition of predetermined quantity (usually in µl quantity) of gold chloride in it. The temperature was monitored and was maintained at 95℃ using thermometer. The mixture was subject to continuous stirring using magnetic stirrer while simultaneously heating it. Tri-sodium citrate was added as a reducing agent and boiled for about 20 minutes. The color change from transparent to ruby red was observed and the colloid was left overnight in the freezer.
Example 3b: Preparation of gold conjugate
Colloidal gold obtained under example 3a is subjected to conjugation using a secondary monoclonal antibody, which is attached/ conjugated to colloidal gold particles having a size of about 40 nm. Following the principle of sandwich Enzyme-linked Immunosorbent Assay, the coating antibody gets bind to the antigen of sample and after that the conjugate antibody binds to the antigen forming a sandwich structure. Due to the colour of gold particle ruby red, when the attachment happens, it shows colour in the particular region of binding in the membrane. It is prepared using gold colloid in amber colored flask where antibody is added in small quantity (µl) and stirred for 60 minutes. Again, bovine serum albumin was added and stirred for 30 minutes. The sample is then collected and centrifuged. Post centrifugation, the pellet obtained is diluted with suspension buffer and the optical density is maintained.
Example 4: Antibody coated device
The COVID 19 or corona virus antigen rapid test kit works on the principle of vertical flow immune-chromatographic test, which relies on capturing gold nanoparticle-labeled antibodies to produce a visible spot on nitrocellulose membrane that is encased in a plastic cassette, hereinafter referred as device (100). The device (100) is comprising of a top plastic cassette (110) configured to hold nitrocellulose membrane (120) in a specific grid (120a) on the top plastic cassette (110), covering the nitrocellulose membrane (120) with a first rigid plastic sheet (130), second absorption sheet (140) and a third absorption pad (150) and close the back plastic cassette (160) by using an adhesive agent – see Figure 1. About 5 drops of the sample prepared using extraction buffer of example 1 is used for testing the presence or absence of virus particle in general and COVID-19 or corona virus in particular.
Example 5: Kit for rapidly detecting corona virus in a given sample
The present disclosure provides a kit; see Figure 2, for rapidly detecting corona virus in a sample. It is a simple, rapid and an in-vitro qualitative screening test to diagnose antigen of novel corona virus (SARS- CoV-2) present in a sample. The test detects the presence of antigen of SARS-CoV-2, the virus which causes the disease COVID-19.
The kit comprises of a gold conjugate comprising of colloidal gold and a secondary monoclonal antibody attached to it as explained under example 3. In addition, it comprises of a bottle with extraction buffer, prepared as per example 1, solution comprising of sodium dodecyl sulphate at a concentration of 0.01% w/w, sodium azide at a concentration of 0.1% w/w, bovine serum albumin at a concentration of 0.2 ± 0.1% w/w in 10mM phosphate buffer having pH of 7.0 ± 0.2. Further, it comprises a bottle with wash buffer, prepared as per example 2, comprising of sodium dodecyl sulphate at a concentration of 0.1% w/w, tween 20 at a concentration of 0.05% w/w, sodium chloride at a concentration of 0.8% w/w, sodium azide at a concentration of 0.1% w/w, non-ionic surfactant with hydrophilic polyethylene oxide chain and an aromatic hydrocarbon group 4-(1,1,3,3-tetramethylbutyl)-phenyl group at a concentration of 2% w/w, polyvinylpyrrolidone at a concentration of 0.5% w/w, hydrophobic-immunoglobulin interaction eliminating reagent at a concentration of 0.2 mg/ml in 10mM of EDTA buffer having pH of 8.0. About, four drops of wash buffer is used in the kit. Furthermore, it comprises an antibody coated, prepared as per example 4, device (100) comprising of a top plastic cassette (110) configured to hold nitrocellulose membrane (120) in a specific grid (120a) on the top plastic cassette (110), covering the nitrocellulose membrane (120) with a first rigid plastic sheet (130), second absorption sheet (140) and a third absorption pad (150) and close the back plastic cassette (160) by using an adhesive agent. Besides, it also contains at least one sterile nasal swab stick and an instruction manual having instructions to use the kit.
Example 6: Test procedure using the kit of the present disclosure
The basic/ fundamental steps involved in testing are as follows:
a) Wash/sanitize your hands. Make sure they are dry before performing the test.
b) Check the expiry date of the box. Do not use if the kit is expired.
c) To perform a test, open the box and remove one cassette, one extraction buffer tube, one extraction buffer bottle, one sterile nasopharyngeal swab, gold conjugate bottle, wash buffer bottle and extraction buffer tube paper stand.
d) Allow the box and its components to attain room temperature prior to use.
e) Keep the extraction buffer tube in the extraction buffer tube paper stand provided in the kit.
f) Keep the wash buffer bottle, extraction buffer bottle and gold conjugate bottle upright on table.
The actual test procedure involved in using the kit for rapidly detecting corona virus is explained as follows:
A sterile nasal swab stick is taken from the kit to systematically collect nasopharyngeal sample from a subject and inserting it into the extraction buffer stored in the bottle. Alternately, an artificial sample can be employed in the laboratory having the virus particles in it. The nasal swab is inserted and rotated three to four times and squeezed to remove the extraction buffer absorbed to the swab. Discard the nasal swab and transfer the sample onto the antibody coated device (100). It is pertinent to state that the specimen collected must be tested immediately after collection. Alternately, the specimen can be stored at room temperature of 2℃to 8℃for four hours or at room temperature for one hour. After the specimen sample (five drops) is added, the device (100) is incubated at room temperature for 50 to 60 seconds and allows the sample to get absorbed onto the membrane. Thereafter, four drops of gold conjugate is added and allowed it to get absorbed. Following this, wash buffer is added and allowed the buffer to soak in the membrane completely. Read the result immediately and discard the device (100) and sample extraction buffer. The test results are read as per Figure 3. Figure 3(a) - more than one distinct red/pink spot appears - positive. One spot should be in the control (C) and the other spot should be in the test region (T) to confirm the presence of virus particle in a given sample. Similarly, from Figure 3(b) one red spot in the control (C) region – negative. No apparent red/pink spot in the test (T) region. Similarly, Figure 3(c) shows invalid result when the control spot fails to appear on the device and this is probably due to contamination of the device (100).
While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.
Reference numerals:
Description Reference number
Device 100
Top plastic cassette 110
Nitrocellulose membrane 120
Grid 120a
First rigid plastic sheet 130
Second absorption sheet 140
Third absorption pad 150
Back plastic cassette 160

 
, Claims:We claim:
1) A kit comprising of:
a) gold conjugate comprising of colloidal gold and a secondary monoclonal antibody attached to it;
b) bottle with extraction buffer solution comprising of sodium dodecyl sulphate at a concentration of 0.01% w/w, sodium azide at a concentration of 0.1% w/w, bovine serum albumin at a concentration of 0.2 ± 0.1% w/w in 10mM phosphate buffer having pH of 7.0 ± 0.2;
c) bottle with wash buffer comprising of sodium dodecyl sulphate at a concentration of 0.1% w/w, tween 20 at a concentration of 0.05% w/w, sodium chloride at a concentration of 0.8% w/w, sodium azide at a concentration of 0.1% w/w, non-ionic surfactant with hydrophilic polyethylene oxide chain and an aromatic hydrocarbon group 4-(1,1,3,3-tetramethylbutyl)-phenyl group at a concentration of 2% w/w, polyvinylpyrrolidone at a concentration of 0.5% w/w, hydrophobic-immunoglobulin interaction eliminating reagent at a concentration of 0.2 mg/ml in 10mM of EDTA buffer having pH of 8.0;
d) antibody coated device (100) comprising of a top plastic cassette (110) configured to hold nitrocellulose membrane (120) in a specific grid (120a) on the top plastic cassette (110), covering the nitrocellulose membrane (120) with a first rigid plastic sheet (130), second absorption sheet (140) and a third absorption pad (150) and close the back plastic cassette (160) by using an adhesive agent;
e) at least one sterile nasal swab stick;
f) at least one plastic disposable bag with self-locking mechanism;
g) a desiccant to absorb any excess moisture in the kit; and
h) an instruction manual having instructions to use the kit.
2) The kit as claimed in claim 1, wherein said colloidal gold is either a solution or dispersion in water and is bright red spherical particles with particle size of less than 100 nm, preferably of 40 nm or blue/ purple spherical nanorods with particle size of more than 100 nm.
3) The kit as claimed in claim 1, wherein said extraction buffer pH is 7.4 ± 0.2 and wash buffer pH is 8.0±0.2.
4) The kit as claimed in claim 1, wherein said wash buffer quantity is four drops.
5) The kit as claimed in claim 1, wherein said kit is used for rapidly detecting COVID-19 antigen in one minute and is stored at a temperature ranging from 2℃ to 8℃.
6) An in-vitro method for identifying a virus particle in a sample using a kit as claimed in claims 1 to 5, comprising steps of:
a) transferring the swab sample to filter cups with extraction buffer to obtain extracted sample with lysed antigen sample components;
b) incubating the extracted sample by adding it onto an antibody coated device (100) comprising of a top plastic cassette (110) configured to hold nitrocellulose membrane (120) in a specific grid (120a) on the top plastic cassette (110), covering the nitrocellulose membrane (120) with a first rigid plastic sheet (130), second absorption sheet (140) and a third absorption pad (150) and close the back plastic cassette (160) by using an adhesive agent; and
c) waiting for a time period of one minute and interpreting the result based on appearance or non-appearance of red/pink spot to confirm the presence or absence of virus particle in a sample.
7) The in-vitro method as claimed in claim 6, wherein five drops of the extracted sample is applied onto the device (100).
8) The in-vitro method as claimed in claim 6, wherein said virus particle includes but not limiting to corona virus, and influenza virus.
9) The in-vitro method as claimed in claim 6, wherein said spot includes control spot and test spot, appearance of both the spots confirm positive and appearance of only control and absence of test spot confirms negative test or absence of virus particle in the sample.
10) The in-vitro method as claimed in claim 9, wherein said control spot fails to appear if the device (100) is not clean or if it is contaminated.