TR202018428A2 - Specialized rapid scan kit for Sars-cov-2 - Google Patents
Specialized rapid scan kit for Sars-cov-2 Download PDFInfo
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- TR202018428A2 TR202018428A2 TR2020/18428A TR202018428A TR202018428A2 TR 202018428 A2 TR202018428 A2 TR 202018428A2 TR 2020/18428 A TR2020/18428 A TR 2020/18428A TR 202018428 A TR202018428 A TR 202018428A TR 202018428 A2 TR202018428 A2 TR 202018428A2
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- 238000001514 detection method Methods 0.000 claims abstract description 18
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims abstract description 16
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 16
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 claims abstract description 15
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 claims abstract description 15
- 238000007397 LAMP assay Methods 0.000 claims abstract description 12
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 10
- 230000003321 amplification Effects 0.000 claims abstract description 9
- 239000007853 buffer solution Substances 0.000 claims abstract description 9
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 9
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- 102000039446 nucleic acids Human genes 0.000 claims abstract description 9
- 238000012360 testing method Methods 0.000 claims description 31
- 108020000999 Viral RNA Proteins 0.000 claims description 12
- 239000007793 ph indicator Substances 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 claims description 2
- XJRPTMORGOIMMI-UHFFFAOYSA-N ethyl 2-amino-4-(trifluoromethyl)-1,3-thiazole-5-carboxylate Chemical compound CCOC(=O)C=1SC(N)=NC=1C(F)(F)F XJRPTMORGOIMMI-UHFFFAOYSA-N 0.000 claims description 2
- 229960003531 phenolsulfonphthalein Drugs 0.000 claims description 2
- 102100036263 Glutamyl-tRNA(Gln) amidotransferase subunit C, mitochondrial Human genes 0.000 claims 1
- 101001001786 Homo sapiens Glutamyl-tRNA(Gln) amidotransferase subunit C, mitochondrial Proteins 0.000 claims 1
- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 13
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- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 208000025721 COVID-19 Diseases 0.000 description 3
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- 108091007433 antigens Proteins 0.000 description 3
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 241000710886 West Nile virus Species 0.000 description 2
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
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- 235000011164 potassium chloride Nutrition 0.000 description 2
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 241000702670 Rotavirus Species 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 241000893930 Swine hepatitis E virus Species 0.000 description 1
- 241000725681 Swine influenza virus Species 0.000 description 1
- 241001223089 Tremovirus A Species 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
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- -1 dNTP Chemical compound 0.000 description 1
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- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
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- 238000012125 lateral flow test Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
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- 210000003296 saliva Anatomy 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
RT-LAMP (Ters Transkriptaz-Döngü Aracılı İzotermal Amplifikasyon Reaksiyonu) yöntemi kullanılarak SARS COV 2 özelinde tespit sağlayan ama primer değişikliği ile diğer tüm viral hastalıklar ya da nükleik asit tanısı için kullanılabilecek bir tespit kiti geliştirilmiştir. Bu buluş için özgün bir amplifikasyon tampon çözeltisi,Bst DNA/RNA polimeraz varyantları (1, 2, 3 vb.) kullanılarak tek reaksiyon ortamında sabit sıcaklıkta amplikasyon metodu, primer setleri dizaynı ve izolasyon çözeltisi optimizasyonu yapılmıştır. Buluş yukarıda listelenen özgün modifikasyonlar ile RT-LAMP yönteminin kolorimetrik olarak 15-30 dakika arasında nükleik asitleri tespit edebileceğini beyan eder.Using the RT-LAMP (Reverse Transcriptase-Loop Mediated Isothermal Amplification Reaction) method, a detection kit has been developed that provides detection specific to SARS COV 2, but can be used for the diagnosis of all other viral diseases or nucleic acid with primary change. For this invention, using a unique amplification buffer solution, Bst DNA / RNA polymerase variants (1, 2, 3 etc.), constant temperature amplification method, primer set design and isolation solution optimization were performed in a single reaction medium. The invention declares that with the specific modifications listed above, the RT-LAMP method can colorimetrically detect nucleic acids in between 15-30 minutes.
Description
TARIFNAME SARS-COV-Z içiN ÖZELLESMIS HIZLI TARAMA KITI Teknik Alan Bu bulus, RT-LAMP sistemini kullanarak kolorimetrik (renkölçümsel) sonuç veren Covid-19 teshis kiti içerigini ve uygulama yöntemini kapsamaktadir. Önceki Teknik COVlD-19 salgini, dünyadaki bütün ülkeleri hazirliksiz yakaladigi için toplumun her alaninda büyük bir etki yaratmistir. Farkli disiplinlerden arastirmacilar bu salginla bas etmek için tespit ve teshis, ilaç gelistirme, iyilestirme, tedavi yöntemi, asi ve koruyucu donanim üretiminde bu salginla mücadele etmek Için çalismaktadirlar. DESCRIPTION SPECIAL FAST SCAN KIT FOR SARS-COV-Z Technical Area This invention is based on Covid-19, which provides colorimetric (colorimetric) results using the RT-LAMP system. Includes diagnostic kit content and application method. Prior Art As the COVID-19 epidemic caught all countries in the world unprepared, it is affecting all areas of society. has made a huge impact. Researchers from different disciplines have determined to cope with this epidemic. and diagnosis, drug development, improvement, treatment method, vaccine and protective equipment production. They are working to combat the epidemic.
Bu süreçte ise dogru ve hizli test prosedürlerinin gelistirilmesi ve yayginlastirilmasi son derece degerlidir. C0vid-19 teshisi için iki tür test uygulanmaktadir. Bunlar protein bazli lateral flow testleri (antijen ya da antikor testleri) ve PZR (Polimeraz Zincir Reaksiyonu, Polymerase Chain Reaction - PCR) tabanli testler olarak ikiye ayrilirlar. In this process, the development and dissemination of accurate and fast test procedures is extremely important. it is valuable. Two types of tests are used to diagnose C0vid-19. These are protein-based lateral flow tests (antigen or antibody tests) and PCR (Polymerase Chain Reaction, Polymerase Chain) They are divided into two as Reaction - PCR) based tests.
PZR tabanli testler yaklasik 2 saatte sonuç veren RT-PCR (Ters Transkriptaz-Polimeraz Zincir Reaksiyonu) ve 30 dakikada sonuç veren RT-LAMP (Ters Transkriptaz-Döngü Aracili lzotermal Amplifikasyon, Reverse Transcryptase-Loop-Mediated Isothermal Amplification) yöntemleridir. Antijen/antikor testleri hizli sonuç vermesine ragmen yalanci negatif üretme oranlari yüksek oldugundan PZR tabanli testlere göre dezavantajlidir. Antikor testleri antijen tabanli testlere oranla daha az tercih edilmektedir: çünkü hastanin virüsü bulastirma durumu ile kesin bir sonuç vermemekte, sadece Covid-19'a karsi antikor üretimi olup olmadigini teshis etmektedir. RT-PCR (Reverse Transcriptase-Polymerase Chain) PCR-based tests give results in approximately 2 hours reaction) and RT-LAMP (Reverse Transcriptase-Loop Mediated Isothermal) with results in 30 minutes Amplification, Reverse Transcryptase-Loop-Mediated Isothermal Amplification) are methods. Antigen/antibody tests do not produce false negatives despite rapid results. Since the rates are high, it is disadvantageous compared to PCR-based tests. Antibody tests antigen It is less preferred than based tests: because the patient's situation of being infected with the virus It does not give a definite result, but only detects whether there is antibody production against Covid-19. is doing.
Türkiye'de su an en çok kullanilan RT-PCR testleridir. RT-PCR testi yüksek güvenilirlige sahiptir. They are the most used RT-PCR tests in Turkey at the moment. The RT-PCR test has high reliability.
Virüse karsi spesifik ve hassas olmasina karsin sonuç alma süresi çok fazla, kullanilan malzemeler pahalidir. Ayrica, RT-PCR yönteminde testin gerçeklestirilmesi ve yorumlanmasi için deneyimli personele ihtiyaç duymakta ve kullanilan malzemelerde siklikla stok ve kapasite sorunu yasanmaktadir. Ayrica testlerin yapilmasi için maliyetli RT-PCR cihazina ihtiyaç duyulmaktadir. Böylece RT-PCR yöntemi güvenilir ve spesifik olmasina ragmen, RT-PCR testine daha uygun bir alternatif olan RT-LAMP testi pahali bir cihaza ihtiyaç duymadan ucuz isitici bloklarla yaklasik 30 dakikada sonuç vermektedir ve maliyeti daha azdir. RT-PCR cihazi ve deneyimli personele ihtiyaç duyulmadan uygulanabilmesinden dolayi sadece hastanelerde ve saglik merkezlerinde degil, tasinabilir olmasi ile birçok yerde egitimi verilen personel ile bu test uygulanabilir. Although it is specific and sensitive to the virus, the result is too long, the used materials are expensive. In addition, performing and interpreting the test in the RT-PCR method Needs experienced personnel for the purpose and frequently stock and capacity in the materials used. problem is occurring. In addition, a costly RT-PCR device is needed to perform the tests. is heard. Thus, although the RT-PCR method is reliable and specific, it is comparable to the RT-PCR test. RT-LAMP test, which is a more suitable alternative, requires an inexpensive heater without the need for an expensive device. It gives results in about 30 minutes with blocks and costs less. RT-PCR device and Since it can be applied without the need for experienced personnel, it is only used in hospitals and This test is carried out not in health centers, but with the personnel trained in many places due to its portability. applicable.
Patent aramalari RT-LAMP ve SARS COV 2 anahtar kelimeleri beraber kullanildiginda herhangi bir patent basvurusu ile eslesmemektedir. Öte yandan RT-LAMP ve diger virüslere iliskin patent basvurularina rastlanmaktadir. Patent searches do not occur when the RT-LAMP and SARS COV 2 keywords are used together. does not match a patent application. On the other hand, related to RT-LAMP and other viruses patent applications are found.
CN101629217A sayili patent basvurusu, domuz gribi virüsü (swine influenza virus) için Özellesmis olup farkli primer setleri, reaksiyon ve izolasyon tampon çözeltileri içermesi, farkli Bst enzimi varyanti kullanmasi, reaksiyon hizlandirici kimyasallar içermemesi açisindan farklidir. Patent application CN101629217A for swine influenza virus It is specialized and contains different primer sets, reaction and isolation buffer solutions, different In terms of using a Bst enzyme variant and not containing reaction accelerating chemicals, is different.
CN103014177A sayili patent basvurusu, rotavirüsün tespiti için gelistirilmis olup primer sekanslari, Bst varyant farkliligi, reaksiyon ve izolasyon tampon çözeltileri açisindan farklidir. Patent application numbered CN103014177A was developed for the detection of rotavirus and the primary sequences, Bst variant diversity, reaction and isolation buffers are different.
CN 102586486B nolu bulus, kus ensefalomiyelit virüsü (avian encephalomyelitis virus) için RT- LAMP yöntemini kullanmayi kapsamaktadir. Primer sekanslari, Bst varyant farkliligi, reaksiyon ve izolasyon tampon çözeltileri degisiktir. Invention CN 102586486B, RT- for avian encephalomyelitis virus It covers using the LAMP method. Primer sequences, Bst variant difference, reaction and isolation buffer solutions are different.
CN103276104A sayili basvuru domuz üreme ve solunum sendromu virüsü 'porcine hepatitis E virüsü, insan immün yetmezligi virüsü (HIV), Bati Nil Virüsü (West Nile Virus), veya özellesmis tespit kitleri olup RT-LAMP yöntemini kullanmaktadir. CN103276104A application swine reproductive and respiratory syndrome virus 'porcine hepatitis E virus, human immunodeficiency virus (HIV), West Nile Virus (West Nile Virus), or They are specialized detection kits and use the RT-LAMP method.
Su ana kadar patent basvurulari arasinda RT-LAMP ile çogaltilmis nükleik asitlerin kolorimetrik (renkölçümsel) olarak görüntülenmesini içeren bir bulus saptanmamistir. Bunun disinda, 2020 yilinda iki adet makale, SARS COV 2 özelinde RT-LAlVIP yönteminin kolaylikla viral RNA tespitinde kullanilabilecegini rapor etmektedir (Kellner vd., 2020; Lalli vd., 2020). Benzer yöntem kullanan bu bulus, kendine özgü Bst enzimi varyantlari, primer sekanslari, amplifikasyon tampon çözeltisi, izolasyon çözeltisi kullanmaktadir. Amplifikasyon tampon çözeltisi kompozisyon olarak Tris-HCI, KCI, MgSO4, dNTP, guanidine HCI, Bst DNA/RNA polimeraz, primer seti, pH indikatörü vb. içermektedir. Among the patent applications so far, colorimetric RT-LAMP-amplified nucleic acids No invention has been found that involves displaying it (colorimetrically). Apart from that, 2020 Two articles in , SARS COV 2, RT-LAlVIP method can be easily viral RNA (Kellner et al., 2020; Lalli et al., 2020). Similar This invention using a method, unique Bst enzyme variants, primer sequences, amplification buffer solution uses isolation solution. Amplification buffer solution as composition Tris-HCl, KCl, MgSO4, dNTP, guanidine HCl, Bst DNA/RNA polymerase, primer set, pH indicator etc. contains.
Referanslar: Kellner, M. J., Ross, J. J., Schnabl, J., Dekens, M. P., Heinen, R., Grishkovskaya, I., & Traugott, M. (2020). A rapid, highly sensitive and open-access SARS-CoV-Z detection assay for laboratory and home testing. References: Kellner, M. J., Ross, J. J., Schnabl, J., Dekens, M. P., Heinen, R., Grishkovskaya, I., & Traugott, M. (2020). A rapid, highly sensitive and open-access SARS-CoV-Z detection assay for laboratory and home testing.
Lalli, M. A., Chen, X., Langmade, S. J., Fronick, C. C., Sawyer, C. S., Burcea, L. C., 8: Mitra, R. Lalli, M. A., Chen, X., Langmade, S. J., Fronick, C. C., Sawyer, C. S., Burcea, L. C., 8: Mitra, R.
D. (2020). Rapid and extraction-free detection of SARS-CoV-Z from saliva with colorimetric LAM P. med Rxiv. D. (2020). Rapid and extraction-free detection of SARS-CoV-Z from saliva with colorimetric LAM P. med Rxiv.
Bulusun Kisa Açiklamasi Bu bulus SARS-COV 2 virüsünün özelinde tüm viral patojenlerin veya nükleik asit sekanslarinin RT-LAMP kullanilarak ayni reaksiyon ortaminda çogaltilmasi ve olusan nükleik asitlerden kaynakli pH degisikliginin kolorimetrik olarak görüntülenmesini kapsamaktadir. Bu bulus Bst varyant farkliligi, reaksiyon ve izolasyon tampon çözeltileri açisindan Iiteratürden farklidir. Brief Description of the Invention This invention shows that all viral pathogens or nucleic acid sequences of SARS-COV 2 virus Proliferation in the same reaction medium using RT-LAMP and the nucleic acids formed It includes colorimetric visualization of the induced pH change. This Invention Bst Variant variation differs from literature in terms of reaction and isolation buffer solutions.
Bunun yani sira kolorimetrik yöntem ile hizli ve kolay tani kolayligi saglamaktadir. In addition, it provides fast and easy diagnosis with the colorimetric method.
Bulusun Ayrintili Açiklamasi Bulus konusu SARS-COV-Z için özellesmis hizli tarama kiti temel olarak RT-LAMP (Ters Transkriptaz-Döngü Aracili Izotermal Amplifikasyon Reaksiyonu) yöntemi PCR cihazi (Thermal Cycler) olmadan sabit sicaklikta nükleik asit çogaltilmasini saglayan bir yönteme iliskindir. Detailed Description of the Invention The rapid screening kit specialized for SARS-COV-Z, the subject of the invention, is basically RT-LAMP (Reverse Transcriptase-Loop Mediated Isothermal Amplification Reaction) method PCR instrument (Thermal It relates to a method that provides nucleic acid amplification at constant temperature without cycles.
Bulus konusu test kiti ters transkriptaz ile RNA'dan DNA sentezi, sonrasinda ise özel dizayn edilmis döngü (Ioop) primer kombinasyonlari ve özel Bst DNA polimerazinin yardimi ile sabit sicaklikta amplifikasyon gerçeklestirmektedir. The inventive test kit is used for DNA synthesis from RNA by reverse transcriptase and then specially designed. fixed loop (Ioop) primer combinations and with the help of special Bst DNA polymerase. It amplifies at temperature.
Reaksiyon yöntemi standart Bst polimerazlari ile çalisabildigi gibi (Bstl ve Bstll) diger Bst varyantlari (Bst 3 ve Bst DNA/RNA polimeraz vb) ile de çalisabilmektedir. Reaksiyon için gelistirilen viral nükleik asit izolasyon tampon çözeltisi burun ve agiz swaplarindan ya da baska sekillerde elde edilmis sürüntü (swap) örneklerinde nükleik asit izole edebilmesinin yani sira amplifikasyon çözeltisine herhangi bir inhibisyon yapmamak üzere dizayn edilmistir. The reaction method can work with standard Bst polymerases (Bstl and Bstll) as well as other Bst It can also work with variants (Bst 3 and Bst DNA/RNA polymerase etc.). for reaction The developed viral nucleic acid isolation buffer solution can be obtained from nose and mouth swabs or other In addition to being able to isolate nucleic acid in swab samples obtained in different shapes, It is designed not to cause any inhibition to the amplification solution.
Bunun disinda guanidine hidroklorür, potasyum klorür, amonyum sülfat vb. kimyasallar reaksiyon hizlandirici olarak görev almaktadir. pH indikatörü olarak fenol kirmizi, nötral kirmizi, bromocresol moru gibi nötral pH indikatörleri kullanilarak sonuç almak mümkündür. Apart from this, guanidine hydrochloride, potassium chloride, ammonium sulfate etc. chemicals It acts as a reaction accelerator. Phenol red as pH indicator, neutral It is possible to get results by using neutral pH indicators such as red, bromocresol purple.
Bu bulus ile pahali spektometrik ya da florometrik PZR tespit cihazlarina ihtiyaç olmadan gözle görülebileceksekilde reaksiyon sonucu veren, heryerde uygulanabilen, pahali cihazlara ihtiyaç duymadan gözlemlenebilen, deneyimli personele ihtiyaç duyulmadan kolay uygulanabilen, hizli taramalara uygun bir yöntem gelistirilmistir. With this invention, it can be observed without the need for expensive spectometric or fluorometric PCR detection devices. There is a need for expensive devices that can be applied everywhere, that give a visible reaction result. can be observed without the need for experienced personnel, can be easily applied, A method suitable for quick scans has been developed.
Bulusun çalismasi için gerekli RT-LAMP primerler hedef nükleik asitin 6-8 bölgesine karsilik gelecek sekilde genelde 4 primer sekansi, optimum 6 primer sekansi içerecek sekilde dizayn edilmistir. Primerler ileri ve geri primerler, iç ileri ve iç geri primerler, dis ileri ve dis geri primerler olarak adlandirilabilir. Dis primerler ayni zamanda döngü primerleri olarak da adlandirilabilir ve kendi üzerine ikincil yapilar olusturma egilimi vardir. Firkete benzeyen bu yapilar sayesinde yüksek sicaklik düsük sicaklik tekrarlarina (thermal cycling) ihtiyaç duymadan devamli olarak DNA çogaltilabilir. The RT-LAMP primers required for the invention to work correspond to regions 6-8 of the target nucleic acid. In the future, it is generally designed to include 4 primer sequences, optimally 6 primer sequences. has been made. Primers forward and backward primers, inside forward and inside reverse primers, tooth forward and tooth backward are called primers. Dis primers are also known as loop primers. It can be named and has a tendency to form secondary structures on itself. This one looks like a hairpin without the need for high temperature low temperature repetitions (thermal cycling) thanks to the structures DNA can be replicated continuously.
Asagida bulus konusu test kiti içerisinde kullanilacak primer sekans dizileri verilmistir.The primer sequence sequences to be used in the test kit of the invention are given below.
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| TR2020/18428A TR202018428A2 (en) | 2020-11-17 | 2020-11-17 | Specialized rapid scan kit for Sars-cov-2 |
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| Application Number | Priority Date | Filing Date | Title |
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| TR2020/18428A TR202018428A2 (en) | 2020-11-17 | 2020-11-17 | Specialized rapid scan kit for Sars-cov-2 |
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