Name: Forfatter Spotlight: Fremskridt inden for multiplex-detektion af luftvejsvira
Uploaded: 2023-11-10T14:50:00
Description: Watch this Scientific Journal Video about Advancements in Multiplex Detection of Respiratory Viruses at JoVE.com

Dette arbejde blev støttet af King Abdullah University of Science and Technology gennem kernefinansiering og National Term Grand Challenge (NTGC) til SMH.

1. Taq polymeraseekspression og oprensning
<ol><li>Konstruer et plasmid med et spaltbart hexa-histidinmærke ved enzymets C-terminal.</li>
	<li>50 ng af ekspressionsvektoren omdannes til E. coli BL21-(DE3)-stamme efter standardprotokol38.</li>
	<li>De transformerede celler podes i fire 6 L-kolber, der hver indeholder 2 liter 2YT-mediebouillon ved 37 °C, omrystes ved 170 omdr./min., indtil OD 600 på 0,8 eller cellenummer 6,4 x 108 er nået.</li>
	<li>Taq-pol...

Multiplex RT-qPCR-analyse til påvisning af respiratorisk virus

Der er en tung økonomisk byrde for sundhedssystemet over hele verden som følge af de høje infektions- og dødelighedsrater på grund af spredningen af almindelige luftvejsvira såsom SARS-CoV-2, influenza A / B og MERS-CoV-varianter 12,19,20. Motiveret af ansvarsfølelsen mod at lette denne byrde indså vi behovet for en hurtig, præcis og tilgængelig diagnostisk analyse såsom RT-qPCR for at skelne mellem disse almindelige ...

Pandemien af den igangværende coronavirus sygdom 2019 (COVID-19) er forårsaget af den nye coronavirus kendt som det alvorlige akutte respiratoriske syndrom coronavirus 2 (SARS-CoV-2)1. På grund af SAR-CoV-2's stærke smitsomhed og kapacitet til hurtig transmission opstod COVID-19-pandemien i Wuhan City, Kina, og spredte sig hurtigt over hele verden. Dette førte til sidst til starten af tegn på åndedrætsbesvær og endda død 2,3,4. COVID-19 er blevet erklæret en pandemi i mere end 213 lande og forventer en kraftig stigning i antallet af bekræftede tilfælde, som det fremgår af papirerne offentliggjort af forskellige forskningsundersøgelser 3,5. COVID-19 overføres primært af små åndedrætsdråber, som inficerede personer frigiver i miljøet og derefter udsættes for sårbare personer gennem indånding eller tæt kontakt med forurenede overflader. Når disse dråber kommer i kontakt med slimhinden i øjne, mund eller næse, kan en person blive smittet6. Statistikker frigivet af Verdenssundhedsorganisationen (WHO) viser, at der har været mere end 76 millioner bekræftede tilfælde af pandemien på verdensplan med svimlende 7 millioner dødsfald7. FN klassificerede således pandemien forårsaget af COVID-19-sygdommen som en katastrofe på grund af dens direkte indvirkning på milliarder af menneskers liv over hele kloden og havde vidtrækkende økonomiske, miljømæssige og sociale virkninger.
Folkesundhedsinitiativer, herunder grundig testning, tidlig påvisning, kontaktsporing og isolering af tilfælde, har alle vist sig at være afgørende for at holde denne pandemi under kontrol 8,9,10,11. Vintermånederne vil øge cirkulationen af andre luftvejsvira som influenza A og B med COVID-19-lignende symptomer, hvilket gør det vanskeligt at identificere, spore og isolere COVID-19-tilfælde tidligt. Hvert år starter influenza A- og B-udbrud i det sene efterår eller begyndelsen af januar med en forudsigelig sæsonudsving12. Talrige epidemiologiske træk deles af SARS-CoV-2- og influenzavirus. Desuden deler ligheder i de modtagelige populationer, der inkluderer børn, ældre, immunkompromitterede og personer med kroniske comorbiditeter som astma, kronisk obstruktiv lungesygdom, hjerte- og nyresvigt eller diabetes12,13. Disse vira deler ikke kun sårbare populationer, men også transmissionsveje for kontakt og åndedrætsdråber14. Det forventes, at patienter sandsynligvis kan indgå mere end en af disse respiratoriske vira, når influenzasæsonen nærmer sig14. Til det skal screeningen af SARS-CoV-2 og influenzavirus udføres på symptomatiske patienter, før de isoleres. Det er ikke muligt at køre separate tests for de tre vira (SARS-CoV-2, influenza A og influenza B) på grund af den globale mangel på ressourcer til nukleinsyreekstraktion og diagnostik. For at screene dem alle i en reaktion skal der udvikles en metode eller test.
Mellemøsten respiratorisk syndrom (MERS)-CoV er et familiemedlem til human coronavirus (CoV). De første MERS-CoV-virusisolater kom fra en indlagt patient i Saudi-Arabien, der var død i september 2012 på grund af akutte åndedrætsproblemer15. Der er beviser, der tyder på, at en fremtrædende reservoirvært for MERS-CoV er dromedarkameler. Det er bevist, at vira fra inficerede dromedarkameler er zoonotiske og dermed kan inficere mennesker16,17. Mennesker inficeret med denne virus kan sprede den til andre gennem tæt kontakt18. Pr. 26. januar 2018 havde der været 2143 laboratoriebekræftede tilfælde af MERS-CoV-infektion, herunder 750 dødsfald globalt19. De mest typiske MERS-CoV symptomer er hoste, feber og åndenød. MERS-CoV-infektioner er også blevet rapporteret at udvise lungebetændelse, diarré og gastrointestinale sygdomssymptomer20. I øjeblikket er der ingen kommerciel vaccine eller specifik behandling for MERS-CoV tilgængelig. Derfor er hurtig og præcis diagnose afgørende for at forhindre de udbredte MERS-CoV-udbrud og differentiere MERS-CoV fra SARS-CoV-2-sygdommen.
Til dato er der foreslået mange tilgange til at detektere disse vira, såsom multiplex RT-PCR 21,22,23,24,25, CRISPR / Cas1226,27, CRISPR / Cas928 og CRISPR / Cas329, lateral flow immunoassay30, papirbaserede biomolekylære sensorer31, SHERLOCK-test i en gryde 32, DNA-aptamer33, loop-medieret isotermisk forstærkning (LAMP)19,34 osv. Hver af de ovennævnte metoder har unikke fordele og ulemper med hensyn til følsomhed og specificitet. Blandt disse metoder er den nukleinsyreamplifikationsbaserede test: multiplex qRT-PCR, den mest almindelige og anses for at være guldstandarden til diagnosticering af SARS-CoV-2, influenza A, influenza B og MERS-CoV.
I denne undersøgelse designede og vurderede vi forskellige primerkombinationer og sonder til effektiv, nøjagtig og samtidig påvisning af SARS-CoV-2, Influenza A, Influenza B og SARS-CoV-2, MERS-CoV ved hjælp af standard twist syntetiske virale RNA'er. De multipleksede assays udviklet til enten MERS-CoV eller SARS-CoV-2 målgener anbefales af Verdenssundhedsorganisationen (WHO). Disse gener koder generelt for proteiner og komplekser, der bidrager til dannelsen af et replikations-/transkriptionskompleks (RTC)35, såsom regionen inden for den åbne læseramme 1a (ORF1a), der anvendes til MERS-CoV-assay. Derudover kodes strukturelle proteiner af de gener, der anvendes i diagnostiske assays, såsom opstrømsregionen af kuvertgenet (upE) og nukleocapsidgenet (N), der anvendes til henholdsvis MERS-CoV- og SARS-Cov-2-assays, 35,36. Vi brugte internt R3T et-trins RT-qPCR-sæt til at etablere RT-qPCR til påvisning af vira37. Virusdetektion, følsomhed, specificitet og dynamisk område af vores R3T et-trins RT-qPCR-sæt og primersæt blev testet og evalueret ved hjælp af 10 gange serielle fortyndinger af standard twist syntetiske RNA'er. Den laveste praktiske detektionsgrænse var ca. 10 udskriftskopier pr. Reaktion. Som et resultat kan det interne R3T et-trins RT-qPCR-sæt og primer-/sondesæt med succes anvendes og implementeres til rutinemæssig samtidig diagnose af SARS-CoV-2, Influenza A, Influenza B og SARS-CoV-2, MERS-CoV.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.45 &#956;m filter cups</td>
			<td>Thermo Scientific</td>
			<td>291-4545</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Tris-Glycine SDS running buffer</td>
			<td>Novex</td>
			<td>LC2675</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culturing plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium sulfate</td>
			<td>Fisher Scientific</td>
			<td>A701-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Corning</td>
			<td>61-238-RH</td>
			<td></td>
		</tr>
		<tr>
			<td>Cation exchange (HiTrap SP HP) 5 mL</td>
			<td>Cytiva</td>
			<td>17-1152-01</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Biotin, 98+%</td>
			<td>Thermo Scientific</td>
			<td>A14207.60</td>
			<td></td>
		</tr>
		<tr>
			<td>DH10Bac competent cells</td>
			<td>Fisher Scientific</td>
			<td>10361012</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis bag (Snakeskin 10,000 MWC)</td>
			<td>Thermo Scientific</td>
			<td>68100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Thermo Scientific</td>
			<td>R0862</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase/Rnase Free Distilled Water</td>
			<td>Ambion</td>
			<td>AM9930</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>Thermo Scientific</td>
			<td>R0192</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli BL21(DE3) competent cells</td>
			<td>Invitrogen</td>
			<td>C600003</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>BP120-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Elution Buffer</td>
			<td>Qiagen</td>
			<td>19086</td>
			<td></td>
		</tr>
		<tr>
			<td>ESF 921 insect cell culture medium (Insect cells media)</td>
			<td>Expression Systems</td>
			<td>96-001-01</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Solution</td>
			<td>Gibco</td>
			<td>A38400-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fugene (transfection reagent)</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Fisher Scientific</td>
			<td>15750060</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G5516-500</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL CA-630</td>
			<td>Sigma Aldrich</td>
			<td>I8896-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma Aldrich</td>
			<td>56750-1Kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Influenza A (H1N1) synthetic RNA</td>
			<td>Twist Bioscience</td>
			<td>103001</td>
			<td></td>
		</tr>
		<tr>
			<td>Influenza A (H3N2)&#160; synthetic RNA</td>
			<td>Twist Bioscience</td>
			<td>103002</td>
			<td></td>
		</tr>
		<tr>
			<td>Influenza B synthetic RNA</td>
			<td>Twist Bioscience</td>
			<td>103003</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Gold Biotechnology</td>
			<td>I3481C100</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Gibco</td>
			<td>11815-032</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Fisher Scientific</td>
			<td>BP1425-500</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth media</td>
			<td>Fisher Scientific</td>
			<td>BP1426-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma Aldrich</td>
			<td>L6876-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>13152-1Kg</td>
			<td></td>
		</tr>
		<tr>
			<td>MERS-CoV synthetic RNA</td>
			<td>Twist Bioscience</td>
			<td>103015</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp Fast Optical 96-well Reaction plates with Barcode (0.1 mL)</td>
			<td>Applied Biosystems</td>
			<td>10310855</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini- PROTEAN TGX Precast Gel</td>
			<td>Bio-Rad</td>
			<td>456-1093</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniprep kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Excel (HisTrap Excel) 5 mL</td>
			<td>Cytiva</td>
			<td>17-3712-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA HP (HisTrap HP) 5 mL</td>
			<td>Cytiva</td>
			<td>17-5248-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Adhesice Covers (PCR Compatible,DNA/Rnase/PCR Inhibitors Free</td>
			<td>Applied Biosystems</td>
			<td>4311971</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Bioreagents</td>
			<td>BP366-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers and Probes</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Mini tablets EDTA-Free</td>
			<td>Thermo Scientific</td>
			<td>A32955</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein marker</td>
			<td>Fermentas</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>RT-qPCR machine (QuantStudio 7 Flex)</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>S.O.C medium</td>
			<td>Fisher Scientific</td>
			<td>15544034</td>
			<td></td>
		</tr>
		<tr>
			<td>SARS-CoV-+A2:C442 synthetic RNA</td>
			<td>Twist Bioscience</td>
			<td>102024</td>
			<td></td>
		</tr>
		<tr>
			<td>Sf9 insect cells</td>
			<td>Gibco</td>
			<td>A35243</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014-1Kg</td>
			<td></td>
		</tr>
		<tr>
			<td>StrepTrap XT 5 mL</td>
			<td>Cytiva</td>
			<td>29401323</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>IBI Scientific</td>
			<td>IB02200</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base Molecular Biology Grade</td>
			<td>Promega</td>
			<td>H5135</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Affymetrix</td>
			<td>22676</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma Aldrich</td>
			<td>P1379-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Gal</td>
			<td>Invitrogen</td>
			<td>B1690</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of multiplex real-time rt-qpcr assays for the detection of sars-cov-2, influenza a/b, and mers-cov

Det alvorlige akutte respiratoriske syndrom coronavirus 2 (SARS-CoV-2), der forårsager Coronavirus sygdom 2019 (COVID-19) er en alvorlig trussel mod offentlighedens sundhed. I influenzasæsoner kan spredningen af SARS-CoV-2 og andre luftvejsvira forårsage en byrde af luftvejssygdomme i hele befolkningen, der er vanskelig at håndtere. Til det skal respiratoriske vira SARS-CoV-2, influenza A, influenza B og Mellemøsten respiratorisk syndrom (MERS-CoV) overvåges nøje i de kommende efterårs- og vintersæsoner, især i tilfælde af SARS-CoV-2, influenza A og influenza B, som deler lignende epidemiologiske faktorer som modtagelige populationer, transmissionsmåde og kliniske syndromer. Uden målspecifikke analyser kan det være udfordrende at skelne mellem tilfælde af disse vira på grund af deres ligheder. Derfor vil et følsomt og målrettet multiplexassay, der let kan skelne mellem disse virale mål, være nyttigt for sundhedspersonale. I denne undersøgelse udviklede vi et realtids revers transkriptase-PCR-baseret assay ved hjælp af et internt udviklet R3T et-trins RT-qPCR-kit til samtidig påvisning af SARS-CoV-2, Influenza A, Influenza B og SARS-CoV-2, MERS-CoV. Med så få som 10 kopier af deres syntetiske RNA'er kan vi med succes identificere SARS-CoV-2, Influenza A, Influenza B og MERS-CoV-mål samtidigt med 100% specificitet. Denne analyse viser sig at være nøjagtig, pålidelig, enkel, følsom og specifik. Den udviklede metode kan bruges som et optimeret SARS-CoV-2, Influenza A, Influenza B og SARS-CoV-2, MERS-CoV diagnostisk assay på hospitaler, medicinske centre og diagnostiske laboratorier samt til forskningsformål.

The pandemic of the ongoing coronavirus disease 2019 (COVID-19) is caused by the novel coronavirus known as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1. Due to SAR-CoV-2&#39;s strong contagiousness and capacity for rapid transmission, the COVID-19 pandemic emerged in Wuhan City, China, and spread quickly throughout the world. This eventually led to the start of respiratory distress signs and even death2,3,4. COVID-19 has been declared a pandemic in more than 213 countries, expecting a steep increase in the number of confirmed cases, as evidenced by the papers published by different research studies3,5. COVID-19 is transmitted primarily by small respiratory droplets that infected individuals release into the environment and then get exposed to vulnerable individuals through inhalation or close contact with contaminated surfaces. When these droplets come into contact with the mucosa of the eyes, mouth, or nose, a person may become infected6. Statistics released by the World Health Organization (WHO) show that there have been more than 76 million confirmed cases of the pandemic worldwide, with a staggering 7 million deaths7. Thus, the United Nations classified the pandemic caused by COVID-19 disease as a disaster because of its direct impact on the lives of billions of people around the globe and had far-reaching economic, environmental, and social effects.
Public health initiatives including thorough testing, early detection, contact tracing, and case isolation have all been shown to be crucial in keeping this pandemic under control8,9,10,11. The winter months will increase the circulation of other respiratory viruses like Influenza A and B with COVID-19-like symptoms making it difficult to identify, track down, and isolate COVID-19 instances early on. Every year, Influenza A and B outbreak starts in the late fall or early January with a predictable seasonality12. Numerous epidemiological traits are shared by SARS-CoV-2 and Influenza viruses. Besides, sharing similarities in the susceptible populations which include children, the elderly, immunocompromised, and individuals with chronic comorbidities such as asthma, chronic obstructive pulmonary disease, cardiac and renal failure, or diabetes12,13. These viruses not only share vulnerable populations but also transmission routes of contact and respiratory droplets14. It is anticipated that patients may likely contract more than one of these respiratory viruses as flu season approaches14. For that, the screening of SARS-CoV-2 and the Influenza viruses needs to be done on symptomatic patients before they are isolated. Running separate tests for the three viruses (SARS-CoV-2, Influenza A, and Influenza B) is not possible due to the global lack of resources for nucleic acid extraction and diagnostics. In order to screen them all in one reaction, a method or test needs to be developed.
Middle East respiratory syndrome (MERS)-CoV is a human coronavirus (CoV) family member. The first MERS-CoV virus isolates came from a hospitalized patient in Saudi Arabia who had died in September 2012 due to acute respiratory issues15. There is evidence that suggests that a prominent reservoir host for MERS-CoV is dromedary camels. It has been proven that viruses from infected dromedary camels are zoonotic and thus can infect humans16,17. Humans infected with this virus can spread it to others through close contact18. As of January 26th,&#160;2018, there had been 2143 laboratory-confirmed cases of MERS-CoV infection including 750 deaths globally19. The most typical MERS-CoV symptoms are coughing, fever, and shortness of breath. MERS-CoV infections have also been reported to exhibit pneumonia, diarrhea and gastrointestinal sickness symptoms20. Currently, no commercial vaccine or specific treatment for MERS-CoV is available. Therefore, prompt and precise diagnosis is essential for preventing the widespread MERS-CoV outbreaks and differentiating MERS-CoV from SARS-CoV-2 disease.
To date, many approaches have been proposed to detect these viruses such as multiplex RT-PCR21,22,23,24,25, CRISPR/Cas1226,27, CRISPR/Cas928, and CRISPR/Cas329, lateral flow immunoassay30, paper-based biomolecular sensors31, SHERLOCK testing in one pot32, DNA aptamer33, loop-mediated isothermal amplification (LAMP)19,34, etc. Each of the aforementioned methods has unique benefits and drawbacks in terms of sensitivity and specificity. Among these methods, the nucleic acid amplification-based test: multiplex qRT-PCR, is the most common and is considered to be the gold standard for the diagnosis of SARS-CoV-2, Influenza A, Influenza B, and MERS-CoV.
In this study, we designed and assessed various primer combinations and probes for the effective, accurate, and simultaneous detection of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV utilizing standard twist synthetic viral RNAs. The multiplexed assays developed for either MERS-CoV or SARS-CoV-2 target genes are recommended by the World Health Organization (WHO). These genes generally encode proteins and complexes that contribute to the formation of a replication/transcription complex (RTC)35 such as the region within the open reading frame 1a (ORF1a) that is used for MERS-CoV assay. In addition, structural proteins are encoded by the genes utilized in diagnostic assays such as the upstream region of envelope gene (upE) and nucleocapsid gene (N) which are used for MERS-CoV and SARS-Cov-2 assays, respectively35,36. We used in-house R3T one-step RT-qPCR kit to establish the RT-qPCR for the detection of viruses37. Virus detection, sensitivity, specificity, and dynamic range of our R3T one-step RT-qPCR kit and primer sets were tested and evaluated using 10-fold serial dilutions of the standard twist synthetic RNAs. The lowest practical detection limit was approximately 10 transcripts copies per reaction. As a result, the in-house R3T one-step RT-qPCR kit and primer/probe sets can be successfully used and implemented for routine simultaneous diagnosis of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV.

Forfatter Spotlight: Fremskridt inden for multiplex-detektion af luftvejsvira 

Udvikling af multiplex realtids RT-qPCR-assays til påvisning af SARS-CoV-2, influenza A / B og MERS-CoV

I de senere år har der været betydelige fremskridt i den diagnostiske tilgang til påvisning af almindelige respiratoriske vira ved hjælp af PCR-tilgange 21,22,23,24,25. På trods af disse fremskridt er den multipleksede tilgang, som gør det muligt at detektere flere vira i en enkelt test, imidlertid ikke blevet implementeret bredt, især i RT-qPCR-platfor...

Watch this Scientific Journal Video about Advancements in Multiplex Detection of Respiratory Viruses at JoVE.com

Vi præsenterer to sondebaserede interne et-trins RT-qPCR-sæt til almindelige luftvejsvira. Det første assay er for SARS-CoV-2 (N), influenza A (H1N1 og H3N2) og influenza B. Den anden er for SARS-Cov-2 (N) og MERS (UpE og ORF1a). Disse analyser kan implementeres med succes i ethvert specialiseret laboratorium.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general ...

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Research

JoVE Journal

Biology

Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general public&#39;s health. During influenza seasons, the spread of SARS-CoV-2 and other respiratory viruses may cause a population-wide burden of respiratory disease that is difficult to manage. For that, the respiratory viruses SARS-CoV-2, Influenza A, Influenza B, and Middle East respiratory syndrome (MERS-CoV) will need to be carefully watched over in the upcoming fall and winter seasons, particularly in the case of SARS-CoV-2, Influenza A, and Influenza B, which share similar epidemiological factors like susceptible populations, mode of transmission, and clinical syndromes. Without target-specific assays, it can be challenging to differentiate among cases of these viruses owing to their similarities. Accordingly, a sensitive and targeted multiplex assay that can easily differentiate between these viral targets will be useful for healthcare practitioners. In this study, we developed a real-time reverse transcriptase-PCR-based assay utilizing an in-house developed R3T one-step RT-qPCR kit for simultaneous detection of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV. With as few as 10 copies of their synthetic RNAs, we can successfully identify SARS-CoV-2, Influenza A, Influenza B, and MERS-CoV targets simultaneously with 100% specificity. This assay is found to be accurate, reliable, simple, sensitive, and specific. The developed method can be used as an optimized SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

We present two probe-based in-house one-step RT-qPCR kits for common respiratory viruses. The first assay is for SARS-CoV-2 (N), Influenza A (H1N1 and H3N2), and Influenza B. The second is for SARS-Cov-2 (N) and MERS (UpE and ORF1a). These assays can be successfully implemented in any specialized laboratory.

Preparation and Testing of Multiplex R3T One-Step RT-qPCR Kit Components

To begin, prepare the 2X buffer for RT-qPCR in DNase/RNase free water. Store the buffer at minus 20 degrees Celsius until further use. Next, mix the primers and the probes in a 1.5 milliliter tube.Make up the volume with elution buffer. Then store the tube at minus 20 degrees Celsius. Prepare an enzyme mix with Taq polymerase and M-MLV RT in a 1.5 milliliter tube on ice.Make up the volume with Taq polymerase storage buffer. Now re-suspend the viral synthetic RNAs in separate tubes with 100 microliters of Tris-EDTA buffer to make stocks of 10 to the sixth RNA copies per microliter. To mix the virus stocks, add five microliters each of SARS-CoV-2 influenza A and B to 50 microliters of Tris-EDTA buffer.Similarly, mix SARS-CoV-2 and MERS-CoV to obtain a second viral mix. Dilute both mixtures tenfold to obtain a final dilution of 10 RNA copies per microliter. To each well of a 96-well plate, pipette 2X buffer, primer, enzyme, DNase/RNase free water, and the corresponding RNA mixtures.Seal the plate with an adhesive plate seal. Then centrifuge the plate for one minute to collect the liquid at the well bottom. Next, program the PCR machine to accept the fast 96-well block type with the experimental type set to standard curve.Set the reagents to TaqMan reagents and select standard run properties. Define the gene targets and the reporter dye. Then define the sample names for each reaction to be tested and assign targets and samples to the plate layout.Now, input the cycle program into the instrument. Transfer the plate to the real-time qPCR machine in the correct orientation and press run to initiate the cycle. Choose the file location to save the experimental data.Then examine the amplification plots provided by the qPCR program. The two developed kits were able to successfully amplify all three target genes simultaneously with as low as 10 RNA copies per reaction. The amplification efficiencies for all viral titrations were above 99%