This work was supported by King Abdullah University of Science and Technology through core funding and the National Term Grand Challenge (NTGC) to S.M.H.

1. Taq polymerase expression and purification
<ol>
	<li>Construct a plasmid with a cleavable hexa-histidine tag at the C-terminus of the enzyme.</li>
	<li>Transform 50 ng of the expression vector into E. coli BL21-(DE3) strain following the standard protocol38.</li>
	<li>Inoculate the transformed cells in four 6 L flasks each containing 2 L of 2YT media broth at 37 &#176;C with shaking at 170 rpm until the OD 600 of 0.8 or cell number 6.4 x 108 is reached.</l...

Multiplex RT-qPCR Assay for Respiratory Virus Detection

There is a heavy economic burden on the healthcare system worldwide resulting from the high infection and mortality rates due to the spread of common respiratory viruses such as SARS-CoV-2, Influenza A/B and MERS-CoV variants12,19,20. Motivated by the sense of responsibility towards alleviating this burden, we realized the need for a quick, precise and accessible diagnostic assay such as RT-qPCR to distinguish between these comm...

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.

<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

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.

Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses 

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

In recent years, there have been significant advances in the diagnostic approach for detecting common respiratory viruses using PCR approaches21,22,23,24,25. However, despite these advancements, the multiplexed approach, which allows for detecting multiple viruses in a single test, has not been widely implemented, particularly in the RT-qPCR platform. This met...

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

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.

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

development-multiplex-real-time-rt-qpcr-assays-for-detection-sars-cov

Research

JoVE Journal

Biology

603 ビュー.  King Abdullah University of Science and Technology (KAUST). 一般的な呼吸器系ウイルス用の2つのプローブベースのワンステップRT-qPCRキットを紹介します。最初のアッセイは、SARS-CoV-2(N)、インフルエンザA(H1N1およびH3N2)、およびインフルエンザBに対するものです。2つ目は、SARS-Cov-2(N)とMERS(UpEとORF1a)です。これらのアッセイは、あらゆる専門ラボでうまく実施できます。 

ビデオ: 著者スポットライト:呼吸器系ウイルスのマルチプレックス検出の進歩 

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.

https://cloudfront.jove.com/CDNSource/teasers/65822_b.jpg

generation, amplification, and titration of recombinant respiratory syncytial viruses

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules...

detection of viruses from bioaerosols using anion exchange resin

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from...

real-time polymerase chain reaction-based detection and quantification of hepatitis b virus dna

Author Spotlight: Advancements and Challenges in Hepatitis B Virus Detection 

Real-time polymerase chain reaction (PCR)-based detection and quantification of hepatitis B virus (HBV) DNA is a sensitive and accurate method for diagnosing and monitoring HBV infection. Here, we present a protocol for HBV DNA detection and the viral load measurement of a...

Detection and Quantification of Hepatitis B Virus DNA in Human Serum

https://cloudfront.jove.com/CDNSource/teasers/66249_b.jpg

multiplex cyclic fluorescent immunohistochemistry

Author Spotlight: Efficient Detection of Immune Cell-Infiltration in Cancer Tissues Using Fluorescent Immunohistochemistry

Multiplex cyclic immunohistochemistry allows in situ detection of multiple markers simultaneously using repeated antigen-antibody incubation, image scanning, and image alignment and integration. Here, we present the operating protocol for identifying immune cell substrates with this technology in lung cancer and paired brain metastasis...

In Situ Immunohistochemical Detection of Immune Cell Markers in Cancer Tissues

https://cloudfront.jove.com/CDNSource/teasers/66136_b.jpg

rapid detection of bacterial pathogens causing lower respiratory tract infections via microfluidic-chip-based loop-mediated isothermal amplification

Author Spotlight: Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical...

Human Respiratory Bacterial Infection Diagnosis Using Isothermal Amplification

https://cloudfront.jove.com/CDNSource/teasers/66677_b.jpg

rapid molecular detection and differentiation of influenza viruses a and b

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to...

technical considerations and approach to redo foregut surgery

Author Spotlight: Recent Advancements in Reoperative Foregut Surgery

Redo foregut surgery is associated with increased patient morbidity and presents a technical challenge for the surgeon. We describe our approach and considerations when performing a redo hiatal hernia repair to provide a guide for other surgeons and improve patient...

Reoperative Hiatal Hernia Repair

https://cloudfront.jove.com/CDNSource/teasers/65532_b.jpg

high-throughput detection of respiratory pathogens in animal specimens by nanoscale pcr

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR...

virwatest, a point-of-use method for the detection of viruses in water samples

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of...

three-dimensional modeling of the left atrium and pulmonary veins with a precise intracardiac echocardiography approach

Author Spotlight: Advancements in Intracardiac Echocardiography for Atrial Anatomy Assessment 

Accurate intracardiac echocardiography (ICE) shows significant accuracy in estimating the left atrial structure, a prospective and promising cardiac structure estimation method. Here we propose a protocol for three-dimensional modeling of the left atrium and pulmonary veins with ICE and fast anatomical mapping (FAM) catheter...

ICE-Based 3D Remodeling for the Left Atrium and Pulmonary Vein

https://cloudfront.jove.com/CDNSource/teasers/65353_b.jpg

simultaneous detection of different antibody classes in a multiplexed serological test

Author Spotlight: Expanding the Scope of Multiplex Immunoassays for Lyme Borreliosis Diagnostics and Pathogen Research 

A three-channel dual-reporter fluorescence flow analysis system was used to develop a bead-based multiplex immunoassay that simultaneously evaluates serum samples for IgG and IgM elicited against multiple antigens of different Borrelia species that cause Lyme borreliosis in Europe and North...

Multiplex Immunoassay for &lt;em&gt;Borrelia&lt;/em&gt;-Specific IgG and IgM in Serum

https://cloudfront.jove.com/CDNSource/teasers/65323_b.jpg

retroperitoneal laparoscopic debridement and drainage for pancreatic abscess

Author Spotlight: Advancements in Retroperitoneal Approach for Necrotizing Pancreatitis

To improve the drainage of patients with necrotizing pancreatitis complicated with peripancreatic abscess and reduce the mortality of patients with necrotizing pancreatitis, we adopted a retroperitoneal approach for peripancreatic abscess debridement and drainage by...

Laparoscopic Retroperitoneal Treatment of Necrotizing Pancreatitis Complications

https://cloudfront.jove.com/CDNSource/teasers/66162_b.jpg

targeted muscle reinnervation: surgical protocol for a randomized controlled trial in postamputation pain

Author Spotlight: Advancements and Challenges in Surgical Treatments for Postamputation Pain

The protocol outlines the surgical procedure&#160;for the treatment of postamputation pain using Targeted Muscle Reinnervation (TMR). TMR will be compared with two other surgical techniques, specifically Regenerative Peripheral Nerve Interface (RPNI) and neuroma excision, followed by immediate burying within muscle under the context of an international, randomized controlled...

Procedure for Targeted Muscle Reinnervation for Treating Postamputation Pain

https://cloudfront.jove.com/CDNSource/teasers/66379_b.jpg

performing repeated intraoperative impedance telemetry measurements during cochlear implantation

Author Spotlight: Advancements in Impedance Monitoring for Cochlear Implant Surgery

Here we present a protocol to conduct repeated impedance telemetry measurements during cochlear implantation (CI). They may allow conclusions on the electrode's and implant's function. Repeated impedance measurements enable objective feedback on whether the electrode is positioned inside the perilymph or outside the inner...

Real-Time Telemetric Impedance Measurements During Cochlear Implantation

https://cloudfront.jove.com/CDNSource/teasers/65600_b.jpg

pseudotyped viruses as a molecular tool to monitor humoral immune responses against sars-cov-2 via neutralization assay

Author Spotlight: Studying Host-Virus Interactions with Pseudotyped Viruses 

Pseudotyped viruses (PVs) are replication-defective virions that are used to study host-virus interactions under safer conditions than handling authentic viruses. Presented here is a detailed protocol that shows how SARS-CoV-2 PVs can be used to test the neutralizing ability of patients' serum after COVID-19...

Assessing Serum Neutralization Against SARS-CoV-2 with Pseudotyped Viruses

https://cloudfront.jove.com/CDNSource/teasers/65658_b.jpg

generation of induced pluripotent stem cell-derived itenocytes via combined scleraxis overexpression and 2d uniaxial tension

Author Spotlight: Advancements in Cell and Tissue Engineering for Tendon Repair 

This article describes a procedure to produce iTenocytes by generating iPSC-derived mesenchymal stromal cells with combined overexpression of Scleraxis using a lentiviral vector and uniaxial stretching via a 2D...

Harnessing iPSC-Derived iTenocytes for Effective Cell-Based Therapy

https://cloudfront.jove.com/CDNSource/teasers/65837_b.jpg

multiplex immunofluorescence combined with spatial image analysis for the clinical and biological assessment of the tumor microenvironment

Author Spotlight: Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment  

In this article, a protocol for manual tyramide signal amplification (TSA) multiplex immunofluorescence (mIF) combined with image analysis and spatial analysis is described. This protocol can be used with formalin-fixed paraffin-embedded (FFPE) sections for the staining of two to six antigens per slide depending on the slide scanner available in the...

Multiplex Immunofluorescence and Spatial Image Analysis of the Tumor Microenvironment

dnazyme 10-23 - based nanomachines for nucleic acid recognition

Author Spotlight: Advancements in DNA Nanosensors &amp;#8211; Addressing Sensitivity and Selectivity Challenges in Molecular Detection

The DNAzyme-based nanomachines can be used for highly selective and sensitive detection of nucleic acids. This article describes a detailed protocol for the design of DNAzyme-based nanomachines with a 10-23 core using free software and their application in the detection of an Epstein-Barr virus fragment as an...

Precision DNAzyme Nanomachines for Discriminative RNA and DNA Analysis

https://cloudfront.jove.com/CDNSource/teasers/66461_b.jpg

ultrafast laser-ablated nanoparticles and nanostructures for surface-enhanced raman scattering-based sensing applications

Author Spotlight: Advancements and Applications in Nanoparticle Synthesis Through Laser Ablation in Liquids 

Ultrafast laser ablation in liquid is a precise and versatile technique for synthesizing nanomaterials (nanoparticles [NPs] and nanostructures [NSs]) in liquid/air environments. The laser-ablated nanomaterials can be functionalized with Raman-active molecules to enhance the Raman signal of analytes placed on or near the...

Synthesizing Nanoparticles and Nanostructures via Laser Ablation

https://cloudfront.jove.com/CDNSource/teasers/65450_b.jpg

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%