a molecular-based assay for quantifying influenza a and b viruses

A Molecular-Based Assay for Quantifying Influenza A and B Viruses

To begin the test process, touch Run Test on the instrument screen. Next, enter the patient ID by using the on-screen keyboard or barcode scanner, and press OK. After that, open the lid and insert the orange test base tube into the orange test base holder. Confirm that the correct test is displayed on the screen, and press OK. Then, insert the blue sample receiver into the blue sample receiver holder and wait for the sample receiver to warm up. Afterward, remove the foil seal by placing two fingers on the edge of the sample receiver to ensure that it stays in place.
Then, place the patient swab to be tested into the sample receiver. Vigorously mix the swab in the liquid for 10 seconds and press the swab head against the side of the sample receiver to help dislodge the specimen from the swab. If the testing swabs are stored in viral transport medium, vortex the viral transport media for 10 seconds, and add 200 microliters of it to the sample receiver. Then, press OK to proceed.
After that, press the white transfer cartridge into the blue sample receiver. Continue pressing on the sample receiver until the orange indicator rises. Then, lift and connect the transfer cartridge to the test base. The orange indicator should descend once the cartridge transfer is attached correctly. Close the lid and do not open until the result screen appears.
Now, touch Run QC Test on the home screen. Select the QC test to be run. Then, confirm the test type to match the QC sample intended for testing by clicking OK and following the on-screen prompts to complete testing. After the test run is complete, observe the results of the test displayed on the screen with interpretations for the presence of influenza A, B, or unknown subtypes and repeat the test for invalid results.

a-molecular-based-assay-for-quantifying-influenza-a-and-b-viruses

Universidad de Cartagena UDEC

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JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Before Running the Assay
NOTE: The Influenza A and B assay is approved for nasopharyngeal swab specimens and for nasopharyngeal swabs stored in viral transport media. Swabs are included in the kit and should be used for optimal performance. However, rayon, foam, flocked swabs, or polyester nasal swabs can also be used to collect nasal swab samples.
<ol>
	<li>To collect a nasal swab sample, insert the swab into the nostril that has the most visible drainage or that is most congested. With gentle rotation, push the swab into the nostril until resistance is met, and rotate several times against the nasal wall before slowly removing it from the nostril. Store the swabs and transport them in vials containing 3 ml of viral transport media (VTM). 
	NOTE: Careful attention to appropriate specimen collection must be taken as inadequate specimen collection may yield erroneous results.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Swab specimens should be tested as soon as possible after collection. However, they may be stored at room temperature for up to 2 hr or refrigerated at 2-8 &#176;C for up to 24 hr if testing is not immediately available. Freshly collected specimens are ideal for optimal test performance. Improper sample handling, storage, and/or transport may yield erroneous results.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: As a precautionary measure, individuals performing this test should treat all specimens as potentially infectious by following universal precautions when handling specimens.</li>
	<li>Bring all samples to room temperature prior to testing. 
	NOTE: All test components are for a single use only and should not be used to perform multiple tests. Do not mix kit components from different lot numbers. Do not use kit reagents past their expiration date.</li>
	<li>Bring the blue sample receiver to room temperature prior to testing. The orange test base can be tested without the need to warm to room temperature.</li>
	<li>Leave all test pieces in the foil wrapping until immediately before use. Turn on the instrument by pressing the power button on the side of the instrument. Enter the user ID and press &#39;OK.</li>
</ol>
2. Running the Test
<ol>
	<li>To begin the test process, touch &#39;Run Test&#39; on the instrument screen. Enter the patient ID by using the on-screen keyboard or barcode scanner and press &#39;OK.&#39; Open the lid and insert the Orange test base into the Orange test base holder.</li>
	<li>Confirm that the correct test is displayed on the screen and press &#39;OK.&#39; Insert the blue sample receiver into the blue sample receiver holder. 
	NOTE: Do not open the sample receiver before placing it in the instrument as this will prevent the elution buffer from reaching the correct operating temperature and may impact test performance.</li>
	<li>Wait for the sample receiver to warm up. When prompted by the instrument, remove the foil seal and place the patient swab to be tested into the sample receiver. 
	NOTE: When removing the foil, place two fingers on the edge of the Sample Receiver to ensure that it stays in place.</li>
	<li>Vigorously mix the swab in the liquid for 10 sec, pressing the swab head against the side of the sample receiver as you mix it to help dislodge the specimen from the swab. Press &#39;OK&#39; to proceed. If testing swabs are stored in a viral transport medium, vortex the VTM for 10 sec then add 200 &#181;l to the sample receiver.</li>
	<li>Press the white transfer cartridge into the blue sample receiver. Continue pressing on the sample receiver until the orange indicator rises.</li>
	<li>Lift and then connect the transfer cartridge to the test base. Observe the orange indicator descend once the cartridge transfer is attached correctly. Close the Lid and do not open until the &#34;Test Complete&#34; message appears on the screen.</li>
</ol>
3. Quality Control (QC)
<ol>
	<li>Touch &#39;Run QC Test&#39; on the home screen. Select the QC Test to be Run. Confirm the test type to match the QC sample intended for testing by touching &#39;OK&#39; and following the on-screen prompts to complete testing.</li>
</ol>
4. Result Interpretation
<ol>
	<li>After the test run is complete, observe the results of the test displayed on the screen with interpretations for the presence of Influenza A, B, and unknown subtypes. For invalid results repeat the test.</li>
</ol>
5. Maintenance and Cleaning
<ol>
	<li>Clean the instrument and the surrounding bench area daily with 70% ethanol or 10% bleach solution. Spray the cleaning solution on a damp, lint-free cloth to clean. Do not pour the solutions directly on the instrument.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alere i Instrument</td>
			<td>Alere</td>
			<td>NAT-000 (Global), NAT-024 (US)</td>
			<td></td>
		</tr>
		<tr>
			<td>Alere i Influenza A &#38; B 24 Test Kit</td>
			<td>Alere</td>
			<td>425-000 (Global), 425-024 (US)</td>
			<td></td>
		</tr>
		<tr>
			<td>Alere i Barcode Scanner</td>
			<td>Alere</td>
			<td>EQ001001</td>
			<td></td>
		</tr>
		<tr>
			<td>Alere Universal Printer</td>
			<td>Alere</td>
			<td>55115 (Global), alereiprinter (US)</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L precision pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L disposable pipette tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Viral transport medium</td>
			<td>Remel</td>
			<td>M4-RT</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Otto, C. C. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/54312">Rapid Molecular Detection and Differentiation of Influenza Viruses A and B.</a> J. Vis. Exp. (2017)
The video demonstrates an assay for quantifying influenza viruses A and B. This assay employs nicking endonuclease amplification with influenza-specific primers and target detection using molecular beacon probes.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Before Running the Assay
NOTE: ...

Take a molecular detection system containing a pre-filled sample receiver, a transfer cartridge, and a test base.&#160;
The test base has reaction tubes containing T1 and T2 primers with nick sites and influenza&#160; A and B recognition regions, an enzyme mix, and molecular beacons with a fluorophore and quencher.
Transfer the virus-containing nasopharyngeal samples in a lysis buffer from the sample receiver to the test base.
Viral lysis releases RNA, followed by the binding of T2 primer to the target region of RNA and extension by reverse transcriptase.
The other T2 primer binds to the same RNA &#8212; displacing the initially extended strand.
The T1 primer binds to the displaced strand, and DNA polymerase extends it. A nicking enzyme cuts its site, with polymerization and strand displacement, forming double-stranded DNA.
The process continues, amplifying duplexes with single-stranded overhangs.
Molecular beacons interact with overhangs, separating the fluorophore from the quencher and producing fluorescence to quantify Influenza A and B.

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Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

鼻湿巾从猪流感病毒检测和​​隔离

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

科研

JoVE Journal

免疫与感染

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

用于病毒感染活成像和定量的路西酶-荧光报告流感病毒

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...

A Molecular-Based Assay for Quantifying Influenza A and B Viruses

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A Molecular-Based Assay for Quantifying Influenza A and B Viruses