TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Purpose</strong></p>
<p>To describe the LN34 real-time RT-PCR assay procedure used for the qualitative detection of lyssavirus RNA in whole RNA extracted from brain tissue samples. The assay detects RNA from diverse lyssaviruses at varying concentrations [1]. </p>

[BEFORE_START]
<p><strong>Definitions/Keywords</strong></p>
<p><strong> </strong></p>
<p>PPE – Personal Protective Equipment</p>
<p>SDS – Safety data sheet</p>
<p>NTC – No template control</p>
<p>Ct – Cycle threshold</p>
<p>PCR – Polymerase Chain Reaction<br />RT-PCR –Reverse Transcription-Polymerase Chain Reaction</p>
<p>RNA – Ribonucleic Acid</p>
<p>RNase – Ribonuclease</p>
<p>βA – Beta-actin <br />Room Temperature – Temperature range of 20°C to 25°C (68°F to 77°F)</p>
<p>DFA – Direct Fluorescent Antibody Test (also known as FAT)</p>
<p>dRIT – Direct, Rapid Immunohistochemical Test</p>
<p> </p>
<p><strong>Reagent Preparation</strong></p>
<ol>
<li>Acceptable surface decontaminants
<ol>
<li>RNase Away (Fisher Scientific)</li>
<li>RNaseZAP (Sigma-Aldrich)</li>
<li>70% EtOH</li>
</ol>
</li>
<li>Reagent Preparation: Vortex and centrifuge all buffers, primers, and probes for a minimum of 5 seconds before use.</li>
<li>Primers and Probes</li>
<li>Preparation of single-use aliquots is recommended.</li>
<li>Store at -20°C in the dark.</li>
<li>Frozen reagents may be used until quality failure or until kit expires</li>
<li>Ag-Path 2X RT-PCR Buffer (Life Technologies): Have thawed and ready for use</li>
<li>25X RT-PCR Enzyme Mix (Life Technologies): Keep on ice or in a cooling block</li>
<li>It is recommended that PCR mastermix reagents be thawed and prepared separately from RNA samples that will be tested</li>
<li>RNA samples<br />
<ul>
<li>Recommended: Use freshly extracted RNA, store on ice until use</li>
<li>Thaw any frozen RNA samples on ice</li>
<li>Thaw single-use aliquot of positive control RNA on ice</li>
</ul>
</li>
<li>Positive control RNA
<ul>
<li>Positive control RNA will be produced and validated at CDC, then shipped to testing laboratories. No dilution is necessary.</li>
<li>Positive control RNA should be stored in single-use aliquots at -80<sup> o</sup>C until use. Avoid freeze-thaw.</li>
<li>Positive control RNA should not be used if the Ct value is out of the expected range determined by CDC for a given lot. Between runs, the Ct value of the positive control RNA from the same batch should not differ by more than 5 Ct.</li>
</ul>
</li>
</ol>
<p><strong>Quality Control/Corrective Action</strong></p>
<ul>
<li>All issues, comments, concerns, or deviations from the protocol are to be documented and brought to the attention of the lab supervisor or technical supervisor immediately if they are thought to affect the quality of the test results in any way.</li>
<li>Technical supervisor will review results prior to issuance of results.</li>
<li>Temperature logs are kept to ensure refrigerators and freezers stay within acceptable temperature range for reagents. Real-time PCR machines are to undergo normal preventative maintenance and quality assessments, as suggested by the manufacturer.</li>
<li>Because of the sensitivity of the fluorogenic 5ʹnuclease assay, special precautions must be taken to avoid false positive amplification. We recommend the following steps to avoid sample contamination:   
<ul>
<li>Wear clean gloves (not previously worn when handling extracted RNA or PCR products) when setting up assays.</li>
<li>Change or decontaminate gloves whenever you suspect they are contaminated.</li>
<li>Keep tubes capped as much as possible.</li>
<li>Before setting up assays and after handling extracted RNA or PCR products, clean equipment and lab benches.</li>
<li>Use aerosol barrier (filter) pipette tips.</li>
<li>Use a clean room or cabinet system to minimize contamination, if possible.</li>
</ul>
</li>
</ul>

[GUIDELINES]
<p><strong>Purpose</strong></p>
<p>To describe the LN34 real-time RT-PCR assay procedure used for the qualitative detection of lyssavirus RNA in whole RNA extracted from brain tissue samples. The assay detects RNA from diverse lyssaviruses at varying concentrations [1].  </p>
<p> </p>
<p><strong>Responsibility</strong></p>
<p><strong> </strong>A trained technician will perform the assay according to the procedural protocol, return samples to storage, and report results to respective supervisors.</p>
<p> </p>
<p><strong>Specimens</strong></p>
<p><strong> </strong>RNA extracted from tissue representing a full cross section of brain stem</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.