TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Patterns of rabies virus spread within the central nervous system suggest that a thorough examination of the brain stem is critical for rabies diagnosis. Viral RNA is widespread in the brain of most animals positive for rabies. However, because virus spread may be unilateral, especially in larger animals, a negative finding for rabies can be made only if a complete cross section of the brain stem is examined. Examination may be made at the level of the pons, medulla, or midbrain of the brain stem.</p>
<p> </p>
<p>While a negative finding for rabies can be made only if a full cross section of brain stem tissue is examined, incomplete or suboptimal specimens should be tested, if possible. Identification of rabies virus RNA in any tissue is diagnostic of rabies infection [1, 2]. Examples of samples that may be tested for rabies virus RNA to rule-in (but not rule-out) rabies infection are hippocampus, cerebellum, cortex, saliva, and nuchal skin biopsy.</p>

[BEFORE_START]
<p> </p>
<p><strong>Definitions and Keywords</strong></p>
<p>BSC – Biological Safety Cabinet</p>
<p>PPE – Personal Protective Equipment</p>
<p>Room Temperature – Temperature range of 20°C to 25°C (68°F to 77°F)</p>
<p> </p>
<p><strong>Quality Control and 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 end product in any aspect.</li>
<li>Temperature levels are to be monitored on applicable equipment to ensure incubators, refrigerators, and freezers stay within criteria set for acceptable temperature range.</li>
<li>Specimen will be assigned based on the test ordered and/or the recommendations of the technical supervisor or lab supervisor.</li>
<li>All samples are to have unique identifiers and corresponding accession labels provided in advance of starting the necropsy procedure.</li>
</ul>
<p><strong> </strong></p>

[GUIDELINES]
<p><strong>Purpose </strong></p>
<p>To describe the procedure of collecting brain stem tissue from a postmortem animal. The collected tissue can be used in the diagnosis of lyssavirus infection by the LN34 real-time RT-PCR assay.</p>
<p> </p>
<p><strong>Responsibility </strong></p>
<p>Laboratorian will perform the procedure according to the protocol. Once sample collection is complete, the work area is disinfected, any biohazardous waste autoclaved or incinerated before discarding, and samples returned to storage.</p>
<p> </p>
<p><strong>Specimens </strong></p>
<p>Postmortem animal brain tissue</p>
<p> </p>
<p><strong>Equipment, Materials and Reagents </strong></p>
<ol>
<li>Necropsy instruments
<ul>
<li>1 set of instruments is needed per sample to prevent cross contamination between samples.</li>
</ul>
</li>
</ol>
<ol start="2">
<li>Autoclave and/or instrument sterilizer
<ul>
<li>All instruments should be cleaned and sterilized before reuse</li>
</ul>
</li>
</ol>
<ol start="3">
<li>Specimen storage containers
<ul>
<li>Container must be large enough for reserved portions of brain stem.</li>
<li>Because of the risk of breakage, glass vials and tubes are unacceptable for specimen storage.</li>
</ul>
</li>
</ol>
<ol start="4">
<li>Freezer
<ul>
<li>Long term storage should be at -70°C or lower</li>
<li>Frost-free freezers should not be used, as heat cycles in such freezers can compromise tissue integrity and, therefore, test results.</li>
</ul>
</li>
</ol>
<ol start="5">
<li>PPE:
<ul>
<li>heavy rubber gloves or other cut-resistant gloves</li>
<li>laboratory gown</li>
<li>waterproof apron</li>
<li>surgical mask</li>
<li>boots</li>
<li>protective sleeves</li>
<li>face shield</li>
<li>disposable gloves</li>
</ul>
</li>
<li>Polyester fiber-tipped applicator swab</li>
<li>Scalpel</li>
<li>Sterile petri dish or paper towel</li>
<li>Disposable sterile screwtop microcentrifuge tubes with o-ring (2 ml)
<ul>
<li>In a clean room, add 1 ml TRIzol reagent to one tube for each sample.</li>
<li>Label tubes with corresponding accessioning information.</li>
</ul>
</li>
<li>Acceptable surface decontaminants
<ol>
<li>Quaternary ammonium disinfectant (1:256)</li>
<li>70% Isopropanol</li>
<li>Iodinophors</li>
</ol>
</li>
</ol>

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