Name: qpcr is a sensitive and rapid method for detection of cytomegaloviral dna in formalin-fixed, paraffin-embedded biopsy tissue
Uploaded: 2014-07-09T20:00:00
Description: Watch this Scientific Journal Video about qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue at JoVE.com

我们承认艾米托马森为她准备这篇稿子帮助和弗雷德里克Skarstedt和Ryan克里斯蒂他们在数字的准备工作。

随着机构审查委员会的批准，进行了搜索的电子实验室的数据库来识别福尔马林固定，石蜡包埋（FFPE）代表巨细胞病毒（CMV）感染的胃肠道病例（GI）的诊断经病理组织学活检（苏木块和伊红（H＆E）和/或免疫组织化学（IHC））。 1，从FFPE胃肠道活检组织组织处理<ol><li>收集FFPE胃肠道活检组织块巨细胞病毒（CMV）定量聚合酶链反应（定量PCR）。这些块被存储在室温下。 </li>...

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	<li>Hodinka, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of Clnical Microbiology. 2, 1558-1574 (2011).</li><li>Lasry, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstrain+variations+in+the+cytomegalovirus+(CMV)+glycoprotein+B+gene+sequence+among+CMV-infected+children+attending+six+day+care+centers.">Interstrain variations in the cytomegalovirus (CMV) glycoprotein B gene sequence among CMV-infected children attending six day care centers.</a> The Journal of infectious diseases. 174, 606-609 (1996).</li><li>Murph, J. R., Baron, J. C., Brown, C. K., Ebelhack, C. L., Bale Jr, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+occupational+risk+of+cytomegalovirus+infection+among+day-care+providers.">The occupational risk of cytomegalovirus infection among day-care providers.</a> JAMA : the journal of the American Medical Association. 265, 603-608 (1991).</li><li>Pass, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccine+prevention+of+maternal+cytomegalovirus+infection.">Vaccine prevention of maternal cytomegalovirus infection.</a> The New England journal of medicine. 360, 1191-1199 (2009).</li><li>McCoy, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=qPCR+increases+sensitivity+to+detect+cytomegalovirus+in+paraffin-embedded,+formalin-fixed+tissue+of+gastrointestinal+biopsies.">qPCR increases sensitivity to detect cytomegalovirus in paraffin-embedded, formalin-fixed tissue of gastrointestinal biopsies.</a> Human Pathology. 45, 48-53 (2014).</li><li>Mills, A. M., Guo, F. P., Copland, A. P., Pai, R. K., Pinsky, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+CMV+detection+in+gastrointestinal+mucosal+biopsies+using+immunohistochemistry+and+PCR+performed+on+formalin-fixed,+paraffin-embedded+tissue.">A comparison of CMV detection in gastrointestinal mucosal biopsies using immunohistochemistry and PCR performed on formalin-fixed, paraffin-embedded tissue.</a> The American journal of surgical pathology. 37, 995-1000 (2013).</li><li>Krech, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement-fixing+antibodies+against+cytomegalovirus+in+different+parts+of+the+world.">Complement-fixing antibodies against cytomegalovirus in different parts of the world.</a> Bulletin of the World Health Organization. 49, 103-106 (1973).</li><li>Chou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newer+methods+for+diagnosis+of+cytomegalovirus+infection.">Newer methods for diagnosis of cytomegalovirus infection.</a> Reviews of infectious diseases. 12 Suppl 7, (1990).</li><li>Brytting, M., Xu, W., Wahren, B., Sundqvist, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytomegalovirus+DNA+detection+in+sera+from+patients+with+active+cytomegalovirus+infections.">Cytomegalovirus DNA detection in sera from patients with active cytomegalovirus infections.</a> Journal of clinical microbiology. 30, 1937-1941 (1992).</li><li>Revello, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cytomegalovirus+in+blood+of+immunocompetent+persons+during+primary+infection:+prognostic+implications+for+pregnancy.">Human cytomegalovirus in blood of immunocompetent persons during primary infection: prognostic implications for pregnancy.</a> The Journal of infectious diseases. 177, 1170-1175 (1998).</li><li>Shinkai, M., Bozzette, S. A., Powderly, W., Frame, P., Spector, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+urine+and+leukocyte+cultures+and+plasma+DNA+polymerase+chain+reaction+for+identification+of+AIDS+patients+at+risk+for+developing+human+cytomegalovirus+disease.">Utility of urine and leukocyte cultures and plasma DNA polymerase chain reaction for identification of AIDS patients at risk for developing human cytomegalovirus disease.</a> The Journal of infectious diseases. 175, 302-308 (1997).</li></ol>

许多实验室方法可用于巨细胞病毒的诊断病毒（CMV）感染，包括血清学，病毒培养，分子血症测定法，活组织检查材料的组织学，并且，最近表明，分子的福尔马林固定，石蜡包埋（FFPE）的活检测定组织5,6。血清学检查，一旦确诊的中流砥柱，只有可靠地识别风险，并不会与急性CMV感染7很好的相关性。病毒培养，常规和早期抗原壳小瓶中，通过长时间的测定时间的阻碍，在后者<...

的巨细胞病毒（CMV）感染在临床标本中的解释是非常重要的。巨细胞病毒是疱疹病毒的betaherpesvirinae亚家族的成员。类似于其他疱疹病毒，巨细胞病毒必须建立持续性或隐性感染者的能力特征是缺乏病毒复制活跃1。在压力或其他免疫抑制的时间可能会发生病毒的再活化，导致病毒复制活跃其特征在于，病毒体释放，这可以伴随疾病表现2-4。胃肠道（GI），巨细胞病毒病的症状通常包括腹部疼痛和/或血便1。 巨细胞病毒性肠胃炎的组织学诊断采用苏木精伊红（H＆E），以确定巨细胞病毒包涵体。在临床上怀疑是高还没有明显的包裹体观察病例，免疫组织化学（IHC）经常作为一种辅助的测试方法。然而，IHC也可以通过稀有非经典出现细胞染色模式（模棱两可）的阻碍，使得难以解释（ 图2）5。它试图利用DNA从福尔马林固定，石蜡包埋（FFPE）的GI活检组织和定量聚合酶链反应（定量PCR）来检测CMV胃肠活检萃取。该技术已被证明是有价值的巨细胞病毒在胃肠活检的检测，并且特别有用的模棱两可的IHC染色例5,6。此外，定量PCR也显示出额外的巨细胞病毒临床试验数据以及相关可用时6。在检测巨细胞病毒使用的qPCR可能会导致早期诊断和治疗的情况下，临床怀疑为巨细胞病毒是很高，但H＆E和CMV免疫组化是否定的。

<table border="0" cellpadding="0" cellspacing="0" width="745">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">1.7 mL microfuge tubes</td>
			<td style="width:195px;">Costar</td>
			<td align="right" style="width:160px;">3620</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">serrated forceps</td>
			<td style="width:195px;">Electron Microscopy Services</td>
			<td style="width:160px;">62086-1S</td>
			<td>Micro Forceps MF-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Tabletop centrifuges (microfuge)</td>
			<td style="width:195px;">Eppendorf</td>
			<td align="right" style="width:160px;">5424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Xylene</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:160px;">Fisher Scientific: X3P</td>
			<td>1 gallon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Ethanol</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:160px;">Fisher Scientific: ET108</td>
			<td>96-100%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">microtome</td>
			<td style="width:195px;">Leica</td>
			<td style="width:160px;">RM2255</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">QIAamp DNA FFPE Tissue Kit</td>
			<td style="width:195px;">Qiagen</td>
			<td align="right" style="width:160px;">56404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">artus CMV LightCycler PCR ASR</td>
			<td style="width:195px;">Qiagen</td>
			<td align="right" style="width:160px;">4500025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">CMV LC PCR Supplement Kit</td>
			<td style="width:195px;">Qiagen</td>
			<td align="right" style="width:160px;">1031873</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">LightCycler centrifuge</td>
			<td style="width:195px;">Roche</td>
			<td align="right" style="width:160px;">75005087</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">LightCycler Cooling Block</td>
			<td style="width:195px;">Roche</td>
			<td align="right" style="width:160px;">10800058001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">LightCycler Capping Tool</td>
			<td style="width:195px;">Roche</td>
			<td align="right" style="width:160px;">3357317</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Color Compensation Kit</td>
			<td style="width:195px;">Roche</td>
			<td align="right" style="width:160px;">2158850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">LightCycler 20 &#956;L Capillaries</td>
			<td style="width:195px;">Roche</td>
			<td style="width:160px;">1 909 339</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">blades</td>
			<td style="width:195px;">Sakura</td>
			<td style="width:160px;">Fisher Scientific: 4689</td>
			<td>Accu-Edge low profile</td>
		</tr>
	</tbody>
</table>

qpcr is a sensitive and rapid method for detection of cytomegaloviral dna in formalin-fixed, paraffin-embedded biopsy tissue

关键的是要确定在胃肠道（GI）的免疫抑制病人的呼吸道巨细胞病毒（CMV）感染，给出了发展为严重感染的风险更大。已经开发了许多实验室方法为巨细胞病毒感染的检测，包括血清学，病毒培养和分子生物学方法。通常情况下，这些方法反映全身受累的巨细胞病毒，不具体确定局部组织的参与。因此，在胃肠道中检测CMV感染是通过活检组织的传统组织学频繁进行。苏木精伊红（H＆E）染色与免疫组织化学（IHC）结合仍然检查这些切片的支柱。 H＆E和免疫组化有时会导致非典型（模棱两可）染色模式，使解释困难。它表明，定量聚合酶链反应（定量PCR）对CMV可以成功地对福尔马林固定，石蜡包埋（FFPE）的活检组织为非常执行高敏感性和特异性。本协议的目的是演示如何在临床实验室的设置进行定量PCR检测巨细胞病毒在FFPE组织活检的检测。这种方法很可能是为病人大有裨益的胃肠活检模棱两可染色巨细胞病毒的病例。

共有228组织块用定量聚合酶链反应（定量PCR），这是由91巨细胞病毒（CMV）阳性例可疑CMV免疫组化的基础上组织学和积极的免疫组织化学（IHC），18，79和阴性对照试验。 如图2，巨细胞病毒阳性病例会表现出典型的巨细胞病毒包涵体（A）和/或阳性IHC染色（B）。模棱两可的情况下会表现出罕见的，非经典的出现染色模式（C），而阴性对照会显示?...

Watch this Scientific Journal Video about qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue at JoVE.com

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

定量PCR是用于检测巨细胞病毒DNA在福尔马林固定，石蜡包埋的活检组织一个灵敏快速的方法

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

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