山羊关节炎-脑炎病毒(CAEV)实时荧光定量PCR检测方法的建立及现场试验

The Open Virology Journal Pub Date : 2012-01-01 Epub Date: 2011-07-27 DOI:10.2174/1874357901206010082
Giovanni Brajon, Daniela Mandas, Manuele Liciardi, Flavia Taccori, Mauro Meloni, Franco Corrias, Caterina Montaldo, Ferdinando Coghe, Cristina Casciari, Monica Giammarioli, Germano Orrù
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引用次数: 18

摘要

山羊和绵羊的山羊关节炎/脑炎(CAE)是由慢病毒(CAEV)引起的持续性病毒感染。这种病毒感染导致成年动物关节炎和儿童脑炎。脑病形式的预后通常很差,给养殖场造成重大经济损失。在这种情况下,CAEV感染的早期/快速实验室诊断可能有助于采取有效的预防措施。在这项工作中,我们对CAEV的env基因进行了定量实时PCR设计,以检测/定量山羊/绵羊样品中CAEV的病毒RNA或前病毒DNA形式。该方法在15只羊身上得到了验证,这些羊被实验性地感染了CAEV或高度相关的慢病毒(maedi visna, MVV);此外,采用血清学分析(Elisa和AGID)对CAEV阳性牧群中37份临床山羊标本进行分析和比较。所有感染MVV的样本结果均为阴性。在实验感染CAEV的绵羊中,感染15天后可检测到前病毒DNA,而血清学方法在40-60天后显示指示性阳性。该方法灵敏度为10(2)个env片段/PCR,定量线性动态范围为10(3)~ 10(7)个env片段/PCR;R2相关系数为0.98。所有临床诊断为山羊关节炎-脑炎(CAE)的受试者均为CAEV DNA阳性。
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Development and Field Testing of a Real-Time PCR Assay for Caprine Arthritis-Encephalitis-Virus (CAEV).

Caprine arthritis/encephalitis (CAE) of goats and occasionally sheep are persistent virus infections caused by a lentivirus (CAEV). This viral infection results in arthritis in adult animals and encephalitis in kids. Prognosis for the encephalitic form is normally poor, with substantial economic loss for the farm. In this context an early/fast laboratory diagnosis for CAEV infection could be useful for effective prophylactic action. In this work we performed a quantitative real time PCR designed on the CAEV env gene to detect/quantify in goat/sheep samples, viral RNA or proviral DNA forms of CAEV. This procedure was validated in 15 sheep, experimentally infected with CAEV or with a highly correlated lentivirus (visna maedi, MVV); in addition, a total of 37 clinical goat specimens recruited in CAEV positive herds were analyzed and compared using serological analysis (Elisa and AGID). All samples infected with MVV resulted negative. In sheep experimentally infected with CAEV, proviral DNA was detectable 15 days post infection, whereas the serological methods revealed an indicative positivity after 40-60 days.This method showed a sensitivity of 10(2) env fragments/PCR) with a linear dynamic range of quantitation from 10(3) to 10(7)env fragments/PCR; the R2 correlation coefficient was 0.98. All subjects with a clinical diagnosis for Caprine Arthritis-Encephalitis (CAE) resulted CAEV DNA positive.

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