Development of real-time PCR array for simultaneous detection of eight human blood-borne viral pathogens.
Development of real-time PCR array for simultaneous detection of eight human blood-borne viral pathogens.
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DOI：
10.1371/journal.pone.0043246
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Lo SC
中科院分区：
文献类型：
--
作者：
Pripuzova N;Wang R;Tsai S;Li B;Hung GC;Ptak RG;Lo SC
用于快速检测多种病毒病原体的实时PCR阵列在样本量和检测时间有限的情况下，即在组织和器官捐献者的资格检测中，应该非常有用。我们开发了能够同时检测8种人类病毒病原体的实时PCR阵列：人类免疫缺陷病毒1型和2型（HIV-1和-2），B肝炎病毒（HBV），丙型肝炎病毒（HCV），人类T细胞白血病病毒-1和-2（HTLV-1和-2），牛痘病毒（VACV）和西尼罗河病毒（WNV）。使用生物信息学方法的组合设计了一百二十（120）个引物，并且在实验测试之后，选择了靶向八种病毒病原体的24个引物组来建立具有SYBR绿色化学的阵列。使用加标至人血浆中的已知病毒量的分析板评价了为阵列选择的病毒特异性引物组的特异性和灵敏度。该阵列检测到：10基因组当量（geq）/ml的HIV-2和HCV，50 geq的HIV-1（亚型B）、HBV（基因型A）和WNV。它检测到100- 1，000 geq/ml的HIV-1亚型（A-G）、N组和CRF（AE和AG）分离株的血浆。对由28株HIV-1和HIV-2临床分离株组成的样本组进行的进一步评价显示，HIV-1或HIV-2特异性引物与另一种HIV无交叉反应性。所有28株病毒分离株均用靶向最保守基因组区域的特异性引物组鉴定。PCR阵列在一组17个先前定量的HIV-1、HCV或HBV阳性的临床血浆样本中正确鉴定出病毒感染，每个PCR反应低至几个geq。本文所述的病毒阵列在检测供体临床样本时表现出足够的性能。目前正在开发进一步提高其对广谱HIV-1亚型的敏感性。
Real-time PCR array for rapid detection of multiple viral pathogens should be highly useful in cases where the sample volume and the time of testing are limited, i.e. in the eligibility testing of tissue and organ donors. We developed a real-time PCR array capable of simultaneously detecting eight human viral pathogens: human immunodeficiency virus types 1 and 2 (HIV-1 and -2), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia virus-1 and -2 (HTLV-1 and -2), vaccinia virus (VACV) and West Nile virus (WNV). One hundred twenty (120) primers were designed using a combination of bioinformatics approaches, and, after experimental testing, 24 primer sets targeting eight viral pathogens were selected to set up the array with SYBR Green chemistry. The specificity and sensitivity of the virus-specific primer sets selected for the array were evaluated using analytical panels with known amounts of viruses spiked into human plasma. The array detected: 10 genome equivalents (geq)/ml of HIV-2 and HCV, 50 geq of HIV-1 (subtype B), HBV (genotype A) and WNV. It detected 100–1,000 geq/ml of plasma of HIV-1 subtypes (A – G), group N and CRF (AE and AG) isolates. Further evaluation with a panel consisting of 28 HIV-1 and HIV-2 clinical isolates revealed no cross-reactivity of HIV-1 or HIV-2 specific primers with another type of HIV. All 28 viral isolates were identified with specific primer sets targeting the most conserved genome areas. The PCR array correctly identified viral infections in a panel of 17 previously quantified clinical plasma samples positive for HIV-1, HCV or HBV at as low as several geq per PCR reaction. The viral array described here demonstrated adequate performance in the testing of donors’ clinical samples. Further improvement in its sensitivity for the broad spectrum of HIV-1 subtypes is under development.
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