Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement.
Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement.
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DOI：
10.1016/s1386-6532(03)00122-7
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发表时间：
2004-03
期刊：
影响因子：
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通讯作者：
Tisdale M
中科院分区：
文献类型：
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作者：
Ward CL;Dempsey MH;Ring CJ;Kempson RE;Zhang L;Gor D;Snowden BW;Tisdale M
背景资料：抗流感药物如扎那米韦的抗病毒作用可以在患者中表现为与安慰剂相比，在治疗的时间过程中病毒载量下降的速率增加。历史上，这是使用空斑测定或培养增强酶联免疫吸附测定（CE-ELISA）来测量的。目的：开发和改进真实的时间定量PCR（qPCR）测定以测量临床样品中的甲型流感和B流感病毒载量，其提供了对现有方法的改进，特别是病毒感染性测定。研究设计：确定了真实的时间qPCR检测试剂盒的动态范围和耐用性，沿着检测试剂盒组分的稳定性。通过平行检测三种不同亚型培养病毒的系列稀释液和一组流感病毒阳性咽拭子标本，对采用CE-ELISA的真实的时间PCR检测试剂盒进行交叉验证。结果：检测试剂盒对甲型和B型流感病毒具有特异性，动态范围至少为7个对数。试验变异性在可接受限度内，但向定量下限增加，即3.33 log 10病毒cDNA拷贝/ml病毒转运培养基（10个病毒RNA拷贝/PCR）。试验组分足够耐用，可耐受长期储存和多次冻融循环。对于真实的时间PCR检测，定量限相当于病毒感染性临界值，相当于灵敏度增加93倍。总结：良好表征的真实的时间PCR测定提供了对用于测量临床样本中的流感A和B的毒株的病毒载量的现有方法的显著改进。
Background: The antiviral effect of anti-influenza drugs such as zanamivir may be demonstrated in patients as an increased rate of decline in viral load over a time course of treatment as compared with placebo. Historically this was measured using plaque assays, or Culture Enhanced Enzyme Linked Immunosorbent Assay (CE-ELISA). Objectives: to develop and characterise real time quantitative PCR (qPCR) assays to measure influenza A and B viral load in clinical samples, that offer improvements over existing methods, in particular virus infectivity assays. Study design: The dynamic range and robustness were established for the real time qPCR assays along with stability of the assay components. Cross validation of the real time PCR assays with CE-ELISA was performed by parallel testing of both serial dilutions of three different subtypes of cultured virus and a panel of influenza positive throat swab specimens. Results: the assays were specific for influenza A and B and the dynamic ranges were at least seven logs. The assay variability was within acceptable limits but increased towards the lower limit of quantification, which was 3.33 log10 viral cDNA copies/ml of virus transport medium (ten viral RNA copies/PCR). The components of the assay were robust enough to withstand extended storage and several freeze–thaw cycles. For the real time PCR assays the limit of quantification was equivalent to the virus infectivity cut off, which equates to a 93-fold increase in sensitivity. Conclusion: Well characterised real time PCR assays offer significant improvements over the existing methods for measuring the viral load of strains of influenza A and B in clinical specimens.