Real time PCR is one of the major tools for molecular diagnosis, however not always reaches the required sensitivity, especially in detecting early infectious disease. To overcome this problem, nested PCR is commonly performed, since it is highly sensitive, but it is time-consuming, prone to cross-contamination and difficult to standardize. Therefore, we developed a sensitive and specific single-tube nested real-time PCR (STN-real-time PCR) assay and evaluated its clinical utility on early infant HIV-1 diagnosis (EID). The STN-real-time PCR enables the simultaneous amplification of four HIV-1 specific amplicons by the use of an internal and external pair of primers targeting ltr/gag region, and another one corresponding to human albumin as an internal control. Thermocycling had different annealing temperatures to favor the sequential use of each pair of primers, and included an initial touchdown step to broaden specificity and increase sensitivity. Finally, HIV-1 was detected by melting curve analysis. A total of 234 samples collected retrospectively and prospectively from HIV-1 exposed infants aged <18 months were used to evaluate the performance of the assay and compare it with a routine diagnostic nested-multiplex PCR. The developed assay had a limit of detection of 3 copies of HIV-1 DNA per reaction and had a sensitivity of 31 % more than routine diagnostic nested-multiplex PCR when testing samples near delivery. In conclusion, we developed a new assay by turning a conventional nested-PCR into a faster, more sensitive and feasible STN-real-time PCR assay for EID and potentially useful for detection of pathogens with variable genomes and present in low copy numbers.

实时荧光定量 PCR 是分子诊断的主要工具之一，但并不总能达到所需的灵敏度，尤其是在检测早期传染病时。为了克服这个问题，通常进行巢式PCR，因为它高度敏感，但耗时、容易交叉污染且难以标准化。因此，我们开发了一种灵敏且特异的单管巢式实时 PCR (STN-real-time PCR) 检测方法，并评估了其在早期婴儿 HIV-1 诊断 (EID) 中的临床效用。STN 实时 PCR 通过使用靶向ltr/gag的内部和外部引物对能够同时扩增四个 HIV-1 特异性扩增子区域，另一种对应于人白蛋白作为内部对照。热循环具有不同的退火温度以有利于每对引物的顺序使用，并包括初始着陆步骤以扩大特异性并提高灵敏度。最后，通过熔解曲线分析检测出HIV-1。从年龄小于 18 个月的 HIV-1 暴露婴儿中回顾性和前瞻性地收集了总共 234 个样本，用于评估检测的性能，并将其与常规诊断性嵌套多重 PCR 进行比较。开发的检测方法限制每次反应检测 3 个 HIV-1 DNA 拷贝，并且在检测交付附近的样品时，其灵敏度比常规诊断性嵌套多重 PCR 高 31%。总之，我们开发了一种新的检测方法，将传统的巢式 PCR 转变为更快、