Key Laboratory of Public Health Detection and Etiological Research of Zhejiang Province, Department of Microbiology, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China.
Shulan International Medical College, Zhejiang Shuren University, Hangzhou 310015, China.
Viruses. 2023 Apr 17;15(4):983. doi: 10.3390/v15040983.
The R294K mutation in neuraminidase (NA) causes resistance to oseltamivir in the avian influenza virus H7N9. Reverse transcription droplet digital polymerase chain reaction (RT-dd PCR) is a novel technique for detecting single-nucleotide polymorphisms. This study aimed to develop an RT-dd PCR method for detecting the R294K mutation in H7N9. Primers and dual probes were designed using the H7N9 NA gene and the annealing temperature was optimized at 58.0 °C. The sensitivity of our RT-dd PCR method was not significantly different from that of RT-qPCR ( = 0.625), but it could specifically detect R294 and 294K in H7N9. Among 89 clinical samples, 2 showed the R294K mutation. These two strains were evaluated using a neuraminidase inhibition test, which revealed that their sensitivity to oseltamivir was greatly reduced. The sensitivity and specificity of RT-dd PCR were similar to those of RT-qPCR and its accuracy was comparable to that of NGS. The RT-dd PCR method had the advantages of absolute quantitation, eliminating the need for a calibration standard curve, and being simpler in both experimental operation and result interpretation than NGS. Therefore, this RT-dd PCR method can be used to quantitatively detect the R294K mutation in H7N9.
神经氨酸酶（NA）中的 R294K 突变导致 H7N9 禽流感病毒对奥司他韦产生耐药性。逆转录液滴数字聚合酶链反应（RT-dd PCR）是一种用于检测单核苷酸多态性的新技术。本研究旨在开发一种用于检测 H7N9 中 R294K 突变的 RT-dd PCR 方法。使用 H7N9 NA 基因设计引物和双探针，并将退火温度优化至 58.0°C。我们的 RT-dd PCR 方法的灵敏度与 RT-qPCR 没有显著差异（ = 0.625），但它可以特异性地检测 H7N9 中的 R294 和 294K。在 89 份临床样本中，有 2 份显示出 R294K 突变。这两株病毒使用神经氨酸酶抑制试验进行评估，结果表明它们对奥司他韦的敏感性大大降低。RT-dd PCR 的灵敏度和特异性与 RT-qPCR 相似，其准确性与 NGS 相当。与 NGS 相比，RT-dd PCR 方法具有绝对定量、无需校准标准曲线、实验操作和结果解释更简单的优点。因此，这种 RT-dd PCR 方法可用于定量检测 H7N9 中的 R294K 突变。
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