Detecting the Neuraminidase R294K Mutation in Avian Influenza A (H7N9) Virus Using Reverse Transcription Droplet Digital PCR Method
Xiuyu Lou,
Hao Yan,
Lingxuan Su,
Yi Sun,
Xinyin Wang,
Liming Gong,
Yin Chen,
Zhen Li,
Zhongbiao Fang,
Haiyan Mao,
Keda Chen,
Yanjun Zhang
Affiliations
Xiuyu Lou
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
Hao Yan
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
Lingxuan Su
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
Yi Sun
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
Xinyin Wang
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
Liming Gong
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
Yin Chen
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
Zhen Li
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
Zhongbiao Fang
Shulan International Medical College, Zhejiang Shuren University, Hangzhou 310015, China
Haiyan Mao
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
Keda Chen
Shulan International Medical College, Zhejiang Shuren University, Hangzhou 310015, China
Yanjun Zhang
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
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 (p = 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.
